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Quality risk assessment following ICH Q9 principles is an important activity to ensure optimal clinical efficacy and safety of a drug product. Typically, risk assessment is focused on product performance wherein critical material attributes, formulation variables, and process parameters are evaluated from a manufacturing perspective. Extending ICH Q9 principles to biopharmaceutics risk assessment to identify factors that can impact in vivo performance is an upcoming area. This is evident by recent regulatory trends wherein a new term critical bioavailability attributes (CBA) has been coined to identify such factors. Although significant work has been performed for biopharmaceutics risk assessment for new molecules, there is a need for harmonized biopharmaceutics risk assessment workflow for generic submissions. In this manuscript, we attempted to provide a framework for performing biopharmaceutics risk assessment for generic regulatory submissions. A detailed workflow for performing biopharmaceutics risk assessment includes identification of initial CBA (iCBA), their confirmatory evaluation followed by definition of the control strategy. Tools for biopharmaceutics risk assessment, i.e., bio-discriminatory dissolution method and physiologically based biopharmaceutics modeling (PBBM) were discussed from a practical perspective. Furthermore, a case study for CBA evaluation using PBBM modeling for an extended-release product for regulatory submission has been described using the proposed workflow. Finally, future directions of integrating CBA evaluation, biopharmaceutics risk assessment to the FDA Knowledge Aided Structured Assessment (KASA) initiative, the necessity of risk assessment templates, and knowledge sharing between industry and academia are discussed. Overall, the work described in this manuscript can facilitate and provide guidance for biopharmaceutics risk assessment for generic submissions., (© 2023. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Background: Mycobacterium tuberculosis is a bacterium that has historically had a substantial impact on human health. Despite advances in understanding and management of tuberculosis (TB), the disease remains a crucial problem that necessitates ongoing work to discover effective drugs, minimize transmission, and improve global health outcomes., Methods: The purpose of this study is to use molecular docking and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analyses to explore the molecular interactions of different proteins that are involved in mycolic acid biosynthesis (HadAB, InhA, KasA, FabD, and beta-ketoacyl-acyl carrier protein synthase III) of M. tuberculosis with Demospongiae metabolites. The docking findings were evaluated using the glide gscore, and the top 10 compounds docked against each protein receptor were chosen. Furthermore, the selected compounds underwent ADMET analysis, indicating that they have the potential for therapeutic development., Results: Among the selected compounds, makaluvamine G showed the highest binding affinity against HadAB, psammaplysin E showed highest binding affinity against InhA, pseudotheonamide D showed the highest binding affinity against KasA protein, dinordehydrobatzelladine B showed the highest binding affinity against FabD, and nagelamide X showed the highest binding affinity against beta-ketoacyl-acyl carrier protein synthase III. Additionally, molecular mechanics generalized born surface area (MM-GBSA) binding free energy and molecular dynamics simulations were used to support the docking investigations., Conclusion: The results of the study suggest that these compounds may eventually be used to treat TB. However, computer validations were included in this study, and more in vitro research is required to turn these prospective inhibitors into clinical drugs., Competing Interests: None

Recent studies have reported the β-ketoacyl-acyl carrier protein KasA as a druggable target for Mycobacterium tuberculosis . This review summarizes the current status of major classes of KasA inhibitors with an emphasis on significant contributions from structure-based design methods leveraging X-ray crystal structures of KasA alone and in complex with inhibitors. The issues addressed within each inhibitor class are discussed while detailing the characterized interactions with KasA and structure-activity relationships. A critical analysis of these findings should lay the foundation for new KasA inhibitors to study the basic biology of M. tuberculosis and to form the basis of new antitubercular molecules of clinical significance with activity against drug-sensitive and drug-resistant infections., Competing Interests: MN and JF are listed as inventors on patent filings pertinent to the indole and indazole compounds mentioned in this manuscript as employees of Rutgers University. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rudraraju, Daher, Gallardo-Macias, Wang, Neiditch and Freundlich.)

Background: The global rise in drug-resistant Mycobacterium tuberculosis (M.tb), and especially the significant prevalence of isoniazid (INH)-resistance constitute a significant challenge to global health. Therefore, the present study aimed to investigate mutations in prevalent gene loci-involved in INH-resistance phenotype-among M.tb clinical isolates from southwestern Iran., Methods: Drug susceptibility testing (DST) was performed using the conventional proportional method on confirmed 6620 M.tb clinical isolates, and in total, 15 INH-resistant and 18 INH-susceptible isolates were included in the study. Fragments of six genetic loci most related to INH-resistance (katG, inhA promoter, furA, kasA, ndh, oxyR-ahpC intergenic region) were PCR-amplified and sequenced. Mutations were explored by pairwise alignment with the M.tb H37Rv genome., Results: The analysis of gene loci revealed 13 distinct mutations in INH-resistant isolates. 60% (n = 9) of the INH-resistant isolates had mutations in katG, with codon 315 predominately (53.3%, n = 8). Mutation at InhA - 15 was found in 20% (n = 3) of resistant isolates. 26.7% (n = 4) of the INH-resistant isolates had kasA mutations, of which G269S substitution was the most common (20%, n = 3). The percentage of mutations in furA, oxyR-ahpC and ndh was 6.7% (n = 1), 46.7% (n = 7), and 20% (n = 3), respectively. Of the mutations detected in ndh and oxyR-ahpC, 5 were also observed in INH-susceptible isolates. This study revealed seven novel mutations, four of which were exclusively in resistant isolates., Conclusions: This study supports the usefulness of katG and inhA mutations as a predictive molecular marker for INH resistance. Co-detection of katG S315 and inhA-15 mutations identified 73.3% (11 out of 15 isolates) of INH-resistant isolates., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Published Mycobacterium tuberculosis β-ketoacyl-ACP synthase KasA inhibitors lack sufficient potency and/or pharmacokinetic properties. A structure-based approach was used to optimize existing KasA inhibitor DG167. This afforded indazole JSF-3285 with a 30-fold increase in mouse plasma exposure. Biochemical, genetic, and X-ray studies confirmed JSF-3285 targets KasA. JSF-3285 offers substantial activity in an acute mouse model of infection and in the corresponding chronic infection model, with efficacious reductions in colony-forming units at doses as low as 5 mg/kg once daily orally and improvement of the efficacy of front-line drugs isoniazid or rifampicin. JSF-3285 is a promising preclinical candidate for tuberculosis., Competing Interests: Declaration of Interests D.I., D.Awasthi, G.C.C., D.Alland, M.B.N., P.K., and J.S.F. are listed as inventors on patent filings pertinent to the indole and indazole compounds disclosed in this manuscript as employees of Rutgers University., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In the course of optimizing a novel indazole sulfonamide series that inhibits β-ketoacyl-ACP synthase (KasA) of Mycobacterium tuberculosis , a mutagenic aniline metabolite was identified. Further lead optimization efforts were therefore dedicated to eliminating this critical liability by removing the embedded aniline moiety or modifying its steric or electronic environment. While the narrow SAR space against the target ultimately rendered this goal unsuccessful, key structural knowledge around the binding site of this underexplored target for TB was generated to inform future discovery efforts.

The enzyme β-Ketoacyl ACP synthase I (KasA) is a potent drug target in mycolic acid pathway of Mycobacterium tuberculosis (Mtb). In the present study, we investigated the structural dynamics of wild-type (WT) and mutants KasA (D66N, G269S, G312S, and F413L) in both monomer and dimer form to provide insight into protein structural stability. To gain better understanding of structural flexibility of KasA, combined molecular dynamics and essential dynamics were employed to analyze the conformational changes induced by non-active site mutations. The results confirm that non-active site mutations lower the structural stability in dimer KasA as compared to WT. The protein network topology and close residue interactions of WT and mutant residues of KasA have been predicted through residue interaction network analysis (RIN). Non-active site mutations distort RIN architecture and subsequently affect the drug binding landscape. T-pad associated with mode vector analysis comprehensively pronounces the structural impact caused by non-active site mutations. It also identified the critical fluctuating residues present in the gate segment (GS) region (115-147). The non-active site mutations altered the structural stability of the mutant protein structures, and these mutations may be a cause for the resistance mechanism of KasA against anti-tuberculosis drugs. Further, it is observed that dimer mutant KasA proteins display much more structural flexibility than WT at the ligand binding site which is evident from the binding site analysis and hydrogen bond interaction patterns. This study provides a better understanding of the structural dynamic behaviour of KasA mutants, thereby facilitating the need to find a novel and potent inhibitor against Mtb., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Tuberculosis (TB) is a leading infectious disease which kills a huge number of people every year over a decade, caused by Mycobacterium tuberculosis. The conventional drugs in the market are no longer effective due to the increasing mycobacterial resistance to antibiotics. Hence, the need of finding efficient drugs to solve this multiple drug resistant factor is becoming an immediate issue. The first-line drugs in current practice for the treatment of TB emphasize on mycolic acid, which protects the bacteria from an immune response generated by the host. A key enzyme involved in this mycolic acid biosynthesis, M. tuberculosis beta-ketoacyl-ACP synthase A (MTB-KasA) is a prime candidate in this study. Thiolactomycin is a natural product inhibitor has shown good inhibitory activity against MTB-KasA. Hence, several thiolactomycin derivatives collected from the literature were taken for absorption, distribution, metabolism, excretion and toxicity prediction, molecular docking and molecular dynamics simulation studies with MTB-KasA. The in silico drug designing methods used in this study suggests that the thiolactomycin derivatives are having a better binding activity against MTB-KasA and among them the ligand C14 is identified as a promising lead molecule to inhibit multidrug resistance of tuberculosis by showing a long time binding activity.

We report GSK3011724A (DG167) as a binary inhibitor of β-ketoacyl-ACP synthase (KasA) in Mycobacterium tuberculosis Genetic and biochemical studies established KasA as the primary target. The X-ray crystal structure of the KasA-DG167 complex refined to 2.0-Å resolution revealed two interacting DG167 molecules occupying nonidentical sites in the substrate-binding channel of KasA. The binding affinities of KasA to DG167 and its analog, 5g, which binds only once in the substrate-binding channel, were determined, along with the KasA-5g X-ray crystal structure. DG167 strongly augmented the in vitro activity of isoniazid (INH), leading to synergistic lethality, and also synergized in an acute mouse model of M. tuberculosis infection. Synergistic lethality correlated with a unique transcriptional signature, including upregulation of oxidoreductases and downregulation of molecular chaperones. The lead structure-activity relationships (SAR), pharmacokinetic profile, and detailed interactions with the KasA protein that we describe may be applied to evolve a next-generation therapeutic strategy for tuberculosis (TB). IMPORTANCE Cell wall biosynthesis inhibitors have proven highly effective for treating tuberculosis (TB). We discovered and validated members of the indazole sulfonamide class of small molecules as inhibitors of Mycobacterium tuberculosis KasA-a key component for biosynthesis of the mycolic acid layer of the bacterium's cell wall and the same pathway as that inhibited by the first-line antitubercular drug isoniazid (INH). One lead compound, DG167, demonstrated synergistic lethality in combination with INH and a transcriptional pattern consistent with bactericidality and loss of persisters. Our results also detail a novel dual-binding mechanism for this compound as well as substantial structure-activity relationships (SAR) that may help in lead optimization activities. Together, these results suggest that KasA inhibition, specifically, that shown by the DG167 series, may be developed into a potent therapy that can synergize with existing antituberculars., (Copyright © 2018 Kumar et al.)

Introduction: Vyaghriharitaki Avaleha (VHA), a polyherbal classical Ayurvedic formulation has been used in Kasa (cough), Swasa (asthma), Rajayakshma (tuberculosis) etc., conditions., Aim: To evaluate the clinical efficacy of VHA in the management of chronic bronchitis., Materials and Methods: Patients of chronic bronchitis were given 10 g of VHA twice a day with lukewarm water before meals for 12 weeks. Improvement in clinical symptoms of chronic bronchitis as the primary outcome measures and St. George's Respiratory Questionnaire scores as secondary outcome measure was studied., Results: Out of 66 enrolled patients, 61 completed the treatment schedule. The results show that VHA provides statistically significant improvement with P < 0.001 in both primary and secondary outcome measures., Conclusion: VHA can be considered as a safe and effective formulation in the management of chronic bronchitis.

The survival of Mycobacterium tuberculosis depends on mycolic acids, very long α-alkyl-β-hydroxy fatty acids comprising 60-90 carbon atoms. However, despite considerable efforts, little is known about how enzymes involved in mycolic acid biosynthesis recognize and bind their hydrophobic fatty acyl substrates. The condensing enzyme KasA is pivotal for the synthesis of very long (C38-42) fatty acids, the precursors of mycolic acids. To probe the mechanism of substrate and inhibitor recognition by KasA, we determined the structure of this protein in complex with a mycobacterial phospholipid and with several thiolactomycin derivatives that were designed as substrate analogs. Our structures provide consecutive snapshots along the reaction coordinate for the enzyme-catalyzed reaction and support an induced fit mechanism in which a wide cavity is established through the concerted opening of three gatekeeping residues and several α-helices. The stepwise characterization of the binding process provides mechanistic insights into the induced fit recognition in this system and serves as an excellent foundation for the development of high affinity KasA inhibitors.

Bordetella pertussis continues to circulate even in countries with good childhood vaccination coverage. Sporadic cases are still observed either due to no vaccination, incomplete vaccination or as a result of failure of vaccination. Though acute in nature it has a longer disease span with multiple life-threatening complications despite adequate management. This study was undertaken to study the effect of Sneha Basti (oil enema) in pertussis in relation to reduction in intensity of cough and complications of the disease when administered along with the conventional pharmacological therapy. Patients with the clinical picture mimicking pertussis were screened, investigated for pertussis and appropriate conventional pharmacological therapy of Erythromycin a preferred antimicrobial agent was started. Concurrently, Sneha Basti was administered to these two children. Both patients showed favourable results in terms of reduction of Kasa (cough) and the disease period with a faster recovery when compared to conventional therapy alone as has been observed routinely. No complications incurred during the complete disease span. It was concluded that if the pharmacological therapy is augmented with the complimentary systems of medicines, it can reduce the prolonged span and intensity of the disease and prevent other complications.

Mineral drugs play an important role in Ayurvedic therapeutics. Looking to the superiority of the metals and minerals to that of herbal and animal drugs, the Rasavaidyas went on experimenting clinical trials over lot of metals and minerals and systematically separated some of them which were exclusively active therapeutically. Swarnamakshika is one such mineral which after proper purification and incineration become highly potent and utilised for diseases like Jwara, Pandu, Prameha, Shwasa, Kasa, Kushtha, Anidra, Apasmar etc.