Rational design, synthesis, antifungal evaluation and docking studies of antifungal peptide CGA-N12 analogues based on the target CtKRE9.

ABSTRACT

Candida tropicalis is a major non-albicans species that causes invasive candidiasis. CGA-N12, an anti-Candida peptide found by our group, disrupted cell wall architecture by inhibiting the activity of the protein killer-resistant 9 (KRE9), a ß-1,6-glucan synthase specific to Candida spp. and plants. Herein, a set of CGA-N12 analogues were rationally designed based on the interaction networks between CGA-N12 and C. tropicalis KRE9 (CtKRE9). Seven CGA-N12 analogues with significantly improved antifungal activity against C. tropicalis were screened by reducing the docking energy of CGA-N12 and CtKRE9 and increasing the number of positive charges on CGA-N12 based on a stable three-dimensional model of CtKRE9. CGA-N12 and its analogues exhibited antifungal activity against C. tropicalis and its persist cells; they also inhibited biofilm formation and eradicated preformed biofilms. Compared with fluconazole, they displayed higher activities against the growth of persister cells and more effective preformed biofilm eradication. Among them, CGA-N12-0801, CGA-N12-0902 and CGA-N12-1002 displayed much higher activity and anti-proteinase digestion stability than CGA-N12. Specifically, CGA-N12-0801 was the optimal analogue, with a minimum inhibitory concentration of 3.46 µg/mL and a therapeutic index of 158.07. The results of electronic microscopy observations and KRE9 activity inhibition assays showed that CGA-N12 and its analogues killed C. tropicalis by disrupting the architecture of the cell wall and the integrity of the cell membrane. In conclusion, for the first time, we provide a simple and reliable method for the rational design of antimicrobial peptides and ideal candidates for treating Candida infections that not effectively eliminated by azole drugs.