Abstract
The yeast Malassezia pachydermatis (M. pachydermatis) is responsible for otitis externa and dermatitis in canine species, and in rare cases life threatening fungemia in immunocompromised people. Current treatment for companion animals involves the use of azole antifungals targeting M. pachydermatis CYP51 (MpCYP51). Despite current treatment efforts, there is evidence to suggest resistance to miconazole and other azoles is occurring in cases of canine otitis externa. Genome sequencing has identified numerous mutations in MpCYP51. However, a lack of published M. pachydermatis structures means the location of mutations has not been identified.
The microbe Helicobacter pylori (H. pylori) is a class one carcinogen, responsible for diseases, such as peptic ulcers as well as gastric cancers. Current treatment involves a triple therapy employing proton pump inhibitors alongside antibiotics. Despite this, infection is often persistent due to the ability of H. pylori to alter the microenvironment. Recently, HtrA proteases have emerged as a drug target, noted as vital for life in H. pylori. It is a bifunctional serine protease, with drugs covalently inhibiting the active site serine residue. However, there is difficulty in targeting HtrA proteases due to the proteolytically active and inactive conformations, as well as significant molecular rearrangement between conformations.
The objective of this study is to model MpCYP51 with azoles and to help understand resistance mutations against azole antifungals in canine otitis externa, as well as design and model covalent small molecule inhibitors targeting H. pylori HtrA (HpHtrA). Homology models of MpCYP51 and HpHtrA were generated using MODELLER, and sequence alignments were provided by T-coffee. Ligands were generated using CHEMDRAW, docking conducted using GOLD and analysis of the proteins was performed using PyMOL.
Docking of azoles including R-miconazole revealed the presence of mutations in proximity of the drugs which may disrupt binding. These results indicate resistance is mediated by A302V and V123A mutations, which are within or adjacent to the active site and bound ligands. The mutants alter the shape of the binding pocket, interfering with ligand binding deep within the active site. Furthermore, using the cleavage specificity profile for HpHtrA helped to guide ligand design, revealing opportunities for arginine mimetics with different covalent scaffolds.
In summary, resistance to miconazole is occurring in cases of canine otitis externa, and the mechanism has been identified. In addition, this study has looked to identify new non peptidic inhibitors with arginine mimetics of HpHtrA via the cleavage specificity profile.