Design and Synthesis of Novel CYP51 Inhibitors as Therapeutics for Candida Albicans Infection

Abstract

Fungal infections are a global concern with C. albicans being one of the most prevalent fungal pathogens that causes infections ranging from superficial to life-threatening invasive infections in immunocompromised patients. Azole antifungal agents remain the prophylactic and first line treatment however, resistance to these drugs has increased with sterol 14α-demethylase (CYP51) mutations being the most common resistance mechanism in C. albicans. The project aimed to design and synthesise novel CYP51 inhibitors that can overcome the azole CYP51 mutation resistance mechanism in C. albicans. Computational methods were performed using MOE for molecular docking and Desmond Maestro for MD simulations to evaluate optimal CaCYP51 active site binding, key amino acid binding interactions and binding stability. All triazole series demonstrated promising MD results with optimal haem positioning and compensatory amide linker interactions (H-bonding, π-π interaction) overcoming Tyr132 mutations in resistant C. albicans. Hydroxyl triazole/tetrazole with long arm moiety (Chapter II), non-hydroxyl triazole/tetrazole propenamide (Chapter III) and non-hydroxyl triazole/tetrazole para-alkoxy biphenyl (Chapter IV) series were selected for synthesis. All novel compounds were synthesised through six to eight synthetic steps and fully characterised (NMR, HPLC-MS, elemental analysis). The compounds 27a-29a and 26b-28b (Chapter II) were active against wild type C. albicans (MIC < 0.03 μg/mL) compared with FLZ MIC = 0.125 μg/mL and PCZ MIC < 0.03 μg/mL. The promising compounds 26b-28b were found to be active against single mutant ScCYP51 strains, Y2301 (Y140F) and Y2513 (Y140H) with MIC≤ 0.03-0.06 μg/mL, comparable to PCZ (0.06 μg/mL) and the difluorophenyl compounds 27a-29a with MIC 0.06-0.5 μg/mL were better than FLZ (32 μg/mL). All compounds 27a-29a and 26b-28b showed comparable antifungal activity to PCZ MIC against Y525 (CaMDR1a) strain, while the antifungal activity was reduced against Y570 (CaCDR1b) strain. Additionally, promising compounds 27a-29a, 26b and 28b showed good selectivity with reduced activity against the human ERG11-overexpressing Y2760 strain (MIC >16 μg/mL). Compound 26b showed greater IC50 against wild type (0.41 ± 0.03 μM) and double mutant CaCYP51 (1.89 ± 0.24 μM) compared with both FLZ (IC50 of 25.72 ± 3.55 μM and lead compound MA6.27 (IC50 of 8.49 ± 1.27 μM). Results provide proposed SAR for CaCYP51 inhibitors optimisation, include 2,4-dichloro phenyl in the short arm (side chain A) that occupies the hydrophobic pocket, CH2-triazole allows optimal haem binding, and amide linker connects both arms and para-alkoxy biphenyl long arm (side chain B) provides optimal positioning with additional binding interactions, such as His132, His310, Met508, Phe380 and Ser378. All azole antifungal agents showed consistent increased MICs against single mutant Tyr132 (Y140F and Y140H) strains, indicating that azoles struggle to overcome these amino acid mutations. In contrast, compound 26b retained strong activity comparable to PCZ and OTZ and exceeded that of FLZ. These findings suggest that compound 26b could be present a potential lead for the development of antifungal agents to overcome amino acid mutation resistance in C. albicans.