Identification Of The Key Amino Acids Responsible For Candida Albicans Sterol 14α-demethylase Selective Interaction With New Azole Aanalogues

Azoles are widely used in the treatment of fungal infections. They act by competitive inhibition of lanosterol 14α-demethylase (P45014DM, CYP51), the key enzyme in sterol biosynthesis, which results in depletion of ergosterol and accumulation of lanosterol and other 14-methyl sterols ultimately leading to the growth inhibition of fungal cells. Inhibition of the ergosterol biosynthetic pathway in fungi has thus attracted intense interests in the application of such antifungal compounds. However, it was found in the past two decades that azole resistance often develops prolonged use of oral imidazole and triazole, or different forms of immunodeficiency in fungal infection patients. Furthermore, cross inhibition of CYP51 in different species also causes undesirable side effects. It is, therefore, of increasingly interest to develop novel antifungal agents with more selective antifungal activity, higher efficiency, broader pectrum, and lower toxicity.Azole antifungal agents inhibit the CYP51 by a mechanism in which the heterocyclic nitrogen atom (N-3 of imidazole and N-4 of triazole) binds to the heme iron atom in the binding site of the enzyme. Because all the fungal CYP51 proteins that had been characterized were membrane-bound and difficult to solve their crystal structures, the study of the interaction between azoles and fungal CYP51 can only be done by methods of molecular modeling. Several three-dimensional (3D) models of CYP51 and their interaction with azole antifungal agents has been reported. Ji et al. built a homologous 3D model of CYP51 from C. albicans based on the crystal coordinates of all four known prokaryotic P450s. With this model they found that the halogenated phenyl group of azole inhibitors is deep in the hydrophobic binding cleft and the long side chains of some inhibitors such as itraconazole and ketoconazole surpass the active site and interact with the residues in the substrate access channel. Another 3D molecular model constructed by Lewis et al. also showed that typical azole inhibitors were able to fit the putative active site of CYP51 by a combination of heme coordination, hydrogen bonding,π–πstacking and hydrophobic interactions within the heme environment of the enzymes. Recently, modelling data of Xiao et al. suggest that the long chain of posaconazole and itraconazole occupies a specific channel within CYP51 and that this additional interaction serves to stabilize the binding of these azoles to the mutated CYP51 proteins. Models generated by Fukuoka et al. predicted that voriconazole was a more potent inhibitor than fluconazole because the additional methyl group of voriconazole resulted in stronger hydrophobic interaction with the aromatic amino acids in the substrate binding site and filled the site more extensively.In our studies, the 3D model of CYP51 from Candida albicans (CACYP51) was constructed. In addition, the binding mode of the substrate with fungal CYP51 was also investigated. In order to search more potent azoles antifungal agents and design lead compounds with higher affinity, the binding mode of azoles antifungal agents with CACYP51 was explored using the previously described molecular docking and 3D-QSAR methods. And a structure-based pharmacophore model was therefore established to guide the rational optimization of the azole antifungal agents. This model predicted a hypothesis that the phenyl group of the C-3 side chain of azole antifungal compounds interacts with the phenol group of Tyr118, a highly conserved residue in CYP51 family, through the formation ofπ-πface-to-edge interaction. On the basis of the previous molecular modeling studies of azole antifungal agents with fungal CYP51, we also found that His310, Ser378 of CACYP51 might play an important role in the inhibitor or subtrate binding. H310 was unique and absolutely conserved throughout the CYP51 family. In a docking model of substrate with CYP51 from Mycobacterium tuberculosis (MTCYP51), the correspondent conserved residue His254 can form hydrogen-bond with OH-group of the substrate. In the docking model of the substrate with CACYP51, H310 can also form hydrogen bond with OH-group of the substrate. It was assumed that the functional role of H310 might be guiding substrate entry by interacting with OH-group of incoming substrate, tabilizing of an oxysterol intermediate in the CYP51 active site and orienting it for the next round of multiple, stereo specific oxidation by donating a hydrogen bond to the newly inserted oxygen atom.Previous docking results showed that our designed azole antifungal compounds could form a hydrogen bond with the side chain of S378 by the substitutions on the para-position of the phenyl group. Also, S378 was used as a hydrogen bond site to design non-azole inhibitors and the results supported our hypothesis. Therefore, we suggest that S378 should be an important hydrogen bond residue in the CACYP51 inhibitor design. To demonstrate this hypothesis and verify the reliability of the derived pharmacophore model, a series of novel azole compounds on the basis of this model were synthesized and tested for their in vitro antifungal activities in our laboratory. One of the major objectives of the present study is to reveal that these azole compounds will have excellent in vitro activities and the structure-activity relationship correlated with the model. As a result, our synthesized compounds may not only be used to perform further biological evaluations to develop new candidates of antifungal drugs, but also can be used as"probes"to investigate the structure-function relationship of CYP51 family.Up to date, the x-ray crystal structure of CYP51 from Mycobacterium tuberculosis has already been solved, but the explicit information about enzyme binding site of pathogenic fungi is not available yet. Even when the structure of a complex is available, it is still very difficult to deduce the relative contribution of each individual residue to the total binding energy. Mutational analysis, therefore, remains to be the mainstay of molecular determination of binding interfaces between receptor-ligand complexes. Although there are a number of papers reporting mutation analyses of current azole antifungal agents, they focused more on the mechanism of drug resistance, and site-directed mutagenesis pinpointing key residues responsible for substrate or inhibitors binding were never reported. In the present study, site-directed mutagenesis of the Y118, H310, S378 residues of C. albicans CYP51 heterologously expressed in S. cerevisiae was undertaken in an attempt to verify our previous molecular modeling by comparative assessment of the strain susceptibility and enzyme binding to a set of azoles antifungals.