Rozprawy doktorskie na temat „Protein-small molecule interactions”

Thesis (Ph.D.)--Boston University
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The binding of small molecules to a protein is among the most important phenomena in the chemistry of life; the activity and functionality of many proteins depend critically on binding small molecules. A deep understanding of protein-small molecule interactions and the interplay between ligation and function can give valuable insight into key systems of interest. The complete characterization of any small molecule-protein interaction requires quantification of many interactions and the pursuit of such information is the purpose of this body of work. The discovery of binding regions on proteins, or "hot spots," is an important step in drug development. To this end, a highly regarded and robust fragment-based protocol has been developed for the detection of hot spots. Firstly, we use this protocol in conjunction with other computation techniques, such as homology modeling, to locate the allosteric binding site of £-phenylalanine in Phenylalanine Hydroxylase. Secondly, computational fragment mapping was employed to locate the site of allostery for Ras, an important signaling protein. Lastly, the identification of hot spots for many unligated protein targets is presented highlighting the importance of a reliable way to predict druggability computationally. The second part of this dissertation shifts focus to the development of electrostatic models of small molecules. It is widely believed that classical potentials can describe neither vibrational frequency shifts in condensed phases nor the response of vibrational frequencies to an applied electric field, the vibrational Stark effect. In this work, an improved classical molecular electrostatic model for the CO ligand was developed to faithfully model these phenomena. This model is found to predict the vibrational Stark effect and Fe-CO binding energy with unprecedented accuracy for such a classical model. As an extension of this work, a geometrically dependent water potential was developed. This work has shown that comparison of results obtained from current water models against experimentally determined proton momentum distributions is an invaluable benchmark
2031-01-01

The solid phase combinatorial method is an excellent tool for the modulation of protein-protein interactions through focused library generations. Nucleophilic aromatic substitution reactions with an iodinated template on solid phase has opened a door for easy and pure libraries of 13-22 membered medium and macrocyclic peptidomimetics. These peptide mimics showed promising activities for tyrosine kinase receptors. Iodine functionality can then be used to modify the products, on the resin, via Sonogashira and Suzuki couplings and presumably through other organometallic catalysis. The coupled products can have conformational biases that differ from the iodinated macrocycles. These coupling reactions also provide a means to introduce additional pharmacophores and to adjust the solubilities of the products. The fluorinated template also gave libraries of cyclic peptidomimetics on solid phase in good yields and purities. These libraries have improved water solubility over the iodinated libraries. The 3-fluorinated template yielded better results than the 5- fluorinated template. Some compounds showed biological activities in cell survival assays providing strong support of our approach to mimic external β-turn sequences in target proteins. Intrasite dimerization with 1,5-hexadiyne gave a homodimer as a byproduct. Solidphase synthesis of bivalent turn mimics with fluorescent tags has been demonstrated. The key feature of this synthetic route is that homo- and hetero-dimers can be formed chemoselectively from unprotected monomeric precursors. The dimerization reaction is very mild and versatile, as only potassium carbonate is required to affect the coupling. Solution phase library synthesis of small molecule mimics is presented. Some monomers of full sequence mimics have been prepared to afford dimer generations. Theses monomers were combined with linker handles to afford diverse length of dimers. Final combination of monomers to make bivalent compounds is in progress.

This thesis describes different aspects of protein interactions. Initially the function of peptides and their conjugates with small molecule inhibitors on the surface of Human Carbonic Anhydrase isoenzyme II (HCAII) is evaluated. The affinities for HCAII of the flexible, synthetic helix-loop-helix motif conjugated with a series of spacered inhibitors were measured by fluorescence spectroscopy and found in the best cases to be in the low nM range. Dissociation constants show considerable dependence on linker length and vary from 3000 nM for the shortest spacer to 40 nM for the longest with a minimum of 5 nM for a spacer with an intermediate length. A rationale for binding differences based on cooperativity is presented and supported by affinities as determined by fluorescence spectroscopy. Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance (HSQC) spectroscopic experiments with 15N-labeled HCAII were used for the determination of the site of interaction. The influence of peptide charge and hydrophobicity was evaluated by surface plasmon resonance experiments. Hydrophobic sidechain branching and, more pronounced, peptide charge was demonstrated to modulate peptide – HCAII binding interactions in a cooperative manner, with affinities spanning almost two orders of magnitude. Detailed synthesis of small molecule inhibitors in a general lead discovery library as well as a targeted library for inhibition of α-thrombin is described. For the lead discovery library 160 members emanate from two N4-aryl-piperazine-2-carboxylic acid scaffolds derivatized in two dimensions employing a combinatorial approach on solid support. The targeted library was based on peptidomimetics of the D-Phe-Pro-Arg showing the scaffolds cyclopropane-1R,2R-dicarboxylic acid and (4-amino-3-oxo-morpholin-2-yl)- acetic acid as proline isosters. Employing 4-aminomethyl-benzamidine as arginine mimic and different hydrophobic amines and electrophiles as D-phenylalanine mimics resulted in 34 compounds showing IC50 values for α-thrombin ranging more than three orders of magnitude with the best inhibitor showing an IC50 of 130 nM. Interestingly, the best inhibitors showed reversed stereochemistry in comparison with a previously reported series employing a 3-oxo-morpholin-2-yl-acetic acid scaffold.

Modern spectroscopic techniques grant various methods for a protein structure determination among with a ligand interaction. This work aims at probing the structural insights of a protein-small molecule interaction with biocrystallography and optical spectroscopies. Two independent systems were investigated in the frame of this thesis. The first one involves flavoenzyme interaction with a natural nucleotide as a cofactor required for its catalytic activity and work was purely based on macromolecular crystallography. The second concerns incorporation of a synthetic fluorescent ligand into a model protein as a solution for hydrophobicity of the probe. Due to the nature of the probe optical spectroscopies (such as absorption, fluorescence lifetime, circular dichroism) were effectively employed together with the crystallographic methodology.

The Cullin-RING E3 ligases (CRLs) are the largest subfamily of E3 ligases that participate in many biological processes determining the fate of proteins by catalysing ubiquitin transfer to specific substrates for proteasomal degradation. SOCS2 is a component of the multisubunit CRL E3 complex (CRL5SOCS2). SOCS2 plays important roles in several cancers and is involved in diabetes and inflammatory diseases. This work aims to understand the substrate recognition mechanism of SOCS2 at an atomic level and provides structural insights to guide the development of small-molecule tools and potential drug leads. Current structural information on SOCS2 is limited to apo form (no ligand bound). In the first part of the work, two novel SOCS2-ElonginB-ElonginC (SBC) structures in complex with substrate peptides of growth hormone receptor (GHR) and erythropoietin receptor (EpoR) were solved by X-ray crystallography with a goal to elucidate the SOCS2 recognition mechanism. Different interactions of the peptides were observed in the structures as a consequence of divergences in the peptide sequences, revealing residues required to catch specific interactions and a protein loop rearrangement as a result of the binding event. An alanine scanning of substrate peptides allowed cross-validation of the structures and identified critical interactions. Based on the crystal structure, five residues that interact with GHR were selected for which single-nucleotide polymorphisms (SNPs) are known in cancer. The results show that the SNPs mutants of SOCS2 located at the phosphotyrosine (pY) pocket are highly disruptive and abolish substrate recognition, suggesting a significant impact to SOCS2 mediated interactions. The second and third part of the work focused on the ligand development at the pY pocket of SOCS2 SH2 domain using a combination of X-ray crystallography and biophysical techniques. Novel crystal structures of SBC in complex with pY and pY analogues were obtained, providing a starting point for compound design. A screening cascade consisting of nuclear magnetic resonance (NMR), surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) was established and validated by an in-house small library of pY analogues. This workflow will facilitate the process of ligand characterisation and design towards a potent binder. The findings of this work unravel interactions of SOCS2 with its substrates in mechanistic detail. Together with the small molecule bound structures and biophysical screening assays, this work provides insights and tools to assist future ligand discovery for CRL5SOCS2.

Small molecules are known to play critical role in understanding most biological mechanisms of cells and organisms. Some examples, such as RNAs, peptides and drug molecules, etc., work by modulating cellular function, but with unknown modes. In most cases, these actions involve the small molecule interacting with proteins serving various functions. In recent years, much effort has been made in the investigation of interactions between small molecules (ligands) and target proteins. In our laboratory, a new technique termed Dynamic Isoelectric focusing Anisotropy Binding Ligand Assay (DIABLA) is being in collaboration with the Tolley Laboratory (SIU) developed to fulfill this task. In this technique, a protein mixture is separated within the capillary using dynamic isoelectric focusing, while a specific small molecule is evenly distributed throughout the capillary. Fluorescence anisotropy is then used to identify target proteins that bind with the ligand. In our research, emphasis has been put on evaluating optimum detection conditions for the fluorescence anisotropy aspects of the measurement. Fluorescence anisotropy has been proven to be an effective and powerful tool in evaluating ligand-protein interactions. In our studies, various protein-ligand systems are investigated, especially inhibitor-cyclooxygenase (COX) systems which include naproxen-COX system, ibuprofen-COX system, resveratrol-COX system and COX inhibitor II-COX system. Other systems include biotin-streptavidin system and progesterone-progesterone receptor system. Several fundamental parameters (concentration, pH, etc.) that affect the detection of fluorescence anisotropy measurement are evaluated. In addition, non-specific binding of the ligands with BSA was also tested as a comparison to specific binding of ligand-COX. By optimizing the binding conditions, the detection limit of using fluorescence anisotropy technique was found to be as low as nanomolar concentrations, which is much improved compared to the current literature reported micromolar regime. A binding curve representing the anisotropy's value as a function of protein concentration was constructed experimentally for each study system. On another study, mathematical calculation of the binding curve was also carried out by Wolfram Mathematica for prediction of the binding curve as well as estimation of the dissociation constant (Kd). By simply curve fitting experimental data to our simulated binding curve, with known ligand concentration, the dissociation constant (Kd) can be obtained with very high accuracy relative to current reported value. Isoelectric focusing coupled fluorescence anisotropy was also performed on the laboratory built system to test the validation of DIABLA. Three standard dyes, rose bengal, erythrosin B and Ru(bpy)3 were used for calibration of the in-laboratory built instrument. Fluorescence measurements were performed in both Horiba Jobin Yvon fluorimeter and our in-laboratory built DIABLA equipment by Cecil Bailey. Good correspondence of data acquired by DIABLA equipment and Horiba fluoremeter was successfully obtained, which proves the validation of DIABLA. Ongoing research is focusing on investigation of the standard dyes as well as some protein mixtures in capillary using DIABLA equipment. In another study, in investigation of inhibitor-COX system, fluorescence properties of most inhibitors were tested for further applications. Fluorescence excitation and emission spectra, fluorescence quantum yield, as well as fluorescence lifetimes were tested with the inhibitors dissolved in both ethanol and water. The difference of fluorescence properties observed in different solvents revealed the solvent effects as well as some possible intramolecular transitions or intermolecular interactions, such as internal charge transfer (ICT) and molecule aggregations.

G-quadruplex secondary structures are four-stranded globular nucleic acid structures that form in specific DNA and RNA G-rich sequences with biological significance, such as those found in human telomeres, oncogene promoter regions, replication initiation sites, and 5’- and 3’-untranslated (UTR) regions, which have been identified as novel drug targets. The non-canonical G-quadruplex secondary structures readily form under physiologically relevant ionic conditions, and exhibit great diversity in their topologies and loop conformations depending on the DNA or RNA sequences at hand. The structural diversity of these unique secondary structures is essential to their specific recognition by different regulatory proteins or small molecule compounds. A significant amount of research has been done in this field that provides compelling evidence for the existence, biological significance, and potential druggability of G-quadruplexes. In this dissertation, I explore G-quadruplex formation in the promoters of BCL2, PDGFR-β and c-Myc oncogenes and their interactions with small molecule compounds or proteins. Firstly, I investigated a newly-identified G-quadruplex (P1G4) forming immediately upstream of the human BCL2 gene, which has been found to be overexpressed in several human tumors. In this research, I have found that P1G4 acts as a transcription repressor, and that its inhibitory effect can be enriched by the G-quadruplex-interactive compound, TMPyP4. Both P1G4 and the previously reported Pu39 G-quadruplexes form independently in adjacent regions within the BCL2 P1 promoter, but P1G4 appears to play a more dominant role in repressing transcriptional activity. NMR and CD studies have shown that the P1G4 G-quadruplex appears to comprise a novel dynamic equilibrium of two parallel structures, one regular, with two 1-nt loops and a 12-nt middle loop, and another broken-stranded, with three 1-nt loops and an 11-nt middle loop; both structures adopt a novel hairpin (stem-loop duplex) conformation in the long central loop. This dynamic equilibrium of two closely-related G-quadruplex structures with a unique hairpin loop conformation may provide a specific target for small molecules to modulate BCL2 gene transcription. I also explored the 3’ end G-quadruplex that forms within the core promoter of PDGFR-β, which has also been observed to be present at abnormal levels in a variety of clinical pathologies, including malignancies. The 3′-end G-quadruplex formed in the PDGFR-β promoter NHE appears to be selectively stabilized by an ellipticine analog, GSA1129, which can shift the dynamic equilibrium in the full-length sequence to favor the 3′-end G-quadruplex, and can repress PDGFR-β activity in cancer cell lines. NMR studies in combination with biophysical experiments have shown that in the wild-type extended 3ʼ-end NHE sequences, two novel intramolecular G-quadruplexes can be formed in a potassium solution, one with a 3’-flanking distant guanine inserted into the 3’-external tetrad (3’-insertion G-quadruplex), and another with a 5’-flanking distant guanine inserted into the 5’-external tetrad (5’-insertion G-quadruplex). Further investigation of the elongated PDGFR-β 3′-end sequence containing both the 5’- and 3’- flanking guanine sequences showed the formation of a combination of the two G-quadruplexes existing in equilibrium. Importantly, it was observed that GSA1129 can bind to and increase the stability of each of the end-insertion G-quadruplexes, raising their Tₘ by 25 degrees. This study highlights the dynamic nature of the 3′-end NHE sequence and the importance of identifying the proper sequence for the formation of biologically relevant G-quadruplex structures. Significantly, the dynamic nature of the 3′-end G-quadruplex suggests that it may be an attractive target for drug regulation. I then analyzed two proteins, Nucleolin and NM23-H2, which interact with the c-Myc G-quadruplex structure that forms in the proximal promoter region of the c-Myc gene; this is one of the most commonly deregulated genes in the human neoplasm. Nucleolin is known to be a transcriptional repressor for c-Myc, binding to and stabilizing the c-Myc G-quadruplex, whereas NM23-H2 is known to be a transcriptional activator that unwinds and destabilizes the c-Myc G-quadruplex. An investigation of the molecular mechanisms of the interaction between the c-Myc G-quadruplex and nucleolin showed that the minimal binding domains required for a tight binding of the protein to the c-Myc G-quadruplex are the four RNA binding domains (RBDs) of nucleolin, referred to as Nuc1234, and that the RGG domain is unnecessary for c-Myc G-quadruplex binding. The stable G-quadruplex formed within Pu27 using G-tract runs I, II, IV and V was determined to be the best substrate (Myc1245T) for nucleolin binding, showing the highest affinity. 3D NMR experiments performed on the free protein Nuc1234 and its complex with the Myc1245T G-quadruplex have shown that upon complex formation, only the disordered linker regions of the protein display significant chemical shift changes, whereas most other residues show chemical shift values similar to those of the free protein. The c-Myc G-quadruplex has three loops that flip outward in a solvent containing K⁺, according to its structure. The hypothesis for this association is that nucleolin wraps around the G-quadruplex and interacts specifically with the flipped-outward loop regions of the c-Myc G-quadruplex via its own inter-RBD linker regions, with little structural change in the RBDs themselves. A definitive determination of the 3D molecular structure of nucleolin and its complex with Myc1245T is currently in development. Biophysical and structural studies were then conducted to investigate the interactions of the protein NM23-H2/NDP kinase B with the c-Myc G-quadruplex. NM23-H2 binds to single-stranded guanine- and cytosine-rich sequences, but not to double-stranded DNA in the NHE III₁ region; the binding therefore appears structure-specific, rather than sequence-specific. Moreover, increasing concentrations of the strong G-quadruplex-interactive compound TMPyP4, a porphyrin-based drug, inhibits the binding of NM23-H2 to the NHE III₁ region; this suggests that the stabilization of the G-quadruplex hinders the recognition and remodeling function of the NM23-H2. By conducting Forster Resonance Energy Transfer (FRET) assays in combination with Circular Dichroism (CD) studies, I demonstrated that NM23-H2 can actively resolve the c-Myc G-quadruplex. Taken together, these results show that the use of small molecules to prevent NM23-H2 from binding to and resolving the NHE III₁ region G-quadruplex may have the potential to inhibit c-Myc transcription for cancer therapeutic purposes. This underlines the importance of understanding the mechanism of function operating between NM23-H2 and the c-Myc G-quadruplex. Understanding molecular mechanism between NM23-H2 and c-Myc is under investigation.

Protein-protein interactions (PPIs) mediate a wide variety of cellular processes, being essential for the regulation of most biological pathways. During the last years, targeting the PPIs complex network has been recognized as an emerging and promising drug discovery strategy for the therapeutic intervention of a number of pathologies. Here, we focus on the 14-3-3 adapter protein PPIs and its modulation through small molecules. This family of conserved proteins is expressed in all eukaryotic cells and maintains essential roles in regulatory processes by binding several hundred identified partners. Due to their involvement in a wide range of physiological and biological events, this protein has been related to numerous diseases, such as cancer and neurodegenerative and metabolic disorders. Despite the great effort made in the modulation of 14-3-3 complexes, only a few successful examples have been reported in the literature being most of them on the disruption of 14-3-3 PPIs. However, the mere identification of these compounds illustrates the feasibility of this approach and encourages the continued development of this discovery strategy. The aim of this thesis is first to provide a summary of the different approaches investigated for the modulation of PPIs and a review of some of the latest examples on inhibition and stabilization with a special focus on the modulation of 14-3-3 adapter protein PPIs. Inside the 14-3-3- PPIs topic, three projects were developed. The first one used the parasitic 14-3-3 as a target, leading to the successful discovery of a family of small molecular inhibitors, considered an innovative strategy for the treatment of tropical neglected diseases. While the second and third ones spotlight the 14-3-3/c-Abl interaction and its relationship with the development of chronic myeloid leukaemia. In this perspective, this thesis has elucidated the 14-3-3 protein as a highly relevant target in drug discovery and proved that modulation of these interactions is a valuable tool for the development of alternative therapies.

La méthode de conception de médicaments à partir de molécules « fragments » (connue sous le nom de « Fragment-Based Drug Design ») a été proposée au milieu des années 90, et a depuis été reconnue comme une alternative tangible aux techniques plus classiques de recherche de médicaments telles que le criblage à haut débit par exemple. La méthode des fragments consiste à cribler un petit nombre (< 10000) de composés organiques de faible poids moléculaire (< 300 Da) afin de détecter ceux qui se lient à la cible (protéine ou acides nucléiques). Du fait de leur faible complexité, les fragments présentent une affinité faible pour la cible, et la détection s'effectue généralement grâce à une technique biophysique (en particulier, résonance magnétique nucléaire (RMN), cristallographie aux rayons X, résonance plasmonique de surface). Les fragments « hits » sont ensuite modifiés par addition de nouvelles fonctions chimiques, ou par liaison de deux fragments, afin d'élaborer, étape par étape, une molécule capable d'établir des interactions plus nombreuses avec la cible, et d'améliorer ainsi l'affinité. Comparée aux méthodes classiques de criblage haut débit, la méthode des fragments offre divers avantages, notamment une meilleure exploration de l'espace chimique, une meilleure efficacité de liaison des molécules « hits », et une plus grande facilité d'optimisation des hits en molécules plus affines. Dans le cadre de ce projet de thèse, plusieurs aspects de la méthode des fragments ont été abordés : dans une première partie, nous étudions un cas concret d'application de la méthode des fragments à la recherche d'un inhibiteur de la peroxiredoxine 5 humaine, en utilisant la RMN comme outil de criblage des fragments ainsi que comme outil d'étude des interactions protéine-fragment. La découverte d'un inhibiteur de cette enzyme représente une avancée importante, qui devrait permettre de mieux comprendre son fonctionnement. Les autres parties de ce projet de thèse abordent des aspects plus méthodologiques de la méthode des fragments : les fragments conservent-ils leur site de liaison, leur efficacité de liaison et leur mode d'interaction au cours de leur élaboration en inhibiteur ? Les fragments peuvent-ils être spécifiques d'une protéine ? D'un site de liaison particulier ? Ces questions, rarement traitées, sont pourtant essentielles à la compréhension du comportement des molécules fragments, et sont abordées d'une part en défragmentant plusieurs inhibiteurs de la protéine Bcl-xL et en étudiant par RMN le comportement de ces fragments vis-à-vis de la protéine en termes d'affinité et de site de liaison, d'autre part en réalisant le criblage par RMN d'une série de fragments sur cinq protéines différentes (peroxiredoxine 5 humaine, sérum albumine humaine et trois protéines homologues de la famille Bcl-2). De manière générale, ce projet de thèse vise à étudier des aspects peu abordés de la méthode des fragments et à proposer des pistes permettant de mieux comprendre le comportement des fragments vis-à-vis de leur cible, au cours du criblage initial comme lors de leur optimisation
Fragment-Based Drug Design (FBDD) has been proposed in 1996 and has since been recognized as a tangible alternative to the more classical drug discovery methods such as High-Throuput Screening. FBDD consists of screening a small number (< 10 000) of low-molecular weight (< 300 Da) compounds and detect those that bind to the target (protein or nucleic acids). Because of their low complexity, fragment molecules usually display low affinities for their target, hence detecting fragment-protein interactions is mostly achieved using a biophysical technique (mostly Nuclear Magnetic Resonance (NMR), X-ray crystallography or Surface Plasmon Resonance). “Hit” fragments are then modified by addition of chemical substituents, or linked together, so as to elaborate a more complex molecule, forming more interactions with the target and hence displaying an improved affinity. As compared to the more classical High Throughput Screening method, fragment screening provides several advantages, including a better exploration of chemical space, highly ligand-efficient hits and easier optimization of the hits into more affine molecules. In this PhD project, several aspects of FDBB have been addressed : first, FBDD approaches were applied to the research of an inhibitor of the human peroxiredoxin 5 protein, using NMR not only as a screening method but also for the characterization of the protein-fragment interactions. The discovery of an inhibitor against this enzyme would allow to better understand its function. Next, methodological aspects of the FBDD method were addressed : Do fragments conserve their binding site, binding efficiency and mode of interaction upon optimization? Can the fragments display specificity towards a given target? Towards a given binding site? These issues, rarely studied, are yet essential to the understanding of the behavior of fragment molecules, and will be addressed firstly by defragmentating several Bcl-xL inhibitors into fragments and studying their behavior towards the protein in terms of a_nity and binding mode, secondly by screening a set of fragments against five different proteins (human peroxiredoxin 5, human serum albumin and three homologous proteins of the Bcl-2 family of proteins). More generally, this PhD project aims at studying less characterized aspects of the fragment methodology and proposing answers to better understand the behavior of fragment molecules towards their targets, both in the initial screening step and then during their optimization

In Chapter 2, bis- and tris- arylsulfonamides, were investigated as possible inhibitors of the p53-MDM2 protein-protein interaction (PPI). The lead compound, 19, inhibited the PPI, in a fluorescence polarisation (FP) based competitive binding assay with ICso 26.4 pM and the most potent analogue, 66, with ICso 3 μM. The active compounds in this series, possess a 5-chloro-4-nitro-2-sulfonamoyl substituted thiophene ring that is very susceptible to SNAr reactions at the 5-position. Analogues of 19 and 66 were prepared to investigate the SAR of these inhibitors. No improvements in activity or structural activity relationship (SAR) consistent with MDM2 binding were observed and no active analogues without the reactive functionality were found. These compound are no longer being investigated. Chapter 3 describes a 3-D shape-based virtual screening campaign to find new lead compounds. Using queries based on the established Nutlin, benzodiazepine and spiro-oxindole inhibitors, the ZINC database was screened using the program ROCS to find compounds that have good shape similarity (measured by 3D Tanimoto) and similar functional group overlap to the query molecules. 155 compounds were purchased and tested 16 of which inhibited the MDM2-p53 PPI in the FP assay at IC50 ranging between 48.22 and 140.42 1tM. Three analogues, 156,168 and 180, induce low levels of p53 induction in cells using a Luciferese based reporter gene assay with most the potent compound, 180, showed 5.75 fold induction at 8.89 μM. A number of the hit compounds warrant further investigation. Chapter 4 describes the investigation 1.5-benzodiazepiene-2-ones and 1,3-dihydrobenzimidazolin-2-ones as novel scaffolds on which to base potential p53 inhibitors. A small series of analogues of each class were prepared and their ability to disrupt the MDM2-p53 PPI determined using an FP assay. None of the 1.5-benzodiazepiene-2-ones showed any inhibition of the PPI at concentrations up to 500 μM. Some of the 1,3 dihydrobenzimidazolin-2-one based compounds showed low levels of inhibition with the most potent analogue, 214 having IC5o 196.18 PM. These inhibitors showed some SAR based on the size of substituents and the presence of a 6-chloro substituent that has been shown to considerably enhance the activity in other classes of inhibitor. Compounds of this type warrant further investigation using a more diverse compound library.

In all kinds of disease models, many proteins involved in protein-protein interactions (PPIs) are mutated and do not function properly. The important role of PPIs in disease makes the design of small molecule inhibition an interesting proposition. This project looks at mouse double minute 2 (MDM2) and mouse double minute X (MDMX) which binds and inhibits the tumour suppressor protein p53. MDM2 and MDMX are therefore attractive therapeutic targets due to their role in tumour progression. The aim is to identify small molecule dual inhibitors that are able to disrupt MDM2 and MDMX from binding to p53. Both N-terminal MDM2 and MDMX were successfully expressed and purified with high purity and decent yield. These proteins were used to develop Fluoresence Polarization (FP) and Capillary Electrophoresis (CE) assays for small molecule inhibitors screening. This work has successfully developed FP and CE assays for detecting weakly interacting fragments. The CE assay is a novel method for detecting weak fragments for protein-protein interactions, which are a challenging target. Two approaches were employed to identify small molecule inhibitors for MDM2- N/p53 interaction. At first, small molecules were identified using in silico screening and these hits were verified using FP and CE assays. Second, analogue exploration was applied to identify fragments from the small molecule inhibitors discovered from the in silico screening. Diphenylamine and oxindole fragments were identified as the most potent. However, diphenylamine fragment was discovered to aggregate MDM2-N and was ranked as a false positive hit. No protein aggregation was found when incubated with the oxindole fragment. Therefore oxindole can provide a good starting point for the design of higher affinity analogues. Studying the interaction of MDMX has only recently been undertaken. MDMX contains a high homology binding site with MDM2. Hence, developing a dual MDM2/MDMX inhibitor has become an attractive target to focus on. FP and CE assays were developed to screen compounds against MDMX-N. In silico screening against MDM2-N and MDMX-N found several hits. One compound was discovered as a dual binder to MDM2-N and MDMX-N with low μM affinity. This novel hit is potentially a good starting point for the design of higher affinity analogues.

Hypoxia-inducible factor (HIF) is a heterodimeric, oxygen-dependent, transcription factor that regulates the cellular response to hypoxia by directing the expression of multiple genes, such as those involved in angiogenesis and glucose transport. HIF activation has been shown to aid the survival of cancer cells in hypoxic regions; hence it is viewed as a potentially important target for cancer therapy. There are two predominant isoforms of HIF, HIF-1 and HIF-2, formed by heterodimerisation of HIF-1 or HIF-2, respectively, with HIF-1. The dimerisation of the two subunits is necessary for DNA-binding and subsequent activation of transcription. Miranda et al. (2013) have recently identified a six amino acid cyclic peptide inhibitor of HIF dimerisation (cyclo-CLLFVY); the Tat-tagged variant is called P1. This has shown activity within several cell-based assays.1 This project sought to identify which amino acid residues of cyclo-CLLFVY were critical to its activity by synthesising five alanine analogues and testing them in cell and biophysical assays. It was not possible to identify an active motif and it could be concluded that the specific conformation of the intact cyclic peptide is required for activity. The functionality of independently bacterially expressed fragments of HIF-1 and HIF-1 was also validated by an EMSA. The Tavassoli group used these proteins to establish the binding location of the inhibitor to the HIF-1-PAS-B domain (work by A. Tavassoli and A. Male).

The MDM2 and MDM4 oncoproteins are both involved in regulating the tumour suppressor, p53. While the MDM2–p53 interface is structurally and biophysically well characterised, the MDM4-p53 interaction has only recently attracted researchers’ attentions. The goal of this project was to establish structural and chemical ground rules for the disruption of the interactions between the N-terminal domains of MDM2/4 and p53, which is an attractive anticancer strategy. In the current work, successful recombinant production and purification protocols for both the N-terminal domains of MDM2 (i.e. MDM2-N, residues 11-118) and MDM4 (MDM4-N, residues 14-111) have been established, yielding protein in sufficient quantity and quality for analysis using nuclear magnetic resonance spectroscopy (NMR). Two screening strategies were employed to identify small-molecule antagonists of the MDM2-N:p53 interaction. First, a virtual screening exercise identified several compounds that were shown (by NMR) to bind to MDM2-N with μM KDs. Docking studies supported by NMR chemical shift perturbation analysis suggested proposals for binding modes. The results are discussed in relation to the previously reported binding to MDM2-N of well-characterised inhibitors of the MDM2:p53 interaction such as Nutlin-3. Second, a fragment-based library was screened against MDM2-N using TROSY-type NMR spectra to monitor binding. Several hits were identified and the results are discussed with regard to the “druggability” of the MDM2-N p53 interaction. To better understand the p53-binding groove of MDM4-N, multidimensional NMR was used to investigate the structure and backbone dynamics of double-isotopically labelled samples of MDM4-N, both free (i.e. apo-MDM4-N) and in complexes with a p53-derived peptide or Nutlin-3. The apo-MDM4-N is more conformationally dynamic than MDM2, since it contains unstructured regions. These regions appear to become structured upon binding of a ligand. MDM4 appears to bind its ligand through conformational selection and/or an induced fit mechanism involving reorganization of key sub-sites within the binding groove. This study highlighted Abstract differences between Nutlin-3 and peptide binding that suggest the rational design of specific inhibitors of the MDM4:p53 interaction.

Small heat-shock proteins (sHsps) are part of a broad cellular sys- tem that functions to maintain a stable proteome under stress. They also perform a variety of regulatory roles at physiological conditions. Despite the multitude of sHsp targets, their interactions with partners are not well understood due to highly dynamical structures. In this thesis, I apply a variety of biophysical and structural approaches to examine distinct interactions made by the abundant human sHsps αβ-crystallin and Hsp27. First, I find that αβ-crystallin binds a cardiac-specific domain of the muscle sarcomere protein titin. A cardiomyopathy-causative variant of αβ-crystallin is shown to disrupt this interaction, with demonstrated implications for tissue biomechanics. Next, I investigate the conformation and unfolding behaviour of another sarcomere-associated protein, filamin C, finding support for the hypothesis that it is mechanosensitive. This leads into an interrogation of the interaction between filamin C and Hsp27, which we find is modulated by phosphorylation of Hsp27. This modulation only manifests during filamin C unfolding, pointing toward a protective chaperoning mode against over-extension during mechanical stress. This finding is bolstered by up-regulation and interaction of both proteins in a mouse model of heart failure. I establish a system for similar studies of a third sHsp, cvHsp, which is muscle-specific and implicated in various myopathies but scantly understood at the molecular level compared to αβ-crystallin and Hsp27. Finally, I probe the stoichiometries and kinetics of complexes formed between αβ-crystallin and Hsp27 themselves, which co-assemble into a highly polydisperse ensemble. This involved the development of a high-resolution native mass spectrometry method for disentangling heterogeneous systems. Together these findings add to our understanding of the roles and mechanisms of ATP-independent molecular chaperones.

Les protéines 14-3-3 sont des protéines adaptatrices qui exercent leurs fonctions biologiques en modulant l’activité de centaines d’autres protéines. De part leur impressionnant interactome, les protéines 14-3-3 sont des acteurs qui influencent de nombreux événements cellulaires et, par conséquent, de maladies associées. La stabilisation ou l’inhibition sélective d’interactions protéine-protéine (IPP) de 14-3-3 sont considérées comme des approches prometteuses pour trouver des thérapies innovantes contre des maladies comme la maladie d’Alzheimer, certains cancers ou la maladie de Parkinson.Notre premier but afin de trouver des petites molécules capables de moduler ces cibles a été d’étudier au niveau moléculaire des IPP de 14-3-3. Dans ce but, nous avons utilisé la Résonance Magnétique Nucléaire (RMN) pour attribuer les déplacements chimiques des atomes du squelette de 14-3-3σ. Nous avons ensuite étudié l’interaction entre 14-3-3 et la protéine Tau phosphorylée. Nous avons découvert que Tau se lie strictement dans la cavité amphipathique de 14-3-3 et peut s’ancrer aux deux monomères du dimère de 14-3-3. Nous avons aussi étudié l’interaction 14-3-3/p53 et avons découvert, en utilisant la RMN, que l’affinité du peptide p53 envers 14-3-3 est liée à des interactions intramoléculaires au niveau du peptide. Nous nous sommes enfin focalisés sur l’optimisation d’expériences RMN visant le criblage et la caractérisation de l’activité des petites molécules qui se lient à 14-3-3 ou à des complexes de 14-3-3 avec des peptides phosphorylés. Nous avons aussi utilisé des peptides phospho-mimétiques pour inhiber l’interaction 14-3-3/Tau. D’autre part, nous avons criblé une bibliothèque de fragments contre 14-3-3σ et trouvé trois hits qui se lient à des régions différentes de la protéine. Des expériences RMN ont ensuite permis de caractériser l’activité de certaines petites molécules actives sur des complexes de 14-3-3 avec, par exemple des peptides de p53 ou p65, et nous avons aussi démontré la capacité de certains de ces composés à stabiliser les complexes
14-3-3 proteins are adapter proteins that exert their biological functions by modulating the activity of hundreds of proteins. This remarkable interactome makes 14-3-3 proteins influent actors in many cellular events and, by consequence, in several pathologies. The selective stabilization or inhibition of 14-3-3 protein-protein interactions (PPIs) are therefore seen as promising approaches for finding innovative therapies for a number of conditions like Alzheimer’s, cancer or Parkinson. Our first objective towards finding small molecule modulators of these targets was to obtain the molecular detail of 14-3-3 PPIs. To this end, using Nuclear Magnetic Resonance (NMR), we assigned the backbone chemical shifts of 14-3-3σ. We then studied the 14-3-3/phosphorylated Tau interaction and found that Tau binds strictly within the amphipathic binding grove of 14-3-3 and can anchor in both monomers of the 14-3-3 dimer. We also studied the 14-3-3/p53 interaction and showed by NMR, that intramolecular interactions within the peptide define a conformation that drives the affinity towards 14-3-3. 2019We then focused on the optimization of NMR assays for screening and characterization of the effect of small-molecules binding to 14-3-3 or 14-3-3 complexes with target’s phosphopeptides. We used, for example, phospho-mimetic peptides to inhibit the Tau/14-3-3 interaction. In a different strategy, we screened a fragment library against 14-3-3σ and found three hits binding to different regions of the protein. Using our NMR assays we further characterized small molecules binding 14-3-3 complexes with, for example, p53 and p65 peptides and demonstrated the stabilization capacity of some compounds

Upregulation of epidermal growth factor receptor (EGFR) has been associated with numerous cancers such as those affecting the mammary glands, lungs and ovaries. We aimed to discover small molecule modulators which potentially promote the protein-protein interaction (PPI) between EGFR and protein tyrosine phosphatase 1B (PTP1B), which is known to dephosphorylate and inactivate the former, as a novel anticancer treatment. A combination of in silico (including protein-protein docking and structure-based virtual screening) and in vitro methods were employed to predict PPI structures and hit compounds, determine compound cytotoxicity, and elucidate possible mechanisms of action. Protein-protein docking results converged for model 17 and was rated as an “acceptable” prediction according to the CAPRI quality assessment criteria. Six hit compounds (CHB112, CHB224, CHB286, CHB331, CHB725, CHB739) which were found by structure-based virtual screening, had docking scores that ranged from -11.0 to -7.8kcal/mol. Lead compounds CHB286 and CHB331 inhibited cell viability with IC50 values of 46.0 μM and 54.5 ± 24.2 μM in HeLa cells, and 38.5 ± 4.3 μM and 35.6 ± in A431 cells, respectively. Furthermore, CHB286, CHB331 as well as CHB739 impaired colony formation in both cell lines. Western blotting analysis showed that treatment with CHB286 decreased EGFR signalling, decreased EGFR expression, as well as decreased phosphorylated c-Raf and MAPK/ERK kinase 1/2 (MEK1/2) levels in HeLa cells, which was indicative of increased formation of the EGFR-PTP1B PPI. On the other hand, all other hit compounds increased EGFR activity which implied PPI inhibition. Convergence of results from different protein-protein docking methods (i.e. ClusPro, HADDOCK and RosettaDock) generated a promising model which potentially represented the PPI between EGFR and PTP1B, which could be in turn utilized as a target for structure-based drug discovery of potential small molecule modulators of the EGFR-PTP1B PPI.

Photopharmacology is an emerging field that relies on the development of photosensitive compounds to enable precise spatiotemporal control over endogenous proteins. Photochromic ligands· are designed to respond to specific wavelengths of light with a reversible change of structure that enhances or diminishes their activity or affinity towards the desired target. Applications are expected to lead to safer treatments in medicine and to innovative tools to investigate complex signalling networks in biology. Thus, in this thesis we aimed at further expanding the spectrum of protein-protein interactions (PPis) and G protein-coupled receptors (GPCRs), that can be addressed by means of photopharmacology. To do so, we focused on resolving different shortcomings that we identified as critical for the actual performance and widespread application of photochromic compounds. For example, Traffic Light 2 (TL2) is a photoswitchable cell-permeable peptide that can inhibit clathrin-mediated endocytosis (CME) in a light regulated manner. Despite proving effective in mammalian cells, the influence of CME over several other cellular processes limited further applications in this biological system. Thus, we validated its activity in a simpler eukaryotic system, Saccharomyces cerevisiae. Using kymography and spheroplast expressing GFP-tagged Sla-1, a coat-associated endocytic protein, we characterized the effect ofTL2 and light over CME dynamics. Studying homologies between mammalian and yeast endocytic proteins we also proposed a mechanism of action for this peptide. The same strategy used to develop Tls was then applied to design new photoswitchable peptides targeting PPis involved in developmental processes. In particular, we focused on Wnt/β-catenin signalling and synthesised a library of light-regulated peptides to reversibly inhibit or activate this pathway with light. The photophysical behaviour of the peptides was characterized and the initial design was validated in vitro by means of circular dichroism and fluorescent-polarization binding assay. The activity was then verified in mammalian cells using a dual-luciferase reporter gene assay. Finally, using Schmidtea mediterranea planarians we set up a model of tissue regeneration to probe in vivo photoregulation of Wnt/β-catenin signalling. The design of photoswitchable peptides was then broadened from PPIs to signalling peptides that bind to and activate receptors. Orexins are neuroexcitatory peptides that modulate through GPCRs neural circuits involved in regulating sleep and arousal. Analysing reported crystal structures of the orexin receptors and NMR solution structures of the peptide we identified a strategy to develop a photochromic analogue of orexins. The derivative was synthesised by standard solid-phase peptide synthesis inserting a non-natural photoswitchable amino acid into the backbone of the peptide. The photochromic behaviour of the analogue was fully characterised, and its activity was assessed by means of different techniques that allow to monitor transient increases in intracellular calcium. Using circular dichroism and molecular modelling simulations we provide a structural model to explain the difference in activity observed between the trans and cis isomers. Finally, in sight of their possible therapeutic relevance, we rationally designed a small library of photochromic ligands to target the adrenergic system. In particular, we used a non-conventional approach, based on azoheteroarenes, to render photoswitchable a class of a-adrenergic ligands. We named these compounds adrenoswitches and characterised their activity in in vitro and in vivo. Affinity towards α-adrenoceptors was assessed through a radioligand binding assay in pre-frontal cortex membranes obtained post-mortem from human brains. In addition, the potency of the compounds was screened in a model of vascular reactivity using ex vivo rat aortic rings. Finally, the most promising compound of the series was used in proof-of-concept applications to manipulate with light locomotor activity in zebrafish and pupillary responses in mice.
La fotofarmacología es una técnica muy prometedora que permite controlar con luz y de manera reversible Ia actividad de compuestos activos biológicamente. En esta tesis doctoral hemos desarrollado péptidos y ligandos fotoconmutables para regular las interacciones entre proteínas y Ia actividad de receptores acoplados a proteínas G (GPCRs). En primer Iugar, validamos Ia actividad de TL2, un péptido fotoconmutable que inhibe Ia endocitosis mediada por clatrina, en un modelo simple de célula eucariota, Ia levadura Saccharomyces cerevisiae. Mediante Ia tecnica de "kimografía" en esferoplastos que expresan el marcador fluorescente GFP-Sia1, caracterizamos el efecto de TL2 en este proceso celular y su posible mecanismo de acción. Luego aplicamos Ia estrategia de diseño de TL2 para crear una librería de péptidos que inhiben o activan con luz Ia vía de señalización mediada por Wnt/β-catenina. Tras su caracterización química y fotocrómica, analizamos el efecto de los ciclos de luz sobre las estructuras secundarias de los péptidos y su union a Ia β-catenina. Para evaluar su actividad, empleamos un ensayo de luminiscencia que permite cuantificar Ia expresión génica y ponemos a punto un modelo in vivo de regeneración de tejidos en planarias Schmidtea mediterranea. A continuación, ampliamos las aplicaciones de los péptidos fotoconmutables desde Ia inhibición de las interacciones proteína-proteína descrita anteriormente, a Ia activación de receptores de neuropéptidos como las orexinas. En particular, creamos un análogo fotoconmutable de Ia orexina insertando un aminoácido con un grupo azobenceno en Ia secuencia lineal del péptido. Validamos este nuevo diseño mediante estudios estructurales de dicroísmo circular y modelado molecular, y su actividad mediante Ia medida de señales intracelulares como el calcio. Por ultimo,diseñamos y sintetizamos varios derivados fotoconmutables de ligandos α-adrenergicos insertando azoheteroarenos en sus estructuras. Mediante ensayos de unión evaluamos Ia afinidad de los compuestos hacia los adrenoreceptores de tipo α,y comprobamos su activad en un modelo de reactividad vascular en aorta de rata. Uno de los compuestos permitió manipular Ia motilidad de peces cebra y el tamaño pupilar en ratones, demostrando por primera vez el control de los adrenoreceptores con luz in vivo.

Inhibition of the ST AT3: ST AT3 protein-protein interaction is an attractive approach for cancer therapy as it can lead to suppression of tumour cell growth and induce apoptosis. The racemic ochromycinone (STA21) is one of the few known small-molecule STAT3:STAT3 inhibitors. Our synthetic efforts focused on synthesis of the natural product YM-181741, which possesses at least three points for chemical variation to prepare compound libraries as potential STAT3:STAT3 inhibitors. Synthesis of the angucycline molecule was achieved employing an approach based on a Gold (lII)-catalysed intramolecular [4+2] benzannulation reaction. A facile and highly efficient route for the preparation of racemic form of the natural product was developed that offers a high degree of flexibility for modification of the scaffold at different stages of its synthesis. The enantioselective synthesis of (S)- YM181741 was successfully carried out through the (R)-diyne building block via an enantioselective copper-catalysed 1,4-conjugate addition reaction on a system bearing a v-coordinating group, in order to install the chirality on the diyne moiety. The optimised reaction conditions afforded the Michael adduct in good yield and high enantiomeric excess (up to 96% ee). Further chemical elaboration of the (±)- YM-181741 natural product was investigated in order to explore the necessary chemical diversity to assess a preliminary model of interaction between the SH2 domain of ST AT3 and the angucycline scaffold. The biological evaluation of the focused library allowed the identification of angucycline derivatives possessing high binding affinity for the SH2 region and the ability to inhibit STAT3 transcriptional activity.

Insulin-like growth factors (IGFs) are important systemic mediators of growth and survival that suppress apoptosis and promote cell cycle progression, angiogenesis and metastatic activities in various cancers by activating IGF-IR tyrosine kinase-mediated signaling. These effects depend on the bioavailability of IGFs, which is regulated by IGF binding proteins (IGFBPs). Increased IGFBP-2 and IGFBP-5 expression observed in castration-resistant prostate cancer is thought to promote tumor progression by enhancing IGF-mediated signaling. IGFBPs have cooperative carboxy-terminal and amino-terminal low and a high affinity IGF binding sites. I hypothesize that blocking the high affinity IGF binding site can affect the bioavailability of IGFs to target tissues and thus be used for treatment of various IGF-responsive diseases including prostate cancer. I initially characterized immunologic reagents capable of being used in sandwich ELISA formats to detect IGF-I and IGFBP-5 and attempted several configurations to establish an IGF-I/IGFBP-5 “bridged” sandwich ELISA platform to measure association and dissociation of IGF-I/IGFBP-5 complex formation. The inability of all bridged ELISA formats tested to measure IGF-I/IGFBP-5 binding, lead me to developed a Bio-Layer Interferometry-based assay that measures IGF-I/ IGFBP-5 binding kinetics that will allow for screening of factors that can affect this intermolecular interaction. I demonstrated that biotinylated IGF-I bound to streptavidin-coated biosensors can be used to measure binding of recombinant IGFBP-5 [2.24 nm shift in optical density (Response)]. I also demonstrated that IGF-I could efficiently disrupt this interaction (0.21 nm shift), while the amino-terminal IGF-I mutant, E3R, exhibits an intermediate competitive activity (1.47 nm shift) and insulin exhibits a low competitive activity (1.83 nm shift). In addition, I demonstrated that IGF-I can competitively disrupted this interaction, resulting in a dissociation rate constant (Kdis 1.5-³ 1/s), In contrast, the amino terminal IGF-I mutant, E3R binds with an intermediate affinity (Kdis 5.6-⁴ 1/s), and buffer free sample results in a (Kdis) of 1.5-⁴ (1/s). These results demonstrate the capacity of this BLI-based assay to differentiate relative competitive activity of compounds that target the high affinity IGF-I binding site of IGFBPs and establish a platform to screen for factors that might be developed as rationale therapeutics to disrupt sequestration of IGF-I by IGFBPs.

Human cytomegalovirus (HCMV) is a leading cause of severe disease in immunocompromised individuals, including AIDS and transplanted patients, and in congenitally infected newborns. Despite the availability of several drugs, pharmacological treatment of HCMV infections is associated with poor bioavailability, toxicity and the emergence of resistant strains. Furthermore, no vaccine is available and no drugs are approved to prevent vertical transmission during pregnancy, therefore, it is essential to identify new potential targets of therapeutic intervention. HCMV DNA polymerase accessory protein UL44 plays an essential role in viral replication, conferring processivity to the DNA polymerase catalytic subunit UL54 by tethering it to the DNA. Binding of UL44 to dsDNA occurs in the absence of ATP and clamp loaders, and depends on UL44 homodimerization. Indeed, our research group previously demonstrated that the protein can dimerize in cells and point mutations disrupting protein self-interaction also prevent DNA binding and abolish viral OriLyt-dependent DNA replication in transcomplementation assay. Therefore, disruption of UL44 homodimerization represents an attractive target for the development of new antivirals. Based on these observations, using the recently published crystal structure of UL44 hodimers our research group previously performed a virtual screening with the Glide software in combination with a library of 1.3 x 106 small molecules (SMs) to identify SMs potentially interfering with UL44 homodimerization. After three rounds of screening (HTVS: high-throughput virtual screening, SP: standard precision, XP: extra precision), followed by an in depth analysis of compounds chemical properties, 18 SMs were selected for further analysis. Selected compounds were obtained from a commercial supplier, to be tested in a variety of assays for their ability to inhibit UL44 homodimerization both in cell and in vitro, as well as on HCMV replication. For this purpose, we applied two in vitro methods to monitor the effect of our candidates on UL44 dimerization such GST-pulldown Assay and Thermal Shift Assay (TSA). Furthermore, Plaque Reduction Assay (PRA) and Fluorescence Reduction Assay (FRA) were performed to study their inhibitory effects on viral replication. Initially we validated that GST-pulldown assay was suitable to study UL44 dimerization and its perturbation caused by SMs. Therefore, we performed a first screening to test the effect of the 18 SMs on UL44 dimerization, and we identified 3 of these which reproducibly inhibited the dimerization. Prompted by these results, we selected such SMs to investigate a possible dose-response relationship between the dimerization inhibition and the compounds concentration. Unfortunately, data analysis revealed a high variability, and lack of correlation between the inhibition and the concentration of SMs used in the assays. In parallel, PRAs were performed to validate the effect of selected SMs, but these were unable to inhibit viral replication with high potency. Subsequently, two recombinant HCMV viruses (TB4-IE2-EYFP and TB4-UL83-EYFP) were obtained from Michael Winkler (Leibniz-Institut für Primatenforschung Goettingen, Germany) in order to study the effect of our molecules on HCMV replication by FRAs. Therefore, after a screening of the 18 SMs, 4 of these were identified as possible inhibitors of viral replication and were selected to further studies. We investigated their and 50% effective dose (ED50) and 50% cytotoxic concentration (CC50) values and their effects on the viral gene expression. Only 2 SMs reproducibly inhibited expression of Early and Late genes. Then we evaluated an alternative in vitro assay and among the various possibilities, we have chosen TSA that is able to inform whether a SM induce the disruption of constitutive oligomeric interfaces. In a first moment we validated that the assay allow the discrimination between monomeric and dimeric forms of UL44, suggesting its potential for the screening of our SMs. As result of the SMs screening by TSA, one molecule revealed a possible inhibition of dimerization.
Cytomegalovirus (CMV) è un importante patogeno di interesse umano. Al momento gli antivirali disponibili ed utilizzati per la terapia contro l’infezione da CMV presentano una serie di problematica quali l’alto costo, bassa biodisponibilità, alta tossicità ed il presentarsi di ceppi virali resistenti. Inoltre non è disponibile un vaccino ed ancora non è stato approvato alcun farmaco per prevenire la trasmissione verticale durante la gravidanza. Per questi motivi, sono necessari nuovi efficaci farmaci antivirali. La proteina accessoria UL44 della DNA polimerasi di CMV, svolge un ruolo essenziale nella replicazione virale, conferendo processività alla subunità catalitica UL54 ancorando il complesso oloenzimatico al DNA. Il legame di UL44 al dsDNA avviene in assenza di ATP e dei clamp loaders, e dipende dalla omodimerizzazione di UL44. Il nostro gruppo di ricerca, infatti ha recentemente dimostrato che la proteina può dimerizzare in cellule e che mutazioni puntiformi in grado di inficiare tale dimerizzazione prevengono il legame con il DNA ed aboliscono la replicazione del DNA virale oriLyt-dipendente in saggi di transcomplementazione transiente. Perciò, la distruzione dell’omodimerizzazione UL44 rappresenta un potenziale ed allettante target per lo sviluppo di nuovi antivirali. Partendo da queste osservazioni ed usando la struttura cristallografica degli omodimeri UL44 che è stata recentemente pubblicata, il nostro gruppo di ricerca ha eseguito un virtual screening con il software Glide in combinazione con una libreria di 1.3 x 10^6 piccole molecole (SMs) per identificare SMs che potenzialmente potessero interferire con l’omodimerizzazione di UL44. Dopo tre rounds di screening (HTVS: high-throughput virtual screening, SP: standard precision, XP: extra precision), seguiti da un’analisi delle proprietà chimiche dei composti, sono state selezione 18 SM per ulteriori analisi. I composti selezionati sono stati acquistati presso un fornitore commerciale, per essere testati in diversi saggi per valutare le loro abilità di inibire l’omodimerizzazione di UL44 in vitro, sia la replicazione virale. Con questo scopo, abbiamo utilizzato due metodi in vitro per monitorare l’effetto dei nostri candidati sulla dimerizzazione di UL44, quali GST-pulldown assay e Thermal Shift Assay (TSA). Inoltre per studiare l’inibizione sulla replicazione virale sono stati eseguiti saggi di Plaque Reduction Assay (PRA) e Fluorescence Reduction Assay (FRA). Inizialmente abbiamo confermato che il GST-pulldown assay fosse idoneo per studiare la dimerizzazione di UL44 e la sua perturbazione causata dalle SMs. Pertanto abbiamo eseguito un primo screening per saggiare l’effetto delle 18 SMs sulla dimerizzazione di UL44, ed abbiamo identificato 3 molecole che inibivano la dimerizzazione in modo riproducibile. Incoraggiati da questi risultati, abbiamo selezionato queste SMs per valutare una possibile relazione dose-risposta tra l’inibizione della dimerizzazione e la concentrazione dei composti. Sfortunatamente, l’analisi dei dati hanno rivelato alta variabilità, e perdita di correlazione tra l’inibizione e la concentrazione delle SMs utilizzate nei saggi. In parallelo, abbiamo eseguito PRAs per validare l’effetto delle SMs precedentemente selezionate, ma queste si sono presentati incapaci di inibire la replicazione virale in modo consistente. Successivamente abbiamo ottenuto due virus CMV ricombinanti (TB4-IE2-EYFP and TB4-UL83-EYFP) da Michael Winkler (Leibniz-Institut für Primatenforschung Goettingen, Germany) per studiare l’effetto delle nostre molecole sulla replicazione di CMV mediante FRAs. Pertanto, dopo uno screening delle 18 SMs, 4 di queste sono state identificate come possibili inibitori della replicazione virale e sono state selezionate per essere ulteriormente studiate. Abbiamo valutato la loro dose effettiva 50% (ED50) e la loro concentrazione citotossica 50% (CC50) e gli effetti relativi all’espressione genica. Solo due SMs in modo riproducibile inibivano l’espressione dei geni Early e Late. Poi abbiamo valutato un saggio in vitro alternativo e tra le varie possibilità, abbiamo scelto il TSA che è un saggio in grado di informare se una SM induce la distruzione delle interfacce di un oligomero costitutivo. In un primo momento abbiamo confermato che il saggio permettesse la discriminazione tra le forme monomeriche e dimeriche di UL44, suggerendo il suo potenziale per lo screening delle nostre SMs. Come risultato di questo screening, una molecola ha rivelato possibile inibizione della dimerizzazione.

In the first part of the Thesis, the synthesis of different classes of peptidomimetics targeting integrin receptors is described. Both simple ligands and drug conjugates were synthesized and tested to assess their biological activity. In cancer therapy, peptides and peptidomimetics targeting integrins are employed as carriers to deliver cytotoxic drugs at the tumor site, taking advantage of integrin over-expression on the surface of tumor cells. Within this frame, synthetic efforts have been focused on the synthesis and on the conjugation to the cytotoxic agent paclitaxel of isoDGR-based integrin ligands to effect drug-targeting to integrin over-expressing tumor cells. The second part of the Thesis deals with the vascular endothelial growth factor receptors and their ligands. In particular, the interaction of VEGF-C with VEGFRs has been considered, focusing the attention on the reason why this growth factor was considered a privileged candidate for the preparation of a small library of VEGFR-selective peptides. These peptides have been synthesized by introducing systematic modifications in the natural portion of the growth factor and evaluated as VEGFR binders and anti-angiogenic agents in vitro.

Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity. As such, mutations in this family of proteins result in a wide variety of diseases from cancers to cardiomyopathies. The sHSPs Beta-2 (HspB2) and alpha-beta crystalline (CryAB) are two of the ten human sHSPs and are both expressed in cardiac and skeletal muscle cells. A heart that cannot properly recover or defend against stressors such as extreme heat or cold, oxidative/reductive stress, and heavy metal-induced stress will constantly struggle to maintain efficient function. Accordingly, CryAB is required for myofibril recovery from ischemia/reperfusion (I/R) and HspB2 is required I/R recovery as well as efficient cardiac ATP production. Despite these critical roles, little is known about the molecular function of these chaperones. We have identified over two hundred HspB2-binding partners through both yeast two-hybrid and copurification approaches, including interactions with myofibril and mitochondrial proteins. There is remarkable overlap between the two approaches (80%) suggesting a high confidence level in our findings. The sHSP, CryAB, only binds a subset of the HspB2 interactome, showing that the HspB2 interactome is specific to HspB2 and supporting non-redundant roles for sHSPs. We have confirmed a subset of these binding partners as HspB2 clients via in vitro chaperone activity assays. In addition, comparing the binding patterns and activity of sHSP variants in comparison to wild type can help to elucidate how variants participate in causing disease. Accordingly, we have used Y2H and in vitro chaperone activity assays to compare the disease-associated human variants R120GCryAB and A177PHspB2 to wild type and have identified differences in binding and chaperone function. These results not only provide the first molecular evidence for non-redundancy of the sHSPs, but provides a useful resource for the study of sHSPs in mitochondrial and myofibril function.

Human cytomegalovirus (HCMV) is a leading cause of congenital defects in humans. Currently available antivirals utilized for the therapy against HCMV infections have a series of contraindications due to their high cost, low bioavailability, high toxicity and the uprising of resistant viral strains. Furthermore no vaccine is available and no drugs are approved to prevent vertical transmission during pregnancy, therefore new, effective antiviral are highly needed. HCMV DNA polymerase accessory protein UL44 plays an essential role in viral replication, conferring processivity to the DNA polymerase catalytic subunit UL54 by tethering it to the DNA. Binding of UL44 to dsDNA occurs in the absence of ATP and clamp loaders, and depends on UL44 homodimerization. Indeed, our research group recently demonstrated that the protein can dimerize in cells and point mutations disrupting protein self-interaction also prevent DNA binding and abolish viral oriLyt-dependent DNA replication in transient transcomplementation assays. Therefore, disruption of UL44 homodimerization represents an attractive target for the development of new anti-virals. Based on these observations, using the recently published crystal structure of UL44 homodimers our research group previously performed a virtual screening with the Glide software in combination with a library of 1.3 x 10^6 small molecules (SMs) to identify SMs potentially interfering with UL44 homodimerization. After three rounds of screening (HTVS: high-throughput virtual screening, SP: standard precision, XP: extra precision), followed by an in depth analysis of compounds chemical properties, 18 SMs were selected for further analysis. Selected compounds were obtained from a commercial supplier, to be tested in a variety of assays for their ability to inhibit UL44 homodimerization both in cells and in vitro, as well as on HCMV replication. To this end, we applied a number of in cells and in vitro assays to monitor the effect of the SMs on UL44 dimerization. In cells methods included Fluorescent Resonant Energy Transfer (FRET) and Bioluminescence Resonant Energy Transfer (BRET), while as GST pull-down assays was chosen as the in vitro method. FRET acceptor photobleaching was capable of detecting both UL44 homodimerization and UL44 binding to the catalytic subunit C-terminal domain (residues 1125-1242). Such interactions were sensitive to point mutations specifically impairing the two processes, highlighting the specificity of this technique. Therefore, we were able to confirm that UL44 forms dimers in cells. However, data acquisition and analysis proved quite time consuming and dependent on high fusion proteins expression levels. BRET assays allowed to quickly and precisely quantitatively monitor UL44 self-interaction and binding to UL54 in living cells, and through saturation experiments allowed to precisely calculate Bmax and B50 values, relative to homodimerization or binding to UL54 for a number of single amino acids substitution derivatives of UL44 impaired for dimerization, binding to UL54 or to dsDNA, as well as for nuclear targeting. These data allowed gaining insights relative to the formation of HMCV DNA polymerase holoenzyme, suggesting conformational changes within UL44 upon DNA binding in complex to UL54. Furthermore, calculation of Bmax and B50 values was used to establish three different cellular systems expressing ideal amount of UL44 for screening purposes, in that they generated a BRET ratio similar to half of the Bmax. These included a fully stable expression system, whereby both RLuc-UL44 and YFP-UL44 are stably expressed in HEKA derived cells, a stable/transient system whereby YFP-UL44 is stably expressed and RLuc-UL44 is transiently expressed, or a fully transient system whereby both fusion proteins are transiently expressed. Competition assays performed overexpressing increasing amounts of FLAG-tagged UL44 as a competitor revealed that no inhibition could be obtained for the fully stably expressing system. This result highlighted the difficulty in disrupting a pre-existing protein complex and suggested to focus our attention on transient systems. Keeping this in mind, a first small-scale screening was performed to study the impact of 18 SMs on UL44 dimerization by BRET using the stable/transient system. The 18 selected SMs were resuspended in DMSO and their toxicity was evaluated in cell culture, before being added at sub-toxic concentration to the YFP-UL44 cell line, six hours post-transfection with RLuc-UL44. Our analysis identified only compounds slightly inhibiting the BRET ratio relative to UL44 homodimerization. Based on these disappointing results, we revaluated the BRET assays setup, by decreasing the time of SM incubation before assaying their effect from 42 to 18h, by switching to a completely transient system, and by decreasing the amount of expressed proteins. The last modification required a change in the substrate used for generation of bioluminescent signal from RLuc-fusion proteins from CTZ to hCTZ. A new screening was performed, which resulted in very similar results to those obtained using the original setup. Furthermore when hit candidates were re-evaluated for their effect using a negative control, their effect on BRET ratio of UL44 proved unspecific. Additionally, BRET - similarly to FRET - failed to detect a specific effect on the UL54/UL44 interaction by a SM known to disrupt such interaction. Therefore we conclude that, with our current settings, BRET and FRET are not the ideal techniques to search for SM inhibitors of protein-protein interactions We then focused on in vitro methods, starting with a GST pull-down assay, which was performed using UL44 C-terminal domain (residues 1-290), either fused to GST of a 6His-tag. Our results indicated that GST pull-down was capable of detecting differences in binding between wild-type UL44 and a dimerization-impaired mutant, suggesting a possible application for the screening of SMs. GST pull-down is currently being implemented for this purpose, and preliminary data suggest that a number of tested SMs could impair UL44 dimerization. In summary, we have developed a number of assays to monitor UL44 dimerization. Whereas in cells RET based assays confirmed that UL44 form dimers in living cells, they proved suboptimal for screening proposes. On the other hand, in vitro methods such as GST pull-down might prove more sensitive and are currently being implemented for the identification of SMs inhibitors of UL44 dimerization
Cytomegalovirus (CMV) è un importante patogeno di interesse umano. Al momento gli antivirali disponibili ed utilizzati per la terapia contro l’infezione da CMV presentano una serie di controindicazioni dovute all’alto costo, bassa biodisponibilità, alta tossicità ed il presentarsi di ceppi virali resistenti. Inoltre non è disponibile un vaccine ed ancora non è ancora stato approvato l’uso di alcun farmaco per prevenire la trasmissione verticale durante la gravidanza. Per questi motivi, sono necessari nuovi ed efficaci farmaci antivirali. La proteina accessoria UL44 della DNA polimerasi di CMV, svolge un ruolo essenziale nella replicazione virale, conferendo processività alla subunità catalitica UL54 ancorando il complesso oloenzimatico al DNA. Il legame di UL44 al dsDNA avviene in assenza di ATP e dei clamp loaders, e dipende dalla omodimerizzazione di UL44. Infatti, il nostro gruppo di ricerca ha recentemente dimostrato che la proteina può dimerizzare in cellule e che mutazioni puntiformi in grado di inficiare tale dimerizzazione prevengono il legame con il DNA ed aboliscono la replicazione del DNA virale oriLyt-dipendente in saggi di transcomplementazione transiente. Perciò, la distruzione dell’omodimerizzazione UL44 rappresenta un potenziale allettante bersaglio per lo sviluppo di nuovi anti-virali. Partendo da queste osservazioni ed usando la struttura cristallografica recentemente pubblicata degli omodimeri UL44, il nostro gruppo di ricerca ha eseguito un virtual screening con il software Glide in combinazione con una libreria di 1.3 x 10^6 piccole molecole (SMs) per identificare SMs che potenzialmente potessero interferire con l’omodimerizzazione di UL44. Dopo tre rounds di screening (HTVS: high-throughput virtual screening, SP: standard precision, XP: extra precision), seguiti da un’analisi delle proprietà chimiche dei composti, sono state selezione 18 SM per ulteriori analisi. I composti selezionati sono stati acquistati presso un fornitore commerciale, per essere testati in diversi saggi per valutare le loro abilità di inibire l’omodimerizzazione di UL44, sia in cellule che in vitro. A questo scopo, abbiamo utilizzato diversi saggi in cellule e in vitro per monitorare l’effetto delle SMs sulla dimerizzazione di UL44. Nei saggi cellulari, le tecniche utilizzate includono Fluorescent Resonant Energy Transfer (FRET) e Bioluminescence Resonant Energy Transfer (BRET), mentre per saggi in vitro è stato utilizzato il saggio GST-pull down. I nostri dati indicano che la metodica FRET acceptor photobleaching è in grado di rilevare sia l’omodimerizzazione di UL44, sia il legame con il dominio C-terminale della subunità catalitica (residui 1125-1242). Queste interazioni sono sensibili all’introduzione di mutazioni puntiformi che alterano i due processi, evidenziando la specificità di questa tecnica. Siamo stati quindi in grado di confermare che UL44 forma dimeri in un contesto cellulare. Purtroppo, l’acquisizione dei dati e la loro analisi richiedono un lungo tempo e dipendono da alti livelli di espressione delle proteine di fusione. Per contro, il saggio BRET permette un rapido e preciso monitoraggio quantitativo dell’omodimerizzazione di UL44 e il legame con UL54 in cellule viventi. Inoltre, attraverso esperimenti di saturazione, che permettono di calcolare in modo preciso i valori di Bmax e B50 relative all’omodimerizzazione o al legame con UL54 per varianti di UL44 che contengono singole sostituzioni amminoacidiche che affliggono la dimerizzazione di UL44, il suo legame a UL54 o il DNA, nonché il trasporto al nucleo della proteina. I dati ottenuti possono aiutare a comprendere il processo di formazione dell’oloenzima della DNA polimerasi di CMV, suggerendo cambiamenti conformazionali nel complesso olenzimatico in seguito a legame con il DNA. Inoltre, il calcolo di Bmax e B50 è stato usato per sviluppare tre differenti sistemi cellulari esprimenti un’ideale quantità di UL44 da utilizzare come piattaforma per lo screening di SM, poiché sono in grado di generare valori di BRET ratio simili al 50% della Bmax .Questi includono un sistema di espressione completamente stabile, in cui sia RLuc-UL44 che YFP-UL44 sono stabilmente espresse in cellule derivate da HEK293 A, un sistema ibrido stabile/transiente, in cui YFP-UL44 è stabilmente espressa e RLuc-UL44 è espressa in transiente, o un sistema completamente transiente in cui entrambe le proteine sono espresse transientemente. Saggi di competizione eseguiti sovra-esprimendo quantità crescenti di UL44 fusa a un FLAG tag hanno evidenziato che nessun inibizione può essere ottenuta per il sistema completamente stabile, probabilmente per la difficoltà di distruggere un complesso proteico preformato piuttosto che prevenirne la formazione. Per questo motive ci siamo focalizzati su sistemi transienti di espressione piuttosto che sul sistema interamente stabile. Sulla base di questi dati, un primo screening su piccolo scala è stato eseguito per studiare l’effetto di 18 SMs sulla dimerizzazione di UL44 usando il sistema BRET stabile/transiente. Le 18 piccole molecole sono state risospese in DMSO e la loro tossicità è stata valutata in coltura cellulare, prima di essere aggiunte a concentrazioni subtossiche alla linea YFP-UL44, sei ore dopo la trasfezione per esprimere RLuc-UL44. La nostra analisi ha identificato solo composti che inibivano blandamente il BRET ratio relativo alla dimerizzazione di UL44. Per questo motivo abbiamo ri-valutato il set up del saggio BRET, diminuendo il tempo d’incubazione delle SM prima di testare i valori BRET da 42 a 18 ore, utilizzando un saggio completamente transiente e diminuendo la quantità di proteine espresse. Per quest’ultima è stato necessario cambiare il substrato utilizzato per generare il segnale bioluminescente da CTZ a hCTZ. È stato eseguito un nuovo screening, con risultati molto simili a quelli ottenuti utilizzando il setup originale. Inoltre, quando i candidati migliori sono stati rivalutati usando un controllo negativo, il loro effetto sul BRET ratio è risultato aspecifico. Ulteriormente, la BRET, come la FRET, non è riuscita a rilevare uno specifico effetto nell’interazione UL44/UL54 di una SM in grado di distruggere questa interazione. Possiamo quindi concludere che BRET e FRET non sono tecniche ideali per la ricerca di SM inibitrici dell’interazione tra le proteine. Ci siamo poi focalizzati su metodi in vitro, partendo dal saggio GST-pull down, il quale è stato effettato utilizzando il dominio C-terminale (residui 1-290) di UL44, fuso o con GST o con 6His-tag. I risultati ottenuti mostrano che la tecnica GST-pull down è in grado di rilevare differenze nel legame tra UL44 wild-type e i mutanti con mutazioni nel sito di dimerizzazione, suggerendo una possibile applicazione di GST-pull down per lo screening delle SMs. Questa tecnica è stata implementata per questo studio, e i dati preliminari suggeriscono che il numero di SMs testate potrebbe portare all’inibizione della dimerizzazione di UL44. In conclusione, abbiamo sviluppato diversi saggi per monitorare la dimerizzazione di UL44. I saggi cellulari basati su RET confermano che UL44 forma dimeri in cellule viventi, e dimostrano di essere subottimali per lo screening. D’altro canto, i metodi in vitro come GST-pull down dimostrerebbero un maggiore sensibilità e sono stati implementati per l’identificazione degli inibitori della dimerizzazione di UL44.

Le travail de thèse consistera à explorer et caractériser l'ensemble conformationnel de protéines intrinsèquement désordonnées (IDPs) en utilisant plusieurs techniques complémentaires, notamment des simulations avancées de dynamique moléculaire et la diffusion des rayons X aux petits angles (SAXS). Les IDPs sont des protéines possédant une ou plusieurs régions n'ayant pas de structures secondaires stables lorsqu'elles sont isolées, mais pouvant en adopter lors de leur association avec de multiples autres protéines. La question, à laquelle ce travail souhaite répondre dans le cas de trois IDPs, est de savoir si ces éléments de structures secondaires, formés à l'interfaces des complexes protéine-protéine, pré-existent de façon transitoire, ou non, à l'état non-lié des IDPs en solution. S'il est possible d'identifier et de caractériser ces éléments de reconnaissance moléculaire dans les IDPs isolées, alors les résultats de ce travail permettront de guider par la suite la détermination des structures de complexes protéiques impliquant des IDPs
The PhD work will consist in exploring and characterizing the conformational ensemble of intrinsically disordered proteins (IDPs), by using several complementary methods, including enhanced molecular dynamics simulations and small angle X-ray scattering (SAXS). IDPs are proteins having one or several regions that lack stable secondary structures in the unbound state, but which can adopt various structured conformations to bind other proteins. In the case of three IDPs, the project aims to answer the question of whether these secondary structures formed at the protein-protein interfaces transiently pre-exist or not in the unbound state of solvated IDPs. If it is possible to identify and characterize these molecular recognition features (MoRFs) in the IDP unbound state, then the results of this work will subsequently help to determine the structures of protein complexes involving IDPs

The development of orally bioavailable small molecule drugs targeting protein-protein interactions (PPIs) has been challenging1. Unlike conventional targets, PPIs’ extended, open surface makes it difficult for small molecules to bind. In order to achieve strong binding, it is frequently necessary to use larger molecules, which traditionally is considered to disfavor druglikeness2. However, PPIs possess great therapeutic potential due to their abundance and regulatory roles in cells3. More extensive studies are needed to identify larger chemotypes that retain good druglike properties and therefore might have utility against PPI targets. NF-κB Essential Modulator (NEMO), interacting with IκB Kinase subunit β (IKKβ), is an important PPI target because of its regulatory role in NF-κB signaling4. Literature suggests that the N-terminal domain of NEMO is intrinsically disordered in the absence of bound ligand5. To test this hypothesis, I developed variants of the NEMO N-terminal domain, and studied their secondary structure, stability, and affinity for IKKβ, showing that the N-terminal domain of NEMO is intrinsically structured (Chapter Two). I also characterized partially peptidic NEMO inhibitors from our collaborator, Carmot Therapeutics. We tested the binding of these compounds and their peptidic fragments to full-length NEMO using fluorescence anisotropy (FA)6 and surface plasmon resonance (SPR). The results provided information about hit validity, binding affinity and kinetics (Chapter Three). Macrocycles are of interest for inhibiting PPIs partly because of their proposed good membrane permeability7. To evaluate this hypothesis, I implemented a membrane permeability assay, tested the permeability of a set of macrocyclic compounds, and used the results to develop a multiple linear regression model to predict permeability from macrocycles’ physicochemical properties. The model suggests that hydrophobicity correlates positively with good permeability, while high polarity or high aromatic ring count renders macrocycles less permeable (Chapter Four). Finally, in a separate project, to elucidate the origins of protein-ligand binding energy between interleukin-2 (IL-2) and its known small molecule inhibitors8, I developed a SPR based binding assay, and validated it by showing that the KD value of known inhibitor Ro26-45508 agrees with the literature value (Chapter Five). The assay will be useful in future studies of IL-2 inhibitors and their fragments.

abstract: Detection of molecular interactions is critical for understanding many biological processes, for detecting disease biomarkers, and for screening drug candidates. Fluorescence-based approach can be problematic, especially when applied to the detection of small molecules. Various label-free techniques, such as surface plasmon resonance technique are sensitive to mass, making it extremely challenging to detect small molecules. In this thesis, novel detection methods for molecular interactions are described. First, a simple detection paradigm based on reflectance interferometry is developed. This method is simple, low cost and can be easily applied for protein array detection. Second, a label-free charge sensitive optical detection (CSOD) technique is developed for detecting of both large and small molecules. The technique is based on that most molecules relevant to biomedical research and applications are charged or partially charged. An optical fiber is dipped into the well of a microplate. It detects the surface charge of the fiber, which does not decrease with the size (mass) of the molecule, making it particularly attractive for studying small molecules. Third, a method for mechanically amplification detection of molecular interactions (MADMI) is developed. It provides quantitative analysis of small molecules interaction with membrane proteins in intact cells. The interactions are monitored by detecting a mechanical deformation in the membrane induced by the molecular interactions. With this novel method small molecules and membrane proteins interaction in the intact cells can be detected. This new paradigm provides mechanical amplification of small interaction signals, allowing us to measure the binding kinetics of both large and small molecules with membrane proteins, and to analyze heterogeneous nature of the binding kinetics between different cells, and different regions of a single cell. Last, by tracking the cell membrane edge deformation, binding caused downstream event – granule secretory has been measured. This method focuses on the plasma membrane change when granules fuse with the cell. The fusion of granules increases the plasma membrane area and thus the cell edge expands. The expansion is localized at the vesicle release location. Granule size was calculated based on measured edge expansion. The membrane deformation due to the granule release is real-time monitored by this method.
Dissertation/Thesis
Doctoral Dissertation Electrical Engineering 2015

Indiana University-Purdue University Indianapolis (IUPUI)
Protein-protein interactions play a crucial role in a wide range of biological processes. Research on the design and synthesis of small molecules to modulate these proteinprotein interactions can lead to new targets and drugs to modulate their function. In Chapter one, we discuss the design and synthesis of small molecules to probe a proteinprotein interaction in a voltage-gated Ca2+ channel. Virtual screening identified a compound (BTT-3) that contained a 3,4-dihydro-3,4’-pyrazole core. This compound had modest biological activity when tested in a fluorescence polarization (FP) assay. The synthetic route to BTT-3 consisted of six steps. In addition, analogs of BTT-3 were made for a structure-activity study to establish the importance of a carboxylate moiety. We also synthesized a biotinylated benzophenone photo-affinity probe and linked it to BTT-3 to identify additional protein targets of the compound. In Chapter two, small-molecule antagonists targeting uPA-uPAR protein-protein interaction are presented. A total of 500 commercially-available compounds were previously identified by virtual screening and tested by a FP assay. Three classes of compounds were found with biological activity. The first class of compounds contains pyrrolidone core structures represented by IPR- 1110, the second class has a novel pyrrolo[3,4-c]pyrazole ring system, represented by xv IPR-1283 and the last series had compounds with a 1,2-disubstituted 1,2- dihydropyrrolo[3,4-b]indol-3(4H)-one core structure, represented by IPR-540. Each of these three compounds were synthesized and assessed by FP and ELISA assays. A binding mode of IPR-1110 with uPA was subsequently proposed. Based on this binding mode, another 61 IPR-1110 derivatives were synthesized by us to illustrate the SAR activity. Analogs of the other two series were also synthesized.

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2014
Therapeutic proteins (TPs) are becoming extremely important as therapeutic agents. Refinements in molecular structure have warranted long-term administration with sig- nificantly increase on efficacy and adhesion to therapeutic regimens by the patients, broadening the scope of indications for this class of therapy. Therefore, some biologics have become the standard of care in several therapeutic areas, such as inflammatory and oncology. A consequence of the increasing biologics indications is the concomitant administration with well-established small-molecule drugs (sMDs). Patent expiration of several biologics, such as monoclonal Antibodies (mAbs), may also contribute to the in- creasing use of TPs since biossimilars become available at more appealing prices. A bet- ter understanding of biologic-drug interactions (BDIs) is necessary to avoid the decrease of therapeutic regimens efficacy. By the existing regulatory guidance, the development of a new sMD includes an evaluation of the potential drug-drug interactions (DDIs) for concomitant medications. Only a few drug interaction studies have been performed with macromolecules since those studies, involving TPs, are inherently complicated. Few clinically relevant animal models available, long elimination half-lives, and complex elim- ination pathways, which differ from the classical cytochrome P450 (CYP450) system, are among the expected difficulties. In spite of the pharmacokinetic (PK) interactions, the most important and well-studied mechanism of interaction, pharmacodynamics (PD) in- teractions may also represent a possible explanation for several BDIs. The present re- view of the current literature encompasses several BDIs involving TPs (either as perpe- trators or as victims) and their specific mechanism of interaction.

Deviation from normal healthy conditions, termed as disease, can often be triggered due to the malfunctioning of proteins. Modulating the functions of proteins by administering therapeutic agents (drugs) may alleviate the disease conditions. The majority of the drugs currently available in the market are small organic molecules due to their pharmacological and commercial advantages. These small molecule drugs interact with the protein targets through specific sites on the surface of the protein structure (binding sites). Thus, the structural data of protein-small molecule complexes forms a crucial starting point for most drug discovery programs. The work reported in this thesis deals with understanding various aspects of protein-small molecule interactions. The thesis begins (Chapter 1) with a general introduction on the implication of proteins structural data in drug discovery programs. Chapter 2 provides a fundamental understanding of the general trend in local quality of protein-small molecule crystal complexes deposited in the Protein Data Bank (PDB). Our results suggest ‘seeing is not always believing’ and aims to sensitize the non-crystallographer user community that high-resolution need not always guarantee confident small molecule binding poses. The study indicates 35% of the inspected ~0.28 million protein-small molecule binding site pairs available from ~66000 PDB entries, need serious attention before using those as input in any important applications. Results reported in Chapter 3 suggest that the stereochemical quality of bound small molecules generally agrees well with their crystallographic quality. The findings from this work could be the stepping-stones for developing structure determination technique-independent ligand pose validation tools. The learning from Chapter 3 is extended to Chapter 4 to investigate the stereochemical quality of the small molecules bound to protein structures determined by cryo-EM. Our data shows that the stereochemical quality of small molecules bound to high-resolution protein structures determined by cryo-EM is comparable to high-quality small molecules bound to protein crystal structures. Chapter 5 presents a computational analysis aimed at providing insights into the molecular basis of the specificity of a novel anti-tubercular compound, NU-6027 (identified in a phenotypic screening by experimental collaborators), towards two out of the eleven known Serine-Threonine Protein Kinases in Mycobacterium tuberculosis (Mtb). Chapter 6 reports the development of a freely available web server that facilitates the identification of new uses of old drugs and aid in drug repurposing. In Chapter 7, the principles of ‘neighborhood behavior’ are exploited to identify potential known drugs that could be repurposed against the main protease of SARS-CoV-2. Chapter 8 discusses a virtual screening strategy to identify potential binders of a novel Mtb target, Rv1636 (or the Universal Stress Protein). Collaborators have experimentally validated some of the compounds shortlisted from the computational studies. Chapter 9 summarizes the findings from work reported in the entire thesis and future applications. Overall, this thesis inspects protein-small molecule complexes from a local perspective, aiding the design of rigorous computational experiments that can contribute to solving global unmet medical needs. Interested readers may contact the author directly for Supplementary data at "sohini@iisc.ac.in"
DST-INSPIRE fellowship

博士
國立交通大學
生物科技系所
102
Protein-protein interactions (PPIs) are essential for biological processes and thus often considered as potentially pharmaceutical drug targets. Targeting the interfaces between proteins has huge therapeutic potential, but discovering small-molecule drugs which block PPIs is an enormous challenge currently. According to our previous studies, β-tubulin:CCT-β complex can serve as an effective chemotherapeutic target for treating clinical tubulin-binding agent-resistant or CCT-β-overexpressing tumors and disrupting XIAP:CASP7 complex represents an effective and safe novel strategy for selectively killing CASP3-deficent cancer cells. We have developed a novel multiple-sites (or modes) virtual screening strategy with site-moiety maps to discover small molecules which can be simultaneously fitted into multiple sites (or modes) to disrupt PPIs. Moreover, we identified three key residues in CCT-β, K250, F252 and R293, which play an important role in interacting with β-tubulin and binding of the small molecules to I-Lys site induces disassociation of XIAP:CASP7 through an allosteric mechanism. Our method discovers the first potent and non-covalent inhibitor, R379603, efficiently induces apoptosis of HEK-293 cells with an EC50 at 4.6 M via targeting two previously uncharacterized and independent sites to block β-tubulin:CCT-β interface and triggering caspase-dependent signaling cascades. Notably, R379603 causes a more severe apoptosis of paclitaxel-resistant MCF-7 breast cancer cells in vitro and in cultured cells due to an increased level of CCT-β as compared with its parental MCF-7 cells. We also discovered a small molecule inhibitor (643943) which can reversibly bind to I-Lys site, an allosteric site away from the interface of the linker region between BIR1 and BIR2 domains of XIAP and CASP7 active site. This compound thus releases XIAP constraint and activated CASP7 to selectively kill CASP3 down-regulated (CASP3/DR) cancer cells. Besides, 643943 effectively sensitizes chemo-resistant cancer cells to chemotherapy at a low-micromolar concentration (≦5 μM). We believe that multiple-sites (or modes) strategy provides an alternative and useful method for discovering potential inhibitors and binding mechanisms for pharmaceutical targets.

Nicotine addiction, opioid use disorder, and COVID-19 have made lasting impacts on every aspect of society. These are complicated conditions, and studies in these fields will likely continue for decades, if not centuries. Here, we make contributions to each of these issues using electrophysiology and microscopy. The first chapter goes into the motivation behind this thesis and the major experiments I used in my graduate career. In the second chapter, we introduce a new amino acid into the mouse muscle nicotinic acetylcholine receptor in an attempt to understand the dynamics of receptor activation. In the third chapter, we continue the Lester lab’s work on the neuroscientific effects of menthol and how it plays a role in nicotine addiction. We found the binding site for menthol on the α4β2 nicotinic acetylcholine receptor, which continues our hypothesis that the neuroscientific effects of menthol are detrimental to cigarette smokers. Fortunately, partly because of our studies, mentholated nicotine products are being phased out of the United States. The fourth and fifth chapters investigate μ-opioid receptor trafficking, both the trafficking from the endoplasmic reticulum and endocytosis from the plasma membrane. Both of these events play a role in inducing opioid use disorder and increasing the danger of using opioids. We hope that these studies will help other researchers understand opioid use disorder and fight the opioid epidemic. Finally, we studied the effects of SARS-COV-2 proteins on epithelial sodium channels. These channels are important for regulating lung fluid levels where their improper function may cause pulmonary edema. Pulmonary edema has been observed in COVID-19 patients. Altogether, we believe that we have made meaningful impacts on these important health concerns in this thesis. We look forward to how the scientific communities continue to build on our results.

Indiana University-Purdue University Indianapolis (IUPUI)
All platinum (Pt)-based chemotherapeutics exert their efficacy primarily via the formation of DNA adducts which interfere with DNA replication, transcription and cell division and ultimately induce cell death. Repair and tolerance of Pt-DNA lesions by nucleotide excision repair and homologous recombination (HR) can substantially reduce the effectiveness of the Pt therapy. Inhibition of these repair pathways, therefore, holds the potential to sensitize cancer cells to Pt treatment and increase clinical efficacy. Replication Protein A (RPA) plays essential roles in both NER and HR, along with its role in DNA replication and DNA damage checkpoint activation. Each of these functions requires RPA binding to single-stranded DNA (ssDNA). We synthesized structural analogs of our previously reported RPA inhibitor TDRL-505, determined the structure activity relationships and evaluated their efficacy in tissue culture models of epithelial ovarian cancer (EOC) and non-small cell lung cancer (NSCLC). These data led us to the identification of TDRL-551, which exhibited a greater than 2-fold increase in in vitro and cellular activity. TDRL-551 showed synergy with Pt in tissue culture models of EOC and in vivo efficacy, as a single agent and in combination with platinum, in a NSCLC xenograft model. These data demonstrate the utility of RPA inhibition in EOC and NSCLC and the potential in developing novel anticancer therapeutics that target RPA-DNA interactions.

Small molecule Zn(II) complexes containing N- and S- donor environments may serve as appropriate models for mimicking Zn protein sites, and thus, their reactions with heavy metal ions such as Pt(II) and W(0) may provide insight into possible adduct formation and zinc displacement. To study such possible interactions between zinc finger proteins and platinum-bound DNA, the ZnN2S2 dimeric complex, N,N?-bis(2- mercaptoethyl)-1,4-diazacycloheptane zinc (II), [Zn-1?]2, has been examined for Znbound thiolate reactivity in the presence of Pt(II) nitrogen ? rich compounds. The reactions yielded Zn/Pt di- and tri- nuclear thiolate-bridged adducts and metalexchanged products, which were initially observed via ESI-mass spectrometry (ESI-MS) analysis of reaction solutions, and ultimately verified by comparison to the ESI-MS analysis, 195Pt NMR spectroscopy, and X-ray crystallography of directly synthesized complexes. The isolation of Zn-(?-SR)-Pt-bridged [(Zn(bme-dach)Cl)(Pt(dien))]Cl adduct from these studies is, to our knowledge, the first Zn-Pt bimetallic thiolatebridged model demonstrating the interaction between Zn-bound thiolates and Pt(II). Additional derivatives involving Pd(II) and Au(III) have been explored to parallel the experiments executed with Pt(II). The [Zn-1?]2 was then modified by cleavage with Na+[ICH2CO2]- to produce (N- (3-Thiabutyl)-N?-(3-thiapentaneoate)-1,4-diazacycloheptane) zinc(II), Zn-1?-Ac or ZnN2SS?O, and 1,4-diazacycloheptane-1,4-diylbis(3-thiapentanoato) zinc(II), Zn-1?-Ac2 or ZnN2S?2O2, monomeric complexes (where S = thiolate, S? = thioether). The [Zn-1?]2 di- and Zn-1?-Ac mono-thiolato complexes demonstrated reactivity towards labile-ligand tungsten carbonyl species, (THF)W(CO)5 and (pip)2W(CO)4, to yield, respectively, the [(Zn-1?-Cl)W(CO)4]- complex and the [(Zn-1?-Ac)W(CO)5]x coordination polymer. With the aid of CO ligands for IR spectral monitoring, the products were isolated and characterized spectroscopically, as well as by X-ray diffraction and elemental analysis. To examine the potential for zinc complexes (or zinc-templated ligands) to possibly serve as a toxic metal remediation agents, Zn-1?-Ac and Zn-1?-Ac2 were reacted with Ni(BF4)2. The formation of Zn/Ni exchanged products confirmed the capability of ?free? Ni(II) to displace Zn(II) within the N-, S-, and O- chelate environment. The Zn/Ni exchanged complexes were analyzed by ESI-MS, UV-visible spectroscopy, IR spectroscopy of the acetate regions, and X-ray crystallography. They serve as foundation molecules for more noxious metal exchange / zinc displacement products.

Protein-protein interactions (PPIs) are attractive targets because of their therapeutic potential. One approach to design small molecules that can disrupt the PPIs is to use structural information of proteins. With this approach, triazole-based peptidomimetics that mimic beta-turn hot-spot regions in neurotrophins were synthesized. The monovalent mimics were assembled into bivalent mimics via a combinatorial method. Three different bivalent mimics were prepared for different studies. Bivalent mimics with long-linkers bound to TrkA or TrkC receptor and showed partial antagonism for the receptors. Other mimics were conjugated with cytotoxic compounds and they were used for TrkC targeted drug delivery. The last group of bivalent mimics previously showed targeted delivery effects for pancreatic cancer cells. In this study, we synthesized Eu-chelated bivalent mimics to perform a competitive binding assay for pancreatic cancer cells. Previous research in our group focused on design of secondary structures' mimics on rigid scaffolds as "minimalist mimics." We sought to establish structural design criteria for the minimalist mimics, and we wanted to propose that sets of such compounds could mimic local pairs of amino acids in any secondary structures as "universal peptidomimetics." Thus, we designed five compounds, such as oxazoline-, pyrrole-, dyine- "kinked" and "linear" bistrizole-based peptidomimetics, and performed molecular modelings, DFT calculations, and QMD for them to validate our hypothesis. On the concepts of "minimalist mimics" and "universal peptidomimetics," we developed the C alpha ? C beta vector matching program to evaluate preferred orientations of C alpha - C beta coordinates for secondary structures. We applied the program to omegatides and pyrrolinone-pyrrolidine oligomers. The compounds matched better with strands than for helices. We expanded the C alpha ? C beta vector matching idea to a method that ranks preferred conformations of small molecules on any combination of three interface side-chains in all structurally characterized PPIs. We developed a PDB mining program (explores key orientation, EKO) to do this, and EKO applied to pyrrolinone-pyrrolidine oligomers to find targets. EKO found several interesting targets, such as AICAR Tfase, GAPDH, and HIV-1 protease. HIV-1 dimerization inhibition and Zhang-Poorman kinetic assays were performed to validate our hypothesis, and the results showed that pyrrolinone-pyrrolidine derivatives inhibited HIV-1 dimerization.

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2016
Ao longo dos anos, vários estudos científicos têm comprovado a importância da proteína supressora de tumor p53 na homeostase celular. Esta proteína encontra-se inativada em aproximadamente 50% dos tumores humanos, quer seja por mutação ou por deleção do seu gene. Por outro lado, a inativação da p53 por inibição reversível é frequentemente observada nos tumores que expressam a forma selvagem da proteína. Esta inativação pode resultar da sobre-expressão dos seus reguladores negativos, tais como a proteínas MDM2 e MDMX, conduzindo a patologias oncogénicas caracterizadas principalmente pela expressão da p53 do tipo selvagem. Vários estudos têm demonstrado que a interação da p53 com as MDM’s envolve três aminoácidos hidrofóbicos (Phe19, Trp23 e Leu26) da proteína p53. Além disso, sabe-se que a reativação da p53 é facilitada pela inibição destas interações. Nos últimos anos, várias famílias de compostos têm sido concebidas e desenvolvidas como moduladoras da atividade da p53. No entanto, o objetivo de desenvolver uma terapia anti-tumoural baseada na inibição das interações p53-MDM’s, e consequentemente na reativação da p53, encontra-se ainda no início, com apenas alguns candidatos em ensaios clínicos. Deste modo, torna-se premente a descoberta e desenvolvimento de inibidores mais potentes e seletivos para a interação entre a p53 e os seus reguladores negativos. Nos últimos anos, o nosso grupo de investigação tem vindo a investigar o potencial de várias famílias de spirooxindoles com anéis de cinco membros para o tratamento do cancro. Trabalhos anteriormente desenvolvidos pelo grupo, mostraram que as spiropirazolinas e spiroisoxazolina oxindoles apresentam propriedades anticancerígenas in vitro. O anel spiro encontrado nestes compostos funciona como uma estrutura heterocíclica rígida, a partir da qual podem ser projetados os três grupos lipofílicos que mimetizam a Phe19, Trp23 e Leu26 da p53. Estudos mais detalhados sobre o mecanismo de ação das spiroisoxazolina oxindoles mostraram que estes compostos inibem a interação p53-MDM2. No entanto, estes compostos apenas induziram moderada atividade anti-proliferativa (IC50 cerca de 35 μM) para a linha celular de cancro colo-retal humano HCT-116. Estudos de química computacional indicaram que esta moderada atividade resulta provavelmente da orientação espacial do oxindole nestes compostos não permitir que mimetizasse o resíduo Trp23 da p53, fundamental para ocorrer a ligação à proteína MDM2. Em particular, os estudos de docking molecular mostraram que o oxindole é projetado para a cavidade que seria ocupada pela Phe19 da proteína p53, enquanto os grupos aromáticos ligados ao anel de isoxazolina são projetados para as cavidades Trp23 e Leu26 da p53. A substituição do átomo de oxigénio do anel de isoxazolina por um grupo N-Ar, poderia permitir não só a inclusão de mais um substituinte no anel de 5 membros, como uma reorientação dos substituintes do anel de 5 membros de forma a ocuparem as cavidades da MDM2 a que se ligam os aminoácidos da p53. Por esse motivo, foi desenvolvida uma família de spiropirazolina oxindoles que foi testada nas linhas celulares de cancro da mama humano, MCF-7 e MDA-MB-231. Os compostos mais ativos apresentaram um valor de IC50 cerca de 7 μM. Adicionalmente, estes compostos apresentaram seletividade para a linha celular MDA-MB-231 e não foram tóxicos em células não tumorais Hek-293T. Nesta dissertação, otimizou-se esta família de compostos (spiropirazolina oxindoles) e avaliou-se em mais detalhe os seus efeitos biológicos. As spiropirazolina oxindoles foram obtidas através de uma reação de cicloadição 1,3-dipolar entre 2-indolinonas e nitrilo iminas (formadas in situ a partir de cloretos de hidrazonoílo). Esta nova biblioteca de compostos foi caracterizada e posteriormente avaliada quanto ao seu potencial anti-proliferativo in vitro, utilizando as linhas celulares humanas de cancro colo¬retal HCT-116 e cancro da mama MCF-7 e MDA-MB-231, e glioma de murino GL-261. Para a linha de cancro colo-retal os compostos apresentaram atividades entre os 11-13 µM, e para as linhas de cancro da mama MCF-7 e MDA-MB-231 apresentaram atividades de 7-12 µM e 6-11 µM, respetivamente. No caso da linha celular de glioma de murino, apenas um composto apresentou valores de atividade anti-proliferativa na mesma gama de valores com um IC50 de 19 µM. De salientar, a inclusão de um grupo N-Ar aumentou para mais do dobro a capacidade anti-proliferativa em HCT-116 p53, quando é feita uma comparação direta entre os compostos equivalentes de spiroisoxazolina oxindoles. Com o intuito de avaliar o potencial citotóxico dos compostos em células saudáveis, foi realizado o ensaio de viabilidade celular em fibroblastos de colon humano normal e em culturas primárias de astroglia de murino. Como resultado, dois compostos (2e e 2m) pertencentes à família spiropirazolina oxindoles não apresentaram citotoxicidade nas células saudáveis, apesar de serem citotóxicos nas linhas celulares de cancro. O mesmo não se verificou para o composto 2q, para o qual foi observada citotoxicidade tanto em células normais como cancerígenas. Os compostos não citotóxicos em células normais foram então avaliados quanto à sua capacidade de indução de apoptose e paragem do ciclo celular na linha celular HCT-116. Os resultados demonstraram que estes compostos induzem tanto a morte por apoptose como a paragem do ciclo celular na fase G0/G1, estando ambos os fenómenos dependentes do tempo de exposição ou concentração de composto utilizada. Com a finalidade de confirmar a capacidade desta família de compostos para induzir a morte celular, foram ainda avaliados os níveis de libertação da enzima citoplasmática lactato desidrogenase (LDH) indicativos do grau de rutura da membrana celular e, por conseguinte, de morte. Os resultados obtidos corroboraram o ensaio de apoptose, confirmando assim a capacidade de indução de morte dependente da concentração. As spiropirazolina oxindoles apresentaram ainda a capacidade de aumentar os níveis de expressão da p53 e induzir a inibição da MDM2, através da diminuição dos seus níveis de expressão proteica. Adicionalmente, a aptidão destes compostos para inibir a interação p53-MDM2 foi observada através do ensaio de complementação de fluorescência bimolecular (BiFC), e foi ainda verificada uma boa estabilidade em PBS (pH 7.4). Por último, foi observado um efeito sinergético entre um agente quimioterapêutico amplamente utilizado na clínica e um dos compostos em estudos. As spiropirazolina oxindoles foram também avaliadas quanto à capacidade de induzir apoptose e paragem do ciclo celular na linha de glioma de murino GL-261. No entanto, os resultados obtidos não demonstraram qualquer evidência de indução de apoptose ou paragem do ciclo celular nestas células, indicando que estes compostos não têm potencial terapêutico neste tipo de tumores. No âmbito desta tese foi também estudado em mais detalhe o mecanismo de ação de três spirotriazolina oxindoles previamente desenvolvidas no grupo como potenciais agentes anticancerígenos. Resultados obtidos anteriormente, tinham demonstrado que esta família apresentava boa seletividade para as linhas de cancro da mama MCF-7 e MDA-MB-231, sem apresentar citotoxicidade na linha não-tumoral Hek-293T. Observou-se ainda a capacidade destes compostos para inibir a interação p53-MDM2, ativar as caspases-3 e -7 e, consequentemente, induzir apoptose. Por último, uma boa estabilidade em plasma já tinha sido observada para os compostos em estudo. De forma a estudar o eventual mecanismo de ação desta família de compostos como potenciais agentes anticancerígenos, na presente dissertação, testaram-se os compostos mais promissores de spirotriazolina oxindoles em células da linha de cancro colo-retal HCT-116, tendo-se verificado que estes compostos induzem apoptose e clivagem da PARP, assim como a paragem do ciclo celular na fase G0/G1, sendo esta dependente do tempo de exposição ou da concentração de composto utilizada. Adicionalmente, avaliou-se a capacidade destes compostos para ativar a p53 e diminuir os níveis de expressão de MDM2, indicando uma potencial inibição da interação p53-MDM2. A indução de apoptose e paragem do ciclo celular foi também avaliada na linha celular de cancro da mama MDA-MB-231. Contudo, os resultados obtidos não demonstraram qualquer indução de apoptose após 72 horas de exposição das células ao composto. Porém, às 48 horas de exposição das células ao composto foi possível verificar a ativação das caspases-3 e -7 e paragem do ciclo celular na fase G2/M. Por último, os compostos mais promissores de spirotriazolina oxindoles foram testados em fibroblastos de colon humano normal, não se tendo verificado citotoxicidade para as concentrações testadas. Em suma, nesta dissertação demonstrou-se o potencial de cinco compostos (2e, 2m, 7f, 7h e 7z) pertencentes a duas famílias de spirooxindoles, contendo anéis heterocíclicos de 5 membros, como agentes anticancerígenos e inibidores da interação proteína-proteína p53-MDM2.
Over the years, several scientific studies have been proving the importance of the p53 tumour suppressor protein, in cellular homeostasis. This protein is found inactivated in approximately 50% of human cancers, by mutation or deletion of its gene. Moreover, the inactivation of p53 by reversible inhibition is frequently observed in various human cancers, especially in those expressing wild type p53. This inactivation results from overexpression of its negative regulators such as MDM2 and MDMX, leading to oncogenic pathologies, mostly characterized by expression of a p53 wild type form. Several studies have shown that p53 interaction with its negative regulators involves three main hydrophobic amino acids (Phe19, Trp23 and Leu26) in the p53 protein. Moreover, it is known that p53 reactivation is facilitated by inhibition of its negative regulators. In recent few years, several families of compounds were designed and developed as modulators of p53. However, the development of a p53 reactivating cancer therapy through inhibition of p53–MDM´s interaction is still at the beginning, with only a few candidates entering clinical trials. Therefore, the discovery of more powerful and selective p53–MDM´s interaction inhibitors are still an unmet need. The work developed in this master thesis aimed at developing new anticancer agents containing a spirooxindole scaffold having a pyrazoline ring. The spiro ring works as the rigid heterocyclic scaffold, from which the three lipophilic groups can be projected to mimic the p53 amino acids. The first goal included the development and optimization of a potential new anticancer agent by 1,3-dipolar cycloaddition reaction, complemented with biological activity studies, such as evaluation of cell death and cell cycle progression, and analysis of p53¬MDM2 interaction. In addition, studies of combination therapy using the newly synthetized molecules and a chemotherapeutic agent commonly used in the clinic, were also performed and finally, the stability of the compound in a saline solution was assessed. The second main goal of this thesis was to understand the effect of changing the carbon atom by a nitrogen atom in position 4´ of the pyrazoline ring. The results presented here reveal the potential anticancer activity of the new molecules, as shown by the ability to induce apoptosis and cell cycle arrest, inhibit the interaction between p53 and MDM2, while presenting good stability in PBS and no cytotoxic effects in normal human cells. Furthermore, a synergistic effect with the chemotherapeutic agent was observed. The change of the carbon atom for a nitrogen atom in position 4´ (pyrazoline ring replaced by a tryazoline ring) led to a new family of small molecules that has the ability to increase the expression of p53, thus leading to apoptosis and cell cycle arrest. In addition, these compounds were not cytotoxic in human normal cells.

E2A-PBX1 is expressed as a consequence of a recurring chromosomal translocation seen in 5% of acute lymphoblastic leukemia cases. We recently reported that substitution of a leucine residue (L20A) within the N-terminal transcriptional activation domain (AD1) of E2A-PBX1 markedly impairs binding to the KIX domain of CBP/p300 and, importantly, leukemia induction in a mouse bone marrow transplantation model. Since both the protein-protein interaction and consequent leukemogenesis rely on a focal contact point and might therefore be susceptible to antagonism by small molecules, we devised a cell-free assay based on fluorescence anisotropy (FA) to detect binding of a fluorescently labeled peptide derived from AD1 of E2A-PBX1 (FITC-E2A) with recombinantly expressed KIX domain. The optimized FA assay reveals a dissociation constant of 2 µM for the wild-type interaction and correctly detects disruption of the complex by naphthol AS-E phosphate, a compound previously shown to antagonize KIX binding. The optimized FA assay was used to screen the Prestwick, Spectrum and Chembridge libraries containing 12400 compounds in total. Of the initial 43 positive hits from the libraries, 10 caused a reproducible decrease in FA. Since intrinsic small molecule fluorescence can produce false positive results in the FA-based screen, intrinsically fluorescent compounds were excluded from further analysis unless they could be shown to bind to KIX. Two hits, L1 and C2, were intrinsically fluorescent but demonstrated KIX interactions and one hit, P9, was not intrinsically fluorescent. These three compounds were tested for their ability to inhibit binding of a larger portion of E2A (residues 1 to 483) to full length CBP in a pull down assay with only compound P9 demonstrating efficacy. Further characterization of P9 by NMR showed no binding to KIX, however evaluation by FA showed binding to FITC-E2A with a 20 µM affinity. A cell-based cytotoxicity assay demonstrated that compound P9 was slightly more toxic on leukemic cells that express E2A-PBX1, compared to leukemic cells lacking E2A-PBX1 expression. Mammalian two-hybrid analysis did not provide details of the effects of P9 on the E2A:KIX interaction. We expect the identification of a novel compound, P9, capable of disrupting the oncogenic E2A-PBX1:CBP interaction, to guide the development of effective, less toxic leukemia drugs and provide new tools for elucidating the molecular mechanisms of leukemia induction by E2A-PBX1.
Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2009-08-31 11:13:19.517

碩士
國立臺灣海洋大學
生物科技研究所
95
Abstract Molecular recognition plays an important regulator in cellular activities. Mapping the interaction sites of protein is of great interest since it contributes much to our understanding of the mechanisms of molecular recognition and provides the basis for rational vaccine design. We employed Nanoprobe-Based Affinity Mass Spectrometry (NBAMS) to rapidly and accurately map small molecule/protein interaction sites. To demonstrate the general applicability of our approach in tackling of small molecule/protein interaction, three biomolecular interaction systems, mannose/Con-A, haparin/A27Laa, and inhibitors/α 1,2-L-fucosidase were investigated in this study. In the first part of this thesis, the mannose/Con-A interaction will be used the assay optimization, kinetic study, and investigation of detection limit. Our result shows our nanoscale probe facilitating the fast interaction of mannose and Con A; the reaction time can be completed within 15 min with detection limit of 15 ng. For epitope mapping, the carhohydrate-binding peptides were identified with various proteases (trypsin, chymotrypsin, and Glu-C), consistent with reported binding sites by X-ray crystallization analysis. Moreover, the subtle structure change of Con-A/mannose in metal environment can be observed. For harparin/A27Laa system, the peptide at m/z 2694.6 (residue 1-32) of A27Laa was identified, which contains basic strip of 12 residues responsible for binding to cell-surface heparan sulfates. Although A27Laa lost its C-terminal, we found the epitope can be bound to the heparin-MNPs in our approach. For α1,2-L-fucosidases/inhibitor system, IV we probed the structural differences of the epitopes for three fuconojirimycin inhibitors: Inhibitor1:(2R,3S,4S,5S)-2-(aminomethyl)-6-methylpiperidine-3,4,5-triol Inhibitor2: 5-(3-aminopropoxy)-2-methylpiperidine-3,4-diol Inhibitor3:1-(3-aminopropyl)-2-methylpiperidine-3,4,5-triol and the effects of different length of linker conjugated on the surface of MNPs. The change of binding epitopes is consistent to the structure modeling of α1,2-L-fucosidase complexed with different inhibitor. Moreover, we found that the effect of digestion time is critical for the epitope mapping. Given the flexibility of nanoprobe derivation, adaptation to various proteases, and the high rapid and high sensitivity analysis, we believe our approach hold great promise in probing binding epitopes of small molecule and proteins.

Signal transducer and activator of transcription 3 (STAT3) is a cancer-driving proto-oncoprotein that represents a novel target for the development of chemotherapeutics. In this study, the functional requirements to furnish a potent STAT3 inhibitor was investigated. First, a series of peptidomimetic inhibitors were rationally designed from lead parent peptides. Prepared peptidomimetics overcame the limitations normally associated with peptide agents and displayed improved activity in biophysical evaluations. Notably, lead peptidomimetic agents possessed micromolar cellular activity which was unobserved in both parent peptides. Peptidomimetic design relied on computational methods that were also employed in the design of purine based STAT3 inhibitory molecules. Docking studies with lead STAT3-SH2 domain inhibitory molecules identified key structural and chemical information required for the construction of a pharmacophore model. 2,6,9-heterotrisubstituted purines adequately fulfilled the pharmacophore model and a library of novel purine-based STAT3 inhibitory molecules was prepared utilizing Mitsunobu chemistry. Several agents from this new library displayed high affinity for the STAT3 protein and effectively disrupted the STAT3:STAT3-DNA complex. Furthermore, these agents displayed cancer-cell specific toxicity through a STAT3 dependant mechanism. While purine agents elicited cellular effects, the dose required for cellular efficacy was much higher than those observed for in vitro STAT3 dimer disruption. The diminished cellular activity could be attributed to the apparent poor cell permeability of the first generation purine library; thus, a second library of purine molecules was constructed to improve cell penetration. Unfortunately, iii 2nd generation purine inhibitors failed to disrupt phosphorylated STAT3 activity and suffered from poor cell permeability. However, a lead sulfamate agent was discovered that showed potent activity against multiple myeloma cancer cells. Investigations revealed potential kinase inhibitory activity as the source of the sulfamate purine’s biological effect. Explorations into the development of a potent STAT3 SH2 domain binder, including the creation of salicylic purine and constrained pyrimidine molecules, are ongoing. Finally, progress towards the creation of a macrocyclic purine combinatorial library has been pursued and is reported herein.