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.

Vaginosis: Advances in new therapeutic development and microbiome restoration.

Vaginosis is a condition experienced by most women at least once in their lifetime. This condition arises due to the imbalance in the microbiome of the vaginal ecosystem. Most of the pathogens of this disease are organisms which are commonly found in a normal healthy vagina. The vaginal microbiome is important as they act as a primary defence against secondary infections and Sexually transmitted diseases and infections (STDs and STIs). The vagina is mostly dominated by Lactobacillus along with other microbes including Gardnerella vaginalis, Atopobium vaginae., Prevotella spp., Mobiluncus spp., etc. Vaginal microbiome also includes Candida albicans and other species of the genus. The ratio in which these species are present varies from person to person and the dominant species decides the whether a vagina is "normal" or not. Lactobacillus dominated vagina is considered normal and if dominated by Gardnerella and such it is considered to be Bacterial vaginosis (BV) and similarly for Vulvovaginal Candidiasis (VVC). The microbiome also undergoes changes during menstrual cycles and menopausal stages. Due to the dynamic nature of this microbiome, it is tough to perfectly restore the balance. But several treatments are currently available with antibiotics like Clindamycin and derivatives of 5-nitroimidazole drugs like Metronidazole. The extensive use and the non-adherence to the treatment regimen has led to drug resistance through biofilm formation, efflux pumps, single nucleotide polymorphisms and resulting recurrent episode of vaginosis in women. Alternative medicines, preparations from plant sources, anti-microbial peptides and nano formulations are also being explored. Most of these medicines tend to focus on reducing the pathogen load rather than restoring the balance of the ecosystem. Vaginal microbiome transplant, an effort to restore the normalcy in the vaginal environment is becoming a popular treatment. In this review we discuss about the types of vaginosis, available treatments, challenges in treating the condition and the new drugs that are under investigation.

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.

Plasmodium vivax liver stage assay platforms using Indian clinical isolates.

BACKGROUND: Vivax malaria is associated with significant morbidity and economic loss, and constitutes the bulk of malaria cases in large parts of Asia and South America as well as recent case reports in Africa. The widespread prevalence of vivax is a challenge to global malaria elimination programmes. Vivax malaria control is particularly challenged by existence of dormant liver stage forms that are difficult to treat and are responsible for multiple relapses, growing drug resistance to the asexual blood stages and host-genetic factors that preclude use of specific drugs like primaquine capable of targeting Plasmodium vivax liver stages. Despite an obligatory liver-stage in the Plasmodium life cycle, both the difficulty in obtaining P. vivax sporozoites and the limited availability of robust host cell models permissive to P. vivax infection are responsible for the limited knowledge of hypnozoite formation biology and relapse mechanisms, as well as the limited capability to do drug screening. Although India accounts for about half of vivax malaria cases world-wide, very little is known about the vivax liver stage forms in the context of Indian clinical isolates. METHODS: To address this, methods were established to obtain infective P. vivax sporozoites from an endemic region in India and multiple assay platforms set up to detect and characterize vivax liver stage forms. Different hepatoma cell lines, including the widely used HCO4 cells, primary human hepatocytes as well as hepatocytes obtained from iPSC's generated from vivax patients and healthy donors were tested for infectivity with P. vivax sporozoites. RESULTS: Both large and small forms of vivax liver stage are detected in these assays, although the infectivity obtained in these platforms are low. CONCLUSIONS: This study provides a proof of concept for detecting liver stage P. vivax and provide the first characterization of P. vivax liver stage forms from an endemic region in India.

Synthesis, antituberculosis studies and biological evaluation of new quinoline derivatives carrying 1,2,4-oxadiazole moiety.

Tuberculosis is the infectious disease caused by mycobacterium tuberculosis (Mtb), responsible for the utmost number of deaths annually across the world. Herein, twenty-one new substituted 1,2,4-oxadiazol-3-ylmethyl-piperazin-1-yl-quinoline derivatives were designed and synthesized through multistep synthesis followed by in vitro evaluation of their antitubercular potential against Mtb WT H37Rv. The compound QD-18 was found to be promising with MIC value of 0.5 µg/ml and QD-19 to QD-21 were also remarkable with MIC value of 0.25 µg/ml. Additionally, we have carried out experiments to confirm the metabolic stability, cytotoxicity and pharmacokinetics of these compounds along with kill kinetics of QD-18. These compounds were found to be orally bioavailable and highly effective. Altogether, these results indicate that QD-18, QD-19, QD-20 and QD-21 are promising lead compounds for the development of a novel chemical class of antitubercular drugs.

Enhanced anticancer efficacy of histone deacetyl inhibitor, suberoylanilide hydroxamic acid, in combination with a phosphodiesterase inhibitor, pentoxifylline, in human cancer cell lines and in-vivo tumor xenografts.

Vorinostat [suberoylanilide hydroxamic acid (SAHA)], a histone deacetylase inhibitor, shows limited clinical activity against solid tumors when used alone. The methyl xanthine drug, pentoxifylline (PENT), has been described to have antitumor properties. The aim of this study was to look for the enhanced anticancer activities of both agents when used in combination at doses lower than their respective efficacy dose when used alone. We investigated the antitumor potential of this novel combination in vitro and in vivo. The combination index was assessed for these two drugs to look for synergistic antiproliferative activity against a broad spectrum of human cancer cell lines. Consistent additive to synergistic interactions were observed in HCT116 cells when PENT was combined with SAHA at all drug tested concentrations. The combination of SAHA and PENT induces chromatin condensation and apoptosis downstream of the pan histone deacetylase inhibition and phosphodiesterase regulation, leading to subsequent cell cycle arrest at their lower tested concentrations. Further, the ability of this combination to inhibit angiogenesis, both in vitro and in vivo, was examined and a significant inhibition in tube formation in HUVEC cells and neovascularization of Matrigel plug was observed. A significant inhibition in tumor growth was observed in severe combined immunodeficient mice bearing HCT116 (colon) and PC3 (prostate) human xenografts treated with SAHA (30 mg/kg, intraperitoneal) in combination with PENT (60 mg/kg, intraperitoneal), with no loss in body weight and 100% survival. In conclusion, these findings indicate the enhanced anticancer activity of SAHA in combination with PENT both in vitro and in vivo.

In vivo effective dibenzo[b,d]furan-1-yl-thiazoles as novel PDE-4 inhibitors.

Herein we report the synthesis, PDE-4B and TNF-α inhibitory activities of a few dibenzo[b,d]furan-1-yl-thiazole derivatives. The hydroxycyclohexanol amide derivatives 14, 18, 24, 29, 31 and 33 exhibited promising in vitro PDE-4B and TNF-α inhibitory activities. Compound 24 showed good systemic availability in preclinical animal models and was also found to be non-toxic (exploratory mutagenicity test). Further it exhibited promising results in in vivo asthma/COPD and Uveitis models.

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).

A novel animal model of metabolic syndrome with non-alcoholic fatty liver disease and skin inflammation.

CONTEXT: Metabolic syndrome and non-alcoholic fatty liver disease (NAFLD) are the emerging co-morbidities of skin inflammation. Occurrence of skin inflammation such as psoriasis is substantially higher in NAFLD patients than normal. Currently, there are no animal models to study the interaction between these co-morbidities. OBJECTIVE: The present study seeks to develop a simple mouse model of NAFLD-enhanced skin inflammation and to study the effect of NAFLD on different parameters of skin inflammation. MATERIALS AND METHOD: Metabolic syndrome and NAFLD were induced in C57BL/6 mice by feeding high-fat diet (HFD, 60% kcal) and high fructose liquid (HFL, 40% kcal) in drinking water. Skin inflammation was induced by repeated application of oxazolone (1% sensitization and repeated 0.5% challenge) in both normal and NAFLD mice and various parameters of skin inflammation and NAFLD were measured. RESULTS: HFD and HFL diet induced obesity, hyperglycemia, hyperinsulinemia, and histological features of NAFLD in mice. Oxazolone challenge significantly increased ear thickness, ear weight, MPO activity, NF-κB activity, and histological features of skin inflammation in NAFLD mice as compared with normal mice. Overall, induction of oxazolone-induced skin inflammation was more prominent in NAFLD mice than normal mice. Hence, HFD and HFL diet followed by topical oxazolone application develops metabolic syndrome, NAFLD, and enhanced skin inflammation in mice. DISCUSSION AND CONCLUSION: This simple model can be utilized to evaluate a therapeutic strategy for the treatment of metabolic syndrome and NAFLD with skin inflammation and also to understand the nexus between these co-morbidities.

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.

Quinolone-based HDAC inhibitors.

HDAC inhibitors emerged as promising drug candidates in combating wide variety of cancers. At present, two of the compounds SAHA and Romidepsin were approved by FDA for cutaneous T-cell lymphoma and many are in various clinical phases. A new quinolone cap structure was explored with hydroxamic acid as zinc-binding group (ZBG). The pan HDAC inhibitory and antiproliferative activities against three human cancer cell lines HCT-116 (colon), NCI-H460 (lung) and U251 (glioblastoma) of the compounds (4a-4w) were evaluated. Introduction of heterocyclic amines in CAP region increased the enzyme inhibitory and antiproliferative activities and few of the compounds tested are metabolically stable in both MLM and HLM.

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.

Discovery of adamantane based highly potent HDAC inhibitors.

Herein, we report the development of highly potent HDAC inhibitors for the treatment of cancer. A series of adamantane and nor-adamantane based HDAC inhibitors were designed, synthesized and screened for the inhibitory activity of HDAC. A number of compounds exhibited GI50 of 10-100 nM in human HCT116, NCI-H460 and U251 cancer cells, in vitro. Compound 32 displays efficacy in human tumour animal xenograft model.

Bioanalytical method development, validation and quantification of dorsomorphin in rat plasma by LC-MS/MS.

The present investigation describes the development and validation of a sensitive liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method for the estimation of dorsomorphin in rat plasma. A sensitive LC-MS/MS method was developed using multiple reaction monitoring mode, with the transition of m/z (Q1/Q3) 400.2/289.3 for dorsomorphin and m/z (Q1/Q3) 306.2/236.3 for zaleplon. Chromatographic separation was achieved on a reverse phase Agilent XDB C18 column (100 × 4.6 mm, 5 µm). The mobile phase consisted of acetonitrile and 5 mm ammonium acetate buffer (pH 6.0) 90:10 v/v, at a flow rate of 0.8 mL/min. The effluence was ionized in positive ion mode by electrospray ionization (ESI) and quantitated by mass spectrometry. The retention times of dorsomorphin and internal standard were found to be 2.13 and 1.13 min, respectively. Mean extraction recovery of dorsomorphin and internal standard in rat plasma was above 80%. Dorsomorphin calibration curve in rat plasma was linear (r(2) ≥ 0.99) ranging from 0.005 to 10 µg/mL. Inter-day and intra-day precision and accuracy were found to be within 85-115% (coefficient of variation). This method was successfully applied for evaluation of the oral pharmacokinetic profile of dorsomorphin in male Wistar rats.

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.

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.

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.