MDFit: automated molecular simulations workflow enables high throughput assessment of ligands-protein dynamics.

Molecular dynamics (MD) simulation is a powerful tool for characterizing ligand-protein conformational dynamics and offers significant advantages over docking and other rigid structure-based computational methods. However, setting up, running, and analyzing MD simulations continues to be a multi-step process making it cumbersome to assess a library of ligands in a protein binding pocket using MD. We present an automated workflow that streamlines setting up, running, and analyzing Desmond MD simulations for protein-ligand complexes using machine learning (ML) models. The workflow takes a library of pre-docked ligands and a prepared protein structure as input, sets up and runs MD with each protein-ligand complex, and generates simulation fingerprints for each ligand. Simulation fingerprints (SimFP) capture protein-ligand compatibility, including stability of different ligand-pocket interactions and other useful metrics that enable easy rank-ordering of the ligand library for pocket optimization. SimFPs from a ligand library are used to build & deploy ML models that predict binding assay outcomes and automatically infer important interactions. Unlike relative free-energy methods that are constrained to assess ligands with high chemical similarity, ML models based on SimFPs can accommodate diverse ligand sets. We present two case studies on how SimFP helps delineate structure-activity relationship (SAR) trends and explain potency differences across matched-molecular pairs of (1) cyclic peptides targeting PD-L1 and (2) small molecule inhibitors targeting CDK9.

Identification of benzothiazole derived monosaccharides as potent, selective, and orally bioavailable inhibitors of human and mouse galectin-3; a rare example of using a S···O binding interaction for drug design.

As a result of our continued efforts to pursue Gal-3 inhibitors that could be used to fully evaluate the potential of Gal-3 as a therapeutic target, two novel series of benzothiazole derived monosaccharides as potent (against both human and mouse Gal-3) and orally bioavailable Gal-3 inhibitors, represented by 4 and 5, respectively, were identified. These discoveries were made based on proposals that the benzothiazole sulfur atom could interact with the carbonyl oxygen of G182/G196 in h/mGal-3, and that the anomeric triazole moiety could be modified into an N-methyl carboxamide functionality. The interaction between the benzothiazole sulfur and the carbonyl oxygen of G196 in mGal-3 was confirmed by an X-ray co-crystal structure of early lead 9, providing a rare example of using a S···O binding interaction for drug design. It was found that for both the series, methylation of 3-OH in the monosaccharides caused no loss in h & mGal-3 potencies but significantly improved permeability of the molecules.

Molecular mechanism of interspecies differences in the binding affinity of TD139 to Galectin-3.

Galectin-3 (Gal-3), a ß-galactoside-binding lectin, has been implicated in a plethora of pathological disorders including fibrosis, inflammation, cancer and metabolic diseases. TD139-a thio-digalactoside inhibitor developed by Galecto Biotech as a potential therapeutic for idiopathic pulmonary fibrosis-is the most advanced small-molecule Gal-3 inhibitor in clinical studies. It binds to human Gal-3 with high affinity but has lower affinity towards mouse and rat homologs, which is also manifested in the differential inhibition of Gal-3 function. Using biophysical methods and high-resolution X-ray co-crystal structures of TD139 and Gal-3 proteins, we demonstrate that a single amino acid change corresponding to A146 in human Gal-3 is sufficient for the observed reduction in the binding affinity of TD139 in rodents. Site-directed mutagenesis of A146V (in human Gal-3) and V160A (in mouse Gal-3) was sufficient to interchange the affinities, mainly by affecting the off rates of the inhibitor binding. In addition, molecular dynamics simulations of both wild-type and mutant structures revealed the sustained favorable noncovalent interactions between the fluorophenyl ring and the active site A146 (human Gal-3 and mouse V160A) that corroborate the finding from biophysical studies. Current findings have ramifications in the context of optimization of drug candidates against Gal-3.

Shared Consensus Machine Learning Models for Predicting Blood Stage Malaria Inhibition.

The development of new antimalarial therapies is essential, and lowering the barrier of entry for the screening and discovery of new lead compound classes can spur drug development at organizations that may not have large compound screening libraries or resources to conduct high-throughput screens. Machine learning models have been long established to be more robust and have a larger domain of applicability with larger training sets. Screens over multiple data sets to find compounds with potential malaria blood stage inhibitory activity have been used to generate multiple Bayesian models. Here we describe a method by which Bayesian quantitative structure-activity relationship models, which contain information on thousands to millions of proprietary compounds, can be shared between collaborators at both for-profit and not-for-profit institutions. This model-sharing paradigm allows for the development of consensus models that have increased predictive power over any single model and yet does not reveal the identity of any compounds in the training sets.

Comparative Study of Activities of a Diverse Set of Antimycobacterial Agents against Mycobacterium tuberculosis and Mycobacterium ulcerans.

A library of compounds covering a broad chemical space was selected from a tuberculosis drug development program and was screened in a whole-cell assay against Mycobacterium ulcerans, the causative agent of the necrotizing skin disease Buruli ulcer. While a number of potent antitubercular agents were only weakly active or inactive against M. ulcerans, five compounds showed high activity (90% inhibitory concentration [IC90], ≤1 µM), making screening of focused antitubercular libraries a good starting point for lead generation against M. ulcerans.

Whole cell screen based identification of spiropiperidines with potent antitubercular properties.

Whole cell based screens to identify hits against Mycobacterium tuberculosis (Mtb), carried out under replicating and non-replicating (NRP) conditions, resulted in the identification of multiple, novel but structurally related spiropiperidines with potent antitubercular properties. These compounds could be further classified into three classes namely 3-(3-aryl-1,2,4-oxadiazol-5-yl)-1'-alkylspiro[indene-1,4'-piperidine] (abbr. spiroindenes), 4-(3-aryl-1,2,4-oxadiazol-5-yl)-1'-alkylspiro[chromene-2,4'-piperidine] (abbr. spirochromenes) and 1'-benzylspiro[indole-1,4'-piperidin]-2(1H)-one (abbr. spiroindolones). Spiroindenes showed ⩾ 4 log10 kill (at 2-12 µM) on replicating Mtb, but were moderately active under non replicating conditions. Whole genome sequencing efforts of spiroindene resistant mutants resulted in the identification of I292L mutation in MmpL3 (Mycobacterial membrane protein Large), required for the assembly of mycolic acid into the cell wall core of Mtb. MIC modulation studies demonstrated that the mutants were cross-resistant to spirochromenes but not to spiroindolones. This Letter describes lead identification efforts to improve potency while reducing the lipophilicity and hERG liabilities of spiroindenes. Additionally, as deduced from the SAR studies, we provide insights regarding the new chemical opportunities that the spiroindolones can offer to the TB drug discovery initiatives.

Discovery of benzothiazoles as antimycobacterial agents: Synthesis, structure-activity relationships and binding studies with Mycobacterium tuberculosis decaprenylphosphoryl-ß-D-ribose 2'-oxidase.

We report the discovery of benzothiazoles, a novel anti-mycobacterial series, identified from a whole cell based screening campaign. Benzothiazoles exert their bactericidal activity against Mycobacterium tuberculosis (Mtb) through potent inhibition of decaprenylphosphoryl-ß-d-ribose 2'-oxidase (DprE1), the key enzyme involved in arabinogalactan synthesis. Specific target linkage and mode of binding were established using co-crystallization and protein mass spectrometry studies. Most importantly, the current study provides insights on the utilization of systematic medicinal chemistry approaches to mitigate safety liabilities while improving potency during progression from an initial genotoxic hit, the benzothiazole N-oxides (BTOs) to the lead-like AMES negative, crowded benzothiazoles (cBTs). These findings offer opportunities for development of safe clinical candidates against tuberculosis. The design strategy adopted could find potential application in discovery of safe drugs in other therapy areas too.

Atropisomerism Observed in Galactose-Based Monosaccharide Inhibitors of Galectin-3 Comprising 2-Methyl-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione.

Galectin-3 (Gal-3) is a carbohydrate binding protein that has been implicated in the development and progression of fibrotic diseases. Proof-of-principal animal models have demonstrated that inhibition of Gal-3 is a potentially viable pathway for the treatment of fibrosis─with small molecule Gal-3 inhibitors advanced into clinical trials. We hereby report the discovery of novel galactose-based monosaccharide Gal-3 inhibitors comprising 2-methyl-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (compound 20) and 4-phenyl-4H-1,2,4-triazole (compound 15). Notably, hindered rotation caused by steric interaction between the 3-thione and ortho-trifluoromethyl group of compounds 20, 21 induced formation of thermodynamically stable atropisomers. Distinct X-ray cocrystal structures of 20 and 21 were obtained, which clearly demonstrated that the configuration of 21 proscribes a key halogen bonding σ-hole interaction of 3-chloro with carbonyl oxygen of Gly182, thereby leading to significant loss in potency. Ultimately, 20 and 15 were evaluated in mouse pharmacokinetic studies, and both compounds exhibited oral exposures suitable for further in vivo assessment.

Discovery of BMS-986308: A Renal Outer Medullary Potassium Channel Inhibitor for the Treatment of Heart Failure.

Recent literature reports highlight the importance of the renal outer medullary potassium (ROMK) channel in renal sodium and potassium homeostasis and emphasize the potential impact that ROMK inhibitors could have as a novel mechanism diuretic in heart failure patients. A series of piperazine-based ROMK inhibitors were designed and optimized to achieve excellent ROMK potency, hERG selectivity, and ADME properties, which led to the identification of compound 28 (BMS-986308). BMS-986308 demonstrated efficacy in the volume-loaded rat diuresis model as well as promising in vitro and in vivo profiles and was therefore advanced to clinical development.

Synthesis and structure activity relationship of imidazo[1,2-a]pyridine-8-carboxamides as a novel antimycobacterial lead series.

Imidazo[1,2-a]pyridine-8-carboxamides as a novel antimycobacterial lead were generated by whole cell screening of a focused library against Mycobacterium tuberculosis. Herein, we describe the synthesis and structure activity relationship evaluation of this class of inhibitors and the optimization of physicochemical properties. These are selective inhibitors of Mycobacterium tuberculosis with no activity on either gram positive or gram negative pathogens.

Investigation of Unusual N-(Triphenyl-λ5-phosphanylidene) Amide Fragmentation Observed upon MS/MS Collision-Induced Dissociation.

A mechanism of unusual tandem (MS/MS) fragmentation of protonated species of N-(triphenyl-λ5-phosphanylidene) derivatives, [M + H]+ to generate triphenylphosphine oxide (TPPO) within the mass spectrometer has been investigated and reported. Collision-induced dissociation of these molecules resulted in the generation of TPPO as a signature fragment. This fragment suggested the presence of a P-O bond in the structure which was contrary to the structure of the compound identified by nuclear magnetic resonance spectrometry (NMR) and single-crystal X-ray diffractometry (SXRD) techniques with a P═N bond rather than a P-O bond. In order to confirm the generation of the TPPO fragment within the mass spectrometer, 14 different N-(triphenyl-λ5-phosphanylidene) derivatives containing amide, 18O-labeled amide, thiamide, and nonacyl phosphazene derivatives were synthesized and their MS/MS behavior was studied by liquid chromatography-high-resolution mass spectrometry. Fragmentation of these amide derivatives generated TPPO/TPPS or their 18O-labeled analogues as the major fragment in almost all cases under similar MS conditions. Based on the outcome of these experiments, a plausible mechanism for such fragmentation, involving the intramolecular shifting of oxygen from carbon to phosphorus, has been proposed. DFT calculations for the protonated species at B3LYP-D3/6-31+G(d,p) further supported the proposed mechanism involving a four-membered ring, P-O-C-N, as the transition state. Details of this work are presented here.

Identification of Monosaccharide Derivatives as Potent, Selective, and Orally Bioavailable Inhibitors of Human and Mouse Galectin-3.

Galectin-3 (Gal-3), a member of the ß-galactoside-binding protein family, is implicated in a wide variety of human diseases. Identification of Gal-3 inhibitors with the right combination of potency (against both human and mouse Gal-3) and pharmacokinetic properties to fully evaluate the potential of Gal-3 for therapeutic intervention has been a major challenge due to the characteristics of its binding pocket: high hydrophilicity and key structural differences between human Gal-3 and the mouse ortholog. We report the discovery of a novel series of monosaccharide-based, highly potent, and orally bioavailable inhibitors of human and mouse Gal-3. The novel monosaccharide derivatives proved to be selective for Gal-3, the only member of the chimeric type of galectins, over Gal-1 and Gal-9, representative of the prototype and tandem-repeat type of galectins, respectively. The proposed binding mode for the newly identified ligands was confirmed by an X-ray cocrystal structure of a representative analogue bound to Gal-3 protein.

Scaffold Morphing To Identify Novel DprE1 Inhibitors with Antimycobacterial Activity.

We report a novel benzimidazole (BI) based DprE1 inhibitor that resulted from scaffold morphing of a 1,4-azaindole series. The clinical progression of the 1,4-azaindole series from our previous work validates the potential of exploring newer chemical entities with antimycobacterial activity driven via a noncovalent inhibition of the decaprenylphosphoryl-ß-d-ribose-2'-epimerase (DprE1). The representative compounds from the new scaffold reported in this study exhibited an improved solubility and higher free plasma fraction, while retaining potent DprE1 inhibition and antimycobacterial activity. A representative compound from the benzimidazole series demonstrated good efficacy in a murine model of tuberculosis. Furthermore, molecular modeling of the BI scaffold suggests plausible modes of binding in the active site of DprE1 enzyme from Mycobacterium tuberculosis that can be used for further exploration of the series.

Explicit ion, implicit water solvation for molecular dynamics of nucleic acids and highly charged molecules.

An explicit ion, implicit water solvent model for molecular dynamics was developed and tested with DNA and RNA simulations. The implicit water model uses the finite difference Poisson (FDP) model with the smooth permittivity method implemented in the OpenEye ZAP libraries. Explicit counter-ions, co-ions, and nucleic acid were treated with a Langevin dynamics molecular dynamics algorithm. Ion electrostatics is treated within the FDP model when close to the solute, and by the Coulombic model when far from the solute. The two zone model reduces computation time, but retains an accurate treatment of the ion atmosphere electrostatics near the solute. Ion compositions can be set to reproduce specific ionic strengths. The entire ion/water treatment is interfaced with the molecular dynamics package CHARMM. Using the CHARMM-ZAPI software combination, the implicit solvent model was tested on A and B form duplex DNA, and tetraloop RNA, producing stable simulations with structures remaining close to experiment. The model also reproduced the A to B duplex DNA transition. The effect of ionic strength, and the structure of the counterion atmosphere around B form duplex DNA were also examined.

Discovery of Imidazo[1,2-a]pyridine Ethers and Squaramides as Selective and Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis.

The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.

Nitroarenes as Antitubercular Agents: Stereoelectronic Modulation to Mitigate Mutagenicity.

Nitroarenes are less preferred in drug discovery due to their potential to be mutagenic. However, several nitroarenes were shown to be promising antitubercular agents with specific modes of action, namely, nitroimidazoles and benzothiazinones. The nitro group in these compounds is activated through different mechanisms, both enzymatic and non-enzymatic, in mycobacteria prior to binding to the target of interest. From a whole-cell screening program, we identified a novel lead nitrobenzothiazole (BT) series that acts by inhibition of decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) of Mycobacterium tuberculosis (Mtb). The lead was found to be mutagenic to start with. Our efforts to mitigate mutagenicity resulted in the identification of 6-methyl-7-nitro-5-(trifluoromethyl)-1,3-benzothiazoles (cBTs), a novel class of antitubercular agents that are non-mutagenic and exhibit an improved safety profile. The methyl group ortho to the nitro group decreases the electron affinity of the series, and is hence responsible for the non-mutagenic nature of these compounds. Additionally, the co-crystal structure of cBT in complex with Mtb DprE1 established the mode of binding. This investigation led to a new non-mutagenic antitubercular agent and demonstrates that the mutagenic nature of nitroarenes can be solved by modulation of stereoelectronic properties.

Structure guided lead generation for M. tuberculosis thymidylate kinase (Mtb TMK): discovery of 3-cyanopyridone and 1,6-naphthyridin-2-one as potent inhibitors.

M. tuberculosis thymidylate kinase (Mtb TMK) has been shown in vitro to be an essential enzyme in DNA synthesis. In order to identify novel leads for Mtb TMK, we performed a high throughput biochemical screen and an NMR based fragment screen through which we discovered two novel classes of inhibitors, 3-cyanopyridones and 1,6-naphthyridin-2-ones, respectively. We describe three cyanopyridone subseries that arose during our hit to lead campaign, along with cocrystal structures of representatives with Mtb TMK. Structure aided optimization of the cyanopyridones led to single digit nanomolar inhibitors of Mtb TMK. Fragment based lead generation, augmented by crystal structures and the SAR from the cyanopyridones, enabled us to drive the potency of our 1,6-naphthyridin-2-one fragment hit from 500 µM to 200 nM while simultaneously improving the ligand efficiency. Cyanopyridone derivatives containing sulfoxides and sulfones showed cellular activity against M. tuberculosis. To the best of our knowledge, these compounds are the first reports of non-thymidine-like inhibitors of Mtb TMK.

Utility of calculated 13C NMR chemical shifts in differentiating conformational isomers: a study of metal-complexed and uncomplexed bispidines.

Calculated 13C NMR chemical shifts were key to assigning the structures of the conformational forms of complexed and uncomplexed bispidine derivatives.

Computer-aided design of chiral ligands. Part I. Database search methods to identify chiral ligand types for asymmetric reactions.

The utility of database searching to identify chiral ligand motifs is outlined. The key elements of three known chiral ligands have been described as bond vectors. The CAVEAT program was then used to screen the Cambridge Structural Database (CSD), portions of the Chemical Abstracts Services three-dimensional database (CAS-3D), and the TRIAD tricyclic structure database for scaffolds containing these elements. Scaffolds corresponding to the known starting points were identified indicating that this method can be used to identify chiral ligand structural motifs. In addition, alternate structural motifs were found that suggested alternative possible ligands.

Biarylmethoxy Nicotinamides As Novel and Specific Inhibitors of Mycobacterium tuberculosis.

A whole cell based screening effort on a focused library from corporate collection resulted in the identification of biarylmethoxy nicotinamides as novel inhibitors of M. tuberculosis (Mtu) H37Rv. The series exhibited tangible structure-activity relationships, and during hit to lead exploration, a cellular potency of 100 nM was achieved, which is an improvement of >200-fold from the starting point. The series is very specific to Mtu and noncytotoxic up to 250 µM as measured in the mammalian cell line THP-1 based cytotoxicity assay. This compound class retains its potency on several drug sensitive and single drug resistant clinical isolates, which indicate that the compounds could be acting through a novel mode of action.