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.

Optimization of pyrrolamides as mycobacterial GyrB ATPase inhibitors: structure-activity relationship and in vivo efficacy in a mouse model of tuberculosis.

Moxifloxacin has shown excellent activity against drug-sensitive as well as drug-resistant tuberculosis (TB), thus confirming DNA gyrase as a clinically validated target for discovering novel anti-TB agents. We have identified novel inhibitors in the pyrrolamide class which kill Mycobacterium tuberculosis through inhibition of ATPase activity catalyzed by the GyrB domain of DNA gyrase. A homology model of the M. tuberculosis H37Rv GyrB domain was used for deciphering the structure-activity relationship and binding interactions of inhibitors with mycobacterial GyrB enzyme. Proposed binding interactions were later confirmed through cocrystal structure studies with the Mycobacterium smegmatis GyrB ATPase domain. The most potent compound in this series inhibited supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC50) of <5 nM, an MIC of 0.03 µg/ml against M. tuberculosis H37Rv, and an MIC90 of <0.25 µg/ml against 99 drug-resistant clinical isolates of M. tuberculosis. The frequency of isolating spontaneous resistant mutants was ∼10(-6) to 10(-8), and the point mutation mapped to the M. tuberculosis GyrB domain (Ser208 Ala), thus confirming its mode of action. The best compound tested for in vivo efficacy in the mouse model showed a 1.1-log reduction in lung CFU in the acute model and a 0.7-log reduction in the chronic model. This class of GyrB inhibitors could be developed as novel anti-TB agents.

Alanine racemase mutants of Mycobacterium tuberculosis require D-alanine for growth and are defective for survival in macrophages and mice.

Alanine racemase (Alr) is an essential enzyme in most bacteria; however, some species (e.g. Listeria monocytogenes) can utilize d-amino acid transaminase (Dat) to generate d-alanine, which renders Alr non-essential. In addition to the conflicting reports on gene knockout of alr in Mycobacterium smegmatis, a recent study concluded that depletion of Alr does not affect the growth of M. smegmatis. In order to get an unambiguous answer on the essentiality of Alr in Mycobacterium tuberculosis and validate it as a drug target in vitro and in vivo, we have inactivated the alr gene of M. tuberculosis and found that it was not possible to generate an alr knockout in the absence of a complementing gene copy or d-alanine in the growth medium. The growth kinetics of the alr mutant revealed that M. tuberculosis requires very low amounts of d-alanine (5-10 µg ml(-1)) for optimum growth. Survival kinetics of the mutant in the absence of d-alanine indicated that depletion of this amino acid results in rapid loss of viability. The alr mutant was found to be defective for growth in macrophages. Analysis of phenotype in mice suggested that non-availability of d-alanine in mice leads to clearance of bacteria followed by stabilization of bacterial number in lungs and spleen. Additionally, reversal of d-cycloserine inhibition in the presence of d-alanine in M. tuberculosis suggested that Alr is the primary target of d-cycloserine. Thus, Alr of M. tuberculosis is a valid drug target and inhibition of Alr alone should result in loss of viability in vitro and in vivo.

Validation of whole genome sequencing from dried blood spots.

BACKGROUND: Dried blood spots (DBS) are a relatively inexpensive source of nucleic acids and are easy to collect, transport, and store in large-scale field surveys, especially in resource-limited settings. However, their performance in whole-genome sequencing (WGS) relative to that of venous blood DNA has not been analyzed for various downstream applications. METHODS: This study compares the WGS performance of DBS paired with venous blood samples collected from 12 subjects. RESULTS: Results of standard quality checks of coverage, base quality, and mapping quality were found to be near identical between DBS and venous blood. Concordance for single-nucleotide variants, insertions and deletions, and copy number variants was high between these two sample types. Additionally, downstream analyses typical of population-based studies were performed, such as mitochondrial heteroplasmy detection, haplotype analysis, mitochondrial copy number changes, and determination of telomere lengths. The absolute mitochondrial copy number values were higher for DBS than for venous blood, though the trend in sample-to-sample variation was similar between DBS and blood. Telomere length estimates in most DBS samples were on par with those from venous blood. CONCLUSION: DBS samples can serve as a robust and feasible alternative to venous blood for studies requiring WGS analysis.

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.

Optimization of Compound Plate Preparation to Address Precipitation Issue in Mammalian A549 Cytotoxicity Assay.

This study illustrates the optimization of low-volume dispensing on a liquid handling system (LHS) to overcome the precipitation of compounds in the mammalian cytotoxicity assay with low dimethyl sulfoxide (DMSO) tolerance. All compounds at AstraZeneca Bangalore are tested in the mammalian cytotoxicity assay. In order to maintain the DMSO levels, serially diluted plates were prepared in DMSO/water. It was observed that some of the compounds precipitated. The IC50 data for such compounds were therefore erratic. To circumvent the problem of compound precipitation, the LHS was optimized to dispense low volumes (<1 µL). The plates were serially diluted using neat DMSO. Since the dilution was done using neat DMSO, there were no issues with precipitation. The serially diluted sample (0.5 µL) from the plate was stamped onto the assay plate to give the desired DMSO concentration. No significant differences in IC50 data were observed for 1 µL dispenses made from DMSO/water and 0.5 µL dispenses from neat DMSO for the samples with no precipitation issues. These data therefore gave us the confidence to switch over to 0.5 µL dispenses for the cytotoxicity assay to address the precipitation issue. However, precipitation of samples in the assay buffer is beyond the scope of this discussion.

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.

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.

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.

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.

N-aryl-2-aminobenzimidazoles: novel, efficacious, antimalarial lead compounds.

From the phenotypic screening of the AstraZeneca corporate compound collection, N-aryl-2-aminobenzimidazoles have emerged as novel hits against the asexual blood stage of Plasmodium falciparum (Pf). Medicinal chemistry optimization of the potency against Pf and ADME properties resulted in the identification of 12 as a lead molecule. Compound 12 was efficacious in the P. berghei (Pb) model of malaria. This compound displayed an excellent pharmacokinetic profile with a long half-life (19 h) in rat blood. This profile led to an extended survival of animals for over 30 days following a dose of 50 mg/kg in the Pb malaria model. Compound 12 retains its potency against a panel of Pf isolates with known mechanisms of resistance. The fast killing observed in the in vitro parasite reduction ratio (PRR) assay coupled with the extended survival highlights the promise of this novel chemical class for the treatment of malaria.

Novel N-linked aminopiperidine-based gyrase inhibitors with improved hERG and in vivo efficacy against Mycobacterium tuberculosis.

DNA gyrase is a clinically validated target for developing drugs against Mycobacterium tuberculosis (Mtb). Despite the promise of fluoroquinolones (FQs) as anti-tuberculosis drugs, the prevalence of pre-existing resistance to FQs is likely to restrict their clinical value. We describe a novel class of N-linked aminopiperidinyl alkyl quinolones and naphthyridones that kills Mtb by inhibiting the DNA gyrase activity. The mechanism of inhibition of DNA gyrase was distinct from the fluoroquinolones, as shown by their ability to inhibit the growth of fluoroquinolone-resistant Mtb. Biochemical studies demonstrated this class to exert its action via single-strand cleavage rather than double-strand cleavage, as seen with fluoroquinolones. The compounds are highly bactericidal against extracellular as well as intracellular Mtb. Lead optimization resulted in the identification of potent compounds with improved oral bioavailability and reduced cardiac ion channel liability. Compounds from this series are efficacious in various murine models of tuberculosis.

Aminoazabenzimidazoles, a novel class of orally active antimalarial agents.

Whole-cell high-throughput screening of the AstraZeneca compound library against the asexual blood stage of Plasmodium falciparum (Pf) led to the identification of amino imidazoles, a robust starting point for initiating a hit-to-lead medicinal chemistry effort. Structure-activity relationship studies followed by pharmacokinetics optimization resulted in the identification of 23 as an attractive lead with good oral bioavailability. Compound 23 was found to be efficacious (ED90 of 28.6 mg·kg(-1)) in the humanized P. falciparum mouse model of malaria (Pf/SCID model). Representative compounds displayed a moderate to fast killing profile that is comparable to that of chloroquine. This series demonstrates no cross-resistance against a panel of Pf strains with mutations to known antimalarial drugs, thereby suggesting a novel mechanism of action for this chemical class.

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.

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.

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.

Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice.

Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthesis pathway in bacteria. Bioinformatics analysis revealed that the Mycobacterium tuberculosis genome contains four genes (ilvB1, ilvB2, ilvG and ilvX) coding for the large catalytic subunit of AHAS, whereas only one gene (ilvN or ilvH) coding for the smaller regulatory subunit of this enzyme was found. In order to understand the physiological role of AHAS in survival of the organism in vitro and in vivo, we inactivated the ilvB1 gene of M. tuberculosis. The mutant strain was found to be auxotrophic for all of the three branched-chain amino acids (isoleucine, leucine and valine), when grown with either C(6) or C(2) carbon sources, suggesting that the ilvB1 gene product is the major AHAS in M. tuberculosis. Depletion of these branched chain amino acids in the medium led to loss of viability of the DeltailvB1 strain in vitro, resulting in a 4-log reduction in colony-forming units after 10 days. Survival kinetics of the mutant strain cultured in macrophages maintained with sub-optimal concentrations of the branched-chain amino acids did not show any loss of viability, indicating either that the intracellular environment was rich in these amino acids or that the other AHAS catalytic subunits were functional under these conditions. Furthermore, the growth kinetics of the DeltailvB1 strain in mice indicated that although this mutant strain showed defective growth in vivo, it could persist in the infected mice for a long time, and therefore could be a potential vaccine candidate.