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.

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.

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.

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.

Lead optimization of 1,4-azaindoles as antimycobacterial agents.

In a previous report, we described the discovery of 1,4-azaindoles, a chemical series with excellent in vitro and in vivo antimycobacterial potency through noncovalent inhibition of decaprenylphosphoryl-ß-d-ribose-2'-epimerase (DprE1). Nevertheless, high mouse metabolic turnover and phosphodiesterase 6 (PDE6) off-target activity limited its advancement. Herein, we report lead optimization of this series, culminating in potent, metabolically stable compounds that have a robust pharmacokinetic profile without any PDE6 liability. Furthermore, we demonstrate efficacy for 1,4-azaindoles in a rat chronic TB infection model. We believe that compounds from the 1,4-azaindole series are suitable for in vivo combination and safety studies.

Aerosol infection model of tuberculosis in wistar rats.

We explored suitability of a rat tuberculosis aerosol infection model for investigating the pharmacodynamics of new antimycobacterial agents. Infection of rats via the aerosol route led to a reproducible course of M. tuberculosis infection in the lungs. The pulmonary bacterial load increased logarithmically during the first six weeks, thereafter, the infection stabilized for the next 12 weeks. We observed macroscopically visible granulomas in the lungs with demonstrable acid-fast bacilli and associated histopathology. Rifampicin (RIF) at a dose range of 30 to 270 mg/kg exhibited a sharp dose response while isoniazid (INH) at a dose range of 10 to 90 mg/kg and ethambutol (EMB) at 100 to 1000 mg/kg showed shallow dose responses. Pyrazinamide (PZA) had no dose response between 300 and 1000 mg/kg dose range. In a separate time kill study at fixed drug doses (RIF 90 mg/kg, INH 30 mg/kg, EMB 300 mg/kg, and PZA 300 mg/kg) the bactericidal effect of all the four drugs increased with longer duration of treatment from two weeks to four weeks. The observed infection profile and therapeutic outcomes in this rat model suggest that it can be used as an additional, pharmacologically relevant efficacy model to develop novel antitubercular compounds at the interface of discovery and development.

Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis.

New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.

Moxifloxacin, ofloxacin, sparfloxacin, and ciprofloxacin against Mycobacterium tuberculosis: evaluation of in vitro and pharmacodynamic indices that best predict in vivo efficacy.

Members of the fluoroquinolone class are being actively evaluated for inclusion in tuberculosis chemotherapy regimens, and we sought to determine the best in vitro and pharmacodynamic predictors of in vivo efficacy in mice. MICs for Mycobacterium tuberculosis H37Rv were 0.1 mg/liter (sparfloxacin [SPX]) and 0.5 mg/liter (moxifloxacin [MXF], ciprofloxacin [CIP], and ofloxacin [OFX]). The unbound fraction in the presence of murine serum was concentration dependent for MXF, OFX, SPX, and CIP. In vitro time-kill studies revealed a time-dependent effect, with the CFU reduction on day 7 similar for all four drugs. However, with a J774A.1 murine macrophage tuberculosis infection model, CIP was ineffective at up to 32x MIC. In addition, MXF, OFX, and SPX exhibited less activity than had been seen in the in vitro time-kill study. After demonstrating that the area under the concentration-time curve (AUC) and maximum concentration of drug in plasma were proportional to the dose in vivo, dose fractionation studies with total oral doses of 37.5 to 19,200 mg/kg of body weight (MXF), 225 to 115,200 mg/kg (OFX), 30 to 50,000 mg/kg (SPX), and 38 to 100,000 mg/kg (CIP) were performed with a murine aerosol infection model. MXF was the most efficacious agent (3.0+/-0.2 log10 CFU/lung reduction), followed by SPX (1.4+/-0.1) and OFX (1.5+/-0.1). CIP showed no effect. The ratio of the AUC to the MIC was the pharmacodynamic parameter that best described the in vivo efficacy. In summary, a lack of intracellular killing predicted the lack of in vivo activity of CIP. The in vivo rank order for maximal efficacy of the three active fluoroquinolones was not clearly predicted by the in vitro assays, however.

Isoniazid pharmacokinetics-pharmacodynamics in an aerosol infection model of tuberculosis.

Limited data exist on the pharmacokinetic-pharmacodynamic (PK-PD) parameters of the bactericidal activities of the available antimycobacterial drugs. We report on the PK-PD relationships for isoniazid. Isoniazid exhibited concentration (C)-dependent killing of Mycobacterium tuberculosis H37Rv in vitro, with a maximum reduction of 4 log10 CFU/ml. In these studies, 50% of the maximum effect was achieved at a C/MIC ratio of 0.5, and the maximum effect did not increase with exposure times of up to 21 days. Conversely, isoniazid produced less than a 0.5-log10 CFU/ml reduction in two different intracellular infection models (J774A.1 murine macrophages and whole human blood). In a murine model of aerosol infection, isoniazid therapy for 6 days produced a reduction of 1.4 log10 CFU/lung. Dose fractionation studies demonstrated that the 24-h area under the concentration-time curve/MIC (r2 = 0.83) correlated best with the bactericidal efficacy, followed by the maximum concentration of drug in serum/MIC (r2 = 0.73).

High-throughput screen for inhibitors of transglycosylase and/or transpeptidase activities of Escherichia coli penicillin binding protein 1b.

Penicillin binding protein (PBP) 1b of Escherichia coli has both transglycosylase and transpeptidase activities, which are attractive targets for the discovery of new antibacterial agents. A high-throughput assay that detects inhibitors of the PBPs was described previously, but it cannot distinguish them from inhibitors of the MraY, MurG, and lipid pyrophosphorylase. We report on a method that distinguishes inhibitors of both activities of the PBPs from those of the other three enzymes. Radioactive peptidoglycan was synthesized by using E. coli membranes. Following termination of the reaction the products were analyzed in three ways. Wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads were added to one set, and the same beads together with a detergent were added to a second set. Type A polyethylenimine-coated WGA-coated SPA beads were added to a third set. By comparison of the results of assays run in parallel under the first two conditions, inhibitors of the transpeptidase and transglycosylase could be distinguished from inhibitors of the other enzymes, as the inhibitors of the other enzymes showed similar inhibitory concentrations (IC(50)s) under both conditions but the inhibitors of the PBPs showed insignificant inhibition in the absence of detergent. Furthermore, comparison of the results of assays run under conditions two and three enabled the distinction of transpeptidase inhibitors. Penicillin and other beta-lactams showed insignificant inhibition with type A beads compared with that shown with WGA-coated SPA beads plus detergent. However, inhibitors of the other four enzymes (tunicamycin, nisin, bacitracin, and moenomycin) showed similar IC(50)s under both conditions. We show that the main PBP being measured under these conditions is PBP 1b. This screen can be used to find novel transglycosylase or transpeptidase inhibitors.