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In the course of optimizing a novel indazole sulfonamide series that inhibits β-ketoacyl-ACP synthase (KasA) of Mycobacterium tuberculosis, a mutagenic aniline metabolite was identified. Further lead optimization efforts were therefore dedicated to eliminating this critical liability by removing the embedded aniline moiety or modifying its steric or electronic environment. While the narrow SAR space against the target ultimately rendered this goal unsuccessful, key structural knowledge around the binding site of this underexplored target for TB was generated to inform future discovery efforts.

[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.

The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis . Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid.

Society urgently needs new, effective medicines for the treatment of tuberculosis. To kick-start the required hit-to-lead campaigns, the libraries of pharmaceutical companies have recently been evaluated for starting points. The GlaxoSmithKline (GSK) library yielded many high-quality hits, and the associated data were placed in the public domain to stimulate engagement by the wider community. One such series, the spiro compounds, are described here. The compounds were explored by a combination of traditional in-house research and open source methods. The series benefits from a particularly simple structure and a short associated synthetic chemistry route. Many members of the series displayed striking potency and low toxicity, and highly promising in vivo activity in a mouse model was confirmed with one of the analogues. Ultimately the series was discontinued due to concerns over safety, but the associated data remain public domain, empowering others to resume the series if the perceived deficiencies can be overcome.

Antifolates are widely used to treat several diseases but are not currently used in the first-line treatment of tuberculosis, despite evidence that some of these molecules can target Mycobacterium tuberculosis (Mtb) bacilli in vitro. To identify new antifolate candidates for animal-model efficacy studies of tuberculosis, we paired knowledge and tools developed in academia with the infrastructure and chemistry resources of a large pharmaceutical company. Together we curated a focused library of 2508 potential antifolates, which were then tested for activity against live Mtb. We identified 210 primary hits, confirmed the on-target activity of potent compounds, and now report the identification and characterization of 5 hit compounds, representative of 5 different chemical scaffolds. These antifolates have potent activity against Mtb and represent good starting points for improvement that could lead to in vivo efficacy studies.

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.

There is an urgent need to develop new and safer antitubercular agents that possess a novel mode of action. We synthesized and evaluated a novel series of 3-aminomethyl 4-halogen benzoxaboroles as Mycobacterium tuberculosis (Mtb) leucyl-tRNA synthetase (LeuRS) inhibitors. A number of Mtb LeuRS inhibitors were identified that demonstrated good antitubercular activity with high selectivity over human mitochondrial and cytoplasmic LeuRS. Further evaluation of these Mtb LeuRS inhibitors by in vivo pharmacokinetics (PK) and murine tuberculosis (TB) efficacy models led to the discovery of GSK3036656 (abbreviated as GSK656). This molecule shows potent inhibition of Mtb LeuRS (IC50 = 0.20 μM) and in vitro antitubercular activity (Mtb H37Rv MIC = 0.08 μM). Additionally, it is highly selective for the Mtb LeuRS enzyme with IC50 of >300 μM and 132 μM for human mitochondrial LeuRS and human cytoplasmic LeuRS, respectively. In addition, it exhibits remarkable PK profiles and efficacy against Mtb in mouse TB infection ...

We report here a dehydropeptidase-deficient murine model of tuberculosis (TB) infection that is able to partially uncover the efficacy of marketed broad-spectrum β-lactam antibiotics alone and in combination. Reductions of up to 2 log CFU in the lungs of TB-infected mice after 8 days of treatment compared to untreated controls were obtained at blood drug concentrations and time above the MIC ( T >MIC ) below clinically achievable levels in humans. These findings provide evidence supporting the potential of β-lactams as safe and mycobactericidal components of new combination regimens against TB with or without resistance to currently used drugs.

Phenotypic screens for bactericidal compounds against drug-resistant tuberculosis are beginning to yield novel inhibitors. However, reliable target identification remains challenging. Here, we show that tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamide (THPP) selectively pulls down EchA6 in a stereospecific manner, instead of the previously assigned target Mycobacterium tuberculosis MmpL3. While homologous to mammalian enoyl-coenzyme A (CoA) hydratases, EchA6 is non-catalytic yet essential and binds long-chain acyl-CoAs. THPP inhibitors compete with CoA-binding, suppress mycolic acid synthesis, and are bactericidal in a mouse model of chronic tuberculosis infection. A point mutation, W133A, abrogated THPP-binding and increased both the in vitro minimum inhibitory concentration and the in vivo effective dose 99 in mice. Surprisingly, EchA6 interacts with selected enzymes of fatty acid synthase II (FAS-II) in bacterial two-hybrid assays, suggesting essentiality may be linked to feeding long-chain fatty acids to FAS-II. Finally, our data show that spontaneous resistance-conferring mutations can potentially obscure the actual target or alternative targets of small molecule inhibitors.

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.

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.

Murine models of M ycobacterium tuberculosis infection are essential tools in drug discovery. Here we describe a fast standardized 9-day acute assay intended to measure the efficacy of drugs against M. tuberculosis growing in the lungs of immunocompetent mice. This assay is highly reproducible, allows good throughput, and was validated for drug lead optimization using isoniazid, rifampin, ethambutol, pyrazinamide, linezolid, and moxifloxacin.

To counter the global threat caused by Plasmodium falciparum malaria, new drugs and vaccines are urgently needed. However, there are no practical animal models because P. falciparum infects human erythrocytes almost exclusively. Here we describe a reliable falciparum murine model of malaria by generating strains of P. falciparum in vivo that can infect immunodeficient mice engrafted with human erythrocytes. We infected NOD(scid/beta2m-/-) mice engrafted with human erythrocytes with P. falciparum obtained from in vitro cultures. After apparent clearance, we obtained isolates of P. falciparum able to grow in peripheral blood of engrafted NOD(scid/beta2m-/-) mice. Of the isolates obtained, we expanded in vivo and established the isolate Pf3D7(0087/N9) as a reference strain for model development. Pf3D7(0087/N9) caused productive persistent infections in 100% of engrafted mice infected intravenously. The infection caused a relative anemia due to selective elimination of human erythrocytes by a mechanism dependent on parasite density in peripheral blood. Using this model, we implemented and validated a reproducible assay of antimalarial activity useful for drug discovery. Thus, our results demonstrate that P. falciparum contains clones able to grow reproducibly in mice engrafted with human erythrocytes without the use of myeloablative methods.

Background Microscopic analysis of blood smears is currently the most frequently used method to measure parasitemias in experiments of drug efficacy in murine models of malaria. However, it is subjective and labour intensive, which preclude its utilization in large-scale evaluation programs. Flow cytometry is an alternative method, but due to the limited specificity achieved with the currently available techniques, it has not been widely used in murine models of malaria during preclinical evaluation. We describe a new flow cytometric method based on the differences of autofluorescence and DNA content measured after staining with YOYO-1 that are observed in infected erythrocytes compared with noninfected erythrocytes. Methods Samples of blood from Plasmodium yoelii–infected animals were fixed with glutaraldehyde, incubated with RNAase, and stained with YOYO-1 in 96-well plate format. After acquisition, erythrocytes gated in logarithmic side/scatter plots were analyzed in bidimensional FL-2/YOYO-1 plots in comparison with unidimensional YOYO-1 analysis. Results The infected erythrocytes showed a characteristic pattern of staining different from that of noninfected erythrocytes. In routine evaluation, the limit of sensitivity was 0.01% and the measurements of parasitemia were linear at parasitemias above 0.1%. Interestingly, using this approach, infected reticulocytes could be differentiated from infected normocytes. Conclusions The method described is robust, increases the specificity and sensitivity of detection in routine testing, and is especially well suited for detection of low parasitemias in murine models of malaria. © 2005 Wiley-Liss, Inc.

The Multistate Tuberculosis Pharmacometric (MTP) model, a pharmacokinetic-pharmacodynamic disease model, has been used to describe the effects of rifampicin on Mycobacterium tuberculosis (M. tuberculosis) in vitro. The aim of this work was to investigate if the MTP model could be used to describe the rifampicin treatment response in an acute tuberculosis mouse model. Sixty C57BL/6 mice were intratracheally infected with M. tuberculosis H37Rv strain on Day 0. Fifteen mice received no treatment and were sacrificed on Days 1, 9 and 18 (5 each day). Twenty-five mice received oral rifampicin (1, 3, 9, 26 or 98 mg·kg−1·day−1; Days 1–8; 5 each dose level) and were sacrificed on Day 9. Twenty mice received oral rifampicin (30 mg·kg−1·day−1; up to 8 days) and were sacrificed on Days 2, 3, 4 and 9 (5 each day). The MTP model was linked to a rifampicin population pharmacokinetic model to describe the change in colony forming units (CFU) in the lungs over time. The transfer rates between the different bacterial states were fixed to estimates from in vitro data. The MTP model described well the change in CFU over time after different exposure levels of rifampicin in an acute tuberculosis mouse model. Rifampicin significantly inhibited the growth of fast-multiplying bacteria and stimulated the death of fast- and slow-multiplying bacteria. The data did not support an effect of rifampicin on non-multiplying bacteria possibly due to the short duration of the study. The pharmacometric modelling framework using the MTP model can be used to perform investigations and predictions of the efficacy of anti-tubercular drugs against different bacterial states. Electronic supplementary material The online version of this article (doi:10.1007/s10928-017-9508-2) contains supplementary material, which is available to authorized users.

Induction of HIV expression through lymphocyte activation has been proposed as a strategy to purge latent reservoirs. Prostratin is a non-tumourogenic phorbol ester that delays HIV replication in vitro, but paradoxically activates HIV expression in latently infected cells. To get a better insight into the mechanisms of action of prostratin, we have analysed the effect of prostratin on HIV activation and HIV receptor and coreceptors’ surface expression in human lymphocytes. Peripheral blood mononuclear cells (PBMCs) were transfected with luciferase expression constructs under the control of wild type HIV-long terminal repeat (LTR) and consensus sequences for transcription factors involved in HIV-LTR transactivation (NF-κB, SP1, NFAT). Prostratin stimulates transactivation of LTR vectors, κB- and SP-1-driven luciferase constructs. In another set of experiments, PBMCs were transfected with a full-length infectious viral clone. Prostratin induced HIV transcription and viral expression as detected by luciferase activity in cellular extracts and p24 levels in culture supernatants, respectively. Expression of the HIV coreceptors CCR5 and CXCR4 was decreased by prostratin and, concomitantly, prostratin inhibited the infection of PBMCs with R5 and X4 strains. However, prostratin did not inhibit infection with a pseudotyped viral clone that enters into the cells independently of HIV receptors. These results help to explain the paradoxical effects of prostratin. On one hand, prostratin induces HIV activation in latently infected cells through the induction of NF-κB and Sp1. On the other hand, strong and persistent downregulation of HIV receptors decreases infection of new targets and delays HIV propagation. These data support the potential use of prostratin to activate HIV from latency and purge viral reservoirs.

Molluscum contagiosum virus (MCV) is a human poxvirus that causes abnormal proliferation of epithelial cells. MCV encodes specific molecules to control host defences, such as MC159L, which as previously shown prevents apoptosis induced by death receptors. However, unlike most poxviruses, MCV lacks a homologue to the E3L and K3L proteins of vaccinia virus, which are involved in the control of the key antiviral and pro-apoptotic dsRNA-dependent protein kinase, PKR. In this study, we analysed the relationship of MC159L to PKR. We found that MC159L is not a direct inhibitor of PKR since it does not associate with PKR and cannot block PKR-induced phosphorylation of eIF-2α. However, expression of MC159L inhibits apoptosis triggered by PKR through death receptor-mediated pathways. In addition, MC159L inhibits NF-κB activation induced in response to PKR. Expression of MC159L cannot counteract the PKR-mediated antiviral action in the context of a poxvirus infection, despite its ability to affect these signalling events. These findings show that MC159L is able to interfere with downstream events triggered by PKR in the absence of a direct physical interaction, and assign a role to MC159L in the control of some PKR-mediated biological effects.