Discovery of Species-Specific Proteotypic Peptides To Establish a Spectral Library Platform for Identification of Nontuberculosis Mycobacteria from Mass Spectrometry-Based Proteomics.

Nontuberculous mycobacteria are opportunistic bacteria pulmonary and extra-pulmonary infections in humans that closely resemble Mycobacterium tuberculosis. Although genome sequencing strategies helped determine NTMs, a common assay for the detection of coinfection by multiple NTMs with M. tuberculosis in the primary attempt of diagnosis is still elusive. Such a lack of efficiency leads to delayed therapy, an inappropriate choice of drugs, drug resistance, disease complications, morbidity, and mortality. Although a high-resolution LC-MS/MS-based multiprotein panel assay can be developed due to its specificity and sensitivity, it needs a library of species-specific peptides as a platform. Toward this, we performed an analysis of proteomes of 9 NTM species with more than 20 million peptide spectrum matches gathered from 26 proteome data sets. Our metaproteomic analyses determined 48,172 species-specific proteotypic peptides across 9 NTMs. Notably, M. smegmatis (26,008), M. abscessus (12,442), M. vaccae (6487), M. fortuitum (1623), M. avium subsp. paratuberculosis (844), M. avium subsp. hominissuis (580), and M. marinum (112) displayed >100 species-specific proteotypic peptides. Finally, these peptides and corresponding spectra have been compiled into a spectral library, FASTA, and JSON formats for future reference and validation in clinical cohorts by the biomedical community for further translation.

Discovery of FNDR-20123, a histone deacetylase inhibitor for the treatment of Plasmodium falciparum malaria.

BACKGROUND: Emergence of anti-malarial drug resistance and perpetual increase in malaria incidence necessitates the development of novel anti-malarials. Histone deacetylases (HDAC) has been shown to be a promising target for malaria, despite this, there are no HDAC inhibitors in clinical trials for malaria treatment. This can be attributed to the poor pharmacokinetics, bioavailability and selectivity of the HDAC inhibitors. METHODS: A collection of HDAC inhibitors were screened for anti-malarial activity, and the best candidate was profiled in parasite-killing kinetics, growth inhibition of sensitive and multi-drug resistant (MDR) strains and against gametocytes. Absorption, distribution, metabolism and excretion pharmacokinetics (ADME-PK) parameters of FNDR-20123 were determined, and in vivo efficacy was studied in a mouse model for Plasmodium falciparum infection. RESULTS: A compound library of HDAC inhibitors (180 in number) was screened for anti-malarial activity, of which FNDR-20123 was the most potent candidate. The compound had been shown to inhibit Plasmodium HDAC with IC50 of 31 nM and human HDAC with IC50 of 3 nM. The IC50 obtained for P. falciparum in asexual blood-stage assay was 42 nM. When compared to atovaquone and pyrimethamine, the killing profiles of FNDR-20123 were better than atovaquone and comparable to pyrimethamine. The IC50 values for the growth inhibition of sensitive and MDR strains were similar, indicating that there is no cross-resistance and a low risk of resistance development. The selected compound was also active against gametocytes, indicating a potential for transmission control: IC50 values being 190 nM for male and > 5 µM for female gametocytes. FNDR-20123 is a stable candidate in human/mouse/rat liver microsomes (> 75% remaining post 2-h incubation), exhibits low plasma protein binding (57% in humans) with no human Ether-à-go-go-Related Gene (hERG) liability (> 100 µM), and does not inhibit any of the cytochrome P450 (CYP) isoforms tested (IC50 > 25 µM). It also shows negligible cytotoxicity to HepG-2 and THP-1 cell lines. The oral pharmacokinetics in rats at 100 mg/kg body weight shows good exposures (Cmax = 1.1 µM) and half-life (T1/2 = 5.5 h). Furthermore, a 14-day toxicokinetic study at 100 mg/kg daily dose did not show any abnormality in body weight or gross organ pathology. FNDR-20123 is also able to reduce parasitaemia significantly in a mouse model for P. falciparum infection when dosed orally and subcutaneously. CONCLUSION: FNDR-20123 may be a suitable candidate for the treatment of malaria, which can be further developed.

In silico-based high-throughput screen for discovery of novel combinations for tuberculosis treatment.

There are currently 18 drug classes for the treatment of tuberculosis, including those in the development pipeline. An in silico simulation enabled combing the innumerably large search space to derive multidrug combinations. Through the use of ordinary differential equations (ODE), we constructed an in silico kinetic platform in which the major metabolic pathways in Mycobacterium tuberculosis and the mechanisms of the antituberculosis drugs were integrated into a virtual proteome. The optimized model was used to evaluate 816 triplets from the set of 18 drugs. The experimentally derived cumulative fractional inhibitory concentration (∑FIC) value was within twofold of the model prediction. Bacterial enumeration revealed that a significant number of combinations that were synergistic for growth inhibition were also synergistic for bactericidal effect. The in silico-based screen provided new starting points for testing in a mouse model of tuberculosis, in which two novel triplets and five novel quartets were significantly superior to the reference drug triplet of isoniazid, rifampin, and ethambutol (HRE) or the quartet of HRE plus pyrazinamide (HREZ).

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.

Pharmacokinetic and pharmacodynamic evaluation of AZD5847 in a mouse model of tuberculosis.

AZD5847, a novel oxazolidinone with an MIC of 1 µg/ml, exhibits exposure-dependent killing kinetics against extracellular and intracellular Mycobacterium tuberculosis. Oral administration of AZD5847 to mice infected with M. tuberculosis H37Rv in a chronic-infection model resulted in a 1.0-log10 reduction in the lung CFU count after 4 weeks of treatment at a daily area under the concentration-time curve (AUC) of 105 to 158 µg · h/ml. The pharmacokinetic-pharmacodynamic parameter that best predicted success in an acute-infection model was an AUC for the free, unbound fraction of the drug/MIC ratio of ≥ 20. The percentage of time above the MIC in all of the efficacious regimens was 25% or greater.

1,4-azaindole, a potential drug candidate for treatment of tuberculosis.

New therapeutic strategies against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis are urgently required to combat the global tuberculosis (TB) threat. Toward this end, we previously reported the identification of 1,4-azaindoles, a promising class of compounds with potent antitubercular activity through noncovalent inhibition of decaprenylphosphoryl-ß-D-ribose 2'-epimerase (DprE1). Further, this series was optimized to improve its physicochemical properties and pharmacokinetics in mice. Here, we describe the short-listing of a potential clinical candidate, compound 2, that has potent cellular activity, drug-like properties, efficacy in mouse and rat chronic TB infection models, and minimal in vitro safety risks. We also demonstrate that the compounds, including compound 2, have no antagonistic activity with other anti-TB drugs. Moreover, compound 2 shows synergy with PA824 and TMC207 in vitro, and the synergy effect is translated in vivo with TMC207. The series is predicted to have a low clearance in humans, and the predicted human dose for compound 2 is ≤1 g/day. Altogether, our data suggest that a 1,4-azaindole (compound 2) is a promising candidate for the development of a novel anti-TB drug.

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.

Homo-BacPROTAC-induced degradation of ClpC1 as a strategy against drug-resistant mycobacteria.

Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti-Mycobacterium tuberculosis (Mtb) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance.

In vitro and in vivo efficacy of ß-lactams against replicating and slowly growing/nonreplicating Mycobacterium tuberculosis.

Beta-lactams, in combination with beta-lactamase inhibitors, are reported to have activity against Mycobacterium tuberculosis bacteria growing in broth, as well as inside the human macrophage. We tested representative beta-lactams belonging to 3 different classes for activity against replicating M. tuberculosis in broth and nonreplicating M. tuberculosis under hypoxia, as well as against streptomycin-starved M. tuberculosis strain 18b (ss18b) in the presence or absence of clavulanate. Most of the combinations showed bactericidal activity against replicating M. tuberculosis, with up to 200-fold improvement in potency in the presence of clavulanate. None of the combinations, including those containing meropenem, imipenem, and faropenem, killed M. tuberculosis under hypoxia. However, faropenem- and meropenem-containing combinations killed strain ss18b moderately. We tested the bactericidal activities of meropenem-clavulanate and amoxicillin-clavulanate combinations in the acute and chronic aerosol infection models of tuberculosis in BALB/c mice. Based on pharmacokinetic/pharmacodynamic indexes reported for beta-lactams against other bacterial pathogens, a cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (%TMIC) of 20 to 40% was achieved in mice using a suitable dosing regimen. Both combinations showed marginal reduction in lung CFU compared to the late controls in the acute model, whereas both were inactive in the chronic model.

A Novel Inhibitor against the Biofilms of Non-Tuberculous Mycobacteria.

Non-tuberculous Mycobacteria (NTM), previously classified as environmental microbes, have emerged as opportunistic pathogens causing pulmonary infections in immunocompromised hosts. The formation of the biofilm empowers NTM pathogens to escape from the immune response and antibiotic action, leading to treatment failures. NF1001 is a novel thiopeptide antibiotic first-in-class compound with potent activity against planktonic/replicating and biofilm forms of various NTM species. It is potent against both drug-sensitive and -resistant NTM. It has demonstrated a concentration-dependent killing of replicating and intracellularly growing NTM, and has inhibited and reduced the viability of NTM in biofilms. Combination studies using standard-of-care (SoC) drugs for NTM exhibited synergetic/additive effects, but no antagonism against both planktonic and biofilm populations of Mycobacterium abscessus and Mycobacterium avium. In summary, the activity of NF1001 alone or in combination with SoC drugs projects NF1001 as a promising candidate for the treatment of difficult-to-treat NTM pulmonary diseases (NTM-PD) and cystic fibrosis (CF) in patients.

Genomic Mutations in SARS-CoV-2 Genome following Infection in Syrian Golden Hamster and Associated Lung Pathologies.

The continuous evolution of the SARS-CoV-2 virus led to constant developments and efforts in understanding the significance and impacts of SARS-CoV-2 variants on human health. Our study aimed to determine the accumulation of genetic mutations and associated lung pathologies in male and female hamsters infected with the ancestral Wuhan strain of SARS-CoV-2. The present study showed no significant difference in the viral load between male and female hamsters and peak infection was found to be on day four post infection in both sexes of the animals. Live virus particles were detected up to 5 days post infection (dpi) through the TCID-50 assay, while qRT-PCR could detect viral RNA up to 14 dpi from all the infected animals. Further, the determination of the neutralizing antibody titer showed the onset of the humoral immune response as early as 4 dpi in both sexes against SARS-CoV-2, and a significant cross-protection against the delta variant of SARS-CoV-2 was observed. Histopathology showed edema, inflammation, inflammatory cell infiltration, necrosis, and degeneration of alveolar and bronchial epithelium cells from 3 dpi to 14 dpi in both sexes. Furthermore, next-generation sequencing (NGS) showed up to 10 single-nucleotide polymorphisms (SNPs) in the SARS-CoV-2 (ancestral Wuhan strain) genome isolated from both male and female hamsters. The mutation observed at the 23014 position (Glu484Asp) in the SARS-CoV-2 genome isolated from both sexes of the hamsters plays a significant role in the antiviral efficacy of small molecules, vaccines, and the Mabs-targeting S protein. The present study shows that either of the genders can be used in the pre-clinical efficacy of antiviral agents against SARS-CoV-2 in hamsters. However, considering the major mutation in the S protein, the understanding of the genetic mutation in SARS-CoV-2 after passing through hamsters is crucial in deciding the efficacy of the antiviral agents targeting the S protein. Importance: Our study findings indicate the accumulation of genomic mutations in SARS-CoV-2 after passing through the Syrian golden hamsters. Understanding the genomic mutations showed that either of the hamster genders can be used in the pre-clinical efficacy of antiviral agents and vaccines.

Novel Baicalein-Derived Inhibitors of Plasmodium falciparum.

Malaria, a life-threatening mosquito-borne disease caused by Plasmodium parasites, continues to pose a significant global health burden. Despite notable progress in combating the disease in recent years, malaria remains prevalent in many regions, particularly in Southeast Asia and most of sub-Saharan Africa, where it claims hundreds of thousands of lives annually. Flavonoids, such as the baicalein class of compounds, are known to have antimalarial properties. In this study, we rationally designed and synthesized a series of baicalein derivatives and identified a lead compound, FNDR-10132, that displayed potent in vitro antimalarial activity against Plasmodium falciparum (P. falciparum), both chloroquine-sensitive (60 nM) and chloroquine-resistant (177 nM) parasites. FNDR-10132 was evaluated for its antimalarial activity in vivo against the chloroquine-resistant strain Plasmodium yoelii N67 in Swiss mice. The oral administration of 100 mg/kg of FNDR-10132 showed 44% parasite suppression on day 4, with a mean survival time of 13.5 ± 2.3 days vs. 8.4 ± 2.3 days of control. Also, FNDR-10132 displayed equivalent activity against the resistant strains of P. falciparum in the 200-300 nM range. This study offers a novel series of antimalarial compounds that could be developed into potent drugs against chloroquine-resistant malarial parasites through further chemistry and DMPK optimization.

A Live Attenuated COVID-19 Candidate Vaccine for Children: Protection against SARS-CoV-2 Challenge in Hamsters.

Children are at risk of infection from severe acute respiratory syndrome coronavirus-2 virus (SARS-CoV-2) resulting in coronavirus disease (COVID-19) and its more severe forms. New-born infants are expected to receive short-term protection from passively transferred maternal antibodies from their mothers who are immunized with first-generation COVID-19 vaccines. Passively transferred antibodies are expected to wane within first 6 months of infant's life, leaving them vulnerable to COVID-19. Live attenuated vaccines, unlike inactivated or viral-protein-based vaccines, offer broader immune engagement. Given effectiveness of live attenuated vaccines in controlling infectious diseases such as mumps, measles and rubella, we undertook development of a live attenuated COVID-19 vaccine with an aim to vaccinate children beyond 6 months of age. An attenuated vaccine candidate (dCoV), engineered to express sub-optimal codons and deleted polybasic furin cleavage sites in the spike protein of the SARS-CoV-2 WA/1 strain, was developed and tested in hamsters. Hamsters immunized with dCoV via intranasal or intramuscular routes induced high levels of neutralizing antibodies and exhibited complete protection against the SARS-CoV-2 wild-type isolates, i.e., the Wuhan-like (USA-WA1/2020) and Delta variants (B.1.617.2) in a challenge study. In addition, the dCoV formulated with the marketed measles-rubella (MR) vaccine, designated as MR-dCoV, administered to hamsters via intramuscular route, also protected against both SARS-CoV-2 challenges, and dCoV did not interfere with the MR vaccine-mediated immune response. The safety and efficacy of the dCoV and the MR-dCoV against both variants of SARS-CoV-2 opens the possibility of early immunization in children without an additional injection.

Expanding the anti-flaviviral arsenal: Discovery of a baicalein-derived Compound with potent activity against DENV and ZIKV.

With approximately 3.8 billion people at risk of infection in tropical and sub-tropical regions, Dengue ranks among the top ten threats worldwide. Despite the potential for severe disease manifestation and the economic burden it places on endemic countries, there is a lack of approved antiviral agents to effectively treat the infection. Flavonoids, including baicalein, have garnered attention for their antimicrobial properties. In this study, we took a rational and iterative approach to develop a series of baicalein derivatives with improved antiviral activity against Dengue virus (DENV). Compound 11064 emerged as a promising lead candidate, exhibiting antiviral activity against the four DENV serotypes and representative strains of Zika virus (ZIKV) in vitro, with attractive selectivity indices. Mechanistic studies revealed that Compound 11064 did not prevent DENV attachment at the cell surface, nor viral RNA synthesis and viral protein translation. Instead, the drug was found to impair the post-receptor binding entry steps (endocytosis and/or uncoating), as well as the late stage of DENV infection cycle, including virus assembly/maturation and/or exocytosis. The inability to raise DENV resistant mutants, combined with significant antiviral activity against an unrelated RNA virus (Enterovirus-A71) suggested that Compound 11064 targets the host rather than a viral protein, further supporting its broad-spectrum antiviral potential. Overall, Compound 11064 represents a promising antiviral candidate for the treatment of Dengue and Zika.

Picolinic acid is a broad-spectrum inhibitor of enveloped virus entry that restricts SARS-CoV-2 and influenza A virus in vivo.

The COVID-19 pandemic highlights an urgent need for effective antivirals. Targeting host processes co-opted by viruses is an attractive antiviral strategy with a high resistance barrier. Picolinic acid (PA) is a tryptophan metabolite endogenously produced in mammals. Here, we report the broad-spectrum antiviral activity of PA against enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), flaviviruses, herpes simplex virus, and parainfluenza virus. Mechanistic studies reveal that PA inhibits enveloped virus entry by compromising viral membrane integrity, inhibiting virus-cellular membrane fusion, and interfering with cellular endocytosis. More importantly, in pre-clinical animal models, PA exhibits promising antiviral efficacy against SARS-CoV-2 and IAV. Overall, our data establish PA as a broad-spectrum antiviral with promising pre-clinical efficacy against pandemic viruses SARS-CoV-2 and IAV.

Evaluation of the Therapeutic Potential of Mesenchymal Stem Cells (MSCs) in Preclinical Models of Autoimmune Diseases.

Autoimmune diseases, chronic in nature, are generally hard to alleviate. Present long-term treatments with available drugs such as steroids, immune-suppressive drugs, or antibodies have several debilitating side effects. Therefore, new treatment options are urgently needed. Stem cells, in general, have the potential to reduce immune-mediated damage through immunomodulation and T cell regulation (T regs) by inhibiting the proliferation of dendritic cells and T and B cells and reducing inflammation through the generation of immunosuppressive biomolecules like interleukin 10 (IL-10), transforming growth factor-ß (TGF-ß), nitric oxide (NO), indoleamine 2,3-dioxygenase (IDO), and prostaglandin E2 (PGE2). Many stem cell-based therapeutics have been evaluated in the clinic, but the overall clinical outcomes in terms of efficacy and the longevity of therapeutic benefits seem to be variable and inconsistent with the postulated benefits. This emphasizes a greater need for building robust preclinical models and models that can better predict the clinical translation of stem cell-based therapeutics. Stem cell therapy based on MSCs having the definitive potential to regulate the immune system and control inflammation is emerging as a promising tool for the treatment of autoimmune disorders while promoting tissue regeneration. MSCs, derived from bone marrow, umbilical cord, and adipose tissue, have been shown to be highly immunomodulatory and anti-inflammatory and shown to enhance tissue repair and regeneration in preclinical models as well as in clinical settings. In this article, a review on the status of MSC-based preclinical disease models with emphasis on understanding disease mechanisms in chronic inflammatory disorders caused by exaggerated host immune response in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) was carried out. We also emphasized various factors that better predict the translation of stem cell therapeutic outcomes from preclinical disease models to human patients.

Development and validation of LC/MS/MS method for Triciribine and its monophosphate metabolite in plasma and RBC: Application to mice pharmacokinetic studies.

Triciribine (TCN) is a tricyclic nucleoside analog of adenosine and an inhibitor of Akt kinase. Triciribine 5'-monophosphate (TCNP) is a water-soluble analog of Triciribine and has progressed to Phase I and II clinical trials in oncology. TCNP is also an endogenous anabolite of TCN similar to other nucleoside phosphates. Clinical development of TCNP has been hampered by high pharmacokinetic variability due to complex interplay of TCN-TCNP conversion and reconversion in plasma, erythrocytes (RBC) and peripheral organs. TCN has been demonstrated to be an efficacious agent in mice models of acute lung injury at low doses (0.5 mg/kg/day) although its pharmacokinetic-pharmacodynamic (PK/PD) relationship remained unclear. We have developed and validated a sensitive, specific and robust LC/MS/MS assay for quantitation of TCN and TCNP in plasma and RBC. Using a simple protein precipitation method, quantitation of these analytes was accomplished with recoveries exceeding 85% and with a run time of 4 min. This assay was used to determine the pharmacokinetic parameters of TCN and TCNP in mice after single dose intravenous administration at 1, 3 and 10 mg/kg. TCNP accumulates in RBC, has low clearance and a half-life of 18 to 23 h. Unlike other nucleoside phosphates, TCNP was found to be relatively stable in mice plasma serving as a secondary depot. TCN levels were low and with high clearance relative to hepatic blood flow. A combination of sustained levels of TCNP in RBC and plasma serves as a depot for TCN to elicit robust therapeutic activity in acute lung injury mice models.

A multi-targeting pre-clinical candidate against drug-resistant tuberculosis.

FNDR-20081 [4-{4-[5-(4-Isopropyl-phenyl)- [1,2,4]oxadiazol-3-ylmethyl]-piperazin-1-yl}-7-pyridin-3-yl-quinoline] is a novel, first in class anti-tubercular pre-clinical candidate against sensitive and drug-resistant Mycobacterium tuberculosis (Mtb). In-vitro combination studies of FNDR-20081 with first- and second-line drugs exhibited no antagonism, suggesting its compatibility for developing new combination-regimens. FNDR-20081, which is non-toxic with no CYP3A4 liability, demonstrated exposure-dependent killing of replicating-Mtb, as well as the non-replicating-Mtb, and efficacy in a mouse model of infection. Whole genome sequencing (WGS) of FNDR-20081 resistant mutants revealed the identification of pleotropic targets: marR (Rv0678), a regulator of MmpL5, a transporter/efflux pump mechanism for drug resistance; and Rv3683, a putative metalloprotease potentially involved in peptidoglycan biosynthesis. In summary, FNDR-20081 is a promising first in class compound with the potential to form a new combination regimen for MDR-TB treatment.

Targeting redox heterogeneity to counteract drug tolerance in replicating Mycobacterium tuberculosis.

The capacity of Mycobacterium tuberculosis (Mtb) to tolerate multiple antibiotics represents a major problem in tuberculosis (TB) management. Heterogeneity in Mtb populations is one of the factors that drives antibiotic tolerance during infection. However, the mechanisms underpinning this variation in bacterial population remain poorly understood. Here, we show that phagosomal acidification alters the redox physiology of Mtb to generate a population of replicating bacteria that display drug tolerance during infection. RNA sequencing of this redox-altered population revealed the involvement of iron-sulfur (Fe-S) cluster biogenesis, hydrogen sulfide (H2S) gas, and drug efflux pumps in antibiotic tolerance. The fraction of the pH- and redox-dependent tolerant population increased when Mtb infected macrophages with actively replicating HIV-1, suggesting that redox heterogeneity could contribute to high rates of TB therapy failure during HIV-TB coinfection. Pharmacological inhibition of phagosomal acidification by the antimalarial drug chloroquine (CQ) eradicated drug-tolerant Mtb, ameliorated lung pathology, and reduced postchemotherapeutic relapse in in vivo models. The pharmacological profile of CQ (C max and AUClast) exhibited no major drug-drug interaction when coadministered with first line anti-TB drugs in mice. Our data establish a link between phagosomal pH, redox metabolism, and drug tolerance in replicating Mtb and suggest repositioning of CQ to shorten TB therapy and achieve a relapse-free cure.

Scaffold Morphing To Identify Novel DprE1 Inhibitors with Antimycobacterial Activity.

We report a novel benzimidazole (BI) based DprE1 inhibitor that resulted from scaffold morphing of a 1,4-azaindole series. The clinical progression of the 1,4-azaindole series from our previous work validates the potential of exploring newer chemical entities with antimycobacterial activity driven via a noncovalent inhibition of the decaprenylphosphoryl-ß-d-ribose-2'-epimerase (DprE1). The representative compounds from the new scaffold reported in this study exhibited an improved solubility and higher free plasma fraction, while retaining potent DprE1 inhibition and antimycobacterial activity. A representative compound from the benzimidazole series demonstrated good efficacy in a murine model of tuberculosis. Furthermore, molecular modeling of the BI scaffold suggests plausible modes of binding in the active site of DprE1 enzyme from Mycobacterium tuberculosis that can be used for further exploration of the series.