Mtb-Selective 5-Aminomethyl Oxazolidinone Prodrugs: Robust Potency and Potential Liabilities.

Linezolid is a drug with proven human antitubercular activity whose use is limited to highly drug-resistant patients because of its toxicity. This toxicity is related to its mechanism of action─linezolid inhibits protein synthesis in both bacteria and eukaryotic mitochondria. A highly selective and potent series of oxazolidinones, bearing a 5-aminomethyl moiety (in place of the typical 5-acetamidomethyl moiety of linezolid), was identified. Linezolid-resistant mutants were cross-resistant to these molecules but not vice versa. Resistance to the 5-aminomethyl molecules mapped to an N-acetyl transferase (Rv0133) and these mutants remained fully linezolid susceptible. Purified Rv0133 was shown to catalyze the transformation of the 5-aminomethyl oxazolidinones to their corresponding N-acetylated metabolites, and this transformation was also observed in live cells of Mycobacterium tuberculosis. Mammalian mitochondria, which lack an appropriate N-acetyltransferase to activate these prodrugs, were not susceptible to inhibition with the 5-aminomethyl analogues. Several compounds that were more potent than linezolid were taken into C3HeB/FeJ mice and were shown to be highly efficacious, and one of these (9) was additionally taken into marmosets and found to be highly active. Penetration of these 5-aminomethyl oxazolidinone prodrugs into caseum was excellent. Unfortunately, these compounds were rapidly converted into the corresponding 5-alcohols by mammalian metabolism which retained antimycobacterial activity but resulted in substantial mitotoxicity.

The Antitubercular Activities of Natural Products with Fused-Nitrogen-Containing Heterocycles.

Tuberculosis (TB) is notorious as the leading cause of death worldwide due to a single infectious entity and its causative agent, Mycobacterium tuberculosis (Mtb), has been able to evolve resistance to all existing drugs in the treatment arsenal complicating disease management programs. In drug discovery efforts, natural products are important starting points in generating novel scaffolds that have evolved to specifically bind to vulnerable targets not only in pathogens such as Mtb, but also in mammalian targets associated with human diseases. Structural diversity is one of the most attractive features of natural products. This review provides a summary of fused-nitrogen-containing heterocycles found in the natural products reported in the literature that are known to have antitubercular activities. The structurally targeted natural products discussed in this review could provide a revealing insight into novel chemical aspects with novel biological functions for TB drug discovery efforts.

Identification and Optimization of Novel Inhibitors of the Polyketide Synthase 13 Thioesterase Domain with Antitubercular Activity.

There is an urgent need for new tuberculosis (TB) treatments, with novel modes of action, to reduce the incidence/mortality of TB and to combat resistance to current treatments. Through both chemical and genetic methodologies, polyketide synthase 13 (Pks13) has been validated as essential for mycobacterial survival and as an attractive target for Mycobacterium tuberculosis growth inhibitors. A benzofuran series of inhibitors that targeted the Pks13 thioesterase domain, failed to progress to preclinical development due to concerns over cardiotoxicity. Herein, we report the identification of a novel oxadiazole series of Pks13 inhibitors, derived from a high-throughput screening hit and structure-guided optimization. This new series binds in the Pks13 thioesterase domain, with a distinct binding mode compared to the benzofuran series. Through iterative rounds of design, assisted by structural information, lead compounds were identified with improved antitubercular potencies (MIC < 1 µM) and in vitro ADMET profiles.

Synthesis and Structure-Activity Relationships of a New Class of Oxadiazoles Targeting DprE1 as Antitubercular Agents.

The continuing prevalence of drug-resistant tuberculosis threatens global TB control programs, highlighting the need to discover new drug candidates to feed the drug development pipeline. In this study, we describe a high-throughput screening hit (4-benzylpiperidin-1-yl)(1-(5-phenyl-1,3,4-oxadiazol-2-yl)piperidin-4-yl)methanone (P1) as a potent antitubercular agent. Structure-activity guided synthesis led to the discovery of several analogs with high in vitro potency. P1 was found to have promising potency against many drug-resistant strains, as well as drug-susceptible clinical isolates. It also showed cidality against Mtb growing in host macrophages. Whole genome sequencing of genomic DNA from resistant mutants raised to P1 revealed mutations in decaprenylphosphoryl-ß-d-ribose 2'-oxidase (DprE1). This novel oxadiazole scaffold expands the set of chemical tools for targeting a well-validated pathway to treat tuberculosis.

MEPicides: α,ß-unsaturated Fosmidomycin N-Acyl Analogs as Efficient Inhibitors of Plasmodium falciparum 1-Deoxy-d-xylulose-5-phosphate reductoisomerase.

Malaria, a mosquito-borne disease caused by several parasites of the Plasmodium genus, remains a huge threat to global public health. There are an estimated 0.5 million malaria deaths each year, mostly among African children. Unlike humans, Plasmodium parasites and a number of important pathogenic bacteria employ the methyl erythritol phosphate (MEP) pathway for isoprenoid synthesis. Thus, the MEP pathway represents a promising set of drug targets for antimalarial and antibacterial compounds. Here, we present new unsaturated MEPicide inhibitors of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), the second enzyme of the MEP pathway. A number of these compounds have demonstrated robust inhibition of Plasmodium falciparum DXR, potent antiparasitic activity, and low cytotoxicity against HepG2 cells. Parasites treated with active compounds are rescued by isopentenyl pyrophosphate, the product of the MEP pathway. With higher levels of DXR substrate, parasites acquire resistance to active compounds. These results further confirm the on-target inhibition of DXR in parasites by the inhibitors. Stability in mouse liver microsomes is high for the phosphonate salts, but remains a challenge for the prodrugs. Taken together, the potent activity and on-target mechanism of action of this series further validate DXR as an antimalarial drug target and the α,ß-unsaturation moiety as an important structural component.

Chemical approaches to unraveling the biology of mycobacteria.

Mycobacterium tuberculosis (Mtb), perhaps more than any other organism, is intrinsically appealing to chemical biologists. Not only does the cell envelope feature one of the most complex heteropolymers found in nature1 but many of the interactions between Mtb and its primary host (we humans) rely on lipid and not protein mediators.2,3 Many of the complex lipids, glycolipids, and carbohydrates biosynthesized by the bacterium still have unknown functions, and the complexity of the pathological processes by which tuberculosis (TB) disease progress offers many opportunities for these molecules to influence the human response. Because of the importance of TB in global public health, chemical biologists have applied a wide-ranging array of techniques to better understand the disease and improve interventions.

Structure Based Discovery of Inhibitors of CYP125 and CYP142 from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) was responsible for approximately 1.6 million deaths in 2021. With the emergence of extensive drug resistance, novel therapeutic agents are urgently needed, and continued drug discovery efforts required. Host-derived lipids such as cholesterol not only support Mtb growth, but are also suspected to function in immunomodulation, with links to persistence and immune evasion. Mtb cytochrome P450 (CYP) enzymes facilitate key steps in lipid catabolism and thus present potential targets for inhibition. Here we present a series of compounds based on an ethyl 5-(pyridin-4-yl)-1H-indole-2-carboxylate pharmacophore which bind strongly to both Mtb cholesterol oxidases CYP125 and CYP142. Using a structure-guided approach, combined with biophysical characterization, compounds with micromolar range in-cell activity against clinically relevant drug-resistant isolates were obtained. These will incite further development of much-needed additional treatment options and provide routes to probe the role of CYP125 and CYP142 in Mtb pathogenesis.

The Myxobacterial Antibiotic Myxovalargin: Biosynthesis, Structural Revision, Total Synthesis, and Molecular Characterization of Ribosomal Inhibition.

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.

Nonhydrolyzable d­phenylalanine-benzoxazole derivatives retain antitubercular activity.

The emergence of drug resistant Mycobacterium tuberculosis, the causative agent of tuberculosis, demands the development of new drugs and new drug targets. We have recently reported that the d-phenylalanine benzoxazole Q112 has potent antibacterial activity against this pathogen with a distinct mechanism of action from other antimycobacterial agents. Q112 and previously reported derivatives were unstable in plasma and no free compound could be observed. Here we expand the structure-activity relationship for antimycobacterial activity and find nonhydrolyzable derivatives with decreased plasma binding. We also show that there is no correlation between antibacterial activity and inhibition of PanG, a putative target for these compounds.

Lysyl-tRNA synthetase, a target for urgently needed M. tuberculosis drugs.

Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold.

Molecular insights into Mmpl3 leads to the development of novel indole-2-carboxamides as antitubercular agents.

Tuberculosis (TB) is an air-borne infectious disease and is the leading cause of death among all infectious diseases globally. The current treatment regimen for TB is overtly long and patient non-compliance often leads to drug resistant TB resulting in a need to develop new drugs that will act via novel mechanisms. In this research work, we selected Mycobacterium membrane protein large (MmpL3) as the drug target and indole-2-carboximide as our molecule of interest for further designing new molecules. A homology model was prepared for the Mycobacterium tuberculosis MmpL3 from the crystal structure of Mycobacterium smegmatis MmpL3. A series of indoles which are known to be MmpL3 inhibitors were docked in the prepared protein and the binding site properties were identified. Based on that, 10 molecules were designed and synthesized and their antitubercular activities evaluated. We identified four hits among which the highest potency candidate possessed a minimum inhibitory concentration (MIC) of 1.56 µM at 2-weeks. Finally, molecular dynamics simulation studies were done with 3b and a previously reported MmpL3 inhibitor to understand the intricacies of their binding in real time and to correlate the experimental findings with the simulation data.

GSK2556286 Is a Novel Antitubercular Drug Candidate Effective In Vivo with the Potential To Shorten Tuberculosis Treatment.

As a result of a high-throughput compound screening campaign using Mycobacterium tuberculosis-infected macrophages, a new drug candidate for the treatment of tuberculosis has been identified. GSK2556286 inhibits growth within human macrophages (50% inhibitory concentration [IC50] = 0.07 µM), is active against extracellular bacteria in cholesterol-containing culture medium, and exhibits no cross-resistance with known antitubercular drugs. In addition, it has shown efficacy in different mouse models of tuberculosis (TB) and has an adequate safety profile in two preclinical species. These features indicate a compound with a novel mode of action, although still not fully defined, that is effective against both multidrug-resistant (MDR) or extensively drug-resistant (XDR) and drug-sensitive (DS) M. tuberculosis with the potential to shorten the duration of treatment in novel combination drug regimens. (This study has been registered at ClinicalTrials.gov under identifier NCT04472897).

Identification of ß-Lactams Active against Mycobacterium tuberculosis by a Consortium of Pharmaceutical Companies and Academic Institutions.

Rising antimicrobial resistance challenges our ability to combat bacterial infections. The problem is acute for tuberculosis (TB), the leading cause of death from infection before COVID-19. Here, we developed a framework for multiple pharmaceutical companies to share proprietary information and compounds with multiple laboratories in the academic and government sectors for a broad examination of the ability of ß-lactams to kill Mycobacterium tuberculosis (Mtb). In the TB Drug Accelerator (TBDA), a consortium organized by the Bill & Melinda Gates Foundation, individual pharmaceutical companies collaborate with academic screening laboratories. We developed a higher order consortium within the TBDA in which four pharmaceutical companies (GlaxoSmithKline, Sanofi, MSD, and Lilly) collectively collaborated with screeners at Weill Cornell Medicine, the Infectious Disease Research Institute (IDRI), and the National Institute of Allergy and Infectious Diseases (NIAID), pharmacologists at Rutgers University, and medicinal chemists at the University of North Carolina to screen ∼8900 ß-lactams, predominantly cephalosporins, and characterize active compounds. In a striking contrast to historical expectation, 18% of ß-lactams screened were active against Mtb, many without a ß-lactamase inhibitor. One potent cephaloporin was active in Mtb-infected mice. The steps outlined here can serve as a blueprint for multiparty, intra- and intersector collaboration in the development of anti-infective agents.

DNA-Dependent Binding of Nargenicin to DnaE1 Inhibits Replication in Mycobacterium tuberculosis.

Natural products provide a rich source of potential antimicrobials for treating infectious diseases for which drug resistance has emerged. Foremost among these diseases is tuberculosis. Assessment of the antimycobacterial activity of nargenicin, a natural product that targets the replicative DNA polymerase of Staphylococcus aureus, revealed that it is a bactericidal genotoxin that induces a DNA damage response in Mycobacterium tuberculosis (Mtb) and inhibits growth by blocking the replicative DNA polymerase, DnaE1. Cryo-electron microscopy revealed that binding of nargenicin to Mtb DnaE1 requires the DNA substrate such that nargenicin is wedged between the terminal base pair and the polymerase and occupies the position of both the incoming nucleotide and templating base. Comparative analysis across three bacterial species suggests that the activity of nargenicin is partly attributable to the DNA binding affinity of the replicative polymerase. This work has laid the foundation for target-led drug discovery efforts focused on Mtb DnaE1.

Synthesis and biological evaluation of orally active prodrugs and analogs of para-aminosalicylic acid (PAS).

Tuberculosis (TB) is one of the world's most deadly infectious diseases resulting in nearly 1.3 million deaths annually and infecting nearly one-quarter of the population. para-Aminosalicylic acid (PAS), an important second-line agent for treating drug-resistant Mycobacterium tuberculosis, has moderate bioavailability and rapid clearance that necessitate high daily doses of up to 12 g per day, which in turn causes severe gastrointestinal disturbances presumably by disruption of gut microbiota and host epithelial cells. We first synthesized a series of alkyl, acyloxy and alkyloxycarbonyloxyalkyl ester prodrugs to increase the oral bioavailability and thereby prevent intestinal accumulation as well as undesirable bioactivation by the gut microbiome to non-natural folate species that exhibit cytotoxicity. The pivoxyl prodrug of PAS was superior to all of the prodrugs examined and showed nearly quantitative absorption. While the conceptually simple prodrug approach improved the oral bioavailability of PAS, it did not address the intrinsic rapid clearance of PAS mediated by N-acetyltransferase-1 (NAT-1). Thus, we next modified the PAS scaffold to reduce NAT-1 catalyzed inactivation by introduction of groups to sterically block N-acetylation and fluorination of the aryl ring of PAS to attenuate N-acetylation by electronically deactivating the para-amino group. Among the mono-fluorinated analogs prepared, 5-fluoro-PAS, exhibited the best activity and an 11-fold decreased rate of inactivation by NAT-1 that translated to a 5-fold improved exposure as measured by area-under-the-curve (AUC) following oral dosing to CD-1 mice. The pivoxyl prodrug and fluorination at the 5-position of PAS address the primary limitations of PAS and have the potential to revitalize this second-line TB drug.

A d-Phenylalanine-Benzoxazole Derivative Reveals the Role of the Essential Enzyme Rv3603c in the Pantothenate Biosynthetic Pathway of Mycobacterium tuberculosis.

New drugs and new targets are urgently needed to treat tuberculosis. We discovered that d-phenylalanine-benzoxazole Q112 displays potent antibacterial activity against Mycobacterium tuberculosis (Mtb) in multiple media and in macrophage infections. A metabolomic profiling indicates that Q112 has a unique mechanism of action. Q112 perturbs the essential pantothenate/coenzyme A biosynthetic pathway, depleting pantoate while increasing ketopantoate, as would be expected if ketopantoate reductase (KPR) were inhibited. We searched for alternative KPRs, since the enzyme annotated as PanE KPR is not essential in Mtb. The ketol-acid reductoisomerase IlvC catalyzes the KPR reaction in the close Mtb relative Corynebacterium glutamicum, but Mtb IlvC does not display KPR activity. We identified the essential protein Rv3603c as an orthologue of PanG KPR and demonstrated that a purified recombinant Rv3603c has KPR activity. Q112 inhibits Rv3603c, explaining the metabolomic changes. Surprisingly, pantothenate does not rescue Q112-treated bacteria, indicating that Q112 has an additional target(s). Q112-resistant strains contain loss-of-function mutations in the twin arginine translocase TatABC, further underscoring Q112's unique mechanism of action. Loss of TatABC causes a severe fitness deficit attributed to changes in nutrient uptake, suggesting that Q112 resistance may derive from a decrease in uptake.

6-Fluorophenylbenzohydrazides inhibit Mycobacterium tuberculosis growth through alteration of tryptophan biosynthesis.

A major constraint in reducing tuberculosis epidemic is the emergence of strains resistant to one or more of clinically approved antibiotics, which emphasizes the need of novel drugs with novel targets. Genetic knockout strains of Mycobacterium tuberculosis (Mtb) have established that tryptophan (Trp) biosynthesis is essential for the bacterium to survive in vivo and cause disease in animal models. An anthranilate-like compound, 6-FABA, was previously shown to synergize with the host immune response to Mtb infection in vivo. Herein, we present a class of anthranilate-like compounds endowed with good antimycobacterial activity and low cytotoxicity. We show how replacing the carboxylic moiety with a hydrazide led to a significant improvement in both activity and cytotoxicity relative to the parent compound 6-FABA. Several new benzohydrazides (compounds 20-31, 33, 34, 36, 38 and 39) showed good activities against Mtb (0.625 ≤ MIC≤6.25 µM) and demonstrated no detectable cytotoxicity against Vero cell assay (CC50 ≥ 1360 µM). The target preliminary studies confirmed the hypothesis that this new class of compounds inhibits Trp biosynthesis. Taken together, these findings indicate that fluorophenylbenzohydrazides represent good candidates to be assessed for drug discovery.

Structure-aided optimization of non-nucleoside M. tuberculosis thymidylate kinase inhibitors.

Mycobacterium tuberculosis thymidylate kinase (MtTMPK) has emerged as an attractive target for rational drug design. We recently investigated new families of non-nucleoside MtTMPK inhibitors in an effort to diversify MtTMPK inhibitor chemical space. We here report a new series of MtTMPK inhibitors by combining the Topliss scheme with rational drug design approaches, fueled by two co-crystal structures of MtTMPK in complex with developed inhibitors. These efforts furnished the most potent MtTMPK inhibitors in our assay, with two analogues displaying low micromolar MIC values against H37Rv Mtb. Prepared inhibitors address new sub-sites in the MtTMPK nucleotide binding pocket, thereby offering new insights into its druggability. We studied the role of efflux pumps as well as the impact of cell wall permeabilizers for selected compounds to potentially provide an explanation for the lack of correlation between potent enzyme inhibition and whole-cell activity.