DESIGN and SYNTHESIS of NOVEL CYP51 INHIBITORS

Abstract

Fungal infections are a global issue affecting over 150 million people worldwide annually with 750,000 of these caused by invasive Candida infections. The outcomes of life threatening systemic infections caused by Candida albicans are poor with mortality rates estimated to be between 46-75%. Azole drugs are the frontline treatment against fungal infections however resistance to current azole antifungals in C. albicans poses a threat to public health. Azole resistance can arise through several mechanisms with point mutations in sterol 14α-demethylase (CYP51) leading to amino acid substitutions a major contributor. The aim of this research is to design and synthesise novel azole inhibitors effective against wild type and fluconazole-resistance Candida strains. The development of potent and selective inhibitors from three azole series were investigated for CYP51 inhibitory activity, binding affinity, and minimum inhibitory concentration (MIC) against C. albicans strains biologically as well as computationally. The first series, short and extended novel imidazole/triazole derivatives were synthesised successfully. The short derivatives were more potent against the C. albicans strains (MIC 0.03 μg/mL) compared with the extended derivatives (MIC 1 μg/mL), while both series showed similar enzyme binding and inhibition (Kd low nM, IC50 submicromolar) and were comparable with the standards fluconazole and posaconazole. The short series had poor selectivity for CaCYP51 over the human homolog, while the selectivity of the extended series was higher (21.5-fold) than posaconazole (4.7- fold) based on Kd values, although posaconazole was more selective (615-fold) compared with the extended series (461-fold) based on IC50 values. Series two, 2-(arylphenyl)-N-(4- ((4-arylphenyl)amido)benzyl)-3-(1H-1,2,4-triazol-1-yl)propanamides derivatives, were synthesised successfully. The novel inhibitors exhibited weak activity against C. albicans strains; however, a slight improvement in the IC50 was shown in chloro derivatives (IC50 4.6 -1.3 µM). A series of N-(2-(arylphenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)butanamide derivatives have been synthesised using an efficient synthetic route and shown to be potent against the C. albicans strains (MIC from <0.03 to 1 µg/mL) and potent inhibitors of CaCYP51 (IC50 0.78 to 1.6 µM) compared with the standard fluconazole. All series were studied computationally using CaCYP51 crystal structure (PDB 5FSA) for molecular modelling and molecular dynamic simulations to determine optimal fit in the active site and binding interactions. Leishmania was also of interest as it has been identified by the WHO as a disease with unmet needs with an estimated 700,000 to one million new cases each year in the endemic regions such as East Africa, North Africa and West Asia. CYP5122A1 an orphan enzyme has been identified as a CYP enzyme specific to leishmania, which could provide a novel target in the treatment of leishmania infections. A CYP5122A1 homology model was developed, as no crystal structure is available, using a combination of homology modelling, molecular dynamics simulations, and molecular docking to understand the active site and the binding interaction of CYP5122A1 and selected ligands complexes. Docking results for CYP5122A1 showed amino acids Glu365, Thr366, Val440 in the haem binding pocket and Tyr175, Phe178, Pro441, Asp584 in the access channel, which could have an important role in the binding interactions with designed ligands. Furthermore, some of the novel compounds synthesised in this research were also tested against Leishmania donovani to investigate the inhibitory potential.