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.

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.

Essentiality Assessment of Cysteinyl and Lysyl-tRNA Synthetases of Mycobacterium smegmatis.

Discovery of mupirocin, an antibiotic that targets isoleucyl-tRNA synthetase, established aminoacyl-tRNA synthetase as an attractive target for the discovery of novel antibacterial agents. Despite a high degree of similarity between the bacterial and human aminoacyl-tRNA synthetases, the selectivity observed with mupirocin triggered the possibility of targeting other aminoacyl-tRNA synthetases as potential drug targets. These enzymes catalyse the condensation of a specific amino acid to its cognate tRNA in an energy-dependent reaction. Therefore, each organism is expected to encode at least twenty aminoacyl-tRNA synthetases, one for each amino acid. However, a bioinformatics search for genes encoding aminoacyl-tRNA synthetases from Mycobacterium smegmatis returned multiple genes for glutamyl (GluRS), cysteinyl (CysRS), prolyl (ProRS) and lysyl (LysRS) tRNA synthetases. The pathogenic mycobacteria, namely, Mycobacterium tuberculosis and Mycobacterium leprae, were also found to possess two genes each for CysRS and LysRS. A similar search indicated the presence of additional genes for LysRS in gram negative bacteria as well. Herein, we describe sequence and structural analysis of the additional aminoacyl-tRNA synthetase genes found in M. smegmatis. Characterization of conditional expression strains of Cysteinyl and Lysyl-tRNA synthetases generated in M. smegmatis revealed that the canonical aminoacyl-tRNA synthetase are essential, while the additional ones are not essential for the growth of M. smegmatis.

An IPTG Inducible Conditional Expression System for Mycobacteria.

Conditional expression strains serve as a valuable tool to study the essentiality and to establish the vulnerability of a target under investigation in a drug discovery program. While essentiality implies an absolute requirement of a target function, vulnerability provides valuable information on the extent to which a target function needs to be depleted to achieve bacterial growth inhibition followed by cell death. The critical feature of an ideal conditional expression system is its ability to tightly regulate gene expression to achieve the full spectrum spanning from a high level of expression in order to support growth and near zero level of expression to mimic conditions of gene knockout. A number of bacterial conditional expression systems have been reported for use in mycobacteria. The utility of an isopropylthiogalactoside (IPTG) inducible system in mycobacteria has been reported for protein overexpression and anti-sense gene expression from a replicating multi-copy plasmid. Herein, we report the development of a versatile set of non-replicating IPTG inducible vectors for mycobacteria which can be used for generation of conditional expression strains through homologous recombination. The role of a single lac operator versus a double lac operator to regulate gene expression was evaluated by monitoring the expression levels of ß-galactosidase in Mycobacterium smegmatis. These studies indicated a significant level of leaky expression from the vector with a single lac operator but none from the vector with double lac operator. The significance of the double lac operator vector for target validation was established by monitoring the growth kinetics of an inhA, a rpoB and a ftsZ conditional expression strain grown in the presence of different concentrations of IPTG. The utility of this inducible system in identifying target specific inhibitors was established by screening a focussed library of small molecules using an inhA and a rpoB conditional expression strain.

Genetic and chemical validation identifies Mycobacterium tuberculosis topoisomerase I as an attractive anti-tubercular target.

DNA topoisomerases perform the essential function of maintaining DNA topology in prokaryotes. DNA gyrase, an essential enzyme that introduces negative supercoils, is a clinically validated target. However, topoisomerase I (Topo I), an enzyme responsible for DNA relaxation has received less attention as an antibacterial target, probably due to the ambiguity over its essentiality in many organisms. The Mycobacterium tuberculosis genome harbors a single topA gene with no obvious redundancy in its function suggesting an essential role. The topA gene could be inactivated only in the presence of a complementing copy of the gene in M. tuberculosis. Furthermore, down-regulation of topA in a genetically engineered strain of M. tuberculosis resulted in loss of bacterial viability which correlated with a concomitant depletion of intracellular Topo I levels. The topA knockdown strain of M. tuberculosis failed to establish infection in a murine model of TB and was cleared from lungs in two months post infection. Phenotypic screening of a Topo I overexpression strain led to the identification of an inhibitor, thereby providing chemical validation of this target. Thus, our work confirms the attractiveness of Topo I as an anti-mycobacterial target.

Triaminopyrimidine is a fast-killing and long-acting antimalarial clinical candidate.

The widespread emergence of Plasmodium falciparum (Pf) strains resistant to frontline agents has fuelled the search for fast-acting agents with novel mechanism of action. Here, we report the discovery and optimization of novel antimalarial compounds, the triaminopyrimidines (TAPs), which emerged from a phenotypic screen against the blood stages of Pf. The clinical candidate (compound 12) is efficacious in a mouse model of Pf malaria with an ED99 <30 mg kg(-1) and displays good in vivo safety margins in guinea pigs and rats. With a predicted half-life of 36 h in humans, a single dose of 260 mg might be sufficient to maintain therapeutic blood concentration for 4-5 days. Whole-genome sequencing of resistant mutants implicates the vacuolar ATP synthase as a genetic determinant of resistance to TAPs. Our studies highlight the potential of TAPs for single-dose treatment of Pf malaria in combination with other agents in clinical development.

Revisiting the essentiality of glutamate racemase in Mycobacterium tuberculosis.

Glutamate racemase (MurI) converts l-glutamate into d-glutamate which is an essential component of peptidoglycan in bacteria. The gene encoding glutamate racemase, murI has been shown to be essential for the growth of a number of bacterial species including Escherichia coli. However, in some Gram-positive species d-amino acid transaminase (Dat) can also convert l-glutamate into d-glutamate thus rendering MurI non-essential for growth. In a recent study the murI gene of Mycobacterium tuberculosis was shown to be non-essential. As d-glutamate is an essential component of peptidoglycan of M. tuberculosis, either Dat or MurI has to be essential for its survival. Since, a Dat encoding gene has not been reported in M. tuberculosis genome sequence, the reported non-essentiality of murI was unexplainable. In order to resolve this dilemma we tried to knockout murI in the presence of single and two copies of murI, in wild type and merodiploid strains respectively. It was found that murI could not be inactivated in the wild type background indicating that it could be an essential gene. Also, inactivation of murI could not be achieved in the presence of externally supplied d-glutamate in 7H9 medium suggesting that M. tuberculosis is unable to take up d-glutamate under the conditions tested. However we could generate murI knockout strains at high frequency when two copies of the gene were present indicating that at least one murI gene is required for cellular viability. The essential nature of MurI in M. tuberculosis H37Rv suggests that it could be a potential drug target.

Roles of the two type II NADH dehydrogenases in the survival of Mycobacterium tuberculosis in vitro.

Most bacteria are able to generate sufficient amounts of ATP from substrate level phosphorylation, thus rendering the respiratory oxidative phosphorylation non-critical. In mycobacteria, including Mycobacterium tuberculosis, ATP generation by oxidative phosphorylation is an essential process. Of the two types of NADH dehydrogenases (type I and type II), the type II NADH dehydrogenase (Ndh) which is inhibited by phenothiazines has been thought to be essential. In M. tuberculosis there are two Ndh isozymes (Ndh and NdhA) coded by ndh and ndhA genes respectively. Ndh and NdhA share a high degree of amino acid similarity. Both the enzymes have been shown to be enzymatically active and are inhibited by phenothiazines, suggesting a functional similarity between the two. We attempted gene knockout of ndh and ndhA genes in wild type and merodiploid backgrounds. It was found that ndh gene cannot be inactivated in a wild type background, though it was possible to do so when an additional copy of ndh was provided. This showed that in spite of its apparent functional equivalence, NdhA cannot complement the loss of Ndh in M. tuberculosis. We also showed that NdhA is not essential in M. tuberculosis as the ndhA gene could be deleted in a wild type strain of M. tuberculosis without causing any adverse effects in vitro. RT-PCR analysis of in vitro grown M. tuberculosis showed that ndhA gene is actively transcribed. This study suggests that despite being biochemically similar, Ndh and NdhA play different roles in the physiology of M. tuberculosis.

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.

The phosphatidyl-myo-inositol mannosyltransferase PimA is essential for Mycobacterium tuberculosis growth in vitro and in vivo.

The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myo-inositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs.

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.

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.

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.

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.

Assessment of Mycobacterium tuberculosis pantothenate kinase vulnerability through target knockdown and mechanistically diverse inhibitors.

Pantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by the coaA gene is an essential pantothenate kinase in Mycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-type M. tuberculosis strain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in a M. tuberculosis knockdown strain with reduced PanK expression levels. Additionally, in vitro and in vivo survival kinetic studies performed with a M. tuberculosis PanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK in M. tuberculosis.

Azaindoles: noncovalent DprE1 inhibitors from scaffold morphing efforts, kill Mycobacterium tuberculosis and are efficacious in vivo.

We report 1,4-azaindoles as a new inhibitor class that kills Mycobacterium tuberculosis in vitro and demonstrates efficacy in mouse tuberculosis models. The series emerged from scaffold morphing efforts and was demonstrated to noncovalently inhibit decaprenylphosphoryl-ß-D-ribose2'-epimerase (DprE1). With "drug-like" properties and no expectation of pre-existing resistance in the clinic, this chemical class has the potential to be developed as a therapy for drug-sensitive and drug-resistant tuberculosis.

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.

Evaluation of CoA biosynthesis proteins of Mycobacterium tuberculosis as potential drug targets.

Coenzyme A biosynthesis pathway proteins are potential targets for developing inhibitors against bacteria including Mycobacterium tuberculosis. We have evaluated two enzymes in this pathway: phosphopantetheine adenylyltransferase (CoaD) and dephospho CoA kinase (CoaE) for essentiality and selectivity. Based on the previous transposon mutagenesis studies, coaD had been predicted to be a non-essential gene in M. tuberculosis. Our bioinformatics analysis showed that there is no other functional homolog of this enzyme in M. tuberculosis, which suggests that coaD should be an essential gene. In order to get an unambiguous answer on the essentiality of coaD, we attempted inactivation of coaD in wild type and merodiploid backgrounds. It was found that coaD could only be inactivated in the presence of an additional gene copy, confirming it to be an essential gene. Using a similar approach we found that CoaE was also essential for the survival of M. tuberculosis. RT-PCR analysis showed that both coaD and coaE were transcribed in M. tuberculosis. Amino acids alignment and phylogenetic analysis showed CoaD to be distantly related to the human counterpart while CoaE was found to be relatively similar to the human enzyme. Analysis of CoaD and CoaE structures at molecular level allowed us to identify unique residues in the Mtb proteins, thus providing a selectivity handle. The essentiality and selectivity analysis combined with the published biochemical characterization of CoaD and CoaE makes them suitable targets for developing inhibitors against M. tuberculosis.

Essentiality and functional analysis of type I and type III pantothenate kinases of Mycobacterium tuberculosis.

Pantothenate kinase, an essential enzyme in bacteria and eukaryotes, is involved in catalysing the first step of conversion of pantothenate to coenzyme A (CoA). Three isoforms (type I, II and III) of this enzyme have been reported from various organisms, which can be differentiated from each other on the basis of their biochemical and structural characteristics. Though most bacteria carry only one of the isoforms of pantothenate kinases, some of them possess two isoforms. The physiological relevance of the presence of two types of isozymes in a single organism is not clear. Mycobacterium tuberculosis, an intracellular pathogen, possesses two isoforms of pantothenate kinases (CoaA and CoaX) belonging to type I and III. In order to determine which pantothenate kinase is essential in mycobacteria, we performed gene inactivation of coaA and coaX of M. tuberculosis individually. It was found that coaA could only be inactivated in the presence of an extra copy of the gene, while coaX could be inactivated in the wild-type cells, proving that CoaA is the essential pantothenate kinase in M. tuberculosis. Additionally, the coaA gene of M. tuberculosis was able to complement a temperature-sensitive coaA mutant of Escherichia coli at a non-permissive temperature while coaX could not. The coaX deletion mutant showed no growth defects in vitro, in macrophages or in mice. Taken together, our data suggest that CoaX, which is essential in Bacillus anthracis and thus had been suggested to be a drug target in this organism, might not be a valid target in M. tuberculosis. We have established that the type I isoform, CoaA, is the essential pantothenate kinase in M. tuberculosis and thus can be explored as a drug target.