Cisplatin Inhibits Protein Splicing, Suggesting Inteins as Therapeutic Targets in Mycobacteria

Abstract

Mycobacterium tuberculosis harbors three protein splicing elements, called inteins, in critical genes and their protein products. Post-translational removal of the inteins occurs autocatalytically and is required for function of the respective M. tuberculosis proteins. Inteins are therefore potential targets for antimycobacterial agents. In this work, we report that the splicing activity of the intein present in the RecA recombinase of M. tuberculosis is potently inhibited by the anticancer drug cisplatin (cis-diamminedichloro-platinum(II)). This previously unrecognized activity of cisplatin was established using both an in vitro intein splicing assay, which yielded an IC 50 of ϳ2 M, and a genetic reporter for intein splicing in Escherichia coli. Testing of related platinum(II) complexes indicated that the inhibition activity is highly structure-dependent, with cisplatin exhibiting the best inhibitory effect. Finally, we report that cisplatin is toxic toward M. tuberculosis with a minimum inhibitory concentration of ϳ40 M, and in genetic experiments conducted with the related Mycobacterium bovis bacillus Calmette-Guérrin (BCG) strain, we show that cisplatin toxicity can be mitigated by intein overexpression. We propose that cisplatin inhibits intein activity by modifying at least one conserved cysteine residue that is required for splicing. Together these results identify a novel active site inhibitor of inteins and validate inteins as viable targets for small molecule inhibition in mycobacteria.Tuberculosis remains a leading cause of death worldwide (1, 2), and the global emergence of multidrug-resistant Mycobacterium tuberculosis portends further challenges in TB Several microbial pathogens, including M. tuberculosis, contain protein self-splicing elements called inteins, which interrupt critical genes and their protein products. These inteins are therefore potential novel targets for antibiotic development, particularly because no inteins are present in the human genome. In addition to M. tuberculosis, self-splicing inteins reside in critical proteins of Mycobacterium leprae, Coxiella burnetii, and Cryptococcus neoformans, the etiological agents of leprosy, Q fever, and cryptococcosis, respectively (6 -8). For these pathogens to survive, the inteins must catalyze a multistep reaction in which they are excised from the precursor protein and the two flanking sequences, called exteins, must be ligated to form a functional product protein (9 -11). Because inteins exert control, via splicing, over the function of pathogen-specific proteins, these self-splicing elements are attractive candidates for inhibition studies. The pursuit of intein inhibitors, whether as mechanistic probes or as an avenue for developing antibacterials, does face a unique challenge; unlike a conventional enzyme inhibitor, an effective antagonist of intein splicing must outcompete a substrate that is covalently tethered to the enzyme.M. tuberculosis has inteins in three distinct genes: in the dnaB helicase gene, in the recA recombinase gene,...

Exploring Intein Inhibition by Platinum Compounds as an Antimicrobial Strategy

“…Appearance of multiand extensively drug-resistant strains of M. tuberculosis highlights the need for novel treatment options targeting different cellular processes (28,29). It was shown previously that the chemotherapeutic agent cisplatin is a potent intein inhibitor that arrests the growth of M. tuberculosis in a targeted fashion (14). Here, through testing of more Pt(II) compounds, we discovered that Pttfbz and Zeise's salt are of equivalent potency in vitro with IC 50 values in the 2 M range.…”

“…The effectiveness of the compounds was tested with a fluorescence-based assay using a split GFP construct (29 kDa) containing a minimized RecA intein (15.3 kDa) as described previously (14,19). Inhibition by the compounds was determined by fitting the data with a dose-response curve and calculating the inhibition constant, IC 50 ( Fig.…”

“…This minimized intein is susceptible to inhibition by cisplatin with an IC 50 value in the low micromolar range. This result was mirrored in M. tuberculosis with cisplatin arresting mycobacterial growth by intein targeting (14). To devise a more rational approach to the design of intein-based therapeutics, we sought to provide structural and mechanistic insights into inhibitor binding.…”

Split intein mediated ultra‐rapid purification of tagless protein (SIRP)

“…However, Asp118 is mutated to Gly in C * to confer C‐terminal cleavage activity in the absence of N‐terminal cleavage (Ramirez et al, ), and this protein as well as proteins lacking Cys + 1 (construct 5–23) also potently inhibited by Zn 2+ . In addition, we tested the ability of cisplatin, an inhibitor of the Rec A intein presumably through targeting the Cys1 (Zhang et al, ), can also inhibit C‐terminal of our intein at low micromolar concentration (data not shown). It is worth noting that our engineered intein has Cys1 mutated to Ala to abolish the first thio‐ester transfer step.…”

Backbone assignments of mini-RecA intein with short native exteins and an active N-terminal catalytic cysteine

“…Preventing splicing, and thus the maturation of non-functional precursor to functional spliced product presents a novel method of eliminating protein activity and suggests inteins as potential antimicrobial targets (Belfort 1998; Lew and Paulus 2002). Indeed, it has been shown that the potent in vitro intein inhibitor cisplatin kills M. tuberculosis in vivo (Zhang et al 2011). This effect relied upon the N-terminal catalytic cysteine residue of the intein.…”