Thèses sur le sujet « Binding ligandi »

Questa tesi è incentrata sulla modellazione del binding ligando-proteina con metodi computazionali basati sulla dinamica molecolare. La comprensione di questo processo è fondamentale per la progettazione e la scoperta di nuovi farmaci e l'uso di metodi computazionali per supportare la ricerca sperimentale in questo campo è in costante crescita. Oggi, grazie alla crescente potenza dei computer, è possibile studiare l'intero processo di binding/unbinding del ligando e ottenere stime sulle proprietà termodinamiche e cinetiche. Alla luce di ciò, durante il mio dottorato, diversi metodi avanzati di dinamica molecolare classica (MD) sono stati impiegati e confrontati per identificare un approccio computazionale efficace per studiare i processi di binding/unbinding dei ligandi. In particolare, è stato sviluppato un protocollo basato sulla combinazione degli approcci steered MD (sMD) e Metadinamica (MetaD) con Path Collective Variables (PCVs) con lo scopo di utilizzare i vantaggi di entrambi i metodi per ottenere una descrizione completa del processo. Mentre il metodo SMD è stato impiegato per studiare diversi percorsi di disassociazione e identificare quello preferito, la MetaD con le PCVs è stato utilizzato per determinare più accuratamente l'energia libera di legame. Il protocollo proposto è stato applicato con successo per studiare il legame del ligando al fattore inducibile dell'ipossia (HIF-2α) e ha dimostrato di essere efficace per le simulazioni effettuate sia su una struttura a raggi X nota del ligando-proteina che su una posa di docking. D'altra parte, la maggior parte dei metodi MD richiede la produzione di diverse repliche o lunghe simulazioni per campionare più volte l'evento di binding/unbinding al fine di ottenere una statistica affidabile del processo. Questo produce la necessità di metodi in grado di analizzare tutti gli eventi simulati in una sola volta e di fornire un quadro chiaramente interpretabile delle differenze nei pathway campionati. Per questo motivo, è stato sviluppato un tool basato sulle mappe auto-organizzanti (SOM). Lo strumento PathDetect-SOM (Pathway Detection on SOM), sfruttando i vantaggi dell'ordinamento topologico della SOM, permette di rappresentare visivamente i percorsi di legame campionati durante diversi eventi/repliche MD in una chiara rappresentazione bidimensionale. Inoltre, possono essere derivati suggerimenti sulle proprietà cinetiche e termodinamiche del processo. Lo strumento è stato applicato con successo a diversi casi di studio per dimostrare la sua applicabilità generale. Inoltre, come parte di un progetto eseguito presso il centro di ricerca Jülich (Istituto di Simulazioni Avanzate e Istituto di Neuroscienze e Medicina) sotto la supervisione del Prof. Paolo Carloni, è stata testata una nuova interfaccia ibrida di meccanica quantistica/meccanica molecolare (QM/MM) (MiMiC). Il codice, che permette simulazioni di dinamica molecolare QM/MM di sistemi biomolecolari, è stato applicato alla proteina chinasi mitogeno-attivata p38 in complesso con il ligando 2g per studiare il processo di unbinding del ligando. L'attenzione si è concentrata sulla prima fase del processo che coinvolge la dinamica del ligando nel suo stato legato. Le simulazioni MD QM/MM sono state efficaci nel descrivere accuratamente le interazioni ligando-proteina. In particolare, monitorando il cambiamento delle cariche atomiche durante la simulazione e calcolando la differenza di densità elettronica tra il ligando nel suo stato legato e nel vuoto, sono stati ottenuti approfondimenti sugli effetti di polarizzazione del campo elettrico della proteina sul ligando. Ci si aspetta che questi effetti, anche se piccoli nello stato legato, diventino molto importanti nelle fasi successive del processo di unbinding.
This thesis focused on modeling of ligand-protein binding with computational methods based on molecular dynamics. Understanding this process is crucial for the design and discovery of new drugs and the use of computational methods to support experimental research in this field is constantly growing. Nowadays, thanks to the increasing computer power, it is possible to study the complete ligand binding/unbinding process and obtain estimate on thermodynamic and kinetic properties. In view of this, during my PhD, different advanced classical molecular dynamics (MD) methods were employed and compared to identify an effective computational approach for studying ligand binding/unbinding processes. Specifically, a protocol based on combination of the steered MD (sMD) and the Metadynamics (MetaD) with Path Collective Variables (PCVs) approaches was developed with the aim of using the advantages of both methods to obtain a complete description of the process. While the sMD method was employed to investigate different unbinding pathways and identify the preferred one, MetaD with PCVs was used to determine more accurately the binding free energy. The proposed protocol was successfully applied to study ligand binding to the Hypoxia Inducible Factor (HIF-2α) and it demonstrated to be effective for simulations performed both on a known ligand-protein X-ray structure and on a docking pose. On the other hand, most of the MD methods requires the production of several replicas or long simulations to sample the binding/unbinding event several times in order to obtain a reliable statistics of the process. This produces the need of methods able to analyze all the simulated events at once and to provide a clearly interpretable picture of the differences in the sampled pathways. For this reason, a tool based on the self-organizing maps (SOMs) was developed. The PathDetect-SOM (Pathway Detection on SOM) tool, exploiting the advantages of the topological ordering of the SOM, allowing to visually represent the binding paths sampled during different MD events/replicas in a clear bidimensional representation. In addition, hints on the kinetic and thermodynamic properties of the process can be derived. The tool was successfully applied to different study-cases to demonstrate its general applicability. Furthermore, as part of a project performed at the Jülich research center (Institute of Advanced Simulations and Institute for Neuroscience and Medicine) under the supervision of Prof. Paolo Carloni, a novel hybrid quantum mechanics/molecular mechanics (QM/MM) interface (MiMiC) was tested. The code, that allows QM/MM molecular dynamics simulations of biomolecular systems, was applied to the mitogen-activated protein kinase p38 in complex with the 2g ligand to investigate the ligand unbinding process. The focus was on the first step of the process involving the dynamics of the ligand in its bound state. QM/MM MD simulations were effective in describing ligand-protein interactions accurately. In particular, by monitoring the change of the atomic charges during the simulation and calculating the electronic density difference between the ligand in its bound state and in vacuum, insights into the polarization effects of the protein electric field onto the ligand were obtained. It is expected that these effects, albeit small in the bound state, become very important in the following steps of the unbinding process.

Da sempre biologi e biochimici hanno focalizzato la loro attenzione in numerosi studi volti a chiarire i processi e le strutture molecolari fondamentali per la vita. Tra le tecnologie utilizzate allo scopo di conoscere la struttura di una biomolecola e la sua interazione con altre strutture, la diffrazione a raggi X, la risonanza magnetica nucleare ela microscopia elettronica permettono misure che richiedono grandi quantit¨¤ di composti purificati, sono spesso eseguite in condizioni non-fisiologiche e sono raramente adatte per osservare le reazioni molecolari in tempo reale. Recenti sviluppi sono stati mostrati sia nel campo dellafluorescenza che in numerosi metodi che consentono la selettivit¨¤ e l¡¯analisi delle interazioni molecolari in condizioni fisiologiche, come nelle cellule vive. L'analisi si concentra sulle interazioni ligando-recettore e sul processo successivo di trasduzione del segnale, che determina la risposta finale cellulare. Generalmente questi processi sono mediati, attraverso le membrane cellulari,da proteine canale o recettori accoppiati a proteina G. I nuovi saggi di fluorescenza sono importanti per spiegarela funzione dei recettori e i processi di trasduzione del segnale, cos¨¬ come per lo screening di nuovi composti terapeutici. In particolare, il metodo della fluorescenza polarizzata (FP), della fluorescenza di risonanza a trasferimento di energia (FRET) e della fluorescenza a tempo risolto (TRFS), sono emersi come soluzione agli high-throughput screening assays(HTS), in quanto pi¨´ rapidi, con minor impatto ambientale e minori costi. Sebbene siano stati riscontrati alcuni problemi analitici, i ligandi fluorescenti sono stati proposti come alternativa ai radioligandi per gli studi di binding sui recettori. Tali composti possono anche dare informazioni sulle caratteristiche bio-fisiche del sito di legame del ligando poich¨¦ alcuni fluorofori mostrano una resa quantica che dipende dalla lipofilicit¨¤ o dal pH dell¡¯ambiente.Inoltre potrebbero essere in grado di chiarire la localizzazione e le funzioni biologiche di alcuni tipi di recettore. Per esempio ligandi fluorescenti hanno permesso la localizzazione dei recettori ¦Á1 adrenergici, dei trasportatori della dopamina, dei recettori A1 adenosinici e dei recettori periferici delle benzodiazepine. Sempre attraverso l¡¯uso di tali composti ¨¨ stato possibile lo studio dell¡¯espressione e del clustering del recettore ionotropico 5-HT3, la visualizzazione in tempo reale del trafficking cellulare e dell¡¯internalizzazione del complesso del ligando con i recettori oppioidi ¦Ì e ¦Ä e infine l¡¯oligomerizzazione dei recettori della somatostatina regolata dal legame con il ligando. Sono stati inoltre sintetizzati composti fluorescenti attivi sui recettori sigma (¦Ò) che potrebbero essere in grado di fornire nuove informazioni e chiarimenti sul loro ruolo fisio-patologico. Tali recettori sono presenti a livello del sistema nervoso centrale (SNC), del fegato, del rene, del sistema immunitario e del tessuto endocrino e le loro funzioni fisiologiche sono ancora oggetto di studio. In particolare, nel SNC i recettori ¦Ò1 sono coinvolti nella modulazione dei canali del potassio e del calcio e nella neurotrasmissione glutammatergica, serotoninergica, dopaminergica e muscarinica, suggerendo un loro potenziale ruolo terapeutico nel trattamento di disturbi cognitivi, depressione e schizofrenia. I recettori ¦Ò2, invece, sono overespressi in linee cellulari tumorali e per questo motivo ligandi ¦Ò fluorescenti potrebbero essere utili alla diagnosi tumorale. Inoltre per meglio chiarire il coinvolgimento fisiologico e patologico dei recettori ¦Ò1 e ¦Ò2 nella crescita delle cellule tumorali, un ligando fluorescente potrebbe rappresentare un potenziale tool molecolare per lo studio della vitalit¨¤ cellulare.

The tetraruthenium cluster H4Ru4(CO)12 has been studied for its reactivity with the unsaturated diphosphine ligands (Z)-Ph2PCH=CHPPh2, 4,5-bis (diphenylphosphino)-4-cyclopenten-1,3-dione, bis(diphenyphosphino)benzene and 1,8- bis(diphenyl phosphino)naphthalene under thermal, near-UV photolysis, and Me3NO-assisted activation. All three cluster activation methods promote loss of CO and furnish the anticipated substitution products that possess a chelating diphosphine ligand. Clusters 1, 2, 3 and 4 have been characterized in solution by IR and NMR spectroscopies, and these data are discussed with respect to the crystallographically determined structures for all new cluster compounds. The 31P NMR spectral data and the solid-state structures confirm the presence of a chelating diphosphine ligand in all four new clusters. Sealed NMR tubes containing clusters 1, 2, 3 and 4 were found to be exceeding stable towards near-UV light and temperatures up to ca. 100°C. The surprisingly robust behavior of the new clusters is contrasted with the related cluster Ru3(CO)10(bpcd) that undergoes fragmentation to the donor-acceptor compound Ru2(CO)6(bpcd) and the phosphido-bridged compound Ru2(CO)6 (µ-PPh2)[µ-C=C(PPh2)C(O)CH2C(O)] under mild conditions. The electrochemical properties have been investigated in the case of clusters 1 and 2 by cyclic voltammetry, and the findings are discussed with respect to the reported electrochemical data on the parent cluster H4Ru4(CO)12.

L’ADN gyrase est une enzyme vitale pour la bactérie grâce à sa capacité de manipuler les molécules d’ADN dans la cellule vivante. Cette capacité fait de l’ADN gyrase une cible idéale pour des composés anti-infectieux. Dans ce travail, l’ADN gyrase a été étudié par des méthodes de modélisatoin moléculaire. Une approche de conception de ligands basée sur la structure a été entreprise sur le sous-domaine N-terminal de 24 kDa de l’ADN gyrase B (domaine GHKL). La flexibilité de deux boucles du site actif du domaine GHKL a été étudiée par des simulations de dynamiques moléculaires en présence de différents ligands. Dans une dernière partie, une analyse des modes normaux du dimère du domaine N-terminal de 43 kDa a été entreprise
DNA gyrase is a vital bacterial enzyme necessary for the handling of the large DNA molecules in the living cell. Therefore DNA gyrase is an ideal target enzyme for anti-infectious compounds. In this work DNA gyrase has been studied by molecular modelling methods. A computational structure-based ligand design approach has been carried out on the N-terminal 24 kDa subdomain of DNA gyrase B (GHKL domain). To further examine the flexibility of two active site loops, molecular dynamics simulations have been carried out on the GHKL domain in different ligand binding conditions. In a final part, normal mode analysis has been carried out on the dimer of the 43 kDa domain of DNA gyrase B

The optimisation of ligand-macromolecule interactions is fundamental to the design of therapeutic agents. The GRID method is a procedure for determining energetically favourable ligand binding sites on molecules of known structure using an empirical energy potential. In this thesis, it has been extended, tested, and then applied to the design of anti-influenza agents. In the GRID method, the energy of a hydrogen-bond is determined by a function which is dependent on the length of the hydrogen-bond, its orientation at the hydrogen-bond donor and acceptor atoms, and the chemical nature of these atoms. This function has been formulated in order to reproduce experimental observations of hydrogen-bond geometries. The reorientation of hydrogen atoms and lone-pair orbitals on the formation of hydrogen-bonds is calculated analytically. The experimentally observed water structures of crystals of four biological molecules have been used as model systems for testing the GRID method. It has been shown that the location of well-ordered waters can be predicted accurately. The ability of the GRID method to assist in the assignment of water sites during crystallographic refinement has been demonstrated. It has also been shown that waters in the active site of an enzyme may be both stabilized and displaced by a bound substrate. Ligands have been designed to block the highly conserved host cell receptor site of the influenza virus haemagglutinin in order to prevent the attachment of the virus to the host cells. The protein was mapped energetically by program GRID and specific ligand binding sites were identified. Ligands, which exploited these binding sites, were then designed using computer graphics and energy minimization techniques. Some of the designed ligands were peptides and these were synthesised and assayed. Preliminary results indicate that they may possess anti-influenza activity.

Throughout the central nervous system (CNS), the Cys-loop superfamily of ligand-gated ion channels {LGICs), including nicotinic acetylcholine, serotonin type-3A, strychnine-sensitive glycine and y-aminobutyric acid A/C receptors, play important roles in synaptic transmission by converting chemical signals into electric signals. Designing potent and subtype-selective ligands with therapeutic value requires knowledge about how ligands interact with their binding sites. y-Aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the mammalian CNS and its binding modes at GABA receptors have not been fully elucidated. GABAc receptors consist of p subunits (p1-p3) and they are known to form homomeric receptors. The five subunits are arranged around a central chloride selective ion channel pore. Each subunit contains a large extracellular N-terminal domain, four transmembrane domains {Ml-M4) of which the second (M2) lines the channel pore and a large M3-M4 intracellular loop. The orthosteric binding site is located at the interface between two subunits in the N-terminal domain and the key residues for ligand binding are found at the five discontinuous loops (A-E). This thesis describes how ligand binding and receptor gating are closely related and explores the effect of receptor conformational changes upon ligand binding. A series of point mutations in the N-terminal domain of the GABAc p1 receptor were created and expressed in Xenopus oocytes. The mutant receptors were then examined using a range of pharmacological tools to probe function which was measured using the two-electrode voltage clamp method. The GABA binding mode was explored at GABA receptors using the enantiomers of 3-fluoro-y-aminobutyric acid (3F-GABA) and stereoisomers of 2,3-difluoro-4-aminobutyric acids as conformational probes. Both enantiomers of 3F­GABA were full agonists, with the R-3F-GABA being approximately 3-fold more potent than 5-3F-GABA at GABAc receptors. In contrast, both enantiomers were partial agonists with similar efficacy and potency at GABAA receptors. These results suggest a different GABA binding mode at GABAc receptors to that found in the related but pharmacologically distinct GABAA receptors. The effect of the different stereoisomers of 2,3-difluoro-4-aminobutyric acids were also examined at GABAA, GABA8 and GABAc receptors. In the study, two enantiomeric GABAc receptor ligands were identified, one of which is an agonist (25,35-2,3-difluoro-4-aminobutyric acid) while the other is an antagonist (2R,3R-2,3-difluoro-4-aminobutyric acid). 4-Amino-3-hydroxybutanoic acid (GABOB) is an endogenous ligand found in the CNS in mammals and a metabolite of GABA. Homology modeling of the GABAc Pi receptor revealed a potential hydrogen (H-bond) interaction between the hydroxyl group of GABOB and threonine 244 (T244) located on loop C of the ligand binding site. Using site-directed mutagenesis, the effect of mutating T244 on the efficacy and pharmacology of GABOB and various ligands were examined. It was found that mutating T244 to amino acids that lacked a hydroxyl group in the side chain produced GABA insensitive receptors. Only by mutating PiT244 to serine (PiT2445) produced a GABA responsive receptor, albeit 39-fold less sensitive to GABA than Pi wild-type. It was also found that this mutation also changed the activity of GABAc receptor partial agonists, muscimol and imidazole-4-acetic acid (I4AA). At the concentrations tested, both muscimol and I4AA antagonized the currents produced by GABA at PiT2445 mutant receptors (Muscimol: PiWild-type, EC50 = 1.4 µM; PiT2445, IC50 = 32.8 µM. I4AA: Pi wild-type, EC50 = 8.6 µM; PiT2445, IC50 = 21.4 µM). This indicates that T244 is predominantly involved in channel gating. R-(-)-GABOB and 5-(+)-GABOB are full agonists at Pi wild-type receptors. In contrast, R-(-)-GABOB was a weak partial agonist at PiT2445 (lmM activates 26 % of the current produced by GABA ECso versus Pi wild-type, EC50 = 19 µM; lmax 100%), and 5-(+)-GABOB was a competitive antagonist at PiT2445 receptors (Pi wild-type, EC50 = 45 µM versus PiT2445, IC50 = 417.4 µM, Ks = 204 µM). This highlights that the interaction of GABOB with T244 is enantioselective. In contrast, the potencies of a range of antagonists tested, 3-aminopropyl(methyl)phosphinic acid (3-APMPA), 3-aminopropylphosphonic acid (3-APA), 5- and R-(3-amino-2-hydroxypropyl)methylphosphinic acid (5-(-)-CGP44532 and R-(+)-CGP44533), were not altered. This suggests that T244 is not critical for antagonist binding. Receptor gating is dynamic and this study highlights the role of loop C in agonist-evoked receptor activation, coupling agonist binding to channel gating. Ligands acting on receptors are considered to induce a conformational change within the ligand-binding site by interacting with specific amino acids. In this study, tyrosine 102 (Y102) located in the GABA binding site of the Pi subunit of the GABAc receptor was mutated to alanine (piY102A), serine (piY102S) and cysteine (piY102C) to assess the role of this amino acid plays on the action of 12 known and 2 novel antagonists. Of the mutated receptors, piY102S was constitutively active providing an opportunity to assess the activity of the antagonists on Pi receptors with a proportion of receptors existing in the open conformational state compared to those existing predominantly in the closed conformational state (pi wild-type, PiY102C and PiY102A). It was found that the majority of antagonists studied were able to inhibit the constitutive activity displayed by PiY1025, thus displaying inverse agonist activity. The exception was (±)-4-aminocyclopent-1-enecarboxamide ((±)-4-ACPAM) not exhibiting any inverse agonist activity, but acting explicitly on the closed conformational state of Pi receptors. It was also found that GABA antagonists were more potent at the closed compared to the open conformational states of Pi receptors suggesting that they may act by stabilizing the closed conformational state and thus reducing activation by agonists. Furthermore, of the antagonists tested, Y102 was found to have the greatest influence on the antagonist activity of gabazine (SR-95531) and its analogue (SR-95813). Our GABAc Pi receptor homology model identified a novel cavity, which extended beyond the GABA binding site. The model predicted phenylalanine 124(F124), one of the residues lining the cavity, was pointing towards the orthosteric binding site. In this study, F124 was mutated to various amino acids and only a modest effect on receptor pharmacology was observed. However, the mutations had a significant effect on the channel deactivation rate ('toeactivation)- This finding suggests that F124 may play a role in channel gating or stabilizing the open conformation of the receptor. Designing potent selective agents are the key for the further understanding of the physiological roles of GABAc receptors. Gabazine (SR-95531) is a potent GABAA receptor competitive antagonist. In this study, a series of novel gabazine analogues were tested at GABAA and GABAc receptors. Of the compounds studied, (p)-methoxy analogue without the butyric acid side-chain was 20-fold more potent at GABAc over GABAA receptors. As there was no butyric acid side chain, it is suggested that the carboxylic acid is not important for gabazine activity at this receptor. Establishing the structure-activity relationship based on this analogue will facilitate the development of selective GABAc receptor antagonists with possible physiological effects including memory-enhancement. Overall, our studies describe agonist and GABAc receptor antagonist induced conformational changes within the ligand binding site. Our findings also highlight the dynamic nature of receptor gating, initiated by ligand binding at a site physically distinct from the ion channel.

Nuclear receptors are ligand activated transcription factors, where upon binding with small molecule ligands, these proteins are involved in the regulation of gene expression. To date there are approximately 48 human nuclear receptors known, involved in multiple biological and cellular processes, ranging from differentiation to maintenance of homeostasis. Due to their critical role in transcriptional regulation, these receptors are implicated in several diseases. Currently, 13% of prescribed drugs in the market are NR ligands for diseases such as cancer, diabetes and osteoporosis. In addition to drug discovery, the mechanism of function, mobility and trafficking of these receptors is poorly understood. Gaining insight into the relationship between the function and /or dysfunction of these receptors and their mobility will aid in a better understanding of the role of these receptors. The green fluorescent protein (GFP) has revolutionized molecular biology by providing the ability to monitor protein function and structure via fluorescence. The fluorescence contribution from this biological marker is the chromophore, formed from the polypeptide backbone of three amino acid residues, buried inside 11-stranded â-barrel protein. Synthesis of GFP derivatives of is based on the structure of the arylmethyleneimidazolidinone (AMI), creating a molecule that is only weakly fluorescent. Characterizing these AMI derivatives for other proteins can provide a powerful visualization tool for analysis of protein function and structure. This development could provide a very powerful method for protein analysis in vitro and in vivo. Development of such fluorescent ligands will prove beneficial for the nuclear receptors. In this work, libraries of AMIs derviatives were synthesized by manipulating various R groups around the core structure, and tested for their ability to serve as nuclear receptor ligands with the ability to fluoresce upon binding. The fluorogens are developed for steroidal and non-steroidal receptors, two general classes of nuclear receptors. Specific AMIs were designed and developed for steroid receptor estrogen receptor á (ERá). These ligands are showed to activate the receptor with an EC50 of value 3 ìM and the 10-fold activation with AMI 1 and AMI 2 in comparison to the 21-fold activation observed with natural ERá ligand, 17â-estradiol. These novel ligands were not able to display the fluorescence upon binding the receptor. However, fluorescence localized in nucleus was observed in case of another AMI derivative, AMI 10, which does not activate the receptor. Such ligands open new avenues for developing fluorescent probes for ERá that do not involve fluorescent conjugates attached to a known ERá ligand core. AMIs were also characterized for non-steroidal receptors,specifically the pregnane x receptor (PXR) and retinoic acid receptor á (RARá). To date, fluorogens which turn fluorescence upon binding and activate the receptor have not been developed for these receptors. With respect to PXR, several AMI derivatives were discovered to bind and activate this receptor with a fold-activation better than the known agonist, rifampicin. The best characterized AMI derivative, AMI 4, activates the receptor with an EC50 of value 6.3 ìM and the 154-fold activation in comparison to the 90-fold activation and an EC50 value of 1.3 ìM seen with rifamipicin. This ligand is not only able to activate PXR but also displays fluorescence upon binding to the receptor. The fluroscence pattern was observed around the nucleus. Besides AMI 4, 16 other AMI derivatives are identified that activate PXR with different activation profiles. Thus, a novel class of PXR ligands with fluorescence ability has been developed. The AMI derivatives able to bind and activate RAR, also displayed activation profiles that were comparable to the wild-type ligand, all trans retinoic acid. These ligands activated the receptor with an EC50 value of 220 nM with AMI 109 in comparison to an EC50 value of 0.8 nM with the natural ligand for RARá. When these ligands were tested for fluorescence in yeast, the yeast were able to fluoresce only in the presence of the receptor and the AMI derivative, indicating that these agonists also have the ability to fluoresce.

The human pentraxin proteins, serum amyloid P component (SAP) and C-reactive protein (CRP) have emerged as potentially important targets in the treatment of amyloidosis and cardiovascular diseases respectively, although their normal physiological functions are unclear. Structurally highly conserved homologous proteins are present in common experimental animals such as the rat, mouse, rabbit and hamster but there are major differences from the human p,entraxins in their normal behaviour as acute phase proteins, fine ligand specificity and capacity to activate the complement system. . SAP binds to amyloid fibrils ofall types and may contribute to their formation, stabilisation and persistence. In order to extend our current knowledge ofligand recognition by SAP, the crystal structures ofSAP complexed to two ligands, Methylmalonic acid and Phosphatidylethanolamine, have been solved to 1.6 Aand 1.4Aresolution respectively. Since important biological functions ofproteins are often conserved among species, the structural differences between the rat and human pentraxins were investigated. The crystal structure ofrat SAP was solved to 2.2 Aresolution by molecular replacement. This pentameric structure displayed subtle differences in the electrostatic properties. It remains to be determined whether this has an effect on avid binding of SAP to DNA, a functional property ofh~manSAP still poorly understood. CRP, a pentraxin traditionally defined by its binding affinity for PC, was studied in complex with PE. The crystal structure ofthe CRP-PE complex at 2.7 Aresolution revealed that the nitrogen end ofPE dips further downwards into the hydrophobic pocket ofCRP than PC. CRP-mediated complement activation can exacerbate ischemic tissue injury in the heart as well as in the brain. Therefore, knowledge ofthe exact stoichiometry and the protein-protein interactions between CRP and C1q may aid the development ofsmall molecules capable ofdisrupting such protein-protein interactions. Purification of C1q has been achieved by ion-exchange chromatography and gel filtration from BPL paste. Crystallisation trials have been performed, however no crystals have been observed that contain the protein-protein complex.

Accurate prediction of binding free energies of protein-ligand system has long been a focus area for theoretical and computational studies; with important implications in fields like pharmaceuticals, enzyme-redesign, etc. The aim of this project was to develop such a predictive model for calculating binding free energies of protein-ligand systems based on the LIE-SASA methods. Many models have been successfully fit to experimental data, but a general predictive model, not reliant on experimental values, would make LIE-SASA a more powerful and widely applicable method. The model was developed such that There is no significant increase in computational time No increase in complexity of system setup No increase in the number of empirical parameters. The method was tested on a small number of protein-ligand systems, selected with certain constraints. This was our training set, from which we obtain the complete expression for binding free energy. Expectedly, there was good agreement with experimental values for the training set On applying our model to a similar sized validation set, with the same selection constraints as for the training set, we achieved even better agreement with experimental results, with lower standard errors. Finally, the model was tested by applying it to a set of systems without such selection constraints, and again found good agreement with experimental values. In terms of accuracy, the model was comparable to a system specific empirical fit that was performed on this set. These encouraging results could be an indicator of generality.

The kinetics of ligand substitution for CO in Co4(CO)10(mu4-PPh2) , 1, have been investigated for the ligands P(OMe)3, P(OEt)3, PPh2H, P(0-i-Pr)3, P(n-Bu)3, PPh3, P(i-Pr)3, and PCy3 over a wide temperature range.

The most favoured amino acid in protein-nucleic acid interfaces is arginine due to the existence of the guanidinium group in its structure that can form good hydrogen-bonding geometries. In addition, the length of the side chain gives it a great deal of torsional freedom and therefore permits the guanidinium group to orient itself to interact with a wide range of targets through bidentate H-bonding. A torsional constraint for the guanidine group could be achieved by its conversion into a bicyclic framework, resulting in unidirectional hydrogen bonding with its targets and reducing the loss of the conformational entropy as a result of its binding. The aim of this research is to synthesise nucleic acid targeting oligopeptides which will include non-natural analogues of arginine as part of their sequence with the purpose of activating or inhibiting biological functions. The synthesis of different sized rings of bicyclic guanidine derivatives could be a means to control the hydrophobic interaction with DNA, with hydrophobicity increasing as the size of the ring of the bicyclic guanidine increases. In addition, the high basicity of guanidine leads to production of the corresponding guanidinium cation upon protonation. Therefore, the formation of guanidinium cation leads to hydrogen bonding and electrostatic interactions with anions like phosphate groups. So, the purpose of preparing differently sized rings of bicyclic guanidine in the current work is to compare their interactions with DNA. There are four themes in our research concerned with the design and synthesis of bicyclic guanidine derivatives and non-natural amino acids. The first theme concerns the synthesis of a protected bicyclic guanidine methylene amine that was achieved by utilising Boc-L-methioninol as a precursor and converting it to the corresponding azide. However, incorporation of this bicyclic guanidine into an amino acid moiety proved difficult and thus themodified amino acid was not formed. The second theme of the project considers a novel strategy to generate a bicyclic guanidine methylene alcohol with a variably sized ring in a high yield starting from an azido alkane amine and Boc-L-methionine as precursors. This modification to the route will open a promising way to produce the functionalised bicyclic guanidine. Bicyclic guanidine methylene chloride was synthesised that gave us a way to insert bicyclic guanidine into a tyrosine amino acid moiety so that a protected L-tyrosine possessing bicyclic guanidine with a differently sized ring could be synthesised. The third theme involved the new strategy towards the synthesis of bicyclic guanidine methylene amine followed by conjugation with Fmoc-Glu-OAll to produce a modified amino acid. The fourth, and final,theme of our research includes the preparation of dimers of bicyclic guanidine. Furthermore,a crystal structure for the protected bicyclic guanidine intermediate in this theme has also been obtained.

Low affinity Fcg receptor III (FcgRIII, CD16) triggers a variety of cellular events upon binding to the Fc portion of IgG. A real-time flow cytometry method was developed to measure the affinity and kinetics of such low affinity receptor/ligand interactions, which was shown as an easily operated yet powerful tool. Results revealed an unusual temperature dependence of reverse rate of CD16aTM dissociating from IgG. Except for a few studies using mammalian cell CD16s, most kinetics analyses use purified aglycosylated extracellular portion of the molecules, making it impossible to assess the importance of the receptor anchor and glycosylation on ligand binding. We used a micropipette adhesion frequency assay to demonstrate that the anchor length affects the forward rate and affinity of CD16s for IgG in a species specific manner, most likely through conformational changes. Receptor glycosylation dramatically reduced ligand binding by 100 folds. T cell receptor (TCR) is arguably the most important receptor in the adaptive human immune system. Together with coreceptor CD4 or CD8, TCR can discriminate different antigen peptides complexed with major histocompatibility complex (MHC) molecule (pMHC), which differ by as few as only one amino acid, and trigger different T cell responses. When T cell signaling was suppressed, TCR had similar affinity and kinetics for agonist and antagonist pMHC whose binding to CD8 was undetectable. TCR on activated T cell had a higher affinity for pMHCs, suggesting that TCRs organize themselves differently on activated T cells than on naïve T cells. In the absence of inhibitors for signaling, TCR binds agonist pMHC with several orders of magnitude higher affinity than antagonist pMHC. In addition, engagement of TCR by pMHC signals an upregulation of CD8 binding to pMHC, which is much stronger than the TCR-pMHC binding. The transition from weak TCR binding to the strong CD8 binding takes place around 0.75 second after TCR in contact with pMHC and can be reduced by several inhibitors of tyrosine and lipid phosphorylation, membrane rafts, and actin cytoskeleton. These results provide new insights to understanding T cell discrimination.

Neuropilin (Nrp) is an essential cell surface receptor with dual functionality in the cardiovascular and nervous systems. The first identified Nrp-ligand family was the Semaphorin-3 (Sema3) family of axon repulsion molecules. Subsequently, Nrp was found to serve as a receptor for the vascular endothelial growth factor (VEGF) family of pro-angiogenic cytokines. In addition to its physiological role, VEGF signaling via Nrp directly contributes to cancer stemness, growth, and metastasis. Thus, the Nrp/VEGF signaling axis is a promising anti-cancer therapeutic target. Interestingly, it has recently been shown that Sema3 and VEGF are functionally opposed to one another, with Sema3 possessing potent endogenous anti-angiogenic activity and VEGF serving as an attractive cue for neuronal axons. We hypothesized that direct competition for an overlapping binding site within the Nrp extracellular domain may explain the observed functional competition between VEGF and Sema3. To test this hypothesis we have separately investigated the mechanisms of VEGF and Sema3 binding to Nrp. Utilizing structural biology coupled with biophysics and biochemistry we have identified both distinct and common mechanisms that facilitate the interaction between Nrp and these two ligand families. Specifically, we have identified an Nrp binding pocket to which these ligands competitively bind. The Sema3 family uniquely requires proteolytic activation in order to engage this overlapping binding site. These findings provide critical mechanistic insight into VEGF and Sema3 mediated physiology. Additionally, these data have informed the development of small molecules, peptides, and soluble receptor fragments that function as potent and selective inhibitors of VEGF/Nrp binding with exciting therapeutic potential for treating cancer.

Synthetic molecules that target the major groove in a sequence-selective way are a major goal in molecular medicine. Recently a major step has been taken toward achieving this goal: a novel cylinder has been developed that binds strongly into the major groove of DNA. Experimental techniques have provided some information regarding the binding strength and preferred binding sites of the cylinder on DNA. From all the experimental data it is clear that the parent cylinder binds in the major groove and is able to induce dramatic conformational changes in the DNA; these are unprecedented effects with synthetic DNA binders. However, gaining molecular level information in such a macromolecular system is challenging. Molecular dynamics (MD) simulations can provide information at the molecular level that is complementary to experiment and therefore are an ideal way to get a better understanding of this system. In this work we present the results of various MD simulations designed to probe the DNA-cylinder system. We have studied the effect of using CHARMM22 and CHARMM27 as the force-field for the simulations. Results showed that uncomplexed DNA simulated with CHARMM22 was less stable in the B-form than the comparable strand of DNA simulated with CHARMM27. Investigations into the effects of the cylinders charge and shape are also reported. Multi-nanosecond simulations were performed using two related synthetic cylinders, one with two Fe(II) metal centers and the other with two Cu(I) centers, and DNA. Finally the role of DNA within the system was investigated by performing a series of simulations of the cylinders with d(ATATATATATAT)2, d(CGCGCGCGCGCG)2 and d(CGCGCATATACG). Simulations with these DNA strands has only produced one system (CCu2+ with d(ATATATATATAT)) where the cylinder causes a conformational change in the DNA.

The presence of μ-, δ- and κ-binding sites in homogenates of guinea-pig brain was demonstrated by the use of selective labelling techniques. In saturation experiments, the tritiated ligands [^3H]-[D-Ala^2, MePhe^4, Gly-ol^5]enkephalin, [^3H]-dihydromorphine, [^3H]-morphine and [^3H]-dihydronormorphine labelled only the μ-binding site. The δ-binding site could be labelled selectively with [^3H]-[D-Pen^2, D-Pen^5]enkephalin. However, the less selective δ-ligand, [³H]-[D-Ala², D-Leu⁵] enkephalin, could only be used when its μ-binding was blocked with the unlabelled μ-ligand [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin. Selective labelling of the κ-binding site was more of a problem since the non-selective ligands [^3H]-etorphine, [^3H]-(±)-ethylketazocine and [³H]-(-)-bremazocine bind to the μ-, δ- and κ-sites. Therefore, the κ-binding site could only be labelled selectively when the binding of the tritiated ligands to the μ- and δ-sites was prevented by addition of the unlabelled μ-ligand [D-Ala^2, MePhe^4, Gly-ol^5]enkephalin and the unlabelled δ-ligand [D-Ala², D-Leu⁵]enkephalin. By analysis of the saturation curves obtained using these selective labelling techniques, the proportion of binding sites in homogenates of guinea-pig brain at 25°C was 24% μ-sites; 32% δ-sites and 44% κ-sites. The selective labelling techniques were also used to label the μ, δ- and κ-sites in displacement assays. The compounds with the highest degree of preference for each binding site were: for the μ-site, [D-Ala^2, MePhe^4, Gly-ol^5]enkephalin and Tyr-Pro-MePhe-D-Pro-NH_2; for the δ-site, [D-Pen², L-Pen⁵]enkephalin, [D-Pen², D-Pen⁵]enkephalin and ICI 174864 and for the κ-site, U-50,488H and U-69,593. As far as antagonists were concerned, naloxone displayed the highest preference for the μ-binding site and Mr 2266 had a preference for the κ-binding site but neither compound was highly selective unlike the δ-antagonist ICI 174864. The effect of pre-incubation with β-funaltrexamine on opioid binding was investigated in homogenates of guinea-pig brain and myenteric plexus-longitudinal muscle.

In the past 30 years, the discovery of capabilities of nucleic acids far beyond their well-known information-bearing capacity has profoundly influenced our understanding of these polymers. The discovery by the Cech and Altman labs that nucleic acids could perform catalytic functions, coupled with the Gold and Szostak groups’ demonstration of the de novo evolution of nucleic acids that bind arbitrary ligands, has resulted in a proliferation of newfound roles for these molecules. Nucleic acids have found utility in both engineered systems, such as aptamer therapeutics, as well as in newly appreciated roles in extant organisms, such as riboswitches. As a result of these discoveries, many have pondered the potential importance of the dual (catalytic and informational) roles of nucleic acids in early evolution. A high-yielding synthetic route for the nonenzymatic polymerization of nucleic acids, based on the aqueous self-assembly of their components, would provide a powerful tool in nucleic acid chemistry, with potential utility in prebiotic and contemporary nucleic acid systems alike – however, such a route remains elusive. In this thesis, I describe several steps towards such a synthetic route. In these systems, a nucleic-acid binding ligand drives the assembly of short DNA and RNA duplexes, promoting the production of long nucleic acid polymers, while suppressing the production of short, cyclic species. Additionally, the use of a reversible covalent linkage allows for the production of long polymers, as well as the incorporation of previously cyclized products into these polymers. I also report several explorations of novel base pairings, nucleic acid-ligand interactions, and nucleic acid-ion interactions that have informed our studies of self-assembling nucleic acid systems.

The affinity of a drug to its target protein is one of the key properties of a drug. Although there are experimental methods to measure the binding affinity, they are expensive and relatively slow. Hence, accurately predicting this property with software tools would be very beneficial to drug discovery. In this thesis, several applications have been developed to model and predict the binding mode of a ligand to a protein, to evaluate the feasibility of that prediction and to perform model interpretability in deep neural networks trained on protein-ligand complexes.
La afinidad de un fármaco a su proteína diana es una de las propiedades clave de un fármaco. Actualmente, existen métodos experimentales para medir la afinidad, pero son muy costosos y relativamente lentos. Así, predecir esta propiedad con precisión empleando herramientas de software sería muy beneficioso para el descubrimiento de fármacos. En esta tesis se han desarrollado aplicaciones de software para modelar y predecir el modo de unión de ligando a proteína, para evaluar cómo de factible es tal predicción y para interpretar redes neuronales profundas entrenadas en complejos proteína-ligando.

Rational conversion of biologically active peptides to nonpeptide compounds with retained activity is an appealing approach in drug development. One important objective of the work presented in this thesis was to use computational modeling to aid in such a conversion of the peptide angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe). An equally important objective was to gain an understanding of the requirements for ligand binding to the Ang II receptors, with a focus on interactions with the AT2 receptor. The bioactive conformation of a peptide can provide important guidance in peptidomimetic design. By designing and introducing well-defined secondary structure mimetics into Ang II the bioactive conformation can be addressed. In this work, both γ- and β-turn mimetic scaffolds have been designed and characterized for incorporation into Ang II. Using conformational analysis and the pharmacophore recognition method DISCO, a model was derived of the binding mode of the pseudopeptide Ang II analogues. This model indicated that the positioning of the Arg side chain was important for AT2 receptor binding, which was also supported when the structure–activity relationship of Ang II was investigated by performing a glycine scan. To further examine ligand binding, a 3D model of the AT2 receptor was constructed employing homology modeling. Using this receptor model in a docking study of the ligands, binding modes were identified that were in agreement with data from point-mutation studies of the AT2 receptor. By investigating truncated Ang II analogues, small pseudopeptides were developed that were structurally similar to nonpeptide AT2 receptor ligands. For further guidance in ligand design of nonpeptide compounds, three-dimensional quantitative structure–activity relationship models for AT1 and AT2 receptor affinity as well as selectivity were derived.

Maltose Binding Protein (MBP) of Escherichia Coli, a kind of periplasmic protein, can bind its ligands interacting predominantly either with their anomeric end (end-on binding) or with the middle of the maltodextrin chain (middle binding). Using NMR spectroscopy, we have studied the modes by which maltose, linear maltodextrin and some derivatives like $ beta$-cyclodextrin bind to MBP. 1D proton difference spectra and 2D HSQC proton-nitrogen correlation spectra were acquired of MBP in the presence of different ligands. Spectra with linear maltodextrins showed many common features and were distinctly different from those of MBP with $ beta$-cyclodextrin. 2D HSQC spectra suggest further that MBP- $ beta$-cyclodextrin adopts an open form conformation similar to that of ligand free MBP because of the surprisingly similarity of their spectra. Ligands such as $ beta$-cyclodextrin, can not be transported into the cyto-plasm but have high affinity for MBP, multiple $ alpha$(1-4) linkages and no reducing end. These ligands bind to MBP mainly by the middle binding mode. This suggests that this mode determines high affinity binding of ligand to MBP, but doesn't produce a physiologically active complex.

Cholesterol and fatty acyl-coenzymeA thioesters are signalling molecules with role in regulation of genes involved in lipid and glucose transport and metabolism. The studies described herein focused on three proteins that bind lipids and have different cellular functions: steroidogenic acute regulatory protein (StAR), hepatocyte nuclear factor-4a (HNF-4a) and acyl-CoA binding protein (ACBP). First, StAR mediates delivery of cholesterol to inner mitochondrial membrane in steroidogenesis by a poorly understood mechanism. In our studies, fluorescent NBD-cholesterol binding assays demonstrate that StAR binds cholesterol at two binding sites with 32 nM Kds and circular dichroism spectra show that cholesterol binding results in changes of StAR secondary structure. Fluorescent sterol exchange assays between donor and acceptor mitochondrial membranes indicate that StAR significantly increased the formation of rapidly transferable cholesterol domains. Second, HNF-4a, a nuclear receptor, had been shown to bind fatty acyl-CoAs as natural ligands with apparent low affinities obtained with radiolabeled ligand binding assays. Our fluorescence spectroscopy studies demonstrate that HNF-4a ligand binding domain (HNF-4aLBD) binds acyl-CoAs at a single binding site with Kds of 1.6-4 nM. Fluorescence resonance energy transfer (FRET) between HNF-4aLBD tryptophan residues and cis-parinaroyl-CoA yielded an intermolecular distance of 42 Â thus pointing to direct molecular interaction. Third, although ACBP has been detected in the nucleus, it is not known whether ACBP may directly and/or functionally interact with a nuclear acyl-CoA binding protein such as HNF-4a to regulate transcription. Our present studies in vitro and in intact cultured cells, including circular dichroism of HNF-4a in the presence of ACBP, coimmunoprecipitation of HNF-4a/ACBP complexes, ACBP and HNF-4a colocalization in nuclei of cells by confocal microscopy demonstrate a physical association of ACBP and HNF-4a. FRET microscopy data indicated an intermolecular distance of 53 Â between ACBP and HNF-4a in rat hepatoma cells. Functional assays (transactivation of an HNF4a-dependent reporter gene) showed significant increase in the presence of ACBP in two different cell lines. Expression of ACBP anti-sense RNA decreased HNF-4a-mediated transactivation, pointing to a role of ACBP in co-regulating HNF-4a-dependent transcription.

El trabajo presentado en esta tesis cubre varios campos de investigación relacionados con el desarrollo de moléculas bioactivas. Se compone de cinco partes distintas que se resumen aquí. Predicción de la utilidad farmacológica de dianas terapéuticas. El desarrollo de fármacos está generalmente dirigido a inhibir la función de una proteína específica. Pero para validar esta proteína como diana terapéutica, al principio de un proyecto de descubrimiento de fármacos se tiene que saber si una molécula de tipo fármaco puede unirse con suficiente afinidad a la proteína como para alterar su función. Existen métodos que predicen si una potencial diana terapéutica es tratable o no por vía farmacológica, lo que se ha dado en llamar ‘druggability’. El problema es que estos métodos no están accesibles libremente y su validación es discutible. En la primera parte de la tesis se ha compilado un conjunto extensivo de datos de cavidades en proteínas cuyo novel de ‘druggability’ es conocido, haciéndolo accesible en una plataforma web pública (http://fpocket.sourceforge.net/dcd). Estos datos pueden ser modificados por cualquier persona que quiera contribuir al desarrollo de este conjunto de datos, aumentando su volumen o mejorando su calidad. En estudios previos, los sitios druggable se han asociado a cavidades profundas e hidrofóbicas, ignorando la importancia que tiene los grupos polares en el sitio de unión y su posible relación con la ‘druggability’. Utilizando el set de datos compilado previamente, hemos encontrado que aunque las cavidades ‘druggables’ son mas hidrofóbicas, también tienen grupos polares más expuestos pero con poca superficie de interacción. Esta observación es objeto de posteriores investigaciones en la segunda parte de la tesis. Finalmente, se ha utilizado un algoritmo de búsqueda de sitios de unión, fpocket, que empecé a desarrollar como proyecto de master. Este programa se ha utilizado para extraer todas las características de las cavidades ‘druggables’ y no ‘druggables’ y estos parámetros se han utilizado para entrenar un modelo logístico capaz de predecir si un sitio es druggable o no. Demostramos que el algoritmo y la función de puntuación desarrollado durante esta primera parte predice la ‘druggability’ de manera fiable. Los resultados son de igual calidad a los obtenidos con el único otro programa accesible con funcionalidad parecida (SiteFinder, de Schrödinger), pero nuestro programa tiene las siguientes importantes ventajas: 1) es libre; 2) es mucho más eficiente computacionalmente; y 3) trabaja sobre cavidades detectadas automáticamente por el programa, lo que permite aplicaciones a gran escala. En otras partes de la tesis se verá como su aplicación al conjunto del PDB permite novedosas aplicaciones en el área del diseño de fármacos. Análisis de movilidad de cavidades de las proteínas Existen una gran variedad de algoritmos que permiten identificar posibles sitios de unión en las estructuras tridimensionales de las proteínas. El trabajo presentado en la sección anterior de esta tesis permitía extender uno de estos algoritmos para caracterizar la ‘druggability’ de las cavidades. Un problema de gran calado en el estado actual de la técnica, tanto de detección de sitios de unión como en diseño de fármacos en general,4 es que las proteínas se tratan como un cuerpo rígido a pesar de que en realidad gozan de una gran movilidad estructural. El objetivo de esta sección era otorgar todavía otra funcionalidad a fpocket, el programa de predicción de sitios de unión, para permitir también la detección y el análisis de cavidades de proteínas en movimiento. Habitualmente, los movimientos de las proteína se pueden simular usando la dinámica molecular (MD). Una herramienta capaz de analizar conjuntos de estructuras derivados de MD u otras fuentes puede ser, por tanto, extremadamente útil para observar la aparición de cavidades transitorias y su plasticidad. Como resultado final de este trabajo, se presenta un nuevo programa informático, llamado MDpocket y que se enmarca dentro del paquete fpocket. Para cada conformación de la proteína, se ejecuta un ciclo de detección de cavidades con fpocket. Los resultados de este proceso se plasman sobre una malla tridimensional superpuesta a la estructura de la proteína. La malla puede entonces ser visualizada o analizada en mayor detalle. Lo primero se puede llevar a cabo con programas de visualización molecular tales como PyMOL, VMD o Chimera. Otra funcionalidad dentro de MDpocket es la de seguir la evolución de las propiedades de una cavidad o zona de interés (definida por el usuario) a lo largo del tiempo. Cabe destacar que MDpocket es igualmente capaz de identificar sitios de unión de moléculas tipo fármaco como pequeños canales en la matriz proteica que pueden ser importantes para la migración de pequeños ligandos como gases o moléculas de agua. Las posibles aplicaciones de MDpocket se ejemplifican en tres casos distintos. El primero es la capacidad de identificar la apertura transitoria de cavidades en el sitio de unión de ATP en la proteína HSP90. En el segundo ejemplo, se muestra como MDpocket permite identificar un canal de migración de moléculas biatómicas en mioglobina, un sistema de referencia bien conocido. Aquí se demuestra que MDpocket puede, no tan solo identificar los sitios internos de unión a Xenón, sino también los canales que se abren de forma transitoria para permitir a los ligandos migrar de un sitio a otro. En el último ejemplo, las propiedades del sitio de unión a ATP de la proteína cinasa P38 se analizaron a lo largo de una trayectoria de MD, evaluando la capacidad de MDpocket para identificar aquellas conformaciones que pueden ser particularmente útiles para realizar docking molecular. Uno de los principales problemas en docking de proteína-ligando es que el receptor generalmente se considera rígido, mientras que si se utilizan múltiples conformaciones (cristalográficas o derivadas de MD) para representar al receptor es difícil decidir a priori cuales de ellas pueden dar mejores resultados. Aquí mostramos que MDpocket se puede utilizar para seleccionar conformaciones concretas de una trayectoria de MD para usarlas en procesos de docking. Concretamente, hemos observado que la densidad hidrofóbica promedio (previamente identificada como un descriptor importante para predecir ‘druggability’) correlaciona bien con la probabilidad de que el modo de unión de ligandos pueda ser predicho correctamente. Tal como en el trabajo anterior, MDpocket se incluye dentro del proyecto fpocket y se está accesible como una herramienta libre y de código abierto. Relaciones estructura-cinética de unión El control de los tiempos en interacciones moleculares es una propiedad esencial de los sistemas bioquímicos, pero poco se conoce sobre los factores estructurales que gobiernan la cinética de los procesos de reconocimiento molecular. Partiendo de una observación realizada durante el trabajo de predicción de ‘druggability’, aquí se ha investigado el papel que átomos con poca superficie expuesta a solvente pueden jugar en los sitios de unión de la proteína. En particular, encontramos que los átomos polares en los sitios druggable son minoritarios en comparación con los átomos apolares, pero si bien pueden tener poca superficie accesible, tienden a ser mas protuberantes, lo que los hace más accesibles para establecer interacciones. Hemos establecido que esta propiedad puede estar relacionada con la cinética de unión/disociación de un ligando a su cavidad en el receptor. En diseño de fármacos, la vida media del complejo formado entre el fármaco y su diana terapéutica determina en gran medida sus efectos biológicos, pero en ausencia de relaciones estructura cinética, se hace imposible optimizar esta propiedad de forma racional. Aquí se muestra que átomos polares prácticamente enterrados (ABPAs) – un elemento comúnmente encontrado en los sitios de unión de proteínas – tienden a formar puentes de hidrógeno que están protegidos de las moléculas de agua. La formación y ruptura de este tipo de puentes de hidrógeno implica un estado de transición penalizado energéticamente porque ocurre de modo asincrónico con el proceso de deshidratación/rehidratación. En consecuencia, los puentes de hidrógeno protegidos se intercambian a velocidades lentas. Estas conclusiones se basan en el estudio computacional del proceso de unión de un pequeño ligando a un sitio de unión modelo. El receptor modelo se construyó para permitir modular tanto el grado de exposición del átomo polar como la curvatura del entorno apolar. Mediante el uso de dinámicas moleculares con constricciones y la relación de Jarzinsky, se obtuvieron los perfiles de energía libre de unión para cada cavidad. La presencia de un estado de transición (y por tanto menor velocidad de asociación/disociación) puede anticiparse mediante un simple análisis estructural tal como la medición de la superficie accesible del átomo polar o su grado de protrusión. Esto constituye una nueva y valiosa clave para interpretar y predecir relaciones estructura-actividad, que se ha puesto a prueba investigando sistemas reales. En primer lugar, analizando tanto estructuras cristalográficas depositadas en el PDB como trayectorias de dinámica molecular, se ha demostrado que aquellas moléculas de agua que forman puentes de hidrógeno con ABPAs tienden a tener menor movilidad e intercambios más lentos. Posteriormente, la validez del principio se ha demostrado en dos pares de inhibidores de la proteína Hsp90, una diana terapéutica para cáncer, para los que se han obtenido datos estructurales, termodinámicos y cinéticos mediante distintas técnicas experimentales. El acuerdo entre observables macroscópicas y los resultados de simulaciones moleculares confirma la función de los puentes de hidrógeno protegidos de solvente como trampas cinéticas e ilustra como nuestro hallazgo puede ser usado para facilitar el proceso de diseño de fármacos basado en estructura. Base de datos de cavidades: hacia el ‘pocketoma’ El trabajo presentado en el principio de la tesis perseguía un objetivo muy especifico, que se enmarca en un proyecto mayor del grupo de investigación. La herramienta de predicción de ‘druggability’ se desarrolló con el fin de cribar grandes bases de datos estructurales, tales como el PDB, identificando complejos proteína-proteína no obligados (transitorios) que contengan en su interfase una cavidad potencialmente capaz de unir moléculas de tipo fármaco. Con ello se pretende estabilizar selectivamente dicha interacción y conseguir un efecto biológico que pueda ser terapéutico. Dado que la información sobre cavidades y su druggability asociada va a ser explotadas por otras personas en el grupo en este y otro tipo de proyectos encaminados a facilitar la explotación de nuevos mecanismos de acción, es necesario crear una base de datos que contenga esta información y sea fácilmente navegable. Para empezar, se ejecutó el programa para cada una de las estructuras depositadas en el PDB, identificando todas las cavidades y extrayendo sus descriptores, entre los que se incluye la función de puntuación de druggability. Esta información se guarda de forma organizada en una base de datos relacional (pocketDB), que se relaciona con otras bases de datos tales como Uniprot y Uniref, que contienen información sobre secuencias. Igualmente, se incluyeron información sobre la estructura cuaternaria y otros recursos tales como Kegg, haciendo de pocketDB un potente recurso para filtrar de modo eficiente millones de cavidades, identificando aquellas que tengan mayor interés para cada proyecto. De modo particular, cabe destacar la aplicación de pocketDB a la identificación de cavidades ‘druggable’ situadas en la interfase de complejos transitorios proteína-proteína, resultando en 39 complejos candidatos, entre los que se recobraron 3 casos conocidos previamente, lo que valida la metodología. Además otros tres sistemas identificados, después de una inspección minuciosa, han sido seleccionados en el grupo como candidatos para realizar la prueba de concepto que valide esta nueva estrategia farmacológica. Uno de ellos está actualmente en fase de validación experimental. El ‘pocketoma’ La última sección de mi tesis presenta un proyecto que hace un uso extensivo de la base de datos de cavidades (pocketDB) previamente presentada. Dos cuestiones importantes aún persisten hoy día en el proceso de descubrimiento de fármacos y, de algún modo, contribuyen a la alta tasa de fracasos en las fases tardías del desarrollo, que normalmente se explican por la baja eficacia o el exceso de efectos secundarios (normalmente tóxicos) para el organismo. Ambas situaciones pueden ser explicadas por un fenómeno común: la falta de selectividad. Las fármacos interaccionan en la célula con una diversa variedad de macromoléculas además de con la diana terapéutica, induciendo así efectos secundarios imprevistos. Asimismo, considerar solamente una diana para nuestro fármaco puede resultar menos efectivo de lo esperado, puesto que la pérdida de función de una sola proteína diana puede ser fácilmente reemplazada debido a los mecanismos homeostáticos controlados por robustas redes de interacciones, derivando en una pérdida de eficacia de nuestra molécula. Este trabajo pretende sentar las bases para poder establecer relaciones entre macromoléculas biológicas en base a su potencial para interaccionar con una misma entidad química (fármaco). El trabajo se basa en la asunción de que cavidades similares son capaces de unir ligandos similares. Existen varios métodos que calculan la similitud entre cavidades de unión, sin embargo, hasta la fecha no se ha realizado un análisis relacional profundo de todas las cavidades en el PDB que permita establecer relaciones entre ellos. Con el fin de cumplir con los requerimientos técnicos de unos objetivos tan ambiciosos, se ha desarrollado un novedoso método de comparación de cavidades. En este particular método se considera una representación muy abstracta de la cavidad. Dicha representación reúne información tanto sobre la forma de la cavidad cómo sobre la distribución por pares de los puntos de interacción en la superficie. No obstante, la información sobre la topología exacta de la cavidad es ignorada. Tal abstracción intenta relacionar cavidades que estructuralmente están alejadas, pero que se parecen entre ellas en términos de forma y propiedades fisicoquímicas globales. Usando varios ejemplos de validación en un conjunto de cavidades de referencia, el método resulta capaz de recuperar de un gran set de cavidades aquellas más similares y relacionadas entre sí. Del mismo modo, el método demuestra ser útil para encontrar relaciones entre cavidades previamente no relacionadas y que sin embargo unen los mismos ligandos o moléculas muy similares. Esta nueva implementación se ha utilizado en un experimento de exploración a gran escala para encontrar (i) las mismas cavidades en diferentes estructuras, (ii) cavidades relacionadas y (iii) cavidades con la misma estructura de ligando. Se obtuvieron excelentes resultados para estas tres categorías. Seguidamente, se compararon entre ellas todas las cavidades encontradas en el PDB. Se desarrolló una herramienta computacional novedosa para permitir la navegación en el 'pocketoma' resultante de las comparaciones, que será posible descargar gratuitamente. Los resultados presentados muestran por primera vez un espacio global interrelacionando cavidad-ligando para todas las cavidades del PDB. Finalmente, se señalan a continuación dos aplicaciones que ponen de manifiesto el posible impacto del ‘pocketoma’ y la herramienta de navegación. En el primer ejemplo, una cavidad no caracterizada encontrada en GSK3-β fue comparada contra todas las cavidades del PDB, permitiendo obtener una variedad de cavidades, y sistemas, supuestamente relacionadas. Entre los resultados se encontraban las subunidades de unión a ADP dhaL y dhaM de la PTS dependiente di-hidroacetona cinasa. Se encontró que el sitio de unión a ADP era muy similar a la cavidad investigada en GSK3-β con una sorprendente similitud estructural entre ambos. En el último ejemplo, se navegó por el ‘pocketoma’ utilizando la herramienta visual desarrollada para tal tarea. Durante la exploración se encontró una curiosa e importante relación entre el sitio de unión de la hormona del receptor de estrógenos y una cavidad no caracterizada en la proteína Caspasa-3. Seguidamente, se realizó un estudio de docking con ligandos similares a la hormona y se procedió a realizar una extensiva dinámica molecular con el ligando en la mejor posición para verificar la estabilidad del compuesto en la cavidad de Caspasa-3. El complejo proteína-ligando estudiado resultó ser muy estable. Actualmente, el compuesto identificado se encuentra bajo validación experimental por nuestros colaboradores.

Accurate calculations of free energies for molecular association and solvation are important for the understanding of biochemical processes, and are useful in many pharmaceutical applications. In this thesis, molecular dynamics (MD) simulations are used to calculate thermodynamic properties for solvation and ligand binding.

The thermodynamic integration technique is used to calculate pK_a values for three aspartic acid residues in two different proteins. MD simulations are carried out in explicit and Generalized-Born continuum solvent. The calculated pK_a values are in qualitative agreement with experiment in both cases. A combination of MD simulations and a continuum electrostatics method is applied to examine pK_a shifts in wild-type and mutant epoxide hydrolase. The calculated pK_a values support a model that can explain some of the pH dependent properties of this enzyme.

Development of the linear interaction energy (LIE) method for calculating solvation and binding free energies is presented. A new model for estimating the electrostatic term in the LIE method is derived and is shown to reproduce experimental free energies of hydration. An LIE method based on a continuum solvent representation is also developed and it is shown to reproduce binding free energies for inhibitors of a malaria enzyme. The possibility of using a combination of docking, MD and the LIE method to predict binding affinities for large datasets of ligands is also investigated. Good agreement with experiment is found for a set of non-nucleoside inhibitors of HIV-1 reverse transcriptase.

Approaches for decomposing solvation and binding free energies into enthalpic and entropic components are also examined. Methods for calculating the translational and rotational binding entropies for a ligand are presented. The possibility to calculate ion hydration free energies and entropies for alkali metal ions by using rigorous free energy techniques is also investigated and the results agree well with experimental data.

Infrared spectroscopy detects molecular vibrations and assesses the properties of molecules and their environment. It is a powerful technique to detect ligand induced changes in biomolecules as it has distinct signals and provides different levels of structural information. An addition of a dialysis accessory to attenuated total reflection infrared spectroscopy makes this technique more universal for ligand binding studies. It facilitates to study ligand binding of substrates, activators, inhibitors and ions to macromolecules as well as effect of pH, ionic strength or denaturants on the structure of macromolecules, which play an important role in drug development. This method was tested with two proteins cyt c and calcium ATPase. We studied phosphoenol pyruvate (PEP) in different ionization states by infrared spectroscopy combined with theoretical analysis. Theoretical calculations helped to assign the bands. The infrared spectrum of labeled PEP and infrared measurement in D2O also helped in band assignment. We used the method dialysis accessory to attenuated total reflection infrared spectroscopy to investigate the binding of PEP and Mg2+ to pyruvate kinase (PK), where conformational changes of PK were revealed upon binding of PEP and Mg2+. Isotopic labeled PEP helped to assign and evaluate the infrared absorption bands. The difference spectrum of bound and free PEP indicates specific interactions between ligand and protein. The quantitative evaluation revealed that the enzyme environment has little influence on the P-O bond strengths, which are weakened by less than 3% upon binding. The carboxylate absorption bands indicate shortening of the C-O bond by as little as 1.3 pm. The binding of PEP to PK in presence of monovalent cations K+ and Na+ showed that the binding interactions are very similar.
doctoral

In this thesis, we use a particle coupled to a phonon bath to accurately model biological and chemical reactions. The path decomposition expansion (PDX) formalism is used to determine the tunneling dynamics of the particle. By decomposing the potential energy landscape into the classically allowed and classically forbidden regions, we can calculate the path integrals associated with each region and connect them to evaluate the full Green's function. We will also discuss how deuteration of ligand molecules may affect enzyme-substrate binding in GPCR systems. It has been theorized that binding may be dependent on a molecular vibrational component. We investigate this in the β-adrenergic receptor system using the deuterated and non-deuterated forms of the ligand epinephrine. The measurement for successful binding is determined by the amounts of second messenger cyclic-AMP produced. However, our results proved inconclusive and a discussion of possible problems as well as recommendations is included.
Science, Faculty of
Physics and Astronomy, Department of
Graduate

The adhesive interactions between cells and surfaces play a key role in many vital physiological processes, such as the innate immune response or wound healing, but also in targeted drug delivery and active control on the adhesion of viruses. Adhesion is often mediated by specific intermolecular bonds, which generally function under considerable mechanical load. Bond properties can be explored by dynamic force spectroscopy, which measures the force required to separate two surfaces connected by small numbers of molecular bonds. Motivated by such experiments, the aim of this thesis is to investigate the adhesive effects of discrete, stochastic binding of clusters of intermolecular bonds, supported by a rigid or flexible substrate. The stochastic adhesion of a cluster of bonds connecting a rigid disk and a flat surface is investigated within the framework of piecewise deterministic Markov processes. The model accounts for the rupture and rebinding of discrete bonds, depending on the disk’s motion under applied force. Hydrodynamic forces in the thin layer of viscous fluid between the two surfaces are described using lubrication theory. Bonds are modeled as identical, parallel springs, and equally share the load. Monte Carlo simulations, capturing the stochastic evolution of clusters with few bonds are complemented by various deterministic approximations. Dynamic force spectroscopy experiments are mimicked under linearly ramped force. The stochastic evolution of a bond population connecting a flexible membrane to a rigid wall within a fluid is also formulated as a Markov process, and spatial effects are considered by allowing the vertical elastic bonds to differentially share the load, depending on their extension. The deterministic motion of the membrane, interrupted by stochastic binding and unbinding of bonds, is formulated as a partial differential equation, derived using lubrication theory. The average population and extension of bonds are shown to be inversely correlated, using a wavelet-based semblance method.

The research in this dissertation is about investigation of steric effect on thestablility of metal-containing peptide nucleic acid (PNA); thermodynamcs of metalbinding to ligand-modified PNA studied by Isothermal titration calorimetry (ITC);incorporation of metal in 2,2’-bipyridine(Bpy) modified PNA triplexes and structuralcharacterization of copper complexed 8-hydroxyquinoline (Q) modified PNA. All theseresearch are necessary to further understand the coordination chemistry in PNAcontext, in comparison with the corresponding coordination of metal and simple ligandfor the construction of hybrid inorganic-nucleic acid nanostructures.The steric effect of ligand can be understood by using similar ligands that carrythe same metal binding site, namely 8-hydroxyquinoline, but are attached differently tothe PNA backbone and having incorporated them into PNA duplexes. We conclude thatthe incorporation of a metal complex with high stability constant into a PNA duplex isnot a sufficient condition for the formation of stable hybrid metal-nucleic acid duplexesand that the steric relationship between the complex and the duplex must beconsidered in the design of metal-containing alternative base pairs.Thermodynamic parameters of metal ions with ligand-containing PNA includingCu2+ with Q-PNA, Ni2+ and Cu2+ with Bpy-PNA have been studied by ITC. This studyestablished that the previously proposed supramolecular chelate effect on metal bindingto PNA duplexes exists and it is entropy-driven. Another major factor that influences the binding affinity of ligand-containing PNA for transition metal ions is the stericinteraction between the metal complex and the PNA. In addition, the sequence of thePNA, the base pair mismatches, and the position of ligands in the PNA also influencethe stability constants of metal complexes formed with ligand-modified PNAs.The incorporation of metal ions in PNA triplexes is observed and confirmed byspectroscopic methods and mass spectrometry. The formations of Ni-tsPNA and CotsPNAare stable. Electron paramagnetic resonance(EPR) results show that in the PNAtriplex, the coordination environment of Cu2+ is similar to those of [Cu(Bpy)3]2+ ortrans-[Cu(Bpy)2]2+.A crystal structure of a 9-base pair PNA with a central Q ligand modification isobtained in the presence of excess Cu2+. Due to the unfavorable condition ofcrystallization at pH 4.0 and with excess Cu2+, metal coordination is outside the PNAduplex. An unusual Cu trinuclear cluster bridges two duplexes and excess Cu2+ iscoordinated to terminal nucleobases. The similarity between the EPR parameters for the[CuQ2] complexes with 8-hydroxyquinoline and ipdq precluded us from distinguishingbetween the possible cis- or trans-geometry of the complex formed by Cu2+ with QmodifiedPNA duplexes.Future research includes investigation of thermodynamics by differentialscanning calorimetry (DSC); analysis of kinetics of ITC results and identification of theintermediates; further characterization of the right-handed PNA induced by hydrogenbonding and structural characterizations of metal-containing PNA.