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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

2-Phenylindole and Arylsulphonamide: Novel Scaffolds Bactericidal against Mycobacterium tuberculosis.

A cellular activity-based screen on Mycobacterium tuberculosis (Mtb) H37Rv using a focused library from the AstraZeneca corporate collection led to the identification of 2-phenylindoles and arylsulphonamides, novel antimycobacterial scaffolds. Both the series were bactericidal in vitro and in an intracellular macrophage infection model, active against drug sensitive and drug resistant Mtb clinical isolates, and specific to mycobacteria. The scaffolds showed promising structure-activity relationships; compounds with submicromolar cellular potency were identified during the hit to lead exploration. Furthermore, compounds from both scaffolds were tested for inhibition of known target enzymes or pathways of antimycobacterial drugs including InhA, RNA polymerase, DprE1, topoisomerases, protein synthesis, and oxidative-phosphorylation. Compounds did not inhibit any of the targets suggesting the potential of a possible novel mode of action(s). Hence, both scaffolds provide the opportunity to be developed further as leads and tool compounds to uncover novel mechanisms for tuberculosis drug discovery.

Pyrazolopyrimidines establish MurC as a vulnerable target in Pseudomonas aeruginosa and Escherichia coli.

The bacterial peptidoglycan biosynthesis pathway provides multiple targets for antibacterials, as proven by the clinical success of ß-lactam and glycopeptide classes of antibiotics. The Mur ligases play an essential role in the biosynthesis of the peptidoglycan building block, N-acetyl-muramic acid-pentapeptide. MurC, the first of four Mur ligases, ligates l-alanine to UDP-N-acetylmuramic acid, initiating the synthesis of pentapeptide precursor. Therefore, inhibiting the MurC enzyme should result in bacterial cell death. Herein, we report a novel class of pyrazolopyrimidines with subnanomolar potency against both Escherichia coli and Pseudomonas aeruginosa MurC enzymes, which demonstrates a concomitant bactericidal activity against efflux-deficient strains. Radio-labeled precursor incorporation showed these compounds selectively inhibited peptidoglycan biosynthesis, and genetic studies confirmed the target of pyrazolopyrimidines to be MurC. In the presence of permeability enhancers such as colistin, pyrazolopyrimidines exhibited low micromolar MIC against the wild-type bacteria, thereby, indicating permeability and efflux as major challenges for this chemical series. Our studies provide biochemical and genetic evidence to support the essentiality of MurC and serve to validate the attractiveness of target for antibacterial discovery.

Diarylthiazole: an antimycobacterial scaffold potentially targeting PrrB-PrrA two-component system.

Diarylthiazole (DAT), a hit from diversity screening, was found to have potent antimycobacterial activity against Mycobacterium tuberculosis (Mtb). In a systematic medicinal chemistry exploration, we demonstrated chemical opportunities to optimize the potency and physicochemical properties. The effort led to more than 10 compounds with submicromolar MICs and desirable physicochemical properties. The potent antimycobacterial activity, in conjunction with low molecular weight, made the series an attractive lead (antibacterial ligand efficiency (ALE)>0.4). The series exhibited excellent bactericidal activity and was active against drug-sensitive and resistant Mtb. Mutational analysis showed that mutations in prrB impart resistance to DAT compounds but not to reference drugs tested. The sensor kinase PrrB belongs to the PrrBA two component system and is potentially the target for DAT. PrrBA is a conserved, essential regulatory mechanism in Mtb and has been shown to have a role in virulence and metabolic adaptation to stress. Hence, DATs provide an opportunity to understand a completely new target system for antimycobacterial drug discovery.

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.

Discovery of pyrazolopyridones as a novel class of noncovalent DprE1 inhibitor with potent anti-mycobacterial activity.

A novel pyrazolopyridone class of inhibitors was identified from whole cell screening against Mycobacterium tuberculosis (Mtb). The series exhibits excellent bactericidality in vitro, resulting in a 4 log reduction in colony forming units following compound exposure. The significant modulation of minimum inhibitory concentration (MIC) against a Mtb strain overexpressing the Rv3790 gene suggested the target of pyrazolopyridones to be decaprenylphosphoryl-ß-D-ribose-2'-epimerase (DprE1). Genetic mapping of resistance mutation coupled with potent enzyme inhibition activity confirmed the molecular target. Detailed biochemical characterization revealed the series to be a noncovalent inhibitor of DprE1. Docking studies at the active site suggest that the series can be further diversified to improve the physicochemical properties without compromising the antimycobacterial activity. The pyrazolopyridone class of inhibitors offers an attractive non-nitro lead series targeting the essential and vulnerable DprE1 enzyme for the discovery of novel antimycobacterial agents to treat both drug susceptible and drug resistant strains of Mtb.

Lead optimization of 1,4-azaindoles as antimycobacterial agents.

In a previous report, we described the discovery of 1,4-azaindoles, a chemical series with excellent in vitro and in vivo antimycobacterial potency through noncovalent inhibition of decaprenylphosphoryl-ß-d-ribose-2'-epimerase (DprE1). Nevertheless, high mouse metabolic turnover and phosphodiesterase 6 (PDE6) off-target activity limited its advancement. Herein, we report lead optimization of this series, culminating in potent, metabolically stable compounds that have a robust pharmacokinetic profile without any PDE6 liability. Furthermore, we demonstrate efficacy for 1,4-azaindoles in a rat chronic TB infection model. We believe that compounds from the 1,4-azaindole series are suitable for in vivo combination and safety studies.

4-aminoquinolone piperidine amides: noncovalent inhibitors of DprE1 with long residence time and potent antimycobacterial activity.

4-Aminoquinolone piperidine amides (AQs) were identified as a novel scaffold starting from a whole cell screen, with potent cidality on Mycobacterium tuberculosis (Mtb). Evaluation of the minimum inhibitory concentrations, followed by whole genome sequencing of mutants raised against AQs, identified decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) as the primary target responsible for the antitubercular activity. Mass spectrometry and enzyme kinetic studies indicated that AQs are noncovalent, reversible inhibitors of DprE1 with slow on rates and long residence times of ∼100 min on the enzyme. In general, AQs have excellent leadlike properties and good in vitro secondary pharmacology profile. Although the scaffold started off as a single active compound with moderate potency from the whole cell screen, structure-activity relationship optimization of the scaffold led to compounds with potent DprE1 inhibition (IC50 < 10 nM) along with potent cellular activity (MIC = 60 nM) against Mtb.

Methyl-thiazoles: a novel mode of inhibition with the potential to develop novel inhibitors targeting InhA in Mycobacterium tuberculosis.

InhA is a well validated Mycobacterium tuberculosis (Mtb) target as evidenced by the clinical success of isoniazid. Translating enzyme inhibition to bacterial cidality by targeting the fatty acid substrate site of InhA remains a daunting challenge. The recent disclosure of a methyl-thiazole series demonstrates that bacterial cidality can be achieved with potent enzyme inhibition and appropriate physicochemical properties. In this study, we report the molecular mode of action of a lead methyl-thiazole, along with analogues with improved CYP inhibition profile. We have identified a novel mechanism of InhA inhibition characterized by a hitherto unreported "Y158-out" inhibitor-bound conformation of the protein that accommodates a neutrally charged "warhead". An additional novel hydrophilic interaction with protein residue M98 allows the incorporation of favorable physicochemical properties for cellular activity. Notably, the methyl-thiazole prefers the NADH-bound form of the enzyme with a Kd of ~13.7 nM, as against the NAD(+)-bound form of the enzyme.