Cyclipostins and Cyclophostin Analogues as Multitarget Inhibitors That Impair Growth of Mycobacterium abscessus

Abstract

Twelve new Cyclophostin and Cyclipostins analogs (CyC19-30) were synthesized, thus extending our series to 38 CyCs. Their antibacterial activities were evaluated against four pathogenic mycobacteria (Mycobacterium abscessus, Mycobacterium marinum, Mycobacterium bovis BCG and Mycobacterium tuberculosis) and two Gram negative bacteria. The CyCs displayed very low toxicity towards host cells and were only active against mycobacteria. Importantly, several CyCs were active against extracellular M. abscessus (CyC17/CyC18β/CyC25/CyC26) or intramacrophage residing mycobacteria (CyC7(α,β)/CyC8(α,β)) with minimal inhibitory concentrations (MIC50) values comparable to or better than those of amikacin or imipenem, respectively. An activity-based protein profiling combined with mass spectrometry allowed identification of the potential target enzymes of CyC17/CyC26, mostly being involved in lipid metabolism and/or in cell wall biosynthesis. Overall, these results strengthen the selective activity of the CyCs against mycobacteria, including the most drug-resistant M. abscessus, through the cumulative inhibition of a large number of Ser-and Cysenzymes participating in key physiological processes.

Dissecting the antibacterial activity of oxadiazolone-core derivatives against Mycobacterium abscessus

“…Such a difference between the intra and extracellular activities has already been reported in our previous works with the OX derivatives [12], as well as with another family of growth inhibitors, the Cyclipostins & Cyclophostin analogs [13,14] acting against M. tuberculosis and M. abscessus [25]. Similar to M. tuberculosis [12], the intracellular and extracellular inhibition of M. abscessus growth may probably result from several different mechanisms of action or penetration of the OX derivatives.…”

“…No TAMRA-FP labeling is detected in the presence of inactivated heat-treated Ag85C Mabs . TAMRA labeling of Ag85C Mabs is impaired in the Ag85C Mabs -iBpPPOX adducts, as evidenced by the loss of fluorescence in the iBpPPOX lanes, presumably resulting from the covalent binding of iBpPPOX to the catalytic serine as previously observed[24,25]. TAMRA-labeled Ag85C Mabs was detected by fluorescent gel scanning (λ ex 557 nm, λ em 583 nm) using the Cy 1 3 filter of a ChemiDoc MP Imager (Bio-Rad) before staining of the gel with Coomassie Brilliant Blue dye.…”

“…Based on these results, purified Ag85C Mabs recombinant protein [25] was further incubated with iBpPPOX, using increasing enzyme/inhibitor molar ratio (E/I) ranging from 1:1 to 1:75, and then treated with ActivX TAMRA-FP fluorescent probe, as reported previously [24,25]. Equal amounts of proteins were separated on SDS-PAGE and visualized by Coomassie staining or in-gel fluorescence for TAMRA detection (Fig 4A).…”

“…The lysate was clarified by centrifugation (12,000 × g, 4˚C, 30 min) prior to purification by nickel affinity chromatography with Ni-NTA sepharose beads and elution with the previous Tris (pH 8.0) buffer containing 500 mM imidazole. Purified protein was concentrated at 1 mg/mL and stored at -80˚C [24,25].…”

“…Indeed, nearly all macrophages were lysed at 24 h post-infection with M. abscessus R variant, making it impossible to quantify the intracellular effect of the OXs. This is, however, not the case with M. abscessus S for which more homogenous bacterial suspensions can be obtained for macrophages infection studies [25,31,32]. Therefore, Raw264.7 cells were infected with M. abscessus S at a multiplicity of infection (MOI) of 10, and then incubated for 24 h with all the OX compounds at a final concentration of 90, 60 and 30 μM, or with imipenem (IMP) used as positive drug control.…”