High content screening strategies for large-scale compound libraries with a focus on high-containment viruses.

A majority of viral diseases do not have FDA-approved drugs. The recent outbreaks caused by SARS-CoV-2, monkeypox, and Sudan ebolavirus have exposed the critical need for rapid screening and identification of antiviral compounds against emerging/re-emerging viral pathogens. A high-content screening (HCS) platform is becoming an essential part of the drug discovery process, thanks to developments in image acquisition and analysis. While HCS has several advantages, its full potential has not been realized in antiviral drug discovery compared to conventional drug screening approaches, such as fluorescence or luminescence-based microplate assays. Therefore, this review aims to summarize HCS workflow, strategies, and developments in image-based drug screening, focusing on high-containment viruses.

Novel fluorobenzothiazole as a dual inhibitor of gyrase B and topoisomerase IV against Gram-positive pathogens.

Aim: The development of a novel inhibitor targeting gyrase B and topoisomerase IV offers an opportunity to combat multidrug resistance. Methods: We investigated the activity of RBx 10080758 against Gram-positive bacteria in vitro and in vivo. Results: RBx 10080758 showed a potent 50% inhibitory concentration of 0.13 µM and 0.25 µM against gyrase B and topoisomerase IV, respectively, and exhibited strong whole-cell in vitro activity with MIC ranges of 0.015-0.06 and 0.015-0.03 µg/ml against Staphylococcus aureus and Streptococcus pneumoniae, respectively. In a rat thigh infection model with methicillin-resistant S. aureus, RBx 10080758 at 45 mg/kg exhibited a >3 log10 CFU reduction in thigh muscles. Conclusion: RBx 10080758 displayed potent activity against multiple multidrug-resistant Gram-positive bacteria with a dual-targeting mechanism of action.

ADME, Pharmacokinetic Scaling, Pharmacodynamic and Prediction of Human Dose and Regimen of Novel Antiviral Drugs.

The search for new drugs is an extremely time-consuming and expensive endeavour. Much of that time and money go into generating predictive human pharmacokinetic profiles from preclinical efficacy and safety animal data. These pharmacokinetic profiles are used to prioritize or minimize the attrition at later stages of the drug discovery process. In the area of antiviral drug research, these pharmacokinetic profiles are equally important for the optimization, estimation of half-life, determination of effective dose, and dosing regimen, in humans. In this article we have highlighted three important aspects of these profiles. First, the impact of plasma protein binding on two primary pharmacokinetic parameters-volume of distribution and clearance. Second, interdependence of primary parameters on unbound fraction of the drug. Third, the ability to extrapolate human pharmacokinetic parameters and concentration time profiles from animal profiles.

Lethal synergy between SARS-CoV-2 and Streptococcus pneumoniae in hACE2 mice and protective efficacy of vaccination.

Secondary infections are frequent complications of viral respiratory infections, but the potential consequence of SARS-CoV-2 coinfection with common pulmonary pathogens is poorly understood. We report that coinfection of human ACE2-transgenic mice with sublethal doses of SARS-CoV-2 and Streptococcus pneumoniae results in synergistic lung inflammation and lethality. Mortality was observed regardless of whether SARS-CoV-2 challenge occurred before or after establishment of sublethal pneumococcal infection. Increased bacterial levels following coinfection were associated with alveolar macrophage depletion, and treatment with murine GM-CSF reduced numbers of lung bacteria and pathology and partially protected from death. However, therapeutic targeting of IFNs, an approach that is effective against influenza coinfections, failed to increase survival. Combined vaccination against both SARS-CoV-2 and pneumococci resulted in 100% protection against subsequent coinfection. The results indicate that when seasonal respiratory infections return to prepandemic levels, they could lead to an increased incidence of lethal COVID-19 superinfections, especially among the unvaccinated population.

Viral PB1-F2 and host IFN-γ guide ILC2 and T cell activity during influenza virus infection.

Functional plasticity of innate lymphoid cells (ILCs) and T cells is regulated by host environmental cues, but the influence of pathogen-derived virulence factors has not been described. We now report the interplay between host interferon (IFN)-γ and viral PB1-F2 virulence protein in regulating the functions of ILC2s and T cells that lead to recovery from influenza virus infection of mice. In the absence of IFN-γ, lung ILC2s from mice challenged with the A/California/04/2009 (CA04) H1N1 virus, containing nonfunctional viral PB1-F2, initiated a robust IL-5 response, which also led to improved tissue integrity and increased survival. Conversely, challenge with Puerto Rico/8/1934 (PR8) H1N1 virus expressing fully functional PB1-F2, suppressed IL-5+ ILC2 responses, and induced a dominant IL-13+ CD8 T cell response, regardless of host IFN-γ expression. IFN-γ-deficient mice had increased survival and improved tissue integrity following challenge with lethal doses of CA04, but not PR8 virus, and increased resistance was dependent on the presence of IFN-γR+ ILC2s. Reverse-engineered influenza viruses differing in functional PB1-F2 activity induced ILC2 and T cell phenotypes similar to the PB1-F2 donor strains, demonstrating the potent role of viral PB1-F2 in host resistance. These results show the ability of a pathogen virulence factor together with host IFN-γ to regulate protective pulmonary immunity during influenza infection.

Disease Tolerance during Viral-Bacterial Co-Infections.

Disease tolerance has emerged as an alternative way, in addition to host resistance, to survive viral-bacterial co-infections. Disease tolerance plays an important role not in reducing pathogen burden, but in maintaining tissue integrity and controlling organ damage. A common co-infection is the synergy observed between influenza virus and Streptococcus pneumoniae that results in superinfection and lethality. Several host cytokines and cells have shown promise in promoting tissue protection and damage control while others induce severe immunopathology leading to high levels of morbidity and mortality. The focus of this review is to describe the host cytokines and innate immune cells that mediate disease tolerance and lead to a return to host homeostasis and ultimately, survival during viral-bacterial co-infection.

Novel FtsZ inhibitor with potent activity against Staphylococcus aureus.

OBJECTIVES: FtsZ is an essential bacterial protein and an unexplored target for the development of antibacterial drugs. The development of a novel inhibitor targeting FtsZ offers a potential opportunity to combat drug resistance. DS01750413, a new derivative of PC190723, is a novel FtsZ inhibitor with improved in vitro and in vivo activity. The objective of this study was to investigate the efficacy of DS01750413 against Staphylococcus spp., including MRSA, in in vitro and in vivo models. METHODS: In vitro activities of DS01750413 and standard-of-care antibiotics were evaluated against clinical isolates of Gram-positive pathogens. The in vivo efficacy was evaluated in a murine systemic infection model caused by MRSA. RESULTS: DS01750413 showed potent in vitro activity against MRSA clinical isolates with MIC ranges of 0.5-1 mg/L and also demonstrated concentration-dependent bactericidal killing. In the murine bacteraemia infection model of MRSA, treatment with DS01750413 resulted in prolonged survival of animals compared with placebo-treated animals and exhibited a significant reduction in the bacterial load in liver, spleen, lungs and kidneys. CONCLUSIONS: DS01750413 showed encouraging in vitro and in vivo activity against MRSA. As a novel chemical class, DS01750413 has the potential to become clinically viable antibiotics to address the drug resistance problem by its unique novel targeting mechanism of action.

Novel azoles with potent antileishmanial activity.

Aim: To investigate the antileishmanial activity of novel azole compounds against Leishmania donovani, which causes deadly visceral leishmaniasis disease. Materials & methods: A focused azole-based library was screened against both promastigotes and amastigotes forms of L. donovani strains in flat-bottomed 96-well tissue culture plates and J774A.1 macrophage cell-line infected with L. donovani. The comprehensive screening of azole-based library against L. donovani strains provided novel hits, which can serve as a good starting point to initiate hit to lead optimization campaign. Results: Hits identified from azole-based library exhibited potent in vitro activity against promastigotes and amastigotes of L. donovani. Conclusion: These potent novel azole hits could be a good starting point to carry out for further medicinal chemistry exploration for antileishmania program.

Lack of active SARS-CoV-2 virus in a subset of PCR-positive COVID-19 congregate care patients.

Highly sensitive nucleic acid amplification tests (NAATs) designed to detect SARS-CoV-2 RNA are the standard of care for the diagnosis of COVID-19. However, the accuracy of these methods for the quantitation of active virus rather than non-infectious RNA fragments that can persist for extended periods of time has been unclear. This issue is particularly relevant for congregate care patients who are unable to return to their home residence until fully negative by NAATs. We tested paired samples from individual patients for the presence of virus at both early and later stages of disease. Culture of nasopharyngeal swab samples for 10 days in Vero E6 cells revealed active virus in only 4 out of 14 (28.6%) patients. The ability to isolate viral plaque-forming units (PFU) correlated with viral RNA loads of >6.79 log genomic copies/ml and only occurred in samples collected from patients early after symptom onset and before development of antibody. Culture in Vero E6 cells lacking the STAT1-dependent interferon signaling pathway increased the numbers of viral PFU detected but did not affect the incidence of positive cultures. We conclude that culturable virus is correlated with SARS-CoV-2 NAATs detection only during early symptom onset and with high viral titers/low antibody titers in non-immunosuppressed patients.

Azithromycin Exhibits Activity Against Pseudomonas aeruginosa in Chronic Rat Lung Infection Model.

Pseudomonas aeruginosa forms biofilms in the lungs of chronically infected cystic fibrosis patients, which are tolerant to both the treatment of antibiotics and the host immune system. Normally, antibiotics are less effective against bacteria growing in biofilms; azithromycin has shown a potent efficacy in cystic fibrosis patients chronically infected with P. aeruginosa and improved their lung function. The present study was conducted to evaluate the effect of azithromycin on P. aeruginosa biofilm. We show that azithromycin exhibited a potent activity against P. aeruginosa biofilm, and microscopic observation revealed that azithromycin substantially inhibited the formation of solid surface biofilms. Interestingly, we observed that azithromycin restricted P. aeruginosa biofilm formation by inhibiting the expression of pel genes, which has been previously shown to play an essential role in bacterial attachment to solid-surface biofilm. In a rat model of chronic P. aeruginosa lung infection, we show that azithromycin treatment resulted in the suppression of quorum sensing-regulated virulence factors, significantly improving the clearance of P. aeruginosa biofilms compared to that in the placebo control. We conclude that azithromycin attenuates P. aeruginosa biofilm formation, impairs its ability to produce extracellular biofilm matrix, and increases its sensitivity to the immune system, which may explain the clinical efficacy of azithromycin in cystic fibrosis patients.

Sequential targeting of interferon pathways for increased host resistance to bacterial superinfection during influenza.

Bacterial co-infections represent a major clinical complication of influenza. Host-derived interferon (IFN) increases susceptibility to bacterial infections following influenza, but the relative roles of type-I versus type-II IFN remain poorly understood. We have used novel mouse models of co-infection in which colonizing pneumococci were inoculated into the upper respiratory tract; subsequent sublethal influenza virus infection caused the bacteria to enter the lungs and mediate lethal disease. Compared to wild-type mice or mice deficient in only one pathway, mice lacking both IFN pathways demonstrated the least amount of lung tissue damage and mortality following pneumococcal-influenza virus superinfection. Therapeutic neutralization of both type-I and type-II IFN pathways similarly provided optimal protection to co-infected wild-type mice. The most effective treatment regimen was staggered neutralization of the type-I IFN pathway early during co-infection combined with later neutralization of type-II IFN, which was consistent with the expression and reported activities of these IFNs during superinfection. These results are the first to directly compare the activities of type-I and type-II IFN during superinfection and provide new insights into potential host-directed targets for treatment of secondary bacterial infections during influenza.

In Vitro ADME, Preclinical Pharmacokinetics and Prediction of Human Pharmacokinetics of RBx14255, a Novel Ketolide with Pharmacodynamics Against Multidrug-Resistant Bacterial Pathogens.

PURPOSE: The preclinical pharmacokinetic and pharmacodynamic properties of a potent fluoroketolide RBx14255 against Streptococcus pneumoniae and Haemophilus influenzae was compared with telithromycin and human clinical dose was predicted for preclinical development. METHODS: The in vitro pharmacokinetic characterization was performed for solubility, Caco-2 permeability, microsomal stability, CYP inhibition and plasma protein binding. In vivo pharmacokinetic studies were performed in Swiss albino mice, Sprague Dawley rats and Beagle dogs. The pharmacodynamic studies were carried out in mouse against S. pneumoniae in systemic infection and against S. pneumoniae and H. influenzae in rat lung infection models. RESULTS: RBx14255 showed superior potency and efficacy in mouse and rat infection models. RBx14255 showed pH dependent solubility (0.41 mg/mL at pH 6.8 and >1 mg/mL at pH 1.2), moderate Caco-2 permeability (A to B: 12 nm/s) with high efflux ratio. It showed high plasma protein binding (>97%) in mouse and low binding (45- 70%) in rat, dog and human. The compound is mainly metabolized through CYP3A4. Pharmacokinetic parameters and absolute bioavailability of both, RBx14255 and telithromycin are similar in mouse. Both the ketolides showed low plasma clearance (18% of the normal hepatic blood flow rate) in mouse, moderate to high clearance in rat and dog. Mean oral bioavailability was high in mouse (≥85%), moderate in rat (RBx14255: 15% and telithromycin: 51%) and high to moderate in dog (RBx14255: 98% and telithromycin: 56%). The predicted efficacious dose for a 70 kg man ranges from 124 mg BID to 226 mg BID. CONCLUSION: RBx14255 displayed significantly better pharmacodynamics which correlates with the pharmacokinetic properties against S. pneumoniae and H. influenzae as compared to telithromycin. The predicted human efficacious doses are in the range of 124-226 mg, making it amenable to oral dosage form drug in human. This could be a promising clinical candidate for future studies.

Potential of the fluoroketolide RBx 14255 against Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae in an experimental murine meningitis model.

BACKGROUND: RBx 14255 is a fluoroketolide in pre-clinical evaluation with potent activity against MDR Gram-positive pathogens. OBJECTIVES: To investigate the efficacy of RBx 14255 against bacterial meningitis caused by Streptococcus pneumoniae, Neisseria meningitidis or Haemophilus influenzae in an experimental murine meningitis model. METHODS: In vitro activity of RBx 14255 was evaluated against clinical isolates of S. pneumoniae, N. meningitidis and H. influenzae. The in vivo efficacy of RBx 14255 was evaluated against bacterial meningitis, induced with S. pneumoniae 3579 erm(B), S. pneumoniae MA 80 erm(B), N. meningitidis 1852 and H. influenzae B1414 in a murine meningitis model. RESULTS: RBx 14255 showed strong in vitro bactericidal potential against S. pneumoniae, N. meningitidis and H. influenzae with MIC ranges of 0.004-0.1, 0.03-0.5 and 1-4 mg/L, respectively. In a murine meningitis model, a 50 mg/kg dose of RBx 14255, q12h, resulted in significant reduction of bacterial counts in the brain compared with the pretreatment control. The concentration of RBx 14255 in brain tissue correlated well with the efficacy in this mouse model. CONCLUSIONS: RBx 14255 showed superior bactericidal activity in time-kill assays in vitro and in vivo in an experimental murine meningitis model. RBx 14255 could be a promising candidate for future drug development against bacterial meningitis.

DS86760016, a Leucyl-tRNA Synthetase Inhibitor with Activity against Pseudomonas aeruginosa.

DS86760016 is a new leucyl-tRNA-synthetase inhibitor at the preclinical development stage. DS86760016 showed potent activity against extended-spectrum multidrug-resistant Pseudomonas aeruginosa isolated from clinical samples and in vitro biofilms. In a murine catheter-associated urinary tract infection model, DS86760016 treatment resulted in significant eradication of P. aeruginosa from the kidney, bladder, and catheter without developing drug resistance. Our data suggest that DS86760016 has the potential to act as a new drug for the treatment of Pseudomonas infections.

Design, preparation, and evaluation of liposomal gel formulations for treatment of acne: in vitro and in vivo studies.

The study highlights the significance of co-application of bioactive components into liposomal gel formulations and their comparison to azithromycin for treatment of Acne. A Design of Experiments (DoE) approach was utilized to obtain optimized liposomal formulation encapsulating curcumin, with size and zeta potential of ∼100 nm and ∼14 mV, respectively, characterized by DLS, HR-TEM, FESEM, and AFM. The curcumin liposomal dispersion depicted excellent stability over the period of 60 days, which was further converted in gel form using Carbopol. Pharmacokinetics of curcumin-loaded liposomal gel showed that Tmax for curcumin was achieved within 1 h of post application in both stratum corneum and skin, indicating quick penetration of nano-sized liposomes. Stratum corneum depicted Cmax of 688.3 ng/mL and AUC0-t of 5857.5 h × ng/mL, while the skin samples displayed Cmax of 203.3 ng/gm and AUC0-t of 2938.1 h × ng/gm. Lauric acid and azithromycin liposomal gel formulations were prepared as per the optimum parameters obtained by DoE. In antibacterial activity using agar diffusion assay, lauric acid gel formulation revealed ∼1.5 fold improved antibacterial effect than curcumin gel formulation. Interestingly, their co-application (1:1) exhibited significantly enhanced antibacterial effect against both macrolide-sensitive (1.81 versus 1.25 folds) and resistant strains of P. acnes (2.93 versus 1.22 folds) than their individual counterparts. The in vivo studies in rat ear model displayed a ∼2 fold reduction in comedones count and cytokines (TNF-α and IL-1ß) on co-application with curcumin and lauric acid liposomal gel compared to placebo treated group.

Synthesis and evaluation of thiomannosides, potent and orally active FimH inhibitors.

FimH is a type I fimbrial lectin located at the tip of type-1 pili of Gram-negative uropathogenic Escherichia coli (UPEC) guiding its ability to adhere and infect urothelial cells. Accordingly, blocking FimH with small molecule inhibitor is considered as a promising new therapeutic alternative to treat urinary tract infections caused by UPEC. Herein, we report that compounds having the S-glycosidic bond (thiomannosides) had improved metabolic stability and plasma exposures when dosed orally. Especially compound 5h showed the potential to inhibit biofilm formation and also to disrupt the preformed biofilm. And compound 5h showed prophylactic effect in UTI model in mice.

Concomitant Delivery of Paclitaxel and NuBCP-9 peptide for synergistic enhancement of cancer therapy.

Paclitaxel (PTX) is a microtubule inhibitor administered as an albumin-bound nanoformulation for the treatment of breast cancer. However, the effectiveness of PTX is limited by resistance mechanisms mediated in part by upregulation of the anti-apoptotic BCL-2 and P-glycoprotein (P-gp). Present investigation was designed to study the synergistic potential of NuBCP-9 and PTX loaded polymeric nanoparticles to minimize the dose and improve the efficacy and safety. PTX and NuBCP-9 loaded polylactic acid-polyethylene glycol-polypropylene glycol-polyethylene glycol [PLA-(PEG-PPG-PEG)] nanoparticles were prepared by double emulsion solvent evaporation method. PTX and NuBCP-9 loaded NPs displayed an average size of 90 nm with spherical morphology. PTX and NuBCP-9 dual loaded NPs reducedIC50 by ~40-fold and acted synergistically. Treatment of the syngeneic EAT mice with PTX-NuBCP-9/NPs resulted in improved efficacy than that alone treated mice. Overall, the concomitant delivery PTX and NuBCP-9 loaded NPs showed superior activity than that of PTX and NuBCP-9 alone treated mice.

In Vitro and In Vivo Activities of DS-2969b, a Novel GyrB Inhibitor, and Its Water-Soluble Prodrug, DS11960558, against Methicillin-Resistant Staphylococcus aureus.

DS-2969b is a novel GyrB inhibitor under clinical development. In this study, the in vitro activity of DS-2969b and the in vivo activities of DS-2969b and its water-soluble prodrug, DS11960558, against methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. DS-2969b inhibited the supercoiling activity of S. aureus DNA gyrase and the decatenation activity of its topoisomerase IV. DS-2969b showed antibacterial activity against Gram-positive aerobes but not against Gram-negative aerobes, except for Moraxella catarrhalis and Haemophilus influenzae DS-2969b was active against MRSA with an MIC90 of 0.25 µg/ml, which was 8-fold lower than that of linezolid. The presence of a pulmonary surfactant did not affect the MIC of DS-2969b. DS-2969b showed time-dependent slow killing against MRSA. The frequency of spontaneous resistance development was less than 6.2 × 10-10 in all four S. aureus isolates at 4× MIC of DS-2969b. In a neutropenic MRSA-induced murine muscle infection model, DS-2969b was more efficacious than linezolid by both the subcutaneous and oral routes. DS-2969b and DS11960558 showed efficacy in a neutropenic murine MRSA lung infection model. The pharmacokinetics and pharmacodynamics of DS-2969b and DS11960558 against MRSA were characterized in a neutropenic murine thigh infection model; the percentage of time during the dosing period in which the free drug concentration exceeded the MIC (fTMIC) correlated best with in vivo efficacy, and the static percent fTMIC was 43 to 49%. A sufficient fTMIC was observed in a phase 1 multiple-ascending-dose study of DS-2969b given orally at 400 mg once a day. These results suggest that DS11960558 and DS-2969b have potential for use as intravenous-to-oral step-down therapy for treating MRSA infections with a higher efficacy than linezolid.

In vivo efficacy and pharmacokinetics of bi-aryl oxazolidinone RBx 11760 loaded polylactic acid-polyethylene glycol nanoparticles in mouse hematogenous bronchopneumonia and rat groin abscess caused by Staphylococcus aureus.

RBx 11760 is a bi-aryl oxazolidinone antibacterial agent active against Staphylococcus aureus but has poor solubility. Here we have encapsulated RBx 11760 in PLA-PEG NPs with an aim to improve physicochemical, pharmacokinetics and in vivo efficacy. The average size and zeta potential of RBx 11760 loaded NPs were found to be 106.4 nm and -22.2 mV, respectively. The absolute size of nanoparticles by HRTEM was found to be approximately 80 nm. In vitro antibacterial agar well diffusion assay showed clear zone of inhibition of bacterial growth. In pharmacokinetic study, nanoparticle showed 4.6-fold and 7-fold increase in AUCinf and half-life, respectively, as compared to free drug. RBx 11760 nanoparticle significantly reduced bacterial counts in lungs and improved the survival rate of immunocompromised mice as compared to free drugs. Thus, RBx 11760 loaded nanoparticles have strong potential to be used as nanomedicine against sensitive and drug resistant Staphylococcus aureus infections.