Design and Development of Inhibitors of Sporulation Targeting Clostridioides difficile Infections

The transmission of Clostridioides difficile (C. difficile) infections (CDI) and the high rates of disease recurrence are underpinned by the ability of the vegetative cells to produce and release highly resistant bacterial spores. A significant gap in current CDI treatment protocols is the lack of an effective inhibitor of the sporulation cycle. Recent studies identified the cephamycin class of antibacterials as effective inhibitors of CdSpoVD, a spore-specific, class B penicillin binding protein (PBP) that is essential in C. difficile sporogenesis.Molecular modelling was used in this dissertation to generate a model to guide synthetic derivatisation of the lead compound cefotetan. The synthetic potential of CdSpoVD inhibitors was assessed by covalent docking to the catalytic serine residue within the transpeptidase active site. Preliminary screening was performed using a comparative homology model of CdSpoVD, in addition to a set of AI generated AlphaFold models, which identified two amide derivatives that produced spore reduction values ten times greater than the lead. Following publication of the X-ray crystal structure of CdSpoVD, the high-resolution structure (7rcz) was utilised to develop a structure-activity relationship (SAR) for the design of new derivatives. The key protein residues identified in intermolecular interactions were Ser311, Gln427, Thr513, Thr515, Tyr559 and Phe559. The orientation of the ligand’s C2 carboxylate (C2COO-) and C7 moieties within the active site are significant determinants of predicted binding affinity. Based on the developed SAR constraints, a novel generation of sixty cephamycin derivatives containing sulfonamide and a-thioacetamide modifications were assessed for their synthetic potential using the 7rcz CdSpoVD model.Attempted synthesis of the identified sulfonamide derivatives from cephem precursor 7-MAC, resulted in formation of an unexpected oxocephalosporin product, formed via an N-sulfonylimine intermediate. Proof of concept sulfonamidation reactions using a nonmethoxylated cephem precursor, 7-ACA, confirmed the role of the C7 MeO as a base promoted leaving group in a proposed mechanism of oxocephem formation. Six a-thioacetamide derivatives were synthesised using a three-step synthetic procedure. Optimisation of reaction repeatability and product yield was conducted by changes to solvent, reaction time and the temperature at which the reaction and purification were performed for each derivative. The a-thioacetamides were produced in 38% to 73% yield and >95% purity.The minimum-inhibitory concentration (MIC) of the α-thioacetamide derivatives against the vegetative cells of C. difficile ranged from 10 μg/mL to 40 μg/mL. The antisporulation activity of the six assessed derivatives ranged from a 100 to 1000 fold reduction in C. difficile spore numbers compared to an untreated control in vitro. Assessment of the broad-spectrum MIC and minimum-bactericidal (MBC) activity of the submitted compounds identified antimicrobial hits against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) bacteria, with MIC/MBC values ranging from 2 μg/mL to 128 μg/mL. Only the pyrimidine α-thioacetamide derivative showed cell growth inhibition in Gram-negative bacteria, with an MIC value of 64 μg/mL against Klebsiella pneumoniae (K. pneumoniae). The α-thioacetamides did not produce antibacterial action against Acinetobacter baumannii (A. baumannii) or Pseudomonas aeruginosa (P. aeruginosa) up to 128 μg/mL in the MIC/MBC assessment.The a-thioacetamide derivative containing a benzonitrile modification showed no haemolysis or cytotoxicity activity against Homo sapiens cell lines and no inhibition activity at >32 μg/mL against two fungal species. The half maximal inhibitory concentration (IC50) of this derivative against the CdSpoVD protein target was calculated to be 7.5 nM compared to the IC50 value of cefotetan of 15.2 nM, indicating strong binding affinity of the a-thioacetamide for the PBP active site.