Antitubercular specific activity of ibuprofen and the other 2-arylpropanoic acids using the HT-SPOTi whole-cell phenotypic assay

Abstract

ObjectivesLead antituberculosis (anti-TB) molecules with novel mechanisms of action are urgently required to fuel the anti-TB drug discovery pipeline. The aim of this study was to validate the use of the high-throughput spot culture growth inhibition (HT-SPOTi) assay for screening libraries of compounds against Mycobacterium tuberculosis and to study the inhibitory effect of ibuprofen (IBP) and the other 2-arylpropanoic acids on the growth inhibition of M tuberculosis and other mycobacterial species.MethodsThe HT-SPOTi method was validated not only with known drugs but also with a library of 47 confirmed anti-TB active compounds published in the ChEMBL database. Three over-the-counter non-steroidal anti-inflammatory drugs were also included in the screening. The 2-arylpropanoic acids, including IBP, were comprehensively evaluated against phenotypically and physiologically different strains of mycobacteria, and their cytotoxicity was determined against murine RAW264.7 macrophages. Furthermore, a comparative bioinformatic analysis was employed to propose a potential mycobacterial target.ResultsIBP showed antitubercular properties while carprofen was the most potent among the 2-arylpropanoic class. A 3,5-dinitro-IBP derivative was found to be more potent than IBP but equally selective. Other synthetic derivatives of IBP were less active, and the free carboxylic acid of IBP seems to be essential for its anti-TB activity. IBP, carprofen and the 3,5-dinitro-IBP derivative exhibited activity against multidrug-resistant isolates and stationary phase bacilli. On the basis of the human targets of the 2-arylpropanoic analgesics, the protein initiation factor infB (Rv2839c) of M tuberculosis was proposed as a potential molecular target.ConclusionsThe HT-SPOTi method can be employed reliably and reproducibly to screen the antimicrobial potency of different compounds. IBP demonstrated specific antitubercular activity, while carprofen was the most selective agent among the 2-arylpropanoic class. Activity against stationary phase bacilli and multidrug-resistant isolates permits us to speculate a novel mechanism of antimycobacterial action. Further medicinal chemistry and target elucidation studies could potentially lead to new therapies against TB.

DNA sequence-selective C8-linked pyrrolobenzodiazepine–heterocyclic polyamide conjugates show anti-tubercular-specific activities

“…was performed using the spot culture growth inhibition assay (SPOTi) in 24 well plates. 26 A seed 104 culture of each bacteria was prepared in Luria Bertani (LB) broth and grown overnight at 37 ºC with 105 shaking at 200 rpm. Rhodococcus sp.…”

“…The 118 antimycobacterial activities of the compounds were tested following the HT-SPOTi guidelines. 26,27 HT-119…”

Early diagnosis and effective treatment regimens are the keys to tackle antimicrobial resistance in tuberculosis (TB): A report from Euroscicon's international TB Summit 2016

“…SPOTi is a solid-culture based technique wherein an optimised number of bacteria are spotted on to agar containing a series of dilutions of inhibitor molecules. [131][132][133] This method has been adapted to 96-well microplate format thereby increasing the throughput.…”

“…[145][146][147] Dr Bhakta and his research group reported the common, over-the-counter non-steroidal anti-inflammatory drugs (NSAIDs), ibuprofen and carprofen to be antitubercular specific. 131,148,149 They were found to be active against drug resistant strains and stationary phase mycobacteria. The endogenous mechanism of action of these drugs is still under debate, however, transcriptomic analyses reveal that the drugs may have pleiotropic effects on the pathogen.…”

Improving the Tuberculosis Drug Development Pipeline

“…However this method depends on the availability of resistant mutants and this can be limited in some cases (Gold et al, 2012a;Guzman et al, 2013). The application of whole genome transcriptomics is another alternative way to examine the mechanism of action of new molecules.…”