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Mechanism-Based Inactivator of Isocitrate Lyases 1 and 2 from Mycobacterium Tuberculosis

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Mechanism-Based Inactivator of Isocitrate Lyases 1 and 2 from Mycobacterium Tuberculosis Mechanism-based inactivator of isocitrate lyases 1 and 2 from Mycobacterium tuberculosis Truc V. Phama, Andrew S. Murkinb, Margaret M. Moynihanb,1, Lawrence Harrisc, Peter C. Tylerc, Nishant Shettya,d, James C. Sacchettinia,e, Hsiao-ling Huange,f, and Thomas D. Meeka,2 aDepartment of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843; bDepartment of Chemistry, University at Buffalo, The State University of New York, Buffalo, NY 14260; cThe Ferrier Research Institute, Victoria University of Wellington, Wellington 5046, New Zealand; dFujifilm Diosynth Biotechnologies Texas, College Station, TX 77845; eDepartment of Chemistry, Texas A&M University, College Station, TX 77843; and fAlbany Molecular Research, Inc., Albany, NY 12203 Edited by Perry Allen Frey, University of Wisconsin, Madison, WI, and approved June 7, 2017 (received for review May 2, 2017) Isocitrate lyase (ICL, types 1 and 2) is the first enzyme of the McFadden (16) demonstrated that the affinity label, 3-bromopyruvate glyoxylate shunt, an essential pathway for Mycobacterium tuberculosis (3BP) (Fig. 1), exerted time-dependent inactivation of Escherichia (Mtb) during the persistent phase of human TB infection. Here, we coli ICL. A crystal structure of Mtb ICL1 treated with 3BP report 2-vinyl-D-isocitrate (2-VIC) as a mechanism-based inactivator revealed that Cys191 had been S-pyruvoylated (10). One could of Mtb ICL1 and ICL2. The enzyme-catalyzed retro-aldol cleavage envision synthetic analogs of D-isocitrate that bear nascent elec- of 2-VIC unmasks a Michael substrate, 2-vinylglyoxylate, which then trophilic substituents at C-2, which are unmasked only following forms a slowly reversible, covalent adduct with the thiolate form of ICL-catalyzed retro-aldol cleavage. We speculated that 2-C-vinyl- active-site Cys191. 2-VIC displayed kinetic properties consistent with D-isocitrate (2-VIC) (numbering in Fig. 1 based on D-isocitrate) covalent, mechanism-based inactivation of ICL1 and ICL2 with high would provide such an inactivator. ICL-catalyzed cleavage of the efficiency (partition ratio, <1). Analysis of a complex of ICL1:2-VIC by C2–C3 bond would produce succinate as well as an enzyme-bound electrospray ionization mass spectrometry and X-ray crystallography Michael acceptor, 2-vinylglyoxylate (2VG), which is poised to re- S confirmed the formation of the predicted covalent -homopyruvoyl act with the thiolate form of the proximal Cys191. Mechanism- adduct of the active-site Cys191. based inactivation of target enzymes to produce drug candidates has a history of proven success (17–19). In this work, we describe BIOCHEMISTRY mechanism-based inactivation | isocitrate lyase | tuberculosis | 2-vinyl the preliminary kinetic and structural analysis of the mechanism- isocitrate | covalent adduct based inactivation of Mtb isocitrate lyases 1 and 2 by 2-VIC. uberculosis (TB) is the leading cause of death from an in- Results Tfectious disease. The World Health Organization reported Time-Dependent Inactivation of Mtb ICL1 and ICL2 with 2-VIC. Pre- that, in 2014, an estimated 9.6 million people became infected with incubation of ICL1 (800 nM) with 0–40 μM2-VIC(5a) (chemistry TB, with 1.5 million deaths [World Health Organization report: described in SI Appendix) over a time course of 0–70 min, followed Global Tuberculosis Report 2015 (1)]. Infection by Mycobacterium by dilution into reaction mixtures containing high concentrations tuberculosis (Mtb), the causative agent of TB, may assume a latent of the substrates glyoxylate and succinate, resulted in a time- state when harbored within macrophage phagosomes during ex- dependent loss of ICL1 activity conforming to first-order kinetics tended, “persistent” stages (2). In this hypoxic environment, fatty (Fig. 2A). More than 95% of ICL1 was inactivated after a 65-min acids provide the primary carbon source, and mycobacteria activate the glyoxylate shunt, a functional abridgement of the tricarboxylic Significance acid cycle (3). Here, D-isocitrate is converted to glyoxylate and succinate by the action of two isocitrate lyases (ICLs) of 27% se- Tuberculosis, caused by Mycobacterium tuberculosis (Mtb)bacte- quence identity, ICL1 (428 aa) and ICL2 (766 aa), encoded by the ria, is the most prevalent infectious disease, affecting one-third of genes icl1 and aceA, respectively (4–7). Glyoxylate is subsequently the global population, especially in developing countries. First-line converted to L-malate by malate synthase, encoded by the gene glcB therapies to treat this disease are losing efficacy due to the (8). Enzymes of the glyoxylate shunt are found in prokaryotes, lower emergence of drug resistance. Accordingly, new therapeutic eukaryotes, and plants, but are absent in mammals (6). Deletion of agents of novel mechanisms of action remain an urgent medical both ICL genes leads to growth impairment of Mtb in infected mice need. The isocitrate lyases (ICL1 and ICL2) comprise metabolically and rapid elimination of bacteria from the lungs (4, 5). The ICL essential enzymes of Mtb, are absent in mammals, and thereby inhibitor, 3-nitropropionate (3NP), inhibited Mtb in cell cultures (4, 9, provide therapeutically important drug targets for tuberculosis. 10). Collectively, these results demonstrated the essentiality of the Here, we describe the first example of a mechanism-based inac- ICLs in persistent-stage Mtb. Despite an absence of drug quality tivator of ICL1 and ICL2 that could provide a starting point for the inhibitors of Mtb ICLs, they remain validated targets for the devel- development of new drugs to treat tuberculosis. opment of new drugs to treat TB. Accordingly, the exploitation of the Author contributions: T.V.P., A.S.M., M.M.M., L.H., P.C.T., H.-l.H., and T.D.M. designed chemical mechanism to discover covalent inactivators could rein- research; T.V.P., A.S.M., M.M.M., L.H., N.S., H.-l.H., and T.D.M. performed research; vigorate drug discovery for the ICLs, particularly because their active T.V.P., A.S.M., L.H., P.C.T., N.S., J.C.S., H.-l.H., and T.D.M. analyzed data; and T.V.P., sites contain conserved, catalytic cysteines. Compounds that form A.S.M., M.M.M., J.C.S., H.-l.H., and T.D.M. wrote the paper. covalent bonds, especially reversible ones, with cysteine residues in or The authors declare no conflict of interest. near the enzymatic active sites have received renewed attention as a This article is a PNAS Direct Submission. strategy for the development of enzyme inactivators (11, 12). Freely available online through the PNAS open access option. The catalytic mechanism of Mtb ICL derived from both struc- Data deposition: The atomic coordinates and structure factors have been deposited in the tural data (10) and kinetic analysis (13–15)isdepictedinFig.1. Protein Data Bank, www.pdb.org (PDB ID code 5DQL). (2R,3S)-Isocitrate (1)[D-isocitrate (IC)] coordinates an active-site 1Present address: Janssen Research and Development, Malvern, PA 19355. magnesium ion and undergoes a base-catalyzed retro-aldol re- 2To whom correspondence should be addressed. Email: [email protected]. a 2 3 action ( ) to form glyoxylate ( )andtheaci-anion ( ) of succinate. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Cys191 then protonates C-2 of 3 to afford succinate (4). Ko and 1073/pnas.1706134114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1706134114 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 ICL1), but 2-VIC displayed no cytotoxicity in human dermal fi- broblasts upon treatment for 72 h at ≤400 μM. Protection of ICL from 2-VIC Inactivation by D-Malate, Glyoxylate, Succinate, and Added Thiols. Concentrations of 0.7 mM succinate or 0.1 mM glyoxylate afforded protection from inactivation by 30 μM 2-VIC. D-Malate, a competitive inhibitor of isocitrate (Ki = 310 ± 30 μM; SI Appendix,Fig.S1), served as an isocitrate surrogate to demonstrate that the inactivation effected by 2-VIC occurs at the active site. In preincubation studies of ICL1 with 50 μM2-VIC,the presence of 0.1–3.0 mM D-malate exhibited a concentration- Fig. 1. Chemical mechanism of isocitrate lyase and structures of inactivators. dependent ablation of ICL1 inactivation by 2-VIC (SI Appendix, Proposed two-step chemical mechanism of Mtb ICL1 based on structural (10) Fig. S2). The inactivation was almost entirely eliminated at 3 mM and kinetic (13–15) data. Structures of 3-bromopyruvate, 2-vinyl D-isocitrate, D-malate, which is 10-fold higher than its inhibition constant. The and 2-vinylglyoxylate. concentration dependence of protection from inactivation by D-malate was evaluated by fitting data (SI Appendix,Eq.S7), resulting in an apparent dissociation constant of K = 360 ± 60 μM, ≥ μ d preincubation with 2-VIC at concentrations of 20 M. Data were nearly equal to its value of K , and a Hill coefficient n = 0.56 (SI 1 i fitted globally to Eq. , resulting in a maximal rate constant of in- Appendix,Fig.S2, Inset). = ± −1 activation, kinact 0.080 0.006 min , and a concentration of Addition of either DTT or glutathione to preincubation mixtures = ± μ 2-VIC leading to half-maximal inactivation, Kinact 22 3 M. diminished inactivation of ICL1 by 2-VIC, presumably by in- Using the method of Kitz and Wilson (20), the apparent first-order 2 terception of an enzyme-generated electrophilic species by reaction rate constant of inactivation, kobs,obtainedfromEq. demon- with the added thiols. Addition of micromolar concentrations of strated a hyperbolic dependence on the concentration of the inac- 3 either DTT or glutathione to preincubation mixtures of ICL1 and tivator upon fitting to Eq. (Fig. 2C), indicating saturation behavior 2-VIC demonstrated concentration-dependent protection from of inactivation. The (2R,3R)-diastereomer of 2-VIC (5b) displayed – μ 2-VIC inactivation of ICL1. As shown in SI Appendix,Fig.S3A, no inactivation of ICL1 when preincubated with enzyme at 1 80 M increasing fixed concentrations of 10–1,000 μM DTT in prein- concentrations for as long as 1,200 min.
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