Whole cell screen based identification of spiropiperidines with potent antitubercular properties

Structure–Function Profile of MmpL3, the Essential Mycolic Acid Transporter from Mycobacterium tuberculosis

“…Importantly, MmpL3 is thought to be the target of a number of anti-TB inhibitors under development. 5,26-35 MmpL3 (MW = 101 kDa) and CmpL1 (MW = 83 kDa) which we purified from Cgl cells were treated with the amine reactive cross-linker dithiobis(succinimidylpropionate) and the cross-linked proteins were analyzed by immunoblotting. High molecular weight species (~ 300 kDa) became apparent with increasing concentrations of the cross-linker suggestive of protein oligomerization or formation of stable complexes.…”

“…23-24 The structure of one of the two soluble periplasmic domains of MmpL11 was solved 6 , a transport mechanism based on dynamics simulation was proposed for MmpL5 25 and the use of prediction algorithms yielded different topological models for MmpL3 and other MmpL proteins. 5-7,11,13,26 Yet, despite their significance in the physiology and virulence of Mtb , and from the perspective of anti-TB drug development, 5,26-35 no full-size MmpL protein has yet been purified and reconstituted in vitro . As a result, the transmembrane topology of no MmpL protein had yet been validated experimentally and even basic biochemical properties of these proteins, such as their oligomeric state, determinants of substrate specificity, mechanism of translocation and putative physical association with other inner membrane, periplasmic and/or OM proteins remain to a large extent unknown.…”

Therapeutic Potential of the Mycobacterium tuberculosis Mycolic Acid Transporter, MmpL3

“…The identification and characterization of MmpL3 inhibitors have thus far rested on the whole-genome sequencing of spontaneous resistant mutants and the metabolic labeling with [1,2-14 C]acetate of inhibitor-treated cells to monitor the transfer of mycolic acids to their cell envelope acceptors (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)17). Because the development and further optimization of MmpL3 inhibitors would greatly benefit from less cumbersome approaches, and because of the technical difficulty of developing an in vitro transport assay for this protein, we next sought to determine whether the M. tuberculosis mc 2 6206 MmpL3-DUC knockdown could be used in the development of a target-based whole-cell screening assay.…”

“…To date, whole-cell screening strategies are the ones that have led to the identification of the greatest number of novel inhibitors of potential therapeutic interest (2)(3)(4). Intriguingly, in the last 4 years, the screening of compound libraries against M. tuberculosis in culture followed by the whole-genome sequencing of spontaneous resistant mutants has identified multiple small molecules, including the TB drug candidate SQ109, as inhibitors of the mycolic acid transporter, MmpL3 (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). MmpL3 is an integral inner membrane transporter of the resistance, nodulation, and division (RND) superfamily, involved in the export of mycolic acids under the form of trehalose monomycolate (TMM) to the periplasmic space, where this glycolipid can then serve as a mycolic acid donor in the building of the mycobacterial outer membrane, otherwise known as the mycomembrane (5,15,16).…”

Targeting the trehalose utilization pathways ofMycobacterium tuberculosis

“…79 The genome sequencing of SQ109 resistant mutants generated using SQ109 analogs showed mutation in the mmpL3 gene, suggesting MmpL3 as target for SQ109. 79, 80 Other notable inhibitors identified using similar genetic mutation studies include: N -(2,4-dichlorobenzyl)-1-propyl-1 H- benzo[ d]imidazole-amine (C215, 40), 86 tetrahydropyrazolo([1,5- a ]pyrimidine-3-carboxamide (THPP, 41), 87 N -benzyl-6′-7′-dihydrospiro[piperidine-4,4′-thienol[3,2- c ]pyran] (SPIROS, 42), 87 and 2418 (43) an indolecarboxamide 88, 89, 90, 91 . Compounds 37, 38, and 41 show broad spectrum antibacterial and antifungal activity, even against pathogens devoid of mycolic acids.…”