Expanding the therapeutic options for Candida infections using novel inhibitors of secreted aspartyl proteases.

For widening the therapeutic options for Candida management, the druggability of Candida proteome was systematically investigated using an innovative pipeline of high-throughput data mining algorithms, followed by in vitro validation of the observations. Through this exercise, HIV-1 protease was found to share structural similarity with secreted aspartyl protease-3 (SAP3), a virulence protein of Candida. Using the molecular fingerprint of HIV-1 protease inhibitor GRL-09510, we performed virtual screening of peptidomimetic library followed by high-precision docking and MD simulations for discovery of SAP inhibitors. Wet-lab validation of the four shortlisted peptidomimetics revealed that two molecules, when used in combination with fluconazole, could significantly reduce the dosage of fluconazole required for 50% inhibition of Candida albicans. The SAP inhibitory activity of these peptidomimetics was confirmed through SAP assays and found to be on par with pepstatin A, a known peptidomimetic inhibitor of aspartyl proteases.

4-Substituted 2-amino-3,4-dihydroquinazolines with a 3-hairpin turn side chain as novel inhibitors of BACE-1.

Herein, we report the identification, design, and synthesis of a series of 4-substituted 2-amino-3,4-dihydroquinazolines with hairpin turn side chains as novel inhibitors of BACE-1. The dihydroquinazoline derivatives were rationally designed by modifying the amide group and relocating the α -hydrophobic substituent on the hairpin turn side chain of lead compound 2 to the C4-position on the 3,4-dihydroquinazoline scaffold to facilitate interactions with the S1, S2 and S1' subsites of BACE-1. Among these derivatives, two compounds exhibited potent BACE-1 inhibitory activity: 4-methyl-substituted (22a, BACE-1 CFA IC50 = 0.38 µM; BACE-1 WCA IC50 = 0.14 µM) and 4-cyclohexylmethyl-substituted (22b, BACE-1 CFA IC50 = 0.49 µM; BACE-1 WCA IC50 = 0.14 µM) 2-amino-3,4-dihydroquinazoline, each bearing a side chain of N-cyclohexyl-N-((1-methyl-1H-pyrazol-4-yl)methyl amide. The results suggest that the structural modifications maintain the hairpin turn topology similar to that of compound 2 and provide an additional interaction with the S2 subsite.

Discovery of potent iminoheterocycle BACE1 inhibitors.

The synthesis of a series of iminoheterocycles and their structure-activity relationships (SAR) as inhibitors of the aspartyl protease BACE1 will be detailed. An effort to access the S3 subsite directly from the S1 subsite initially yielded compounds with sub-micromolar potency. A subset of compounds from this effort unexpectedly occupied a different binding site and displayed excellent BACE1 affinities. Select compounds from this subset acutely lowered Aß40 levels upon subcutaneous and oral administration to rats.

Combination of Antifungal Drugs and Protease Inhibitors Prevent Candida albicans Biofilm Formation and Disrupt Mature Biofilms.

Biofilms formed by the fungal pathogen Candida albicans are resistant to many of the antifungal agents commonly used in the clinic. Previous reports suggest that protease inhibitors, specifically inhibitors of aspartyl proteases, could be effective antibiofilm agents. We screened three protease inhibitor libraries, containing a total of 80 compounds for the abilities to prevent C. albicans biofilm formation and to disrupt mature biofilms. The compounds were screened individually and in the presence of subinhibitory concentrations of the most commonly prescribed antifungal agents for Candida infections: fluconazole, amphotericin B, or caspofungin. Although few of the compounds affected biofilms on their own, seven aspartyl protease inhibitors inhibited biofilm formation when combined with amphotericin B or caspofungin. Furthermore, nine aspartyl protease inhibitors disrupted mature biofilms when combined with caspofungin. These results suggest that the combination of standard antifungal agents together with specific protease inhibitors may be useful in the prevention and treatment of C. albicans biofilm infections.