Exploring Intein Inhibition by Platinum Compounds as an Antimicrobial Strategy

Abstract

Inteins, self-splicing protein elements, interrupt genes and proteins in many microbes, including the human pathogen Mycobacterium tuberculosis. Using conserved catalytic nucleophiles at their N-and C-terminal splice junctions, inteins are able to excise out of precursor polypeptides. The splicing of the intein in the mycobacterial recombinase RecA is specifically inhibited by the widely used cancer therapeutic cisplatin, cis-[Pt(NH 3 ) 2 Cl 2 ], and this compound inhibits mycobacterial growth. Mass spectrometric and crystallographic studies of Pt(II) binding to the RecA intein revealed a complex in which two platinum atoms bind at N-and C-terminal catalytic cysteine residues. Kinetic analyses of NMR spectroscopic data support a two-step binding mechanism in which a Pt(II) first rapidly interacts reversibly at the N terminus followed by a slower, first order irreversible binding event involving both the N and C termini. Notably, the ligands of Pt(II) compounds that are required for chemotherapeutic efficacy and toxicity are no longer bound to the metal atom in the intein adduct. The lack of ammine ligands and need for phosphine represent a springboard for future design of platinum-based compounds targeting inteins. Because the intein splicing mechanism is conserved across a range of pathogenic microbes, developing these drugs could lead to novel, broad range antimicrobial agents.Inteins, self-splicing protein elements that invade a host gene, have been of great interest to the field of biotechnology. The unique ability of an intein to break and form peptide linkages has led to their use in applications (1, 2) ranging from sensors of small molecules (3, 4) and environmental conditions (5) to single step protein purification platforms (6, 7). However, there are still underexplored reactions involving native inteins, notably their susceptibility as targets for antimicrobial drugs (8). In nature, inteins reside in key host proteins across several bacterial and fungal pathogens, including Mycobacterium tuberculosis, Mycobacterium leprae, and Cryptococcus neoformans (9).These splicing elements divide the host protein into two parts, termed N-and C-exteins on the corresponding ends of the intein. In the precursor form, the host protein is usually non-functional because of the tendency of an intein to insert into highly conserved functional regions of the protein. In M. tuberculosis, inteins occur in three genes, recA, sufB, and dnaB (9). The functionality of these proteins is key to the survival of the bacterium and relies upon the splicing of the intein from the respective host proteins. Because inteins do not occur in multicellular organisms, prevention of protein splicing provides a promising strategy for the development of novel antimicrobial therapeutics.Canonical intein splicing occurs by a multistep process (10). First, a nucleophilic attack is initiated by the N-terminal cysteine residue (C1) of the intein on the preceding peptide bond, resulting in thioester formation (Fig. 1A, step 1). A second nucleophilic a...

Structure of an engineered intein reveals thiazoline ring and provides mechanistic insight

“…Expression of the ΔΔI-TM intein mutant indicated that splicing, N-terminal cleavage and C-terminal cleavage were arrested (Supporting Information Figure S2), yet no evidence of metal binding could be detected despite the presence of the putative metal binding residues. Platinum binding has been shown to be mediated mainly by the sidechains of Cys1 and Cys + 1 of the RecA intein (Chan et al, 2016;Zhang, Zheng, Callahan, Belfort, & Liu, 2011), suggesting, among other possibilities, that one or both Cys may be perturbed spatially or otherwise modified. Interestingly, the ΔΔI-TM (T70A/ N139A) mutant exhibited the most intense signal at exactly 18 Da less than the expected mass when measured by ESI-MS (monoisotopic M obs = 16,454.60 Da; Figure 2).…”

“…the sidechains of Cys1 and Cys + 1 of the RecA intein(Chan et al, 2016;Zhang, Zheng, Callahan, Belfort, & Liu, 2011), suggesting, among other possibilities, that one or both Cys may be perturbed spatially or otherwise modified. Interestingly, the ΔΔI-TM (T70A/ N139A) mutant exhibited the most intense signal at exactly 18 Da less than the expected mass when measured by ESI-MS (monoisotopic M obs = 16,454.60 Da;Figure 2).…”

Spliceosomal Prp8 intein at the crossroads of protein and RNA splicing

“…Metal binding has been reported for other inteins, and often engages catalytic residues, which would stall protein splicing (47,(51)(52)(53)(54)(55)(56). Therefore, we chose to follow up on the observed copper and zinc inhibition by running in vitro MIG Prp8 A-1V assays to further assess effects on protein splicing over time (Fig.…”

“…Around a dozen intein structures have been solved so far, comprising mainly bacterial and archaeal inteins (63). These have proven useful for studying inteins as novel drug targets (51). As inteins often invade essential proteins in pathogens, inhibiting them from splicing out is an attractive option for developing new antimicrobials (51,64,65).…”

“…Progress towards this goal has been made in prokaryotes using the mycobacterial RecA recombinase intein. A co-crystal of the RecA intein and the antineoplastic compound, cisplatin, helped resolve the mechanism of protein splicing inhibition (51). This showed that the platinum ions of cisplatin bind to the RecA intein at its two catalytic cysteines, C1 and C+1.…”