Malaria, a disease caused by the parasite Plasmodium, continues to be one of the deadliest diseases worldwide. The WHO reported over 627,000 deaths in 2020, and over 1 billion people are at risk of infection. Even though Artemisinin-based Combination Therapies (ACT) are the current standard of care for malaria, the emergence of drug resistance generates a constant need to develop and synthesize new drugs. Tetrahydro-β-carboline acid (THβC) 1-(2,4-dichlorophenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-2-ium-3-carboxylate (MMV008138) has promising antimalarial properties; it was discovered by screening the Malaria Box with the so-called IPP Rescue assay. This assay identified MMV008138 as an inhibitor of the MEP pathway, which produces essential isoprenoid precursors (IPP and DMAPP) in the malaria parasite P. falciparum (EC50 250 ± 70 nM, IPP rescue 100% @ 2.5 μM). Subsequent investigation revealed that (1R,3S)-configuration and 2',4'-dihalogen substitution were critical for the activity of this compound, and that substitution of the non-aromatic ring was not tolerated. To search for new antimalarial structures, our collaborator Dr. Max Totrov constructed a generalized 3D pharmacophore-based on MMV008138 and 92 of its analogs and used it for a virtual ligand screen (VLS) of the 13K compound hit set from which MMV008138 had been selected. This exercise identified TCMDC-140230, a THβC, 1-(3,4-dichlorophenyl)-8-methyl-N-(2-(methylamino)ethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxamide (undefined stereochemistry) reported having nearly the same potency of MMV008138. Synthesis of the stereoisomers of compound TCMDC-140230 was accomplished via Pictet-Spengler reaction of (S)- and (R)-7-methyl tryptophan methyl ester and 3,4-dichlorobenzaldehyde. The individual stereoisomeric esters were converted to the corresponding amides, but none of the stereoisomers of TCMDC-140230 were potent antimalarials (IC50 = 1,300 – 3,700 nM). However, a significant amount of oxidized byproduct 1-(3,4-dichlorophenyl)-8-methyl-N-(2-(methylamino)ethyl)-9H-pyrido[3,4-b]indole-3-carboxamide (MMV1803522) was observed in the synthesis of (1S,3S)- and (1R,3R)-TCDMC-140230. This achiral β-carboline amide (PRC1584, IC50 = 108 ± 7 nM) proved more potent towards P. falciparum than MMV008138 and its toxicity was not reversed by co-application of IPP. Thus, the antimalarial target of MMV1803522 is distinct from that of MMV008138. Most importantly, MMV1803522 at 40 mg/kg/day (oral) cured P. berghei malaria infection in mice. The lead compound also was found to have a good safety profile. Medicines for Malaria Venture (MMV) has expressed interest in this compound which is now also known as MMV1803522. The results from these biological assays gave the insight to develop new analogs that have better asexual blood stage inhibition potency. Extensive structure-activity relationship studies were conducted by synthesizing analogs of the compound MMV1803522. The studies were mainly focused on analyzing the effect of aliphatic substitutions, how well the potency can be improved with different D-ring substitutions, and amide substitutions. In addition to this structural optimization, several metabolism studies were also conducted on this new lead compound. The potency study results of C1 alkyl-substituted analogs of MMV1803522 showed that aromatic substitutions are required at C1 for maintaining good inhibition potency. The heteroaryl substituents at C1 were found to be slightly less potent than the lead compound MMV1803522. Synthesis of analogs without C8 methyl group as in lead compound showed an EC50 < 100 nM is possible with a C8 hydrogen substitution. Most noteworthy is 3,4,5-trichlotophenyl-bearing compound 3.20a, which had an EC50 of 54 ± 8 nM. This compound is twice as potent as MMV1803522. Equipotent analogs to MMV1803522 were also synthesized with different amide substituents. The metabolism studies showed low solubility for compounds having an EC50 less than or close to 100 nM. Unfortunately, the intrinsic clearance rate of several selected compounds was found to be higher than MMV1803522. These results left us with scope for the development of new analog compounds. The emerging structure-activity relationship within this scaffold and outline of remaining challenges to improve potency sub-100 nM without compromising moderate solubility and good metabolic stability are in progress. / Doctor of Philosophy / Malaria is a global health problem that causes significant sickness and death annually in the developing world. The emergence of resistant parasite strains of malaria massively challenges efforts to eliminate this threat. To control the spread of malaria, there is a continuous need for the development of new antimalarial drugs that ideally offer a single-dose cure and new mechanism of action. One such promising target, called, Methyl Erythrytol Phosphate (MEP) pathway which produces IPP and DMAPP, are important isoprenoid precursors required in living beings. A compound MMV008138 was identified from a collection of compounds that exhibited antimalarial activity, the so-called "Malaria Box", and this compound was further analyzed for several biological assays. Unfortunately, MMV008138 was unsuccessful Since it was found toxic in mice when ingested orally. The efforts to develop structurally similar analogs of MMV008138 resulted in the accidental discovery of a compound that inhibits the parasites' growth much better than the former compound. This compound has a similar molecular structure to MMV008138, and the Medicines for Malaria organization (MMV) has designated it as MMV1803522. The newly obtained compound and its analogs were investigated and found to have promising potency to inhibit the growth of the malarial parasite Plasmodium falciparum. Multiple biological assays were conducted and found that even though MMV1803522 is toxic to malarial parasites, it does not show toxicity to other cells. The studies in mice showed that it was not toxic orally. Also, it was found to be non-toxic towards several mammalian cell lines. The development of structurally similar analogs can help in improving the potency of the compound, make a better orally bioavailable compound, and improve oral efficacy. Analyzing these results will help to determine the mechanism of action of the compound.

Lanthionine synthetase C-like protein 2 (LANCL2) is a member of the LANCL protein family, which is broadly expressed throughout the body. LANCL2 is the molecular target of abscisic acid (ABA), a compound with insulin-sensitizing and immune modulatory actions. LANCL2 is required for membrane binding and signaling of ABA in immune cells. Direct binding of ABA to LANCL2 was predicted in silico using molecular modeling approaches and validated experimentally using ligand-binding assays and kinetic surface plasmon resonance studies. The therapeutic potential of the LANCL2 pathway ranges from increasing cellular sensitivity to anticancer drugs, insulin-sensitizing effects and modulating immune and inflammatory responses in the context of immune-mediated and infectious diseases. A case for LANCL2-based drug discovery and development is also illustrated by the anti-inflammatory activity of novel LANCL2 ligands such as NSC61610 against inflammatory bowel disease in mice. This dissertation discusses the value of LANCL2 as a novel therapeutic target for the discovery and development of new classes of orally active drugs against chronic metabolic, immune-mediated and infectious diseases and as a validated target that can be used in precision medicine. Specifically, in Chapter 2 of the dissertation, we performed homology modeling to construct a three-dimensional structure of LANCL2 using the crystal structure of LANCL1 as a template. Our molecular docking studies predicted that ABA and other PPAR - agonists share a binding site on the surface of LANCL2. In Chapter 3 of the dissertation, structure-based virtual screening was performed. Several potential ligands were identified using molecular docking. In order to validate the anti-inflammatory efficacy of the top ranked compound (NSC61610) in the NCI Diversity Set II, a series of in vitro and pre-clinical efficacy studies were performed using a mouse model of dextran sodium sulfate (DSS)-induced colitis. In Chapter 4 of the dissertation, we developed a novel integrated approach for creating a synthetic patient population and testing the efficacy of the novel pre-clinical stage LANCL2 therapeutic for Crohn's disease in large clinical cohorts in silico. Efficacy of treatments on Crohn's disease was evaluated by analyzing predicted changes of Crohn's disease activity index (CDAI) scores and correlations with immunological variables were evaluated. The results from our placebo-controlled, randomized, Phase III in silico clinical trial at 6 weeks following the treatment shows a positive correlation between the initial disease activity score and the drop in CDAI score. This observation highlights the need for precision medicine strategies for IBD. / Ph. D.

Kinases are involved in a multitude of signaling pathways, such as cellular growth, proliferation, and apoptosis, and have been discovered to be important in numerous diseases including cancer, Alzheimer's disease, cardiovascular health, rheumatoid arthritis, and fibrosis. Due to the involvement in a wide variety of disease types, kinases have been studied for exploitation and use as targets for therapeutics. There are many limitations with developing kinase target therapeutics due to the high similarity of kinase active site composition, making the utilization of new techniques to determine kinase exploitability for therapeutic design with high specificity essential for the advancement of novel drug strategies. In silico approaches have become increasingly prevalent for providing useful insight into protein structure-function relationships, offering new information to researchers about drug discovery strategies. This work utilizes streamlined computational techniques on an atomistic level to aid in the identification of orthologue and isoform exploitability, identifying new features to be utilized for future inhibitor design. By exploring two separate kinases and kinase targeting domains, we found that orthologues and isoforms contain distinct features, likely responsible for their biological roles, which can be utilized and exploited for selective drug development. In this work, we identified new exploitable features between kinase orthologues for treatment in Human African Trypanosomiasis and structural morphology differences between two kinase isoforms that can potentially be exploited for cancer therapeutic design. / Master of Science in Life Sciences / Numerous diseases such as cancer, Alzheimer's disease, cardiovascular disease, rheumatoid arthritis, and fibrosis have been attributed to different cell growth and survival pathways. Many of these pathways are controlled by a class of enzymes called kinases. Kinases are involved in almost every metabolic pathway in human cells and can act as molecular switches to turn on and off disease progression. Due to the involvement of these kinases' in a wide variety of disease types, kinases have been continually studied for the development of new drugs. Developing effective drugs for kinases requires an extensive understanding of the structural characteristics due to the high structural similarity across all kinases. In silico, or computational, techniques are useful strategies for drug development practices, offering new information into protein structure-function relationships, which in turn can be utilized in drug discovery advancements. Utilizing computational methods to explore structural features can help identify specific protein structural features, thus providing new strategies for protein specific inhibitor design. In this work, we identified new exploitable features between kinase orthologues for treatment in Human African Trypanosomiasis and structural morphology differences between two kinase isoforms that can potentially be exploited for cancer therapeutic design.

Peroxisome proliferator-activated receptor γ (PPARγ) is associated with a wide range of diseases, including type 2 diabetes mellitus (T2D). Thiazolidinediones (TZDs) are agonists of PPARγ which have an insulin sensitizing effect, and are therefore used as a treatment for T2D. However, TZDs cause negative side effects in patients, such as weight gain, edema, and increased risk of bone fracture. Partial agonists could be an alternative to TZD-based drugs with fewer side effects. However, there is a lack of understanding of the types of PPARγ partial agonists and how they differ from full agonists. In silico techniques, like virtual screening, molecular docking, and pharmacophore modeling, allow us to determine and characterize markers of varying levels of agonism. An extensive search of the RCSB Protein Data Bank found 62 structures of PPARγ resolved with partial agonists. Cross-docking was performed and found that two PDB structures, 3TY0 and 5TWO, would be effective as receptor structures for virtual screening. By clustering known partial agonists by common pharmacophore features, we found several distinct groups of partial agonists. Interaction and pharmacophore models were created for each group of partial agonists. Virtual screening of FDA-approved compounds showed that the models were able to predict potential partial agonists of PPARγ. This study provides additional insight into the different binding modes of partial agonists of PPARγ and their characteristics. These models can be used to assist drug discovery efforts for intelligently designing novel therapeutics for T2D which have fewer negative side effects. / Master of Science in Life Sciences / The peroxisome proliferator-activated receptor γ (PPARγ) protein is associated with a wide range of diseases, including type 2 diabetes mellitus (T2D). Thiazolidinediones (TZDs) are compounds that activate PPARγ, and increase insulin sensitivity in patients with T2D. However, TZDs cause negative side effects in patients, such as weight gain, increased fluid retention, and increased risk of bone fracture. Partial agonists could be an alternative to TZD-based drugs with fewer side effects. However, there is a lack of understanding of the types of PPARγ partial agonists and how they differ from full agonists. Computational techniques allow us to investigate common features between known partial agonists. An extensive search of the RCSB Protein Data Bank found 62 structures of PPARγ which contained partial agonists. Each known partial agonist was docked into twelve complete PPARγ structures, and it was found that two structure models would be effective as receptor structures for virtual screening. A set of known partial agonists were grouped based on common chemical features, and three distinct groups of partial agonists were found. Binding criteria for each of these three groups were developed. A library of FDA-approved compounds was screened using the criteria for binding to identify potential novel partial agonists. Three potential novel partial agonists were found in the screening. This study provides additional insight into how different compounds activate PPARγ. These methods can be used to assist drug discovery efforts for intelligently designing novel therapeutics for T2D which have fewer negative side effects.

No / Since the development of the first cytotoxic agents, synthetic organic chemistry has advanced enormously. The synthetic and medicinal chemists of today are at the centre of drug development and are involved in most, if not all, processes of drug discovery. Recent decreases in government funding and reformed educational policies could, however, seriously impact on drug discovery initiatives worldwide. Not only could these changes result in fewer scientific breakthroughs, but they could also negatively affect the training of our next generation of medicinal chemists.

No / Aldehyde dehydrogenases (ALDHs) belong to a superfamily of 19 isozymes that are known to participate in many physiologically important biosynthetic processes including detoxification of specific endogenous and exogenous aldehyde substrates. The high expression levels of an emerging number of ALDHs in various cancer tissues suggest that these enzymes have pivotal roles in cancer cell survival and progression. Mapping out the heterogeneity of tumours and their cancer stem cell (CSC) component will be key to successful design of strategies involving therapeutics that are targeted against specific ALDH isozymes. This review summarises recent progress in ALDH-focused cancer research and discovery of small-molecule-based inhibitors.

Les biothérapies (anticorps monoclonaux, récepteurs solubles) ciblant les cytokines IL-6 etTNF-alpha pour le traitement des maladies inflammatoires chroniques ont constitué un succèsmajeur de l’industrie pharmaceutique. Elles présentent néanmoins des inconvénientsimportants : résistances, mode d’administration contraignant, coût élevé.Notre équipe travaille sur l’identification de petites molécules inhibant directement cescytokines, afin d’élargir l’offre thérapeutique existante. Administrées par voie orale, ellesconstitueraient une alternative particluièrement favorable aux patients.Durant ma thèse, j’ai réalisé le criblage expérimental (tests cellulaires et tests biochimiquesde liaison) des meilleurs composés identifiés par criblage virtuel d’un grande chimiothèque dediversité, ainsi que de composés dérivés de pyridazine issus d’une chimiothèque médicinale. J’aiainsi pu identifier plusieurs inhibiteurs directs du TNF-alpha et de l’IL-6. De plus, mon travail apermis d’affiner les procédures de criblage du Laboratoire.Ces travaux ouvrent de nouvelles pistes pour le développement de médicaments anti-cytokines. / Anti-cytokine biologics (monoclonal antibodies, soluble receptors) targeting TNF-alpha and IL-6in chronic inflammatory diseases have been a major success for pharmaceutical industry.However, they exhibit several drawbacks : resistance, difficult administration, high costs.Our team works on the discovery of small molecule inhibitors of cytokines suck as TNF-alphaand IL-6, in order to widen the range of therapeutic drugs. Orally active drugs would represent ahighly beneficial alternative for patients.During my PhD, I have performed an experimental screening (using cellular and biochemicalbinding testings) of the best compounds identified through virtual screening of a large chemicallibrary, and on pyridazine compounds of a medicinal chemical library. I have been able toidentify several small molecules inhibiting the interaction of TNF-! and IL-6 with their receptor.Moreover, my work will have an impact on the laboratory screening strategies.Overall, this work opens new avenues for anti-cytokine drug discovery.

Les biothérapies (anticorps monoclonaux, récepteurs solubles) ciblant les cytokines IL-6 etTNF-alpha pour le traitement des maladies inflammatoires chroniques ont constitué un succèsmajeur de l’industrie pharmaceutique. Elles présentent néanmoins des inconvénientsimportants : résistances, mode d’administration contraignant, coût élevé.Notre équipe travaille sur l’identification de petites molécules inhibant directement cescytokines, afin d’élargir l’offre thérapeutique existante. Administrées par voie orale, ellesconstitueraient une alternative particluièrement favorable aux patients.Durant ma thèse, j’ai réalisé le criblage expérimental (tests cellulaires et tests biochimiquesde liaison) des meilleurs composés identifiés par criblage virtuel d’un grande chimiothèque dediversité, ainsi que de composés dérivés de pyridazine issus d’une chimiothèque médicinale. J’aiainsi pu identifier plusieurs inhibiteurs directs du TNF-alpha et de l’IL-6. De plus, mon travail apermis d’affiner les procédures de criblage du Laboratoire.Ces travaux ouvrent de nouvelles pistes pour le développement de médicaments anti-cytokines. / Anti-cytokine biologics (monoclonal antibodies, soluble receptors) targeting TNF-alpha and IL-6in chronic inflammatory diseases have been a major success for pharmaceutical industry.However, they exhibit several drawbacks : resistance, difficult administration, high costs.Our team works on the discovery of small molecule inhibitors of cytokines suck as TNF-alphaand IL-6, in order to widen the range of therapeutic drugs. Orally active drugs would represent ahighly beneficial alternative for patients.During my PhD, I have performed an experimental screening (using cellular and biochemicalbinding testings) of the best compounds identified through virtual screening of a large chemicallibrary, and on pyridazine compounds of a medicinal chemical library. I have been able toidentify several small molecules inhibiting the interaction of TNF-! and IL-6 with their receptor.Moreover, my work will have an impact on the laboratory screening strategies.Overall, this work opens new avenues for anti-cytokine drug discovery.

When applied to drug discovery, modern computational systems can provide insight into the highly complex systems underlying drug activity and predict compounds or targets of interest. Many tools have been developed for computer aided drug discovery (CADD), focusing on small molecule ligands, protein targets, or both. The aim of this thesis is the improvement of CADD tools for describing small molecule properties and application of CADD to several stages of drug discovery regarding two targets for the treatment of obesity and related diseases: the neuropeptide Y4 receptor (Y4R) and the melanocortin-4 receptor (MC4R). In the first chapter, the major categories of CADD are outlined, including descriptions for many of the popular tools and examples where these tools have directly contributed to the discovery of new drugs. Following the introduction, several improvements for encoding stereochemistry and signed property distribution are introduced and tested in scenarios meant to simulate applications in virtual high-throughput screening. Y4R and MC4R are both class A G-protein coupled receptors (GPCRs) with endogenous peptide ligands that play critical roles in the signaling of satiety and energy metabolism. So far, no structures from either receptor family have been experimentally elucidated. CADD was combined with high-throughput screening (HTS) to discover the first small molecule positive allosteric modulators (PAMs) of Y4R. Secondly, CADD techniques were used to model the interaction of Y4R and pancreatic polypeptide based on experimental results that elucidate specific binding contacts. Similar SB-CADD approaches were used to model the interaction of MC4R with its high affinity peptide agonist α-MSH. Due to its role in monogenic forms of obesity, these models were used to predict which residues directly participate in binding and correlate mutated residues with their potential role in the binding site.

This thesis is focused on enzymatic studies of the secreted aspartic proteases (Saps) from Candida albicans as a tool for discovery of anti-candida drugs. C. albicans causes infections in a number of different locations, which differ widely in the protein substrates available and pH. Since C. albicans needs Saps during virulent growth, these enzymes are good targets for drug development. In order to investigate the catalytic characteristics of Saps and their inhibitor affinities, substrate-based kinetic assays were developed. Due to the low sensitivity of these assays, especially at the sub-optimal pH required to mimic the different locations of infections, these assays were not satisfactory. Therefore, a biosensor assay was developed whereby, it was possible to study interaction between Saps and inhibitors without the need to optimise catalytic efficacy. Furthermore, the biosensor assay allowed determination of affinity, as well as the individual association and dissociation rates for inhibitor interactions. Knowledge about substrate specificity, Sap subsite adaptivity, and the pH dependencies of catalytic efficacy has been accumulated. Also, screening of transition-state analogue inhibitors designed for HIV-1 protease has revealed inhibitors with affinity for Saps. Furthermore, the kinetics and pH dependencies of their interaction with Saps have been investigated. One of these inhibitors, BEA-440, displayed a complex interaction with Saps, indicating a conformational change upon binding and a very slow dissociation rate. A time dependent interaction was further supported by inhibition measurements. The structural information obtained affords possibilities for design of new more potent inhibitors that might ultimately become drugs against candidiasis. The strategy to combine substrate specificity studies with inhibitor screening has led to complementary results that generate a framework for further development of potent inhibitors.