We report the synthesis and evaluation of 5-halogenated-1,2,3-triazoles as inhibitors of biotin protein ligase from Staphylococcus aureus. The halogenated compounds exhibit significantly improved antibacterial activity over their nonhalogenated counterparts. Importantly, the 5-fluoro-1,2,3-triazole compound 4c displays antibacterial activity against S. aureus ATCC49775 with a minimum inhibitory concentration (MIC) of 8 μg/mL.
Clostridium difficile causes life-threatening diarrhea and is the leading cause of healthcare-associated bacterial infections in the United States. TcdA and TcdB bacterial toxins are primary determinants of disease pathogenesis and are attractive therapeutic targets. TcdA and TcdB contain domains that use UDP-glucose to glucosylate and inactivate host Rho GTPases, resulting in cytoskeletal changes causing cell rounding and loss of intestinal integrity. Transition state analysis revealed glucocationic character for the TcdA and TcdB transition states. We identified transition state analogue inhibitors and characterized them by kinetic, thermodynamic and structural analysis. Iminosugars, isofagomine and noeuromycin mimic the transition state and inhibit both TcdA and TcdB by forming ternary complexes with Tcd and UDP, a product of the TcdA- and TcdB-catalyzed reactions. Both iminosugars prevent TcdA- and TcdB-induced cytotoxicity in cultured mammalian cells by preventing glucosylation of Rho GTPases. Iminosugar transition state analogues of the Tcd toxins show potential as therapeutics for C. difficile pathology.
Replacing the labile adenosinyl-substituted phosphoanhydride of biotinyl-5′-AMP with a N1-benzyl substituted 1,2,3-triazole gave a new truncated series of inhibitors of Staphylococcus aureus biotin protein ligase (SaBPL). The benzyl group presents to the ribose-binding pocket of SaBPL based on in silico docking. Halogenated benzyl derivatives (12t, 12u, 12w, and 12x) proved to be the most potent inhibitors of SaBPL. These derivatives inhibited the growth of S. aureus ATCC49775 and displayed low cytotoxicity against HepG2 cells. A number of analogues of biotinyl-5′-AMP have recently been reported as inhibitors of BPL as shown in Figure 2. Some of these compounds have potential as antibacterial agents by inhibiting BPL from clinically important pathogens such as Staphylococcus aureus, 6 Escherichia coli, 7,8 and Mycobacterium tuberculosis. 9,10 A range of bioisosteres have been investigated as replacements for the labile phosphoanhydride of biotinyl-5′-AMP 3, including phosphodiester 4, 11,12 hydroxyphosphonate 5, 13 ketophosphonate 6, 13 acylsulfamate 7, 11 and sulphonmyl amide 8 10 (Figure 2). We have also reported biotin triazoles (e.g., 9−11) as a novel class of BPL inhibitor that selectively targets BPL from the clinically important bacterial pathogen Staphylococcus aureus over the human homologue. 3,14,15 Without exception, all isostere-based BPL inhibitors reported to date contain a biotin and an adenine group, or analogue thereof, as discussed above and as shown in Figure 2. These two groups occupy well-defined binding pockets in the enzyme as per biotinyl-5′-AMP 3, as supported by X-ray crystallographic and mutagenesis studies. 3,16 The ribose group of the triazole series can be removed as in 10, and the adenine can be modified as in 11, which has improved stability and >1000-fold specificity for the BPL from S. aureus over the human homologue. 3 We now report the first examples of truncated 1,2,3-triazole-based BPL inhibitors with a 1-benzyl substituent
Biotin protein ligase (BPL) represents a promising target for the discovery of new antibacterial chemotherapeutics. Here we review the central role of BPL for the survival and virulence of clinically important Staphylococcus aureus in support of this claim. X-ray crystallography structures of BPLs in complex with ligands and small molecule inhibitors provide new insights into the mechanism of protein biotinylation, and a template for structure guided approaches to the design of inhibitors for antibacterial discovery. Most BPLs employ an ordered ligand binding mechanism for the synthesis of the reaction intermediate biotinyl-5´-AMP from substrates biotin and ATP. Recent studies reporting chemical analogs of biotin and biotinyl-5´-AMP as BPL inhibitors that represent new classes of anti-S. aureus agents are reviewed. We highlight strategies to selectively inhibit bacterial BPL over the mammalian equivalent using a 1,2,3-triazole isostere to replace the labile phosphoanhydride naturally present in biotinyl-5´-AMP. A novel in situ approach to improve the detection of triazole-based inhibitors is also presented that could potentially be widely applied to other protein targets.
Toxins TcdA and TcdB from Clostridioides difficile glucosylate human colon Rho GTPases. TcdA and TcdB glucosylation of RhoGTPases results in cytoskeletal changes, causing cell rounding and loss of intestinal integrity. Clostridial toxins TcdA and TcdB are proposed to catalyze glucosylation of Rho GTPases with retention of stereochemistry from UDP-glucose. We used kinetic isotope effects to analyze the mechanisms and transition-state structures of the glucohydrolase and glucosyltransferase activities of TcdB. TcdB catalyzes Rho GTPase glucosylation with retention of stereochemistry, while hydrolysis of UDP-glucose by TcdB causes inversion of stereochemistry. Kinetic analysis revealed TcdB glucosylation via the formation of a ternary complex with no intermediate, supporting an S N i mechanism with nucleophilic attack and leaving group departure occurring on the same face of the glucose ring. Kinetic isotope effects combined with quantum mechanical calculations revealed that the transition states of both glucohydrolase and glucosyltransferase activities of TcdB are highly dissociative. Specifically, the TcdB glucosyltransferase reaction proceeds via an S N i mechanism with the formation of a distinct oxocarbenium phosphate ion pair transition state where the glycosidic bond to the UDP leaving group breaks prior to attack of the threonine nucleophile from Rho GTPase.
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