Tesi sul tema "Inhibition covalente"

Les inhibiteurs covalents ciblés (TCI) sont très prometteurs pour la recherche de nouveaux médicaments. Ils offrent un certain nombre d’avantages par rapport aux inhibiteurs réversibles traditionnels, comme un temps de séjour prolongé, une puissance accrue et la possibilité d’apporter des modifications pour une conception efficace. Les inhibiteurs de kinases sont les exemples les plus courants d’ITC. Les enzymes phosphoinositide 3-kinase (PI3K) sont des cibles médicamenteuses importantes en oncologie car elles sont impliquées dans la voie de signalisation de nombreuses fonctions cellulaires telles que le contrôle de la croissance, le métabolisme et l’initiation de la traduction. Les résidus lysine (Lys) ont suscité un intérêt croissant comme alternative pour l’inhibition covalente ciblée. Récemment, les premiers inhibiteurs sélectifs et irréversibles avec des groupes esters comme tête électrophile ciblant le résidu Lys779 et inactivant de manière covalente l’enzyme PI3Kδ ont été rapportés. L’objectif principal de cette thèse est d’élucider le mécanisme de l’inhibition covalente de PI3Kδ par ces inhibiteurs ester afin d’aider à la conception future de nouveaux inhibiteurs avec des activités supérieures. Avant les études mécanistiques sur l’enzyme, nous avons d’abord effectué des calculs ab initio et DFT sur la réaction modèle entre la méthylamine et les acétates de méthyle, phényle et p-NO2 phényle en solution aqueuse. Les mêmes systèmes modèles ont ensuite été étudiés par l’approche de dynamique moléculaire QM/MM "à double niveau". Pour l’option "bas niveau", les simulations QM/MM ont été conduites au niveau PM3/TIP3P, et elles ont permis d’obtenir l’échantillonnage du système dans le cadre de la technique « umbrella sampling ». Les structures obtenues ont ensuite été utilisées pour obtenir des corrections perturbatives à l’énergie libre avec une région QM de "haut niveau" décrite par la méthode M06-2X/6-311+G(d,p). Les résultats montrent que la première étape implique la formation d’un intermédiaire tétraédrique zwittérionique. Ensuite, pour des esters suffisamment électrophiles, tels que le dérivé p-NO2, la réaction se déroule par dissociation du zwittérion sous forme de paire d’ions, suivie d’un transfert de protons conduisant à la formation des produits attendus. Nous avons utilisé des outils théoriques similaires pour étudier les mécanismes d’inhibition dans le cas de l’enzyme. Tout d’abord, un modèle de site actif de l’enzyme a été construit par des simulations classiques de dynamique moléculaire. Ensuite, l’approche ONIOM QM:QM au niveau M06-2X/6-31+G(d,p):PM6 a été appliquée pour obtenir les mécanismes de réaction envisageables dans ce site actif. Ces calculs nous ont permis d’affiner les mécanismes de réaction dans l’environnement enzymatique qui confirment globalement les étapes obtenues à partir du petit système modèle. Nous avons finalement utilisé ces informations pour aborder une étude QM/MM dynamique sur l’enzyme en utilisant le même protocole "double niveau" établi pour le petit système modèle, ce qui nous a permis d’obtenir le profil d’énergie libre du mécanisme d’inhibition de PI3Kδ pour le dérivé p-NO2 de l’inhibiteur ester. La barrière calculée est en bon accord avec les données cinétiques expérimentales disponibles, ce qui valide l’approche théorique proposée et les mécanismes obtenus. Grâce à l’élucidation du mécanisme d’inhibition de composés précédemment testés expérimentalement, notre étude ouvre la voie à la découverte de nouveaux inhibiteurs à activité améliorée à l’aide des outils de la chimie théorique
Targeted Covalent Inhibitors (TCIs) hold great promise for search of new drugs. They offer a number of potential advantages over traditional reversible inhibitors, such as extended residence time, increased potency, and the ability to make modifications for effective design. Kinase inhibitors are the most common examples of TCIs. Phosphoinositide 3-kinase (PI3K) enzymes are important drug targets in oncology as they are involved in the signaling pathway for many cellular functions such as growth control, metabolism and translation initiation. Lysine (Lys) residues have gained increasing interest as an alternative for targeted covalent inhibition. Recently, the first selective and irreversible inhibitors with ester groups as electrophilic head targeting the Lys779 residue and covalently inactivating the PI3Kδ enzyme were reported. The main objective of this thesis is to elucidate the mechanism of the covalent inhibition of PI3Kδ by these ester inhibitors in order to assist future design of new inhibitors with superior activities. Prior to the mechanistic studies on the enzyme, initially, we performed ab initio and DFT calculations on the model reaction between methylamine and methyl, phenyl and p-NO2 phenyl acetates in aqueous solution. The same model systems were then studied by the "dual-level" QM/MM molecular dynamics approach. For the “low-level” option, PM3/TIP3P umbrella sampling QM/MM simulations were applied for the sampling. The obtained structures were then used to obtain perturbative corrections to the free energy with a “high-level” QM region at the M06-2X/6-311+G(d,p) level. The results show that thefirst step involves the formation of the zwitterionic tetrahedral intermediate. Then, for sufficiently electrophilic esters, such as the p-NO2 derivative, the reaction proceeds by dissociation of the zwitterion as an ion pair, followed by proton transfer leading to the formation of the expected products. We, then, employed similar computational tools to shed light on the mechanistic aspects of the enzyme. First, an active site model of the enzyme was built through classical molecular dynamics simulations. Then, ONIOM QM:QM approach at the M06-2X/6 -31+G(d,p):PM6 level was applied to get possible reaction mechanisms in this active site. These calculations guided us to refine the reaction mechanisms in enzyme environment which globally confirm the steps obtained from the small model system. We finally used this information to approach a dynamic QM/MM study on the enzyme using the same“dual-level” protocol established for the small model system, which allowed us to obtain the free energy profile of the inhibition mechanism of PI3Kδ for p-NO2 derivative of the ester inhibitor. The calculated barrier is in good agreement with the available experimental kinetic data, which validates the proposed theoretical approach and the obtained mechanisms. Through the elucidation of the inhibition mechanism of previously experimentally tested compounds, our study paves the way for the discovery of new inhibitors with improved activity with the help of theoretical chemistry tools

Transglutaminases are a family of enzymes expressed in various tissues of our body. Some are expressed ubiquitously while others are specific to a tissue. Their primary catalytic activity is to crosslink substrates via an isopeptidic bond. The work described in this thesis focuses on two of these transglutaminases; human tissue transglutaminase (hTG2) and human factor XIIIa (FXIIIa). Divided into two projects for each enzyme, the main objective of this thesis was directed towards the discovery of potent and selective covalent inhibitors for each isozyme, namely hTG2 and hFXIIIa. The first project was concentrated on the inhibition of hTG2 activity. Ubiquitously expressed in tissues, hTG2 is a multifunctional enzyme. Its primary activity is the formation of isopeptide bonds between glutamine and lysine residues found on the surface of proteins or substrates. In addition to its catalytic activity, hTG2 is also a G-protein, distinguishing it from other members of the transglutaminase family. Much evidence illustrates that hTG2’s multifunctional abilities are conformationally regulated between its “open” and “closed” forms. Overexpression and unregulated hTG2 activity has been associated with numerous human diseases; however, most evidence has been collected for its association with fibrosis and celiac sprue. More recently, elevated hTG2 expression has been linked to cancer stem cell survival and metastatic phenotype in certain cancer cells. These findings call for the development of suitable and potent inhibitors that selectivity inactivate human hTG2 as a potential therapeutic target. Starting with previously designed acrylamide based peptidomimetic irreversible inhibitors, a structure-activity relationship (SAR) study was conducted. In this work, >20 novel irreversible inhibitors were prepared and kinetically evaluated. Our lead inhibitors allosterically inhibited GTP binding by locking the enzyme in its open conformation, as demonstrated both in vitro and in cells. Furthermore, our most potent and efficient irreversible inhibitors revealed selectivity for hTG2 over other relevant members of the transglutaminase family (hTG1, hTG3, hTG6 and hFXIIIa), providing higher confidence towards our goal of developing an ideal drug candidate. The second project was concentrated on the inhibition of hFXIIIa activity. In the blood, coagulation factor XIII (FXIII) is a tetrameric protein consisting of two catalytic A subunits (FXIII-A2) and two carrier/inhibitory B (FXIII-B2) subunits. It is a zymogen, which is converted into active transglutaminase (FXIIIa) in the final phase of coagulation cascade by thrombin proteolytic activity and Ca2+ binding. hFXIII is essential for hemostasis and thus its deficiency results in severe bleeding conditions. Further, hFXIIIa mechanically stabilizes fibrin and protects it from fibrinolysis. Due to the enzyme’s involvement in the stability of blood clots, inhibition of hFXIIIa activity has been linked to thrombotic diseases. Furthermore, inhibitors of the enzyme have the therapeutic potential to be used as anticoagulant agents. The current number of selective and potent inhibitors of hFXIIIa are few, mainly due to the similarity between its catalytic pockets and hTG2. Inspired by a poorly reactive hTG2 inhibitor discovered in this work’s hTG2 SAR study, we synthesized a small library of covalent inhibitors for hFXIIIa. Our kinetic results from this pioneering SAR study will pave the way for future hFXIIIa inhibitor SAR studies.

La spectrométrie de masse MALDI-TOF (matrix-assisted laser desorption/ionization- time of flight) et ESI-TOF (electrospray ionization- time of flight) permettent d'accéder à des méthodologies performantes pour l'étude de la structure primaire des protéines. Ce travail a mis en oeuvre ces deux modes d'ionisation pour étudier les modifications covalentes de trois protéines impliquées dans des contextes biologiques différents. En premier lieu, les modifications post-traductionnelles de deux nitrile hydratases (NHases), métalloenzymes bactériennes catalysant l'hydrolyse de nitriles en amides, ont été caractérisées. Des études sur les protéines intactes ont mis en évidence deux modifications post-traductionnelles--un acide sulfénique et un acide sulfinique--sur les cystéines du centre actif de deux NHases de bactéries différentes. Ensuite, nous avons caractérisé les modifications post-traductionnelles d'une protéine intervenant dans la coagulation sanguine, le facteur IX (FIX), dans le but d'établir une référence structurale du FIX plasmatique. Cette protéine comporte un ensemble de modifications complexes incluant en particulier N-glycosylations, O-glycosylations, phosphorylation et sulfatation, localisées sur une région de la protéine, le peptide d'activation. En dernier lieu, nous avons étudié les mécanismes d'inhibition suicide de deux cytochromes P450. Pour cela, nous avons caractérisé, grâce à des techniques de marquage par des isotopes stables, les métabolites produits lors de l'hydroxylation de l'acide tiénilique par le P450 2C9, réaction conduisant également à l'inactivation de l'enzyme. Ces expériences ont conduit à proposer deux mécanismes réactionnels différents pour ce composé. Le mécanisme de l'inhibition suicide d'un P450 d'origine végétale a été abordé en cherchant à identifier les peptides marqués pendant la réaction. Cette identification n'a pas été obtenue, vraisemblablement en raison de l'instabilité du marquage dans nos conditions d'analyse.

Le domaine de l'épigénétique couvre l'ensemble des phénomènes héritables et transmissibles qui interviennent dans l'expression du génome sans modifier la séquence nucléotidique. L'information épigénétique est régulée par les modifications de la chromatine impliquant les histones et l'ADN. La méthylation de l'ADN est un phénomène réversible jouant un rôle crucial dans l'expression des gènes puisque la méthylation des promoteurs de gènes empêche leur transcription. La modulation aberrante de cette marque épigénétique est associée à diverses pathologies telles que le cancer. Cette méthylation étant réversible, elle peut être ciblée afin de reprogrammer la cellule cancéreuse. Les méthyltransferases d'ADN (DNMT), étant les enzymes responsables de la méthylation, représentent la cible principale de notre stratégie de recherche. Leur inhibition par des petites molécules est au centre de nos recherches de thérapies anticancéreuses dont les bases sont représentées par deux catégories d'inhibiteurs de DNMT existant. Les premiers sont des analogues de cytosine qui est la cible de la méthylation. Ils sont connus pour s'intégrer dans l'ADN et former un complexe covalent irréversible avec l’enzyme (complexe suicide) mais ils souffrent d'un manque de stabilité et de certains effets indésirables dus à leur incorporation dans l’ADN. Les seconds sont les inhibiteurs non nucléosidiques qui sont divers et parfois connus pour cibler d’autres enzymes. Ils ont l’avantage de pouvoir être utilisés comme sondes pour comprendre plus précisément le mécanisme d'inhibition mais ils manquent de spécificité et de sélectivité. Au cours de cette thèse, une banque de molécules a été criblée à partir de la combinaison d'un test enzymatique et d'un test cellulaire visant à inhiber ces enzymes. Les synthèses de trois familles de molécules potentiellement inhibitrices de DNMT issus de ce criblage sont décrites en expliquant le chemin de drug design emprunté pour obtenir des informations mécanistiques d’inhibition de la méthylation d’ADN, notamment de réactivité avec la cible. Les découvertes ont été inspirées par des études de modélisation permettant de mettre en évidence une sélectivité de certains inhibiteurs. La synthèse chimique a également abouti à une nouvelle voie de synthèse d’accès aux diaminopyrimidines dont l’impact permet de faciliter les études chimiques de dérivés quinazolines comme inhibiteur non nucléosidiques utiles pour les thérapies anticancéreuses
Epigenetic is defined as the study of heritable changes in the genes expression without altering the DNA sequence. Two main processes are implicated in this field, the histones modifications and the DNA methylation. By introducing an acetyl or a methyl group on the histone tails or by methylation of DNA, the chromatin state is modified and the gene expression is controlled. Aberrant epigenetic modifications are associated with several diseases, in particular with cancer. In cancer cells, the whole DNA is hypomethylated, thus promoting genome instability, while the promoter region is hypermethylated, inducing silencing of these genes. Overall, these observations indicate that DNA methylation is a central epigenetic process in cancerogenesis. Since DNA methylation is reversible, it is possible to target the methylation process in order to reactivate tumor suppressor genes. The DNA methyltransferases (DNMTs), the enzymes responsible for DNA methylation, use the SAM co-factor at specific CpG sites to product 5-methylcytosine. Three main isoforms (DNMT1, DNMT3A and DNMT3B) are described to ensure efficient methylation process during replication. Two families of DNMT inhibitors already exist, the nucleosidiques analogues are cytidine derivatives and are toxic molecules because of their incorporation into DNA, and the non-nucleosidic analogues are less toxic but also less potent. Our strategy of drug design is based on docking study and high throughput screening (HTS) information. First, novel potent derivatives of reference inhibitors are designed from molecular modelling. Then, three different families of compounds from HTS are described with appropriate structure-activity relationship studies. Mechanistic information on DNA methylation process are described through the discovery of a reactive inhibitor of DNMT3A. The study on a family of hydrazone derivatives of gallic acid is depicted and shows its selectivity for DNMT3A, compared to DNMT1, based on docking study. An alternative chemical pathway to diaminopyrimidines is described and extended to the synthesis of quinazolone in order to synthesize new quinazoline derivatives as potent inhibitors of DNMT. Promising informations are described in this thesis to enrich epigenetic knowledge of tumor genesis and to provide new molecules for anticancer therapy

京都大学
0048
新制・課程博士
博士(工学)
甲第22412号
工博第4673号
新制||工||1729(附属図書館)
京都大学大学院工学研究科合成・生物化学専攻
(主査)教授 浜地 格, 教授 森 泰生, 教授 生越 友樹
学位規則第4条第1項該当
Doctor of Philosophy (Engineering)
Kyoto University
DGAM

Parkinson's disease (PD) is a prevalent neurodegenerative disorder which affects over a million people in the United States. This disease is marked by the selective loss of dopaminergic neurons in the substantia nigra, leading to a decrease in the important neurotransmitter dopamine (DA), which is essential for the initiation and execution of coordinated movement. Currently, the pathogenesis behind PD is unknown, but there is evidence that both exogenous causes, such as pesticides and metals, as well as endogenous causes, such as reactive oxygen species or reactive metabolism intermediates, may play a role in the onset and progression of the disease. DA is catabolized by monoamine oxidase to 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is further metabolized by aldehyde dehydrogenase and aldehyde reductase to the acid and alcohol products, respectively. Studies have demonstrated the reactivity of DOPAL with peptides and proteins, leading to covalent modification which may be detrimental to protein action. Furthermore, studies have shown that DOPAL is toxic, leading to a decrease in cell viability. Due to this, it was of interest to further study DOPAL and how it may play a role in the onset and progression of PD. It was of particular interest to determine protein targets of DOPAL modification. Until recently, no protein targets were identified and the cellular consequence of elevated DOPAL had not been fully studied. It has been previously shown that the important enzyme, tyrosine hydroxylase (TH) is inhibited by other catechols, including DA. This enzyme catalyzes the rate-limiting step in DA synthesis, oxidizing tyrosine to L-DOPA which is further metabolized to DA. Therefore, it was of interest to determine the effect of DOPAL on TH activity. It was hypothesized that DOPAL modifies and inhibits TH, leading to a decrease in the production of L-DOPA and DA. This work employed the use of a dopaminergic cell model (PC6-3 cells), to positively identify TH as a protein target of DOPAL modification. It also used both cell lysate as well as PC6-3 cell studies to investigate the effect of DOPAL modification on TH activity. Mass spectrometry was also utilized to determine sites of protein modification on TH. Results show that TH is potently inhibited by DOPAL modification, leading to a significant decrease in both L-DOPA and DA. Furthermore, DOPAL inhibition appears to be slowly-irreversible, with enzyme activity showing a time- and concentration dependent in recovery after preincubation with DOPAL. A novel cloning and purification procedure was used to clone human recombinant TH, which was used in mass spectrometry studies in which five sites of DOPAL modification were discovered. Furthermore, a real-time assay for TH activity was developed using a plate reader to spectrophotometrically observe the formation of L-DOPA over time. These data demonstrate the toxicity and potent enzyme inhibition by DOPAL and implicate DOPAL as a neurotoxin relevant in the pathogenesis of PD.

Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Applied Biological Sciences, 1986.
MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE.
Bibliography: leaves 157-166.
by Sew-Wah Tay.
Sc.D.

Human tissue transglutaminase (TG2) is a calcium-dependent multifunctional enzyme that natively catalyzes the post-translational modification of proteins, namely by the formation of isopeptide bonds between protein- or peptide-bound glutamine and lysine residues. This ubiquitously expressed enzyme plays important roles in cellular differentiation, extracellular matrix stabilization, and apoptosis, to name a few. However, its unregulated activity has been associated with many pathologies such as fibrosis, cancer, neurodegenerative disorders and celiac disease. Most of these disorders are associated with unregulated acyl-transferase activity. As such, the Keillor group has directed its efforts towards the development of TG2 inhibitors. Over the years, the Keillor group has synthesized large libraries of targeted covalent inhibitors against TG2. These compounds have undergone pharmacodynamic testing in order to examine their kinetic parameters of inhibition. Having gained knowledge of their enzyme kinetics, the logical next step was to consider their pharmacokinetic profiles. In the context of this thesis, we considered two important pharmacokinetic properties: membrane permeability and off-target reactivity. Firstly, we aimed to evaluate our inhibitors for their ability to permeate the cell membrane. In efforts to do so, we were able to adapt, optimize, and validate a parallel artificial membrane permeability assay (PAMPA) utilizing hexadecane as our artificial membrane. We were able to test a few of our own inhibitors and found that compounds NC9, VA4 and AA9 possess Log Pe values of -5.26 ± 0.01, -4.66 ± 0.04 and -6.5 ± 0.5 respectively. Secondly, we sought to investigate the susceptibility of our inhibitors to glutathione addition reactions under physiological conditions. We adapted and optimized a colorimetric assay using Ellman’s reagent (DTNB) and found that our inhibitors are minimally reactive with glutathione. The methods developed over the course of this work provide protocols that can be adopted for the characterization of future inhibitors in the Keillor group, along the process of developing TG2 inhibitors into drug candidates.

In the last decades targeted drugs have improved cancer treatment, but revealed to be ineffective mainly in the treatment of solid tumors, largely because of tumor heterogeneity, activation of redundant pathways, and drug resistance. A common and central signal transduction mechanism in many oncogenic pathways is the phosphorylation of proteins at serine or threonine residues followed by proline (S/T-P). Importantly, the phospho-S/T-P motifs of these proteins are recognized by the peptidyl-prolyl cis/trans isomerase (PPIase) PIN1, which catalyzes the cis-trans or trans-cis conformational change around the S-P or T-P bond. Among PPIases, PIN1 is the only enzyme able to efficiently bind proteins containing phosphorylated S/T-P motifs. As a consequence, the phosphorylation dependent prolyl-isomerase PIN1 acts as a critical modifier of multiple signaling pathways. It is overexpressed in the majority of cancers and its activity strongly contributes to tumor initiation and progression. Conversely, inactivation of PIN1 function curbs tumor growth and cancer stem cell expansion, restores chemosensitivity and blocks metastatic spread, thus providing the rationale for a therapeutic strategy based on PIN1 inhibition. Notwithstanding, potent PIN1 inhibitors are still missing from the arsenal of anti-cancer drugs. By a mechanism-based screening we have identified a novel covalent PIN1 inhibitor, KPT-6566, able to selectively inhibit PIN1 among other prolyl-isomerases, and target it for degradation. We demonstrate that KPT-6566 covalently binds to the catalytic site of PIN1. This interaction results in the release of a quinine-mimicking drug that generates reactive oxygen species and DNA damage inducing cell death specifically in cancer cells. Accordingly, KPT-6566 treatment impairs PIN1-dependent cancer phenotypes in vitro and growth of lung metastasis in vivo.

The human 20S proteasome activity and malfunction has been related to numerous diseases and validated as a protein target for inhibition in the treatment of cancer, with three proteasome inhibitors approved as a drug. But these compounds could be improved, since usually the molecular mechanism of action is unknown. Thus, computational studies can clarify the mode of action of proteasome inhibitors, helping to understand the system and improve the inhibition process The present thesis is devoted to understand the mode of action of two classes of covalent inhibitors of the 20S proteasome, α,β-epoxyketones and γ-lactam-β-lactones. Molecular dynamics simulations with hybrid QM/MM potentials have been used to characterize the free energy landscape for the inhibition mechanism of these compounds and to provide the structures necessary to analyze and understand the inhibition process in the β5 active site of the proteasome, providing valuable knowledge to optimize the compounds into more efficient inhibitors.
Programa de Doctorat en Química Teòrica i Modelització Computacional

In recent years an increased number of covalent protein kinase inhibitors has been approved for cancer therapy and many more are undertaking clinical trials. Covalent binding is usually obtained by introducing in these drugs an electrophilic warhead able to bind specific nucleophilic sites in the protein target. Covalent inhibition of oncogenic protein kinases allows to obtain a stronger and prolonged therapeutic effect compared to reversible inhibition; however, the choice of the dose to be administered to patients and the evaluation of the selectivity among the kinase family are mandatory to reach pharmacological results reducing possible side effects. From the DMPK perspective for covalent inhibitors, in vitro and in vivo data extrapolation, to obtain human pharmacokinetic projection, can be challenging and numerous efforts have to be undertaken in developing methods to accurately and quantitively determine inhibitor target engagement in preclinical and more importantly in clinical studies. Chemoproteomic approaches, taking advantage of the use of chemical probes, provide powerful tools to analyze binding characteristics between small molecules and proteins and validate the mode of action of these drug candidates. Click chemistry is a cycloaddition reaction between an azide and alkyne group to generate a 1,4-disubstituted 1,2,3-triazole ring. In this way, it is possible to functionalize the inhibitor under development with an alkyne moiety that could be labelled with a fluorescent-azide for target engagement detection and quantitation in native environments and a desthiobiotin-azide molecule for identification of the inhibitor off-target binders and proteome-wide selectivity. The functionalization of the inhibitor with a small group such as the alkyne group (the molecule obtained is called probe) allows the incubation of the compound directly in situ, with minimal alteration of inhibitor features (e.g. potency and permeability). Thus, the aim of this project is the optimization and application of click chemistry reaction in order to study covalent mechanism of action of ibrutinib, a covalent inhibitor of the tyrosine protein kinase BTK, approved by FDA in 2013 for the treatment of B cell malignancies. We used ibrutinib as case study, but the developed protocols can be applied to the study of other covalent inhibitors. Target engagement conditions were initially optimized on the recombinant BTK protein using an ibrutinib derivative, bearing an alkyne group, and then transferred to cell extracts. Competition experiments were set up on extracts and then an in cell target engagement experiment was conducted, treating cells with 5 μM ibrutinib. The significant decreasing of the signal intensity of fluorescent probe labeled BTK, after pre-incubation of cells with ibrutinib, suggested a full target occupancy of BTK. Nevertheless, with the aim to precisely calculate this target occupancy, a chemoproteomic workflow coupled to quantitative mass spectrometry analysis has been optimized and set. In this case, click chemistry reaction was performed coupling to ibrutinib probe an azide functionalized with a desthiobiotin moiety in order to capture desthiobiotinylated peptides with a streptavidin resin, taking advantage of high affinity between streptavidin and desthiobiotin. The developed protocols have been validated in cells treated with ibrutinib and, in addition to BTK, two additional protein kinases JAK3 and BLK have been identified modified on cysteine 909 and 319, respectively. These cysteines are located in the ATP-binding site of the two kinases in a position corresponding to the cysteine 481 in BTK. Interaction with ibrutinib for JAK3 and BLK was confirmed by additional analyses on recombinant protein and cell lysates. Total proteome analysis, both in label free quantitation and TMT mode, has been undertaken to additionally characterize ibrutinib treated cells. The work allowed to better understand the preclinical profile of an oncological target therapy drug in term of potency and selectivity in living cells. The process is transferable to other covalent drugs and applied not only in preclinical models but also in clinical trials, helping in the definition of the optimal dose for patients to obtain the best efficacy, limiting side effects.

Acetylcholinesterase (AChE) is an essential enzyme with an evolutionary conserved function: to terminate nerve signaling by rapid hydrolysis of the neurotransmitter acetylcholine. AChE is an important target for insecticides. Vector control by the use of insecticide-based interventions is today the main strategy for controlling mosquito-borne diseases that affect millions of people each year. However, the efficiency of many insecticides is challenged by resistant mosquito populations, lack of selectivity and off-target toxicity of currently used compounds. New selective and resistance-breaking insecticides are needed for an efficient vector control also in the future. In the work presented in this thesis, we have combined structural biology, biochemistry and medicinal chemistry to characterize mosquito AChEs and to develop selective and resistance-breaking inhibitors of this essential enzyme from two disease-transmitting mosquitoes.We have identified small but important structural and functional differences between AChE from mosquitoes and AChE from vertebrates. The significance of these differences was emphasized by a high throughput screening campaign, which made it evident that the evolutionary distant AChEs display significant differences in their molecular recognition. These findings were exploited in the design of new inhibitors. Rationally designed and developed thiourea- and phenoxyacetamide-based non-covalent inhibitors displayed high potency on both wild type and insecticide insensitive AChE from mosquitoes. The best inhibitors showed over 100-fold stronger inhibition of mosquito than human AChE, and proved insecticide potential as they killed both adult and larvae mosquitoes.We show that mosquito and human AChE have different molecular recognition and that non-covalent selective inhibition of AChE from mosquitoes is possible. We also demonstrate that inhibitors can combine selectivity with sub-micromolar potency for insecticide resistant AChE.

Ce projet de thèse décrit la conception d'inhibiteurs de deux enzymes de l'agent du paludisme et de modulateurs de la sous-unité α5 des récepteurs nicotiniques de l'acétylcholine (nAChR) impliqués dans les dépendances. La subtilase de Plasmodium vivax (SUB1), nécessaire pour la sortie des parasites des cellules a été ciblée avec des inhibiteurs covalents réversibles. Nous avons effectué un docking covalent de peptidomimétiques candidats et étudié leur cyclisation. Plusieurs mimétiques ont montré une activité sub-micromolaire et leur structure a pu être résolue par co-cristallisation. Nous avons ciblé la lactate déshydrogénase, essentielle au métabolisme de Plasmodium falciparum avec des inhibiteurs conçus par analogie du tandem cofacteurs-substrat. Nous avons construit une bibliothèque combinatoire que nous avons criblé in silico, en évitant de cibler les isoenzymes humaines. Nous avons sélectionné une cinquantaine de molécules, en cours de synthèse pour tests ex vivo. Enfin, pour lutter contre les dépendances, une chimère α5-α4 de l'AChBP a été utilisée dans une approche multidisciplinaire. La structure en complexe avec les premiers ligands connus d'α5 a été résolue et nous l'avons utilisée avec deux modèles comparatifs pour un criblage in silico. Nous avons introduit l'interaction cation-π dans le logiciel FlexX, autorisé des chaînes latérales flexibles dans le site de liaison et développé un pipeline interactif pour l'analyse des résultats de criblage virtuel. Les molécules obtenues ont été confirmées par des expériences STD-RMN. Des modèles de réseaux neuronaux profonds ont également été construits pour prédire la bioactivité sur cible et hors cible
This PhD project describes the design of inhibitors of two essential malaria enzymes and of novel modulators of specific nicotinic acetylcholine receptors (nAChRs). Plasmodium vivax subtilase SUB1 is required for parasite egress. We focused our efforts on the design of reversible covalent inhibitors of PvSUB1. We performed covalent docking of potential peptide and peptidomimetic candidates and studied peptide cyclization. Several peptides have shown activity in the submicromolar range and could be resolved after co-crystalization. Plasmodium falciparum lactate dehydrogenase is critical for parasite metabolism. We targeted it by design on the basis of inhibitory cofactor analogs. We have built a combinatorial library aiming to bridge the cofactor and the substrate binding site, while avoiding affecting the human isoenzymes. We screened it in silico and selected about fifty molecules that are under synthesis for ex vivo testing. We also targeted α5 subunit containing nAChRs to address addiction. A multidisciplinary approach has been established. It uses an AChBP engineered chimera, which structure was solved in complex with the first known 5 ligands. This structure, and two comparative modeling models were used to perform in silico screening. A cation-π interaction definition was introduced in the FlexX software and side chain flexibility was allowed in the binding site. An interactive pipeline was developed for the analysis of the virtual screening results and hit molecules have been confirmed by STD-NMR experiments. Deep neural networks models were also built to assess on- and off-target bioactivity prediction in a panel of nAChRs and putative off-targets

This dissertation describes the research carried out as part of a PhD program in Chemistry from the 1st October 2017 until 30th November 2020. The PhD project investigated the development of inhibitors of enzymes involved in important metabolic pathways, with the final aim to produce an antiproliferative effect. The present thesis combines the works performed at the University of Milan and Vrije Universiteit of Amsterdam. Part A describes the research performed in Amsterdam, NL during my period abroad from January to September 2019 in the research group of Professor Rob Leurs, at the Division of Medicinal Chemistry of the Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit of Amsterdam. In particular, this part outlines the design, synthesis and pharmacological evaluation of two novel series of potent antitrypanosomal agents, identified through SAR exploration and scaffold hopping approach starting from cyclic nucleotide Trypanosoma brucei phosphodiesterase (PDE) inhibitors. PDE enzymes provide a fine control on several biochemical pathways and have recently been demonstrated to be essential for parasite proliferation. Their disruption by RNA interference (RNAi) dramatically increase intracellular cAMP and, consequently, causes complete mortal trypanosome cell lysis. Part B describes the research done at the Department of Pharmaceutical Sciences, University of Milan, under the supervision of Professor Paola Conti, on the design and synthesis of novel covalent inhibitors targeting the glycolytic enzyme Glyceraldehyde-3- phosphate dehydrogenase (GAPDH). Due to its pivotal role in the glycolysis, GAPDH represents a rate-limiting enzyme in those cells that mostly, or exclusively rely on this pathway for energy production. In this context, GAPDH inhibition represents a valuable approach for the development of anticancer and antiparasitic drugs. Due to the presence of a druggable nucleophilic cysteine residue in the catalytic pocket of the target, I focused my attention on the development of covalent GAPDH inhibitors, presenting an electrophilic warhead with a finely tuned reactivity. In particular, Section B2 reportsthe work conducted on the development of Plasmodium falciparum GAPDH inhibitors and the in vitro antiplasmodial activity. Section B3 shows the work performed on the design and synthesis of human GAPDH inhibitors, with in vitro antitumor activity.

Le mélanome cutané métastatique et les syndromes myélodysplasiques (SMD) sont deux cancers incurables développant des résistances à leurs traitements antitumoraux de référence. Les cellules résistantes à ces thérapies sont caractérisées par une reprogrammation métabolique qui influence et facilite la progression tumorale. Par conséquent, l’inhibition des voies métaboliques semble être une stratégie thérapeutique prometteuse dans ces deux pathologies.Les deux équipes impliquées dans ce projet de thèse collaborent de longue date dans le domaine du cancer. Dans ce contexte et en partenariat avec l’Institut de Chimie de Nice, nos deux équipes ont mis au point des composés innovants ciblant les balances énergétiques intra cellulaires et la voie de l’AMPK. Parmi ces composés nous nous sommes intéressés plus précisément à l’AICAR (Acadésine). Un criblage d’efficacité et des études de structure-activité, nous ont permis d’optimiser la structure de nos composés. Sur la base de leur solubilité, de leur stabilité et sur leur capacité à induire la mort cellulaire de cellules tumorales, nous avons identifié le HA 344 comme composé « lead ». Cette étude propose de caractériser et de valider un nouvel inhibiteur covalent, le HA 344, dérivé de l’Acadésine, efficace sur des lignées de mélanomes et SMD sensibles et résistants à leur traitement de référence mais également sur cellules de patients. En combinant des techniques de click chimie, protéomique et métabolomique, nous avons identifié cette molécule comme un inhibiteur covalent de deux hubs métaboliques différents au sein des cellules tumorales. HA 344 inhibe l’étape finale et limitante de la glycolyse par sa liaison covalente à l’enzyme pyruvate kinase M2 (PKM2), et bloque simultanément l’activité de l’inosine monophosphate déshydrogénase (IMPDH), l’enzyme limitante de la synthèse de novo de guanylate. HA 344 bloque la croissance tumorale in vitro et in vivo de cellules de mélanome sensibles et résistantes aux inhibiteurs de BRAF. Ainsi, ce mécanisme d'action spécifique du HA 344 offre une nouvelle voie thérapeutique potentielle pour les patients atteints de mélanome cutané métastatique et d’autres cancers
Cutaneous metastatic melanoma (CMM) and myelodysplastic syndromes (MDS) are two incurable cancers developing resistance to their reference antitumor treatments. Cells resistant to these therapies are characterized by a metabolic reprogramming which profoundly influences and promotes tumor progression. Therefore, inhibition of metabolic pathways seems to be a promising therapeutic strategy to overcome resistance in these two pathologies.The two teams involved in this thesis project have a long-lasting collaboration in the field of cancer. In this context and in partnership with the Nice Institute of Chemistry, our two teams have developed innovative compounds targeting intra-cellular energy balances and the AMPK pathway. Among these compounds, we were more specifically interested in AICAR (Acadesine). Screening efficiency and structure-activity studies enabled us to optimize the structure of our compounds. Based on their solubility, their stability, and their ability to induce tumor cells death, we have identified HA 344 as a lead compound.This study describes the characterization and validation of a new covalent inhibitor, HA 344, derived from Acadesine, effective on CMM and MDS cell lines either sensitive or resistant to their reference treatment but also on CMM and MDS patient cells. By combining click chemistry, proteomics, and metabolomics approaches, we have identified this molecule as a covalent inhibitor of two different metabolic hubs within cancer cells. HA 344 inhibits the final and rate-limiting step of glycolysis through its covalent binding to the pyruvate kinase M2 (PKM2) enzyme, and concurrently blocks the activity of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme of de novo guanylate synthesis. HA 344 efficiently eliminates tumor growth of BRAF inhibitor sensitive- and resistant-CMM cells both in vitro and in vivo. Thus, this specific mechanism of action of HA 344 provides potential therapeutic avenues not only for patients with CMM but also a broad range of cancers

碩士
國立交通大學
生物資訊及系統生物研究所
105
The Nucleoprotein exonuclease ( NP exonuclease ) of Lassa virus is involved in viral genomic RNA encapsidation, viral RNA synthesis and host immune evasion. NP exonuclease is constituted by N-terminal and C-terminal domains, of which the main function of N-terminal domain is to capture the 5' cap of mRNA in the host cell for carrying out transcription and replication of its own viral RNA; the C-terminal domain of NP exonuclease belongs to the DEDDh exonuclease family. The C-terminal domain is used to degrade pathogen associated molecular patterns generated by infecting host cells, like RNA; and thus further make IRF-3 transcription factor unable to enter the nucleus to induce interferon synthesis of immune system. If the highly conserved amino acids in the C-terminal domain are mutated, it will cause a decline in ability for Lassa virus to escape the host immune system, in other words, the C-terminal domain plays an important role in virus infections; hence the C-terminal domain of NP exonuclease can be used as a target for anti-viral drug research. In this study, we found the inhibitor candidates for NP exonuclease by literature reviews and computational molecular docking program. Through the nuclease activity assay, we identified several inhibitors with high inhibition efficiency, such as ATA、PCMPS、PHMB、PV6R and NCI35. We also determined the crystal structures of apo-NP exonuclease protein structure and NP exonuclease-PCMPS complex in which PCMPS was covalently bound to the cysteine ( C409 ) in the C-terminal domain, indicating that the covalent bond is critical to suppress the activation of NP exonuclease. Our biochemical experiments and two crystal structures reveal a unique inhibitory mechanism of PCMPS through covalent linkage to the NP exonuclease, and this work could be applied to the development of antiviral drugs.

Nitric oxide synthases (NOS) are responsible for the production of nitric oxide (NO), an essential cell-signaling molecule, in mammals. There are three isoforms of NOS with widely different tissue distribution. The overproduction of NO is marked in many human disease states and cancers, however due to the similarities of the enzyme isoforms, targeting NOS for inhibition has proven challenging. Endogenously, the methylated arginines, N[superscript ω]-monomethyl-L-arginine (NMMA) and asymmetric N[superscript ω], N[superscript ω]-dimethyl-L-arginine (ADMA), inhibit NOS. N[superscript ω], N[superscript ω]-Dimethylarginine dimethylaminohydrolase (DDAH1) metabolizes these methylated arginines and thus relieves NOS inhibition. The role of DDAH1 in the regulation of diseases such as cancer and septic shock is still being elucidated. It is thought that targeting DDAH1 for inhibition rather than NOS may circumvent many of the current problems with the treatment of NO overproduction such as isoform selectivity. My PhD studies focus on the synthesis of a series of irreversible inhibitors of DDAH1, an extensive study of their in vitro mode of inhibition, a comparison of analytical fitting methods, and the viability and efficacy of the inactivators in a human cell line. I also studied a potential endogenous inactivator of DDAH1, nitroxyl (HNO), a one-electron reduction product of NO.
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Indiana University-Purdue University Indianapolis (IUPUI)
The extracellular matrix (ECM) provides a structural and signaling platform for cells that comprise various organs, playing a critical role in tissue maintenance, injury, and repair. Hyaluronan (also known as hyaluronic acid, HA) is a ubiquitous ECM polysaccharide consisting of a repeating disaccharide backbone that can be covalently modified by the heavy chains (HC) of the serum protein inter-alpha-inhibitor (IαI) during inflammation. Known as the only covalent modification of HA, the HC linking of HA is exclusively mediated by the inflammation-induced secreted enzyme TNFα-stimulated gene-6 (TSG-6). Mice deficient for HC-HA formation, due to the lack of either TSG-6 or IαI, display reduced survival during systemic lipopolysaccharide (LPS)-induced endotoxic shock and its associated acute lung injury. We therefore hypothesized that HC-HA should play an important protective role against acute lung injury induced by intratracheal LPS or Pseudomonas aeruginosa (PA) gram-negative bacteria. We also identified that lung instillation of LPS or PA caused rapid induction of lung parenchymal HC-HA that was largely cleared during resolution of injury, indicative of a high rate of HA turnover and remodeling during reversible lung injury. However, using TSG-6 knockout mice, we determined that HC-HA exerted minimal protective effects against intratracheal LPS or PA-induced acute lung injury. To better address the differential roles of HC-HA during systemic versus localized intratracheal exposure to LPS, we characterized and compared the induction of HC-HA in plasma and lung in these two models. While lung parenchymal HC-HA formed in both injury models, intravascular HC-HA and TSG-6 were exclusively induced during systemic LPS exposure and were associated with improved outcomes, including decreased number of circulating neutrophils and plasma TNFα levels. Our results suggest that LPS induces HC-HA formation in various tissues depending on the route of exposure and that the specific intravascular induction of HCHA during systemic LPS exposure may have a protective role during endotoxic shock.

Insecticide resistance is a global concern that threatens human health and agricultural productivity. Understanding the molecular basis of resistance will help to manage future insecticide use to ensure that effective, safe and inexpensive pest control is available. In the Australian sheep blowfly Lucilia cuprina, a single mutation (Gly137Asp) in the αE7 carboxylesterase gives rise to resistance by converting the enzyme into an organophosphate (OP) hydrolase. This emergence of new activity provides a unique opportunity to investigate the molecular basis for enzyme evolution. In this thesis, I investigated the structure, function, evolution and inhibition of αE7. Chapter two describes the role of structural diversity in the function of wild type αE7. I applied new methods for extracting information about structural diversity from X-ray diffraction data to explore the changes in structure that accompany high affinity OP binding in αE7. In chapter three, I investigated the molecular basis for the evolution of catalytic OP detoxification in the blowfly. I determined the structure of the Gly137Asp variant by X-ray crystallography, which, along with molecular dynamics simulations and enzyme activity assays, revealed the role of Asp137 in the new catalytic mechanism. The new sidechain is disordered, and potentially only displays a fraction of its catalytic potential. Chapter four explores this catalytic potential through the laboratory-directed evolution of αE7 for increased OP hydrolase activity. I performed detailed kinetic and structural analysis of the evolutionary trajectory and characterized the structural changes responsible for the 8000-fold increase in OP hydrolase activity. The analysis unmasked a hidden, catalytically relevant, conformation of the active site. Furthermore, the results revealed the role of conformational diversity in the evolutionary optimization of αE7 and highlight the challenges to satisfying the competing demands of substrate binding and catalysis in the tightly packed environment of an enzyme’s active site. This work establishes that only a fraction of the evolutionary potential of αE7 has been explored in nature. In chapter five, I combined structural knowledge of αE7 with a computational screen to discover new potent and selective inhibitors of αE7. These compounds, based on a boronic acid scaffold, act as synergists to reduce the amount of OP required to kill L. cuprina by up to 16-fold, and abolish resistance. The broad-spectrum potential for the compounds as a new class of synergist was demonstrated by their low toxicity to animals and their ability to potentiate OP insecticides against another common insect pest, the peach-potato aphid Myzus persicae. These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy. More broadly, this thesis makes contributions to characterizing structural protein heterogeneity using X-ray diffraction, to understanding the molecular basis of enzyme evolution and to the use of in silico screens for the discovery of enzyme inhibitors. The results from this thesis will assist the of control insect pests and the management of insecticide resistance.

碩士
國立臺灣大學
生化科學研究所
107
Previously, we reported the protein-protein interaction (PPI) between β-tubulin and CCT-β complex as a potential anti-cancer chemotherapeutic target. Through virtual screening, a compound 3112210 from Sigma-Aldrich compound bank was identified to be a reversible inhibitor of the PPI by docking into hot spots on this PPI interface of β- tubulin. In this study, 3112210 was tested on a highly metastatic non-small cell lung cancer (NSCLC) cell line, CL1-5. The co-IP experiments showed that, in 3112210-treated cancer cells, β-tubulin and CCT-β complex was disrupted. Furthermore, 3112210 caused CL1-5 cell death through ER stress and apoptosis. In addition to verifying its toxicity toward CL1-5, we performed migration and invasion assays using dosage at about IC20. The results indicated that 3112210 also inhibited cancer cell migration and invasion, and MMP-2, -9 were also inhibited. These anti-metastatic effects were endowed via integrin- related pathways and EMT transcriptional factors, as demonstrated by western blot experiments. To sum, 3112210 is a novel non-covalent inhibitor for β-tubulin:CCT-β complex in CL1-5 lung adenocarcinoma cells to induce cancer cell death and impeded cell metastasis.