Antigen 85C-mediated acyl-transfer between synthetic acyl donors and fragments of the arabinan

Abstract

Antigen 85 (ag85) is a complex of acyltransferases (ag85A-C) known to play a role in the mycolation of the D-arabino-D-galactan (AG) component of the mycobacterial cell wall. In order to better understand the chemistry and substrate specificity of ag85, a trehalose monomycolate mimic p-nitrophenyl 6-O-octanoyl-beta-D-glucopyranoside (1) containing an octanoyl moiety in lieu of a mycolyl moiety was synthesized as an acyl donor. Arabinofuranoside acceptors, methyl alpha-D-arabinofuranoside (2), methyl beta-D-arabinofuranoside (3), and methyl 2-O-beta-D-arabinofuranosyl-alpha-D-arabinofuranoside (9) were synthesized to mimic the terminal saccharides found on the AG. The acyl transfer reaction between acyl donor 1 and acceptors 2, 3, and 9 in the presence of ag85C from Mycobacterium tuberculosis (M. tuberculosis) resulted in the formation of esters, methyl 2, 5-di-O-octanoyl-alpha-D-arabinofuranoside (10), methyl 5-O-octanoyl-beta-D-arabinofuranoside (11), and methyl 2-O-(5-O-octanoyl-beta-D-arabinofuranosyl)-5-O-octanoyl-alpha-D-arabinofuranoside (12) in 2 h, 2 h and 8 h, respectively. The initial velocities of the reactions were determined with a newly developed assay for acyltransferases. As expected, the regioselectivity corresponds to mycolylation patterns found at the terminus of the AG in M. tuberculosis. The study shows that D-arabinose-based derivatives are capable of acting as substrates for ag85C-mediated acyl-transfer and the acyl glycoside 1 can be used in lieu of TMM extracted from bacteria to study ag85-mediated acyl-transfer and inhibition leading to the better understanding of the ag85 protein class.

Inactivation of the Mycobacterium tuberculosis Antigen 85 Complex by Covalent, Allosteric Inhibitors

“…The enzymes of the Ag85 complex appear to have redundant activity in vivo based on the proposed ability to use TMM as a mycolyl donor and subsequent transfer to a variety of substrates (21,46). Additionally, Backus et al (47) have shown that trehalosebased compounds can be amended at various positions and still function as acyl donors.…”

“…The three homologous enzymes catalyze the transfer of mycolic acids from trehalose monomycolate (TMM) to either another TMM molecule to generate trehalose dimycolate (TDM) or to arabinose to mycolate the AG complex (20,21). A deletion mutant of fbpC, the gene encoding Ag85C, reduces by 40% the cell wall-bound mycolic acids but does not affect the production of non-covalently linked mycolates, implying that Ag85C transfers mycolic acid to the AG complex in vivo (22).…”

The Mycobacterial Cell Envelope--Lipids

“…4). High-throughput screening assays were reported for FadD32 (Galandrin et al 2013) and the antigens 85 Elamin et al 2009;Sanki et al 2009;Favrot et al 2013). The latter assays led to the identification of a number of inhibitors of antigens 85, one of them, known as ebselen, also shows activity against Mtb in culture (MIC of 20 mg/mL).…”

The mycolyltransferase 85A, a putative drug target of Mycobacterium tuberculosis: Development of a novel assay and quantification of glycolipid-status of the mycobacterial cell wall

“…Antigen 85 proteins are important for the survival of MTB in the host macrophage. An antigen 85C knockout strain of MTB has a 40% decrease in the amount of cell wall linked mycolic acid (Jackson et al, 1999;Sanki et al, 2008 Despite the knowledge concerning protein/substrate interactions and co-crystal structure of Antigen 85 (Klegerman et al, 1994;Ronning et al, 2004) the prerequisite for designing potent inhibitors to the mycolyltransferase activity is still the design of rapid and easy assays, suitable for HTS. Several synthetic analogues of trehalose and TMM, putative competitive inhibitors of their metabolism, have been shown to have dramatic effects on the glycolipid metabolism of mycobacteria.…”