457504 Structural Insights into Protein Splicing Inhibition By Platinum Therapeutics As Potential Anti-Microbials
Inteins, self-splicing protein elements, interrupt genes and proteins in many microbes, including the human pathogen Mycobacterium tuberculosis. The intein in the mycobacterial recombinase, RecA, is specifically inhibited by cisplatin, cis-[Pt(NH3)2Cl2], widely used to treat a variety of cancers. Cisplatin also inhibits mycobacterial growth by targeting inteins. Structural and kinetic analyses of cisplatin binding to the RecA intein were performed to investigate the inhibition of protein splicing by this platinum(II) compound. Mass spectrometric and crystallographic studies of cisplatin binding revealed a complex in which two platinum atoms bind to the intein at N- and C-terminal catalytic cysteine residues in the presence of the reducing agent tris(2-carboxyethyl)phosphine (TCEP). Kinetic analyses of NMR spectroscopic data support a two-step mechanism for cisplatin binding, in which a Pt(II) unit first rapidly interacts at the N-terminus, followed by a slower, first-order binding event involving both the N-terminus (Pt) and C-terminus (Pt+TCEP). Notably, the ammine ligands of cisplatin, which remain bound to platinum in DNA adducts formed as an early step in the mechanism responsible for its anticancer activity, are no longer bound to the metal atom in the intein adduct, as revealed by crystallography. We also explored two additional platinum-based compounds with and without ammine ligands for their ability to inhibit intein splicing. A platinum 4,4,4-trifluorobenzoylacetonate (tfbz) complex, [Pt(tfbz)(NH3)2](NO3), and K[Pt(Cl3(CH2=CH2)] (Zeise’s salt) displayed similar inhibitory potency to cisplatin, in the low micromolar range. These findings should inform the design of additional platinum-based compounds for targeting inteins in microbial pathogens.