Development of biotin protein ligase inhibitors from Staphylococcus aureus as new antibiotics

Abstract

Biotin protein ligase (BPL) catalyses the ordered reaction of biotin and ATP to give biotinyl-5’-AMP 1.03, which then activates a number of biotin dependent enzymes that are critical to cell survival. Research undertaken in this thesis highlights strategies to selectively inhibit Staphylococcus aureus biotin protein ligase (SaBPL) over the mammalian equivalent using 1,2,3-triazole and acylsulfonamide isosteres to replace the phosphoroanhydride linker found in biotinyl-5’-AMP 1.03. Chapter one describes the structure and catalytic mechanism of the target enzyme SaBPL, along with an overview of chemical analogues of biotin and biotinyl-5’-AMP 1.03 as BPL inhibitors reported to date. Preliminary studies on the utility of a 1,2,3-triazole as a bioisostere of the phosphoroanhydride linker of biotinyl-5’-AMP 1.03 are also discussed. Chapter two further examines 1,2,3-triazole analogues of lead SaBPL bisubstrate inhibitors 1.22 and 1.23. Specific chemical modifications were carried out at the ribose of biotinyl-5’- AMP 1.03, and a new class of purine analogues was developed to mimic the adenine group as in 1.03. In silico docking experiments using our x-ray structure of SaBPL aided in the design of these analogues by predicting optimal binding conformations. A structure activity relationship for the ribose and adenine mimics was developed and this revealed limited improvement in potency against SaBPL on modification at these two sites. Chapter three reports the first examples of truncated 1,2,3-triazole based BPL inhibitors with a 1-benzyl substituent designed to interact with the ribose binding pocket of SaBPL. In silico docking studies using a crystal structure of SaBPL aided in the selection of benzyl groups that present in the ribose-binding pocket of SaBPL. The halogenated benzyl derivatives 3.20, 3.21, 3.23 and 3.24 provided the most potent inhibitors of SaBPL with the respective Kᵢ value of 0.28, 0.6, 0.39 and 1.1 μM. These compounds also inhibited the growth of S. aureus ATCC49775 (MIC = 4 – 16 μg/ml), while possessing low cytotoxicity against HepG2 cells. Chapter four builds upon the active 1,2,3-triazole based inhibitors of SaBPL described in chapter two and three with an investigation at C5 of the triazole ring to generate 1,4,5- trisubstituted 1,2,3-triazoles. A class of 5-iodo 1,2,3-triazoles was synthesised from 1- iodoacetylene 4.02 and azides using CuAAC. Subsequent halogen exchange reaction allowed conversion of iodide to other halogens. 5-Fluoro-1,2,3-triazole 4.07, the lead compound from this series of inhibitors, proved to be a potent and selective inhibitor of SaBPL (Kᵢ = 0.42 ± 0.06 μM) and it significantly reduced S. aureus growth with no cell growth apparent at 16 μg/mL. Chapter five investigates the use of acylsulfonamide as a bioisostere of the phosphoroanhydride linker as in biotinyl-5’-AMP 1.03. Acylsulfonamide 5.05 was found as the most active and selective inhibitor of SaBPL (Kᵢ = 0.72 x 10⁻³ μM) and MtbBPL (Kᵢ = 0.74 x 10⁻³ μM) reported to date. Antibacterial studies revealed that 5.05 was active against susceptible S. aureus (MIC = 0.5-1.0 μg/mL), methicillin-resistant S. aureus ((MIC = 0.5- 1.0 μg/mL) and Mycobacterial tuberculosis ((MIC = 51 μg/mL). Finally, the x-ray structure 5.05 bound to SaBPL was solved to reveal important molecular interactions critical to the potency of 5.05 and emphasized the acylsulfonamide moiety as an effective bioisostere of phosphoroanhydride linker. Chapter six discusses the use of in situ click chemistry as an alternative approach for the synthesis of 1,2,3-triazoles. The target enzyme SaBPL was directly involved in the selection of its optimum triazole based inhibitor by catalysing the reaction of biotin acetylene and organic azides without copper as a catalyst. The use of high throughput LC/MS provided improved efficiency and sensitivity of detection of triazole-based inhibitors and allowed the in situ approach to be widely applied to BPLs from other bacteria. Chapter seven details the experimental procedures for compounds described in chapter 2 – 6, and the chromatographic analysis of in situ click experiments described in chapter 6.