Mechanism of irreversible inhibition of Mycobacterium tuberculosis shikimate kinase by ilimaquinone

Ilimaquinone (IQ), a marine sponge metabolite, has been considered as a potential therapeutic agent for various diseases due to its broad range of biological activities. We show that IQ irreversibly inactivates Mycobacterium tuberculosis shikimate kinase (MtSK) through covalent modification of the protein. Inactivation occurred with an apparent second-order rate constant of about 60 M ⁻¹ s ⁻¹. Following reaction with IQ, LC-MS analyses of intact MtSK revealed covalent modification of MtSK by IQ, with the concomitant loss of a methoxy group, suggesting a Michael-addition mechanism. Evaluation of tryptic fragments of IQ-derivatized MtSK by MS/MS demonstrated that Ser and Thr residues were most frequently modified with lesser involvement of Lys and Tyr. In or near the MtSK active site, three residues of the P-loop (K15, S16, and T17) as well as S77, T111, and S44 showed evidence of IQ-dependent derivatization. Accordingly, inclusion of ATP in IQ reactions with MtSK partially protected the enzyme from inactivation and limited IQ-based derivatization of K15 and S16. Additionally, molecular docking models for MtSK-IQ were generated for IQ-derivatized S77 and T111. In the latter, ATP was observed to sterically clash with the IQ moiety. Out of three other enzymes evaluated, lactate dehydrogenase was derivatized and inactivated by IQ, but pyruvate kinase and catalase-peroxidase (KatG) were unaffected. Together, these data suggest that IQ is promiscuous (though not entirely indiscriminant) in its reactivity. As such, the potential of IQ as a lead in the development of antitubercular agents directed against MtSK or other targets is questionable.