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Bisubstrate Adenylation Inhibitors of Biotin Protein Ligase from Mycobacterium Tuberculosis

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Bisubstrate Adenylation Inhibitors of Biotin Protein Ligase from Mycobacterium Tuberculosis View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Chemistry & Biology Article Bisubstrate Adenylation Inhibitors of Biotin Protein Ligase from Mycobacterium tuberculosis Benjamin P. Duckworth,1 Todd W. Geders,2 Divya Tiwari,3 Helena I. Boshoff,4 Paul A. Sibbald,1 Clifton E. Barry, III,4 Dirk Schnappinger,3 Barry C. Finzel,2 and Courtney C. Aldrich1,* 1Center for Drug Design, University of Minnesota, Minneapolis, MN 55455, USA 2Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA 3Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065, USA 4Tuberculosis Research Section, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA *Correspondence: [email protected] DOI 10.1016/j.chembiol.2011.08.013 SUMMARY the type II fatty acid synthase (FAS-II) in Mtb (Slayden et al., 2000; Timm et al., 2003). Acyl coenzyme A carboxylases The mycobacterial biotin protein ligase (MtBPL) (ACCs) catalyze the first committed step in fatty acid biosyn- globally regulates lipid metabolism in Mtb through thesis through synthesis of the monomeric malonyl-CoA building the posttranslational biotinylation of acyl coenzyme blocks. However, unlike most bacteria that encode for a single A carboxylases involved in lipid biosynthesis that ACC complex, Mtb encodes for multiple ACCs responsible for catalyze the first step in fatty acid biosynthesis and the biosynthesis of malonyl coenzyme A (CoA), (methyl)malonyl pyruvate coenzyme A carboxylase, a gluconeogenic CoA, and (C22–C24) malonyl CoA for construction of the struc- turally diverse lipids in Mtb, including simple linear fatty acids, enzyme vital for lipid catabolism. Here we describe methyl-branched lipids, and mycolic acids (Gago et al., 2006; the design, development, and evaluation of a ratio- Kurth et al., 2009; Portevin et al., 2005). In order for the ACCs nally designed bisubstrate inhibitor of MtBPL. This to become functionally active, the biotin-carboxylase carrier inhibitor displays potent subnanomolar enzyme protein (BCCP) domains of these proteins must be posttransla- inhibition and antitubercular activity against multi- tionally modified to their active holo biotinylated form by a biotin drug resistant and extensively drug resistant Mtb protein ligase (BPL) encoded by birA in Mtb (MtBPL) (Purusho- strains. We show that the inhibitor decreases in vivo thaman et al., 2008). The biotin serves as the cofactor that medi- protein biotinylation of key enzymes involved in ates carboxyl group transfer onto the acetyl CoA substrates. In fatty acid biosynthesis and that the antibacterial addition to fatty acid biosynthesis, fatty acid degradation is activity is MtBPL dependent. Additionally, the gene also an essential process, and mycobacteria are thought to be encoding BPL was found to be essential in primarily lipolytic in vivo deriving their energy via fatty acid catab- olism (Timm et al., 2003). MtBPL may also play a key role in fatty M. smegmatis. Finally, the X-ray cocrystal structure acid degradation, because this enzyme is also responsible for of inhibitor bound MtBPL was solved providing biotinylation of the BCCP domain of pyruvate carboxylase, detailed insight for further structure-activity analysis. which can channel oxaloacetate to the phosphoenolpyruvate Collectively, these data suggest that MtBPL is carboxykinase, a gluconeogenic enzyme that is essential for a promising target for further antitubercular thera- growth on fatty acids and critical for mycobacterial pathogenesis peutic development. (Marrero et al., 2010). MtBPL thus globally regulates lipid metab- olism and represents an attractive target for development of new antitubercular agents. INTRODUCTION There are two classes of biotin protein ligases (BPLs): the monofunctional enzymes represented by the BPL from Tuberculosis (TB) caused by the bacterium Mycobacterium Pyrococcus horikoshii (PhBPL), which solely catalyze posttrans- tuberculosis (Mtb) continues to burden human health on a global lational biotinylation, and the bifunctional enzymes represented scale. Despite an impressive amount of effort directed at eradi- by the prototypical BPL from Escherichia coli (EcBPL), which cating tuberculosis, TB is still the second leading cause of also act as transcriptional repressors (Brown et al., 2004; mortality among infectious diseases superseded only by HIV. Chapman-Smith and Cronan, 1999). Recent biochemical char- The emergence of multidrug-resistant (MDR) and extensively acterization of the BPL from Mtb (MtBPL) has revealed it to be drug-resistant (XDR) TB strains coupled with the high incidence a monofunctional enzyme that exists as a monomer in both the of HIV-TB coinfection and the lack of any new approved chemo- unliganded apo-and holo-forms as well as in the biotinyl adeny- therapeutic agents for TB in over four decades, demands the late (Bio-AMP, 2) liganded form (Purushothaman et al., 2008). development of new antitubercular agents, ideally with novel BPLs catalyze the transfer of biotin onto a conserved lysine mechanisms of action, to combat this devastating pathogen. residue of the biotin-carboxylase carrier protein (BCCP) domain Fatty acid biosynthesis is a validated pathway for antituber- of ACCs. This reaction is accomplished in two steps: in the first- cular drug discovery, and the first-line agent isoniazid inhibits half reaction, BPLs bind biotin (1, Figure 1A) and ATP and 1432 Chemistry & Biology 18, 1432–1441, November 23, 2011 ª2011 Elsevier Ltd All rights reserved Chemistry & Biology Inhibition of M. tuberculosis Biotin Protein Ligase O NH2 O HN NH N PPi O O O N HN NH O P A O O N N ATP S O OH S BPL 1 2 HO OH O O NH 2 HN NH O HN NH N AMP O N B O O NH2 P NH N S O O N BCCP S O BPL BCCP 3 2 HO OH 4 O NH2 NH2 O N HN NH N N O O O N HN NH O C S O O N N N X N N S S H O N O O S H HO OH HO OH 5, X = O 7 8 6, X = NH Figure 1. Enzyme Reaction Catalyzed by BPL (A) First half-reaction to form biotin adenylate. (B) Second half-reaction involving transfer of biotin to acetyl CoA carboxylases. (C) Bisubstrate inhibitors of BPL and degradation products of 5. See also Figure S1. catalyze the nucleophilic attack of the biotin carboxylate onto the RESULTS a-phosphate of ATP to yield the acyl-adenylate Bio-AMP 2 (Figure 1A) and pyrophosphate. In the second-half reaction, Inhibitor Design and Synthesis BPLs transfer biotin from Bio-AMP 2 onto a conserved lysine Bisubstrate inhibitors that mimic acyl-adenylate reaction inter- residue within the BCCP domain 3 to provide biotinylated- mediates typically bind much more tightly than the individual BCCP 4 (Figure 1B). The bisubstrate inhibitor 5 (Figure 1C) has substrates owing to the large number of interactions between been described, which mimics the intermediate acyl-adenylate the inhibitor and both substrate binding sites (Ferreras et al., Bio-AMP 2 by replacement of the labile acylphosphate linkage 2005; Somu et al., 2006). The reported lack of potency of 5 in Bio-AMP 2 with the bioisosteric acylsulfamate moiety toward EcBPL is surprising (Brown and Beckett, 2005), so we (Brown and Beckett, 2005; Brown et al., 2004). Interestingly, synthesized this compound as described in Supplemental the bisubstrate inhibitor 5 was found to bind 1.4-fold more Experimental Procedures. We immediately observed that it is weakly to EcPBL than the single substrate biotin, suggesting chemically unstable and decomposes through cyclonucleoside the acylsulfamate was a poor isostere for the native acylphos- formation to afford N-biotinylsulfamic acid 7 (Figure 1C) and phate linkage. 3,50-cyclo-50-deoxyadenosine 8 (experimental details and com- Here we describe the design, synthesis, biochemical, struc- plete characterization are found in the Supplemental Informa- tural, and biological evaluation of a bisubstrate inhibitor of tion). We hypothesized that a more stable compound would MtBPL that we term Bio-AMS (6, Figure 1C) which binds more result if the 50-oxygen atom in sulfamate 5 were replaced with than 1700-fold more tightly than biotin and possesses potent a nitrogen atom of sulfamide 6 as a result of the decreased nucle- and selective antimicrobial activity against MDR- and XDR-TB ofugality of the sulfamide (Broeckaert et al., 2008). Compound 6 strains of Mtb. We used M. smegmatis to confirm that the (Bio-AMS) was synthesized, and as predicted, it did not undergo BPL-encoding gene, birA, is essential as well as to assess cyclonucleoside formation. Because of its improved stability, susceptibility of MtBPL to inhibition in vivo. Support for the de- Bio-AMS represents an excellent probe to chemically validate signed mechanism of action is provided using a combination MtBPL as a target for antitubercular therapeutic development. of proteomic profiling and genetic studies. Finally, we describe the detailed molecular interactions present between MtBPL Biochemical Evaluation of Bio-AMS and Bio-AMS that establish the molecular basis for potent inhibi- We next evaluated Bio-AMS in a kinetic continuous coupled tion and provides a framework for future structure-aided drug assay with 250 nM MtBPL to determine the concentration- design. response curve for inhibition of MtBPL at fixed saturating Chemistry & Biology 18, 1432–1441, November 23, 2011 ª2011 Elsevier Ltd All rights reserved 1433 Chemistry & Biology Inhibition of M. tuberculosis Biotin Protein Ligase Time, min A 1.0 B 0 20 40 60 80 100 120 9.6 0.8 ce 9.4 s /l 0.6 acµ 9.2 0 9.0 /v i v 0.4 t 8.8 n a t 0 0.2 cej nifol -2 -4 0.0 o m/l -6 0 250 500 750 1000 ac k Bio-AMS, nM 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 180 Molar Ratio of Bio-AMS:MtBPL C D 116 97 100 57 75 d% ensity ze 40 t 50 normali 29 in band 25 20 0 0 0.1 1.0 14 Bio-AMS, µM M 1 2 3 Figure 2.
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