Search

In search of novel drugs against tuberculosis, we previously discovered and profiled a novel hydantoin-based family that demonstrated highly promising in vitro potency against M. tuberculosis. The compounds were found to be non-covalent inhibitors of DprE1, a subunit of decaprenylphosphoryl-β-D-ribose-2′-epimerase. This protein, localized in the periplasmic space of the mycobacterial cell wall, was shown to be an essential and vulnerable antimycobacterial drug target. Here, we report the further SAR exploration of this chemical family through more than 80 new analogues. Among these, the most active representatives combined sub-micromolar cellular potency and nanomolar target affinity with balanced physicochemical properties and low human cytotoxicity. Moreover, we demonstrate in vivo activity in an acute Mtb infection model and provide further proof of DprE1 being the target of the hydantoins. Overall, the hydantoin family of DprE1 inhibitors represents a promising non-covalent lead series for the discovery of novel antituberculosis agents.

Screening of a GSK-proprietary library against intracellular Mycobacterium tuberculosis identified 1, a thioalkylbenzoxazole hit. Biological profiling and mutant analysis revealed that this compound is a prodrug that is bioactivated by the mycobacterial enzyme MymA. A hit-expansion program including design, synthesis, and profiling of a defined set of analogues with optimized drug-like properties led to the identification of an emerging lead compound, displaying potency against intracellular bacteria in the low micromolar range, high in vitro solubility and permeability, and excellent microsomal stability.

[Image: see text] In this study, a series of 49 five-membered heterocyclic compounds containing either a pyridine- or a pyrrole-type nitrogen were synthesized and tested against Mycobacterium tuberculosis. Among them, only the 1,3,5-trisubstituted pyrazoles 5–49 exhibited minimum inhibitory concentration values in the low micromolar range, and some also exhibited an improved physicochemical profile without cytotoxic effects. Three pyrazoles were subjected to an animal tuberculosis efficacy model, and compound 6 induced a statistically significant difference in lung bacterial counts compared with untreated mice. Moreover, to determine the target of this series, resistors were generated, and whole genome sequencing revealed mutations in the mmpL3 gene.

Shiga toxin is an AB5 toxin produced by Shigella species, while related toxins are produced by Shiga toxin-producing Escherichia coli (STEC). Infection by Shigella can lead to bloody diarrhea followed by the often fatal hemolytic uremic syndrome (HUS). In the present paper, we aimed for a simple and effective toxin inhibitor by comparing three classes of carbohydrate-based inhibitors: glycodendrimers, glycopolymers, and oligosaccharides. We observed a clear enhancement in potency for multivalent inhibitors, with the divalent and tetravalent compounds inhibiting in the millimolar and micromolar range, respectively. However, the polymeric inhibitor based on galabiose was the most potent in the series exhibiting nanomolar inhibition. Alginate and chitosan oligosaccharides also inhibit Shiga toxin and may be used as a prophylactic drug during shigella outbreaks.

Tres Cantos Open Lab is a collaborative initiative that integrates teams from academia and GlaxoSmithKline to enable rapid testing of innovative therapeutic hypotheses for endemic infectious diseases. Here, we provide an overview of the key scientific achievements in its first decade. Tres Cantos Open Lab is a collaborative initiative that integrates teams from academia and GlaxoSmithKline to enable rapid testing of innovative therapeutic hypotheses for endemic infectious diseases. Here, we provide an overview of the key scientific achievements in its first decade.

Tuberculosis is the leading cause of death worldwide from infectious diseases. With the development of drug-resistant strains of Mycobacterium tuberculosis, there is an acute need for new medicines with novel modes of action. Herein, we report the discovery and profiling of a novel hydantoin-based family of antimycobacterial inhibitors of the decaprenylphospho-β-d-ribofuranose 2-oxidase (DprE1). In this study, we have prepared a library of more than a 100 compounds and evaluated them for their biological and physicochemical properties. The series is characterized by high enzymatic and whole-cell activity, low cytotoxicity, and a good overall physicochemical profile. In addition, we show that the series acts via reversible inhibition of the DprE1 enzyme. Overall, the novel compound family forms an attractive base for progression to further stages of optimization and may provide a promising drug candidate in the future.

In recent years, several small natural cyclopeptides and cyclodepsipeptides were reported to have antimycobacterial activity. Following this lead, a synthetic pathway was developed for a small series of 12-membered ring compounds with one amide and two ester bonds (cyclotridepsipeptides). Within the series, the ring system proved to be necessary for growth inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis in the low micromolar range. Open-chain precursors and analogues were inactive. The compounds modulated autophosphorylation of the mycobacterial protein kinase B (PknB). PknB inhibitors were active at µM concentration against mycobacteria while inducers were inactive. PknB regulates the activity of the mycobacterial reductase InhA, the target of isoniazid. The activity of the series against Mycobacterium bovis BCG InhA overexpressing strains was indistinguishable from that of the parental strain suggesting that they do not inhibit InhA. All substances were not cytotoxic (HeLa > 5 µg/ml) and did not show any significant antiproliferative effect (HUVEC > 5 µg/ml; K-562 > 5 µg/ml). Within the scope of this study, the molecular target of this new type of small cyclodepsipeptide was not identified, but the data suggest interaction with PknB or other kinases may partly cause the activity.

BM635 is the hit compound of a promising anti-TB compound class. Herein we report systematic variations around the central pyrrole core of BM635 and we describe the design, synthesis, biological evaluation, pharmacokinetic analysis, as well as in vivo TB mouse efficacy studies of novel BM635 analogues that show improved physicochemical properties. This hit-to-lead campaign led to the identification of a new analogue, 4-((1-isopropyl-5-(4-isopropylphenyl)-2-methyl-1H-pyrrol-3-yl)methyl)morpholine (17), that shows excellent activity (MIC = 0.15 μM; SI = 133) against drug-sensitive Mycobacterium tuberculosis strains, as well as efficacy in a murine model of TB infection.

A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.

One way to speed up the TB drug discovery process is to search for antitubercular activity among compound series that already possess some of the key properties needed in anti-infective drug discovery, such as whole-cell activity and oral absorption. Here, we present MGIs, a new series of Mycobacterium tuberculosis gyrase inhibitors, which stem from the long-term efforts GSK has dedicated to the discovery and development of novel bacterial topoisomerase inhibitors (NBTIs). The compounds identified were found to be devoid of fluoroquinolone (FQ) cross-resistance and seem to operate through a mechanism similar to that of the previously described NBTI GSK antibacterial drug candidate. The remarkable in vitro and in vivo antitubercular profiles showed by the hits has prompted us to further advance the MGI project to full lead optimization.

Due to the rise of drug-resistant forms of tuberculosis, there is an urgent need for novel antibiotics to effectively combat these cases and shorten treatment regimens. Recently, drug screens using whole-cell analyses have been shown to be successful. However, current high-throughput screens focus mostly on stricto sensu life/death screening that give little qualitative information. In doing so, promising compound scaffolds or nonoptimized compounds that fail to reach inhibitory concentrations are missed. To accelerate early tuberculosis (TB) drug discovery, we performed RNA sequencing on Mycobacterium tuberculosis and Mycobacterium marinum to map the stress responses that follow upon exposure to subinhibitory concentrations of antibiotics with known targets, ciprofloxacin, ethambutol, isoniazid, streptomycin, and rifampin. The resulting data set comprises the first overview of transcriptional stress responses of mycobacteria to different antibiotics. We show that antibiotics can be distinguished based on their specific transcriptional stress fingerprint. Notably, this fingerprint was more distinctive in M. marinum . We decided to use this to our advantage and continue with this model organism. A selection of diverse antibiotic stress genes was used to construct stress reporters. In total, three functional reporters were constructed to respond to DNA damage, cell wall damage, and ribosomal inhibition. Subsequently, these reporter strains were used to screen a small anti-TB compound library to predict the mode of action. In doing so, we identified the putative modes of action for three novel compounds, which confirms the utility of our approach.

Despite its great potential, the target-based approach has been mostly unsuccessful in tuberculosis drug discovery, while whole cell phenotypic screening has delivered several active compounds. However, for many of these hits, the cellular target has not yet been identified, thus preventing further target-based optimization of the compounds. In this context, the newly validated drug target CTP synthetase PyrG was exploited to assess a target-based approach of already known, but untargeted, antimycobacterial compounds. To this purpose the publically available GlaxoSmithKline antimycobacterial compound set was assayed, uncovering a series of 4-(pyridin-2-yl)thiazole derivatives which efficiently inhibit the Mycobacterium tuberculosis PyrG enzyme activity, one of them showing low activity against the human CTP synthetase. The three best compounds were ATP binding site competitive inhibitors, with Ki values ranging from 3 to 20 μM, but did not show any activity against a small panel of different prokaryotic a...

Background: InhA is a promising and attractive target in antimycobacterial drug development. InhA is involved in the reduction of long-chain trans-2-enoyl-ACP in the type II fatty acid biosynthesis pathway of Mycobacterium tuberculosis. Recent studies have demonstrated that InhA is one of the targets for the second line antitubercular drug ethionamide. Results: In the current study, we have generated quantitative pharmacophore models using known InhA inhibitors and validated using a large test set. The validated pharmacophore model was used as a query to screen an in-house database of 400,000 compounds and retrieved 25,000 hits. These hits were further ranked based on its shape and feature similarity with potent InhA inhibitor using rapid overlay of chemical structures (OpenEye™) and subsequent hits were subjected for docking. Based on the pharmacophore, rapid overlay of chemical structures model and docking interactions, 32 compounds with more than eight chemotypes were selected, purchased and assayed for InhA inhibitory activity. Out of the 32 compounds, 28 demonstrated 10–38% inhibition against InhA at 10 µM. Conclusion: Further optimization of these analogues is in progress and will update in due course.

The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery., Encoded Library Technology (ELT) has streamlined the identification of chemical ligands for protein targets in drug discovery. Here, the authors optimize the ELT approach to screen multiple proteins in parallel and identify promising targets and antibacterial compounds for S. aureus, A. baumannii and M. tuberculosis.

Two new families of closely related selective, non-cytotoxic, and potent antitubercular agents were discovered: thioquinolines and thiazoloquinolines. The compounds were found to possess potent antitubercular properties in vitro, an activity that is dependent on experimental conditions of MIC determination (resazurin test and the presence or absence of Tween-80). To clarify the therapeutic potential of these compound families, a medicinal chemistry effort was undertaken to generate a lead-like structure that would enable murine efficacy studies and help elucidate the in vivo implications of the in vitro observations. Although the final compounds showed only limited levels of systemic exposure in mice, modest levels of efficacy in vivo at nontoxic doses were observed.

In this study, a new series of more than 60 quinoline derivatives has been synthesized and evaluated against Mycobacterium tuberculosis (H37Rv). Apart from the SAR exploration around the initial hits, the optimization process focused on the improvement of the physicochemical properties, cytotoxicity, and metabolic stability of the series. The best compounds obtained exhibited MIC values in the low micromolar range, excellent intracellular antimycobacterial activity, and an improved physicochemical profile without cytotoxic effects. Further investigation revealed that the amide bond was the source for the poor blood stability observed, while some of the compounds exhibited hERG affinity. Compound 83 which contains a benzoxazole ring instead of the amide group was found to be a good alternative, with good blood stability and no hERG affinity, providing new opportunities for the series. Overall, the obtained results suggest that further optimization of solubility and microsomal stability of the series could provide a strong lead for a new anti-TB drug development program.

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis β-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related β-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis., Screens for bactericidal compounds have resulted in promising anti-tubercular hits. Here, the authors analyse in detail the target of an indazole sulfonamide (GSK3011724A), and find that it has a different mode of inhibition compared to other Kas inhibitors of fatty acid biosynthesis in bacteria.

While our understanding of the molecular events leading to disease onset and progression have increased exponentially, our capacity to therapeutically intervene in these events with new chemical diversity has clearly fallen short of that pace. In the quest to readdress this situation, the drug discovery sector is slowly but increasingly exploring sources of alternative chemical matter, such as the ones provided by material science and nanotechnology. While new functional nano-sized materials hold great promise for the future, our lack of understanding of the long term safety implications associated with systemic exposure as well as the unclear regulatory path ahead hamper their present impact in drug development. Paradoxically, the exploitation of novel, functionally active micron-sized, synthetic, non-absorbable chemical matter, for the treatment or prevention of a number of epidemiologically significant conditions remains clearly underexplored. A combination of pre-existing evidence and future potential indicates that micron-sized mesoporous silica materials could be an untapped source of new drug candidates. These are free from both the dreaded high attrition associated with small molecule drug discovery and the uncertainties of nano-size technologies. This, together with the coming of age of synthetic methodologies to control particle size and shape; pore size and geometry; surface chemistry, bioconjugation and formulation, open up exciting possibilities to exploit this novel chemistry-biology therapeutic interface.

To the Editor: New treatments are needed to combat the worldwide increase in resistance to antituberculosis drugs.1 The outlook for patients with tuberculosis who do not show a response to the key agents used in treatment — isoniazid, rifampin, fluoroquinolones, and aminoglycosides — is grim and reminiscent of the plight of patients with cancer in the era before chemotherapy.2 New agents are emerging, but the obligatory evaluation of their safety and efficacy in combination with other antituberculosis and antiretroviral agents slows the pace of progress. Repurposing or combining commercially available products may offer a faster track to new antituberculosis regimens. . . .

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-ca​rboxamide(THPP) and N-benzyl-6′,7′-dihydrospiro[piperidine-4,​4′-thieno[3,2-c]pyran](Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This ‘genetic phenotype’ was further confirmed by a ‘chemical phenotype’, whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice.

International audience; 1,5-Diphenyl pyrroles were previously identified as a class of compounds endowed with high in vitro efficacy against M. tuberculosis. To improve the physical chemical properties and drug-like parameters of this class of compounds, a medicinal chemistry effort was undertaken. By selecting the optimal substitution patterns for the phenyl rings at N1 and C5 and by replacing the thiomorpholine moiety with a morpholine one, a new series of compounds was produced. The replacement of the sulfur with oxygen gave compounds with lower lipophilicity and improved in vitro microsomal stability. Moreover, since the parent compound of this family has been shown to target MmpL3, mycobacterial mutants resistant to two compounds have been isolated and characterized by sequencing the mmpL3 gene; all the mutants showed point mutations in this gene. The best compound identified to date was progressed to dose-response studies in an acute murine TB infection model. The resulting ED(99) of 49 mg/Kg is within the range of commonly employed tuberculosis drugs, demonstrating the potential of this chemical series. The in vitro and in vivo target validation evidence presented here adds further weight to MmpL3 as a druggable target of interest for anti-tubercular drug discovery.

Open Lab as a source of hits and leads against tuberculosis, malaria and kinetoplastid diseases

The multiple parallel synthesis of a series of N,S-bis-alkylated thiopyrazolo[3,4-d]pyrimidines, based on sequential S- then N-alkylation, is reported. These compounds showed significant anti-mycobacterial activity (MICs down to 2mug/ml) and their potential as significant drug-like leads is substantiated through cytotoxicity evaluation and in silico profiling.

A rapid, efficient and versatile microwave-assisted dibutylstannylene-mediated 3-O-alkylation method for galactosides was established. The alkylation reaction was significantly enhanced by microwave irradiation and allowed the use of an alkylating agent too unstable for incorporation using conventional heating.

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. Through the use of high throughput whole cell screening of an extensive compound library a number of imidazo[1,2-a]pyridine (IP) compounds were obtained as potent lead molecules active against M. tuberculosis and Mycobacterium bovis BCG. The IP inhibitors (1-4) demonstrated minimum inhibitory concentrations (MICs) in the range of 0.03 to 5 µM against a panel of M. tuberculosis strains. M. bovis BCG spontaneous resistant mutants were generated against IP 1, 3, and 4 at 5× MIC and subsequent whole genome sequencing identified a single nucleotide polymorphism (937)ACC>(937)GCC (T313A) in qcrB, which encodes the b subunit of the electron transport ubiquinol cytochrome C reductase. This mutation also conferred cross-resistance against IP 1, 3 and 4 demonstrating a common target. Gene dosage experiments confirmed M. bovis BCG QcrB as the target where over-expression in M. bovis BCG led to an increase in MIC from 0.5 to >8 µM for IP 3. An acute murine model of TB infection established bacteriostatic activity of the IP series, which await further detailed characterization.