orlistat and Tuberculosis

Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) still remains the deadliest infectious disease worldwide with 1.5 million deaths in 2018, of which about 15% are attributed to resistant strains. Another significant example is Mycobacterium abscessus (M. abscessus), a nontuberculous mycobacteria (NTM) responsible for cutaneous and pulmonary infections, representing up to 95% of NTM infections in cystic fibrosis (CF) patients. M. abscessus is a new clinically relevant pathogen and is considered one of the most drug-resistant mycobacteria for which standardized chemotherapeutic regimens are still lacking. Together the emergence of M. tb and M. abscessus multi-drug resistant strains with ineffective and expensive therapeutics, have paved the way to the development of new classes of anti-mycobacterial agents offering additional therapeutic options. In this context, specific inhibitors of mycobacterial lipolytic enzymes represent novel and promising antibacterial molecules to address this challenging issue. The results highlighted here include a complete overview of the antibacterial activities, either in broth medium or inside infected macrophages, of two families of promising and potent anti-mycobacterial multi-target agents, i.e. oxadiazolone-core compounds (OX) and Cyclophostin & Cyclipostins analogs (CyC); the identification and biochemical validation of their effective targets (e.g., the antigen 85 complex and TesA playing key roles in mycolic acid metabolism) together with their respective crystal structures. To our knowledge, these are the first families of compounds able to target and impair replicating as well as intracellular bacteria. We are still impelled in deciphering their mode of action and finding new potential therapeutic targets against mycobacterial-related diseases.

Topics: Antitubercular Agents; Carboxylic Ester Hydrolases; Drug Design; Enzyme Inhibitors; Humans; Lactones; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Mycolic Acids; Organophosphorus Compounds; Orlistat; Tuberculosis; Tuberculosis, Multidrug-Resistant

Tetrahydrolipstatin (THL) is a covalent inhibitor of many serine esterases. In mycobacteria, THL has been found to covalently react with 261 lipid esterases upon treatment of Mycobacterium bovis cell lysate. However, the covalent adduct is considered unstable in some cases because of the hydrolysis of the enzyme-linked THL adduct resulting in catalytic turnover. In this study, a library of THL stereoderivatives was tested against three essential Mycobacterium tuberculosis lipid esterases of interest for drug development to assess how the stereochemistry of THL affects respective enzyme inhibition and allows for cross enzyme inhibition. The mycolyltransferase Antigen 85C (Ag85C) was found to be stereospecific with regard to THL; covalent inhibition occurs within minutes and was previously shown to be irreversible. Conversely, the Rv3802 phospholipase A/thioesterase was more accepting of a variety of THL configurations and uses these compounds as alternative substrates. The reaction of the THL stereoderivatives with the thioesterase domain of polyketide synthase 13 (Pks13-TE) also leads to hydrolytic turnover and is nonstereospecific but occurs on a slower, multihour time scale. Our findings suggest the stereochemistry of the β-lactone ring of THL is important for cross enzyme reactivity, while the two stereocenters of the peptidyl arm can affect enzyme specificity and the catalytic hydrolysis of the β-lactone ring. The observed kinetic data for all three target enzymes are supported by recently published X-ray crystal structures of Ag85C, Rv3802, and Pks13-TE. Insights from this study provide a molecular basis for the kinetic modulation of three essential M. tuberculosis lipid esterases by THL and can be applied to increase potency and enzyme residence times and enhance the specificity of the THL scaffold.

Topics: Enzyme Inhibitors; Esterases; Humans; Hydrolysis; Kinetics; Lactones; Lipase; Lipids; Mycobacterium tuberculosis; Orlistat; Tuberculosis

Mycobacterial ES-31 serine protease has been reported to be a drug target using protease and lipase inhibitors in axenic and macrophage cultures. Simple screening techniques are needed for rapid testing of anti-tubercular drugs.. To demonstrate the usefulness of ELISA protocol based on antigenic reactivity of mycobacterial serine protease by indirect ELISA for detecting anti-tubercular activity.. Indirect ELISA for assessment of antigenic reactivity of mycobacterial ES-31 serine protease was standardized using ES-31Ag and anti-DSS-goat-serum and assessed the inhibition of the antigenic reactivity by isoniazid, an anti-tubercular drug and serine protease inhibitor and orlistat, a lipase inhibitor.. Optimal antigenic reactivity of mycobacterial ES-31 serine protease was observed at 5 microg/well of ES-31 antigen and at 1:25 dilution of anti-DSS-goat-serum. Isoniazid showed 42% inhibition of ES-31 serine protease at 0.4 microg/well, while orlistat showed inhibition of 60% at 0.5 microg/well. Inhibition of Mtb H,37Ra bacilli is further confirmed in axenic culture. 35% and 29% inhibition by isoniazid at 0.4 microg/well and orlistat at 0.5 microg/well were observed respectively on bacterial growth.. Simple ELISA protocol based on assay of antigenic reactivity of mycobacterial ES-31 serine protease, a drug target, has been standardized for rapid screening of potential anti-tubercular drugs.

Topics: Antitubercular Agents; Axenic Culture; Bacterial Proteins; Drug Resistance, Microbial; Enzyme-Linked Immunosorbent Assay; Humans; Isoniazid; Lactones; Mycobacterium tuberculosis; Orlistat; Serine Proteases; Tuberculosis

Mycobacterial excretory secretory-31 (SEVA TB ES-31) antigen is shown to possess protease and lipase activities.. To study the effect of commonly used HIV-protease inhibitors and lipase inhibitor Orlistat if any on mycobacterial ES-31 serine protease in vitro enzyme activity and on the growth of M.tb H37Ra bacilli in axenic culture.. Effect of HIV-protease inhibitors namely Ritonavir, Lopinavir and Indinavir and Orlistat on protease activity of ES-31 was assessed using azocasein assay and on bacillary growth in axenic culture of Mycobacterium tuberculosis H37Ra. The concentration of ES-31 antigen in culture filtrate was determined by sandwich peroxidase ELISA using anti ES-31 antibody and the growth of bacilli by CFU count.. HIV-protease inhibitors such as Ritonavir, Lopinavir and Indinavir and lipase inhibitor Orlistat inhibited serine protease activity by 41.3 - 69.7% in vitro. These inhibitors also showed decreased bacterial growth in axenic culture and further confirmed by decreased concentration of ES-31 serine protease secretion in the culture fluid. Ritonavir showed maximum inhibition of 77% on the growth of the bacilli in axenic culture while anti obesity drug Orlistat showed 61% inhibition.. SEVA TB ES-31 with serine protease and lipase activities may be a potential drug target in tuberculosis management.

Topics: Antigens, Bacterial; Bacterial Proteins; Enzyme-Linked Immunosorbent Assay; HIV Protease Inhibitors; Humans; Lactones; Lopinavir; Mycobacterium tuberculosis; Orlistat; Pyrimidinones; Ritonavir; Serine Endopeptidases; Tuberculosis