Browsing by Author "Singh, Vinayak"
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- ItemOpen AccessIdentification of new therapeutic compounds against carbapenemase-producing Klebsiella pneumoniae using a novel agar-based high-throughput microarray compound screening approach(2024) Mahlangu, Zandile; Singh, VinayakKlebsiella pneumoniae, a Gram-negative bacterium, is a common cause of hospital-acquired pneumonia, urinary tract infections (UTIs), bacteraemia, septicaemia, and wound/surgical site infections. Over the years, the pathogen developed resistance to most β-lactam antibacterial drugs used in clinical care. The most important of these drugs are carbapenems – a class of broad-spectrum antibiotics reserved for the treatment of infections caused by multidrug-resistant (MDR) bacteria. The emergence of carbapenem resistance has resulted in limited or ineffective treatment options for MDR K. pneumoniae. This prompted a global initiative to prioritize this pathogen for new drug discovery and development to reduce the severity of such infections. Therefore, this study aimed to develop a cost-effective high-throughput antibacterial microarray screening platform to accelerate drug screening efforts against carbapenemresistant K. pneumoniae. To achieve this, an agar-based microarray assay, modeled based on the standard disc diffusion assay, was developed by optimizing the assay parameters, including the cellulose nanofiber (CNF) immobilization matrix, polymer component of the printing solution, inoculum, agar thickness, and agar concentration. The optimized parameters for effectively running the high-throughput antibacterial screen were set at 1.5% (w/v) CNF immobilization matrix into which spots were printed, 1.6% hydroxypropyl methylcellulose printing solution – serving as a binding agent for compounds, inoculum density ranging between ~1 x 105 and 2 x 105 CFU/mL, 0.8% (w/v) seeding agar which formed a ~0.4 mm layer into which printed compounds diffused into and affected bacteria, and 1.5% (w/v) plain agar which formed a ~1.6 mm layer overlaying the thin seeded agar layer. A proof-of-concept of the developed assay was demonstrated by screening a panel of 12 FDA-approved antibacterial drugs against five bacterial strains with varying degrees of drug susceptibility (i.e., K. pneumoniae 1705, K. pneumoniae 700603, K. pneumoniae 33495, Staphylococcus aureus 25923, and Escherichia coli 25922). Varying patterns and levels of bacterial susceptibility were observed on the platform, with three (polymyxin B sulfate, colistin sulfate, and gentamicin sulfate) and two (polymyxin B sulfate and colistin sulfate) test drugs showing antibacterial activity against K. pneumoniae 1705 and 700603, respectively. Drugsusceptible K. pneumoniae 33495, E. coli 25922, and S. aureus 25923 exhibited susceptibilities to six (polymyxin B sulfate, colistin sulfate, gentamicin sulfate, kanamycin sulphate, G418 sulfate, and tobramycin), seven (polymyxin B sulfate, colistin sulfate, gentamicin sulfate, kanamycin sulphate, G418 sulfate, tobramycin, neomycin sulfate), and five (kanamycin sulphate, neomycin sulfate, gentamicin sulfate, G418 sulfate, tobramycin) drugs, respectively. The optimised assay was used to screen a diverse library of 4154 compounds against a carbapenem-resistant K. pneumoniae (ATCC BAA 1705) and a drug-susceptible K. pneumoniae (ATCC 33495) strains. The primary screen data was mined for antibacterial hits using an assay specific two-step data analysis method (i.e., combination of Z-score data analysis method and image-based analysis). The screening did not yield hits against the carbapenemresistant K. pneumoniae. A secondary screen of 5% of inactive compounds, with Z-scores ranging between standard deviations (SD) of -1 and -2, selected from the primary screen against K. pneumoniae 1705 and 33495, was conducted to test the accuracy and reliability the of the two-step data analysis method and the hit selection criteria. A percentage agreement of >99% was obtained between the results obtained in the primary screen (using the agar-based microarray assay) and those obtained in the secondary screen (using the broth microdilution assay). The developed assay showed great potential as a rapid primary screening tool for anti-K. pneumoniae compounds.
- ItemOpen AccessReal-time investigation of tuberculosis transmission: developing the Respiratory Aerosol Sampling Chamber (RASC)(Public Library of Science, 2016) Wood, Robin; Morrow, Carl; III, Clifton E Barry; Bryden, Wayne A; Call, Charles J; Hickey, Anthony J; Rodes, Charles E; Scriba, Thomas J; Blackburn, Jonathan; Issarow, Chacha; Mulder, Nicola; Woodward, Jeremy; Moosa, Atica; Singh, Vinayak; Mizrahi, Valerie; Warner, Digby FKnowledge of the airborne nature of respiratory disease transmission owes much to the pioneering experiments of Wells and Riley over half a century ago. However, the mechanical, physiological, and immunopathological processes which drive the production of infectious aerosols by a diseased host remain poorly understood. Similarly, very little is known about the specific physiological, metabolic and morphological adaptations which enable pathogens such as Mycobacterium tuberculosis ( Mtb ) to exit the infected host, survive exposure to the external environment during airborne carriage, and adopt a form that is able to enter the respiratory tract of a new host, avoiding innate immune and physical defenses to establish a nascent infection. As a first step towards addressing these fundamental knowledge gaps which are central to any efforts to interrupt disease transmission, we developed and characterized a small personal clean room comprising an array of sampling devices which enable isolation and representative sampling of airborne particles and organic matter from tuberculosis (TB) patients. The complete unit, termed the Respiratory Aerosol Sampling Chamber (RASC), is instrumented to provide real-time information about the particulate output of a single patient, and to capture samples via a suite of particulate impingers, impactors and filters. Applying the RASC in a clinical setting, we demonstrate that a combination of molecular and microbiological assays, as well as imaging by fluorescence and scanning electron microscopy, can be applied to investigate the identity, viability, and morphology of isolated aerosolized particles. Importantly, from a preliminary panel of active TB patients, we observed the real-time production of large numbers of airborne particles including Mtb , as confirmed by microbiological culture and polymerase chain reaction (PCR) genotyping. Moreover, direct imaging of captured samples revealed the presence of multiple rod-like Mtb organisms whose physical dimensions suggested the capacity for travel deep into the alveolar spaces of the human lung.
- ItemOpen AccessSynthesis and evaluation of D-cycloserine analogues against mycobacterium tuberculosis(2020) Renga, Shoneeze Simone; Chibale, Kelly; Singh, VinayakTuberculosis (TB) continues to be the leading cause of death from a single infectious agent worldwide. The rapid emergence of multidrug-resistant and extremely drug-resistant underpin the urgent need for novel, safe and efficacious drugs. D-Cycloserine (DCS) is an oral bacteriostatic anti-tubercular drug used for the treatment of drug-resistant TB. Despite attractive properties, DCS displays significant toxicity at effective dosages. However, a synthetic analogue of DCS, terizidone, has shown an improved safety profile. This study focused on the synthesis of two new classes of DCS analogues: isoxazolidin-3-oneimines (ISIs) and 3-isoxazolidin-4-yl amides (ISAs). These analogues were designed to act as potential prodrugs. In an effort to explore structure-activity-relationships, a total of 17 compounds were synthesized, fully characterized, and evaluated in vitro for their antimycobacterial activity against the drug-susceptible Mtb H37Rv strain cultured in 7H9/ADC media. Of these, two compounds displayed noteworthy antimycobacterial activity (MIC99 ≤ 10 μM). In addition to this, all 17 compounds manifested low cytotoxicity (IC50 > 25 μM) when tested in vitro against the chinese hamster ovarian cells and the human hepatocytes cells. Furthermore, a selected potent compound displayed high microsomal metabolic stability in rat, mouse and human liver microsomes. The kinetic solubility of the target compounds was determined using a HPLC-based method. The solubility data obtained was then correlated with melting point, tPSA and cLogP in order to establish structuresolubility relationships across the two compounds series. Solubility was strongly correlated to melting point in the IZAs series (R2 = 0.9318) and moderately correlated to tPSA for IZIs (R2 = 0.164, whereas there was no correlation between solubility and cLogP (lipophilicity) in either class of compounds (R2 = 0.085 for IZIs and R2= 0.0004 for ISA).
- ItemOpen AccessTotal Synthesis of the Antimycobacterial Natural Product Chlorflavonin and Analogs via a Late-Stage Ruthenium(II)-Catalyzed ortho-C(sp2)-H-Hydroxylation(2022-08-10) Berger, Alexander; Knak, Talea; Kiffe-Delf, Anna-Lene; Mudrovcic, Korana; Singh, Vinayak; Njoroge, Mathew; Burckhardt, Bjoern B.; Gopalswamy, Mohanraj; Lungerich, Beate; Ackermann, Lutz; Gohlke, Holger; Chibale, Kelly; Kalscheuer, Rainer; Kurz, ThomasThe continuous, worldwide spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) endanger the World Health Organization’s (WHO) goal to end the global TB pandemic by the year 2035. During the past 50 years, very few new drugs have been approved by medical agencies to treat drug-resistant TB. Therefore, the development of novel antimycobacterial drug candidates to combat the threat of drug-resistant TB is urgent. In this work, we developed and optimized a total synthesis of the antimycobacterial natural flavonoid chlorflavonin by selective ruthenium(II)-catalyzed ortho-C(sp2)-H-hydroxylation of a substituted 3′-methoxyflavonoid skeleton. We extended our methodology to synthesize a small compound library of 14 structural analogs. The new analogs were tested for their antimycobacterial in vitro activity against Mycobacterium tuberculosis (Mtb) and their cytotoxicity against various human cell lines. The most promising new analog bromflavonin exhibited improved antimycobacterial in vitro activity against the virulent H37Rv strain of Mtb (Minimal Inhibitory Concentrations (MIC90) = 0.78 μm). In addition, we determined the chemical and metabolic stability as well as the pKa values of chlorflavonin and bromflavonin. Furthermore, we established a quantitative structure–activity relationship model using a thermodynamic integration approach. Our computations may be used for suggesting further structural changes to develop improved derivatives.