Discovery and development of novel anti-infective and anti-diabetic agents

Pratik Rajesh Chheda

doi:10.25820/etd.007194

The process of new drug development is complicated, convoluted and long. It takes nearly 10-15 years for a small molecule drug to reach the market upon identification. The first stage of this process is basic research which involves the process of target identification, target validation, hit identification, hit optimization, and lead identification. The identified lead compound is then passed on to the second stage of Preclinical development where Pharmacokinetic, pharmacodynamic and ADME parameters of the lead are evaluated and/or optimized. The aim of my thesis research was the successful application of various medicinal, synthetic, and computational chemistry strategies for the identification and development of novel hit and lead molecules for the management of different diseases. The goal of the first study was to identify selective inhibitors of apicoplast DNA polymerase, which is an antimalarial drug target. Malaria is a pandemic disease caused by parasites belonging to Plasmodium species, which contains an essential organelle referred to as the apicoplast. A single DNA polymerase, apPOL, is targeted to the apicoplast, where it is responsible for replicating and repairing the genome. apPOL has no direct orthologs in mammals; therefore, it is considered to be a promising drug target for the treatment and/or prevention of malaria. To identify novel inhibitors of apPOL, the compounds available in the Malaria Box were screened using a fluorescence based biochemical assay, which lead to the identification of MMV666123 as an inhibitor of apPOL DNA polymerase activity. In the absence of structural data, traditional medicinal and synthetic chemistry optimizations were employed to synthesize analogs of the original hit, and to gain structure-activity relationships information. Several inhibitors of apPOL were identified, and then employed to resolve the crystal structure of apPOL bound to the inhibitor. The structure indicated a putative allosteric binding pocket approximately 20 Å from the active site. Based on the common mechanism shared amongst A-family DNA polymerases, it is likely that compound binding to this allosteric site prevents the polymerase from adopting the closed conformation required for catalytic activity. The findings from our study provide a potential strategy for the design of high potency, specific inhibitors of apPOL that can be developed for the management of malaria. In the second study, the principles of molecular docking and synthetic medicinal chemistry were employed to identify novel modulators of SWELL1 protein and volume regulated anion channel (VRAC) followed by biological studies to understand the basis for their action and potential for use in the management of Type-2-diabetes (T2D). This study was based on the evidence that SWELL1-mediated currents and SWELL1 protein are reduced in adipocytes and pancreatic β-cells in the setting of T2D and hyperglycemia, suggesting that dysfunctional SWELL1-mediated signaling might contribute to T2D pathogenesis via insulin resistance and impaired insulin secretion. Next by literature precedence, DCPIB (SN-401) was used as a tool compound to decipher the correlation between SWELL1 protein and VRAC. Using the recent cryo-EM structure of SN-401 bound to its target SWELL1/LRRC8a, I employed molecular docking to identify key binding interactions of SN-401 with the ion channel and used the data to design novel SN-401 analogs with proposed enhanced or reduced on-target activity. Evaluation of compounds prepared in this study revealed that SN-401 and active analogs function as molecular chaperones (bound to closed channel) to augment SWELL1 expression and plasma membrane trafficking at concentrations >10- fold lower than the IC50 of ~5 µM for ICl,SWELL. In vivo, SN-401 and its active analogs, normalize glucose tolerance by increasing insulin sensitivity and secretion in insulin-resistant T2D mouse models, while augmenting tissue glucose uptake, suppressing hepatic glucose production, and reversing hepatic steatosis in obese T2D mice. Importantly, while SN-401 and active analogs normalize glycaemia in diabetic mice, they have very mild effects on non-obese euglycemic mice – indicating a low risk of hypoglycemic events associated with other commonly used anti-diabetic therapies, including sulfonylureas and insulin. These effects are not observed with structurally similar but inactive analogs of SN-401. Based on the findings, small molecule SWELL1 modulators may represent a “first-in-class” therapeutic approach to treat metabolic syndrome and associated diseases by restoring SWELL1 signaling across multiple organ system that are dysfunctional in T2D. The goal of my third study was to employ virtual screening to identify novel inhibitors of H. pylori glutamate racemase for the management of H. pylori infections. The challenge with the project was the high flexibility of glutamate racemase (GR), which makes employing general docking protocols useless due to the possibility of generating excessive false positives. To overcome this challenge, a virtual screening regime was developed specifically for highly flexible enzyme targets like H. pylori GR. The key step is selection of the “best-performing” combination of receptor and docking program for virtual screening. The workflow employs a hybrid Molecular Dynamics-Docking approach involving all atom classical MD simulations followed by structural clustering of MD snapshots to generate different receptor forms. Following this, known actives and decoys were docked into each form of the receptor using different docking protocols. The results were then analyzed by generating Receiver Operating Characteristic (ROC) curves to select the best performing receptor-docking program pair that is best able to enrich for known actives. Virtual screening of AnalytiCon’s MEGx natural products library using the identified best performing system provided 177 hits, which were then analyzed by a modified version of our previously reported computationally expensive all-atom simulated annealing energy minimization (referred to as SAEM) docking protocol. The resulting top five virtual hits were visually inspected and their affinity to bind to H. pylori GR was evaluated by Surface Plasmon Resonance followed by their ability to inhibit the enzymatic activity of the protein in a coupled-enzyme assay. Four of the five compounds bound to the protein with micromolar affinity and inhibited H. pylori GR with varying degrees of potency. Based on our success of identifying natural product inhibitors of glutamate racemase, compounds of enamine advance database, a collection of 500K drug like molecules, was screened. The method of fingerprint-based clustering was employed to reduce the size of the screening library and the screen lead to the identification of several novel small molecule binders of H. pylori glutamate racemase. Combined, the work in this dissertation showcases the successful application of different computational and synthetic medicinal chemistry strategies, along with comprehensive biological studies to address unmet therapeutic needs with three drug targets. The identification of novel inhibitors of apPOL for the management of malaria, novel modulators of SWELL1 protein for the management of T2D and novel inhibitors of H. pylori GR for the management of H. pylori gastric infections is reported.

Discovery and development of novel anti-infective and anti-diabetic agents

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