Finding of the Low Molecular Weight Inhibitors of Resuscitation Promoting Factor Enzymatic and Resuscitation Activity

Abstract

BackgroundResuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs.Methodology/Principal FindingsHere we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC50 1–7 µg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC50. The candidate compounds suppressed resuscitation of dormant (“non-culturable”) cells of M. smegmatis at 1 µg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 µg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations.Conclusions/SignificanceNPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins.

Resuscitation Promoting Factors: a Family of Microbial Proteins in Survival and Resuscitation of Dormant Mycobacteria

“…Several experimental results confirmed that Rpf possesses cell wall hydrolysing activity but role of all the five rpf-like genes in M. tuberculosis remains speculative. A low molecular weight class of compounds, 2-nitrophenylthiocyanates (NPT), was investigated with anti-Rpf activity [41]. Inhibitors of Rpf activity open a new approach to attempt the problem of reactivation of tuberculosis.…”

“…Demina et al reported several nitrophenylthiocyanates (NPT) with anti-Rpf activity. They have illustrated a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC 50 1-7 lg/ml [41]. These low molecular weight compounds interact with the internal regions of the Rpf molecule and suppressed resuscitation of dormant cells of M. smegmatis at 1 lg/ml or delayed resuscitation of dormant M. tuberculosis.…”

Resuscitation-promoting Factors (Rpf): In Search of Inhibitors

“…It was found that some NPTs interacts with Rpf molecule according to fluorescence quenching of tryptophan fluorescence by compounds 2E however, negatively charged compound 2B did not influence fluorescence [59]. The ability of phenyl groups of NPTs to inhibit RpfB catalytic action well correlates with the high level of hydrophobicity of the catalytic site cleft.…”

Resuscitation-promoting factors are important determinants of the pathophysiology inMycobacterium tuberculosisinfection

“…They are important virulence factors, they lack human homologs ensuring a high degree of drug specificity and their extracellular nature means small molecule inhibitors do not need to enter the cell for biological activity (Kana et al, 2008;Machowski et al, 2014). Inhibitors of Rpf, such as 2-nitrophenylthiocyanates, could be given to patients with LTBI who need treatment with anti-TNFa inhibitors to prevent the reactivation of tuberculosis (Demina et al, 2009;Kaprelyants et al, 2012). Contrarily, Rpf could be administered to patients with active infection, reactivating and sensitizing quiescent bacilli which are thought to be responsible for the requirement for long treatment courses so shortening the duration of treatment (Davies et al, 2008;Zhang et al, 2010).…”