title: LC-MS based assay to measure intracellular compound levels in Mycobacterium smegmatis: linking compound levels to cellular potency.
abstract: Dihydrofolate reductase (DHFR) plays a central role in maintaining cellular pool of tetrahydrofolic acid, a cofactor necessary for DNA, RNA and protein synthesis. The clinical validation of DHFR as antibacterial target was established by the success of trimethoprim (TMP). DHFR is also an attractive target for identifying anti-tuberculosis molecules however, due to observed weak cellular potency, no DHFR inhibitors have been developed as drugs so far. TMP and its analogs have poor cellular potency on Mycobacterium tuberculosis and Mycobacterium smegmatis cells. We found a mutant strain of M. smegmatis, mc²155 to be sensitive to TMP whereas wild type strain was not inhibited by TMP. We utilized this system to probe if poor or lack of activity of TMP is a consequence of poor intracellular compound levels. An LC-MS based method was developed for measuring TMP and rifampicin (RIF) in M. smegmatis. Using the assay, equivalent RIF levels were observed in both strains however, TMP was detected only in mc²155 cells, hence proving a positive correlation between potency and compound levels. To the best of our knowledge this is the first time LC-MS method has been used to measure compound levels in mycobacterial cells. We propose it to be a valuable tool to understand the lack of potency or resistance mechanisms in antimycobacterial drug development.
authors.affiliation: AstraZeneca India Pvt Ltd., Bellary Road, Hebbal, Bangalore 560024, India.
authors.forename: Jyothi
authors.initials: J
authors.lastname: Bhat
authors.affiliation: AstraZeneca India Pvt Ltd., Bellary Road, Hebbal, Bangalore 560024, India.
authors.forename: Ashwini
authors.initials: A
authors.lastname: Narayan
authors.affiliation: AstraZeneca India Pvt Ltd., Bellary Road, Hebbal, Bangalore 560024, India.
authors.forename: Janani
authors.initials: J
authors.lastname: Venkatraman
authors.affiliation: AstraZeneca India Pvt Ltd., Bellary Road, Hebbal, Bangalore 560024, India. Electronic address: monalisa.chatterji@astrazeneca.com.
authors.forename: Monalisa
authors.initials: M
authors.lastname: Chatterji
mesh_terms: D000904:Antibiotics, Antitubercular / Q000032:analysis* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005493:Folic Acid Antagonists / Q000032:analysis / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods*; D020102:Mycobacterium smegmatis / Q000737:chemistry* / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D012293:Rifampin / Q000032:analysis / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014295:Trimethoprim / Q000032:analysis / Q000494:pharmacology
chemical_list: D000904:Antibiotics, Antitubercular; D001426:Bacterial Proteins; D005493:Folic Acid Antagonists; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D012293:Rifampin
keywords: Anti-infective agents; Antibiotic accumulation; Antimicrobial susceptibility; Antimycobacterial agents; Permeability