Design, Synthesis and Testing of Novel Classes of Inhibitors Against Metallo-β-lactamases: Towards Drug Leads to Combat Antibiotic Resistance

Yusof, Yusralina (2015). Design, Synthesis and Testing of Novel Classes of Inhibitors Against Metallo-β-lactamases: Towards Drug Leads to Combat Antibiotic Resistance PhD Thesis, School of Chemistry and Molecular Biosciences, The University of Queensland. doi:10.14264/uql.2016.473

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Author Yusof, Yusralina
Thesis Title Design, Synthesis and Testing of Novel Classes of Inhibitors Against Metallo-β-lactamases: Towards Drug Leads to Combat Antibiotic Resistance
School, Centre or Institute School of Chemistry and Molecular Biosciences
Institution The University of Queensland
DOI 10.14264/uql.2016.473
Publication date 2015-07-18
Thesis type PhD Thesis
Supervisor Ross McGeary
Gary Schenk
Total pages 284
Total colour pages 71
Total black and white pages 213
Language eng
Subjects 0305 Organic Chemistry
0304 Medicinal and Biomolecular Chemistry
Formatted abstract
Overprescription of antibiotics is one of the causes of the increasing incidence of antibiotic resistance. Bacteria are constantly evolving and able to develop resistance to new antibiotics by producing β-lactamases, thus causing the standard treatments to be ineffective. Furthermore, genes for antibiotic resistance can be transferred between unrelated species. β-Lactamases are classified into serine-β-lactamases (SBLs) and metallo-β-lactamases (MBLs). Clavulanic acid is a well-known inhibitor that can effectively inhibit serine β-lactamases (SBLs). Nonetheless, society is in urgent need of metallo-β-lactamase (MBL) inhibitors as clinically approved inhibitors for MBLs are not yet available. IMP-1, a clinically significant MBL has been reported to cause antibiotic resistance associated with infections such as pneumonia and wound infections. IMP-1 shows the characteristics of MBLs with di-Zn ions in the active site. One of the zinc ions in IMP-1 is ligated by three imidazoles of histidines, while the other zinc ion is ligated by the carboxylate of an aspartic acid, the imidazole of a histidine and the thiolate of a cysteine.

In this research, novel inhibitors against IMP-1 MBL were designed based on the excellent competitive inhibition properties of L-captopril and D-captopril, compound RS-4.2, and compound 5.1. Modelling studies were done by docking these newly designed molecules into IMP-1 enzyme (PDB code: 1JJT) using Molegro Virtual Docker (MVD) software. Thiolate group of the designed compounds was predicted to bind to both zinc ions and the carboxylate and amide carbonyl groups of the compounds were predicted to form hydrogen bond or electrostatic interactions with the amino acid residues of the IMP-1 active site. Due to these favourable interactions, the MVD software predicted that the designed compounds would have high inhibition potencies against the enzyme.

Syntheses of the designed molecules were completed via esterification of the starting materials, coupling reaction and lastly hydrolysis to remove both methyl ester and thioester. Eight series of compounds were synthesised including L- and D-proline derivatives (thiols and dicarboxylic acids), D-captopril derivatives, pipecolinic acid derivatives, phenylglycine derivatives, 2-aminopyridine derivatives, 2-amino-6-picolinic acid derivatives and diaminobenzoic acid derivatives. All of the synthesised compounds including the novel inhibitors and their precursors were tested against freshly expressed and purified IMP-1 enzyme. The expression process includes transformation, inoculation, enzyme expression and cell harvesting. The enzyme was purified via two-steps purification by using SP-Sepharose cation exchange column and Sepharacyl S-200 gel filtration size exclusion column. The yield of the enzyme obtained was 12 mg per 2 litres of culture. Among the compounds synthesised in this study, thiols 2.1.1 – 2.1.3 (Ki = 2.2 ± 0.6 to 9.9 ± 4.2 μM) and compounds 5.2 – 5.4 (Ki = 2.1 ± 0.8 to 4.3 ± 2.0 μM) are the most potent compounds against IMP-1. All of the compounds tested inhibit IMP-1 by competitive inhibition mode except compounds 5.2 – 5.4, which uncompetitively inhibit the enzyme.

Screening of compounds synthesised as purple acid phosphatase (PAP) inhibitors were also performed against IMP-1 enzyme since PAPs, similar to IMP-1, are metallohydrolases with two metal ions in the active site. Only compounds 6.10 and 6.14 are potent enough to be further tested for their Ki values determination. Compound 6.10 has low inhibition potency against IMP-1 and inhibits the enzyme by uncompetitive inhibition (Ki = 71 ± 50 μM). Compound 6.14 inhibits IMP-1 competitively with a Ki value of 5.4 ± 2.5 μM.
Keyword antibiotic resistance

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Created: Wed, 06 Jul 2016, 14:08:20 EST by Yusralina Yusof on behalf of Learning and Research Services (UQ Library)