Harnessing random peptide mixtures to combat multidrug-resistant fungal infections

Invasive fungal infections are associated with high mortality and are increasingly difficult to treat due to a limited antifungal arsenal and the rapid emergence of drug resistance. Novel therapeutic strategies that combine potent antifungal activity, low host toxicity, in vivo stability, and a reduced propensity for resistance development are urgently needed. Antimicrobial peptides (AMPs) stand out as a promising class of compounds to combat antimicrobial resistance. Leveraging the unique properties of AMPs, we previously developed a novel approach to synthesize random peptide mixtures (RPMs) with robust bactericidal activity against drug-resistant bacteria. Here, we evaluate the antifungal potential of RPMs and demonstrate species-dependent, broad-spectrum activity of FK20 (L-phenylalanine–L-lysine, 20-mer) against major human fungal pathogens, including Candida spp., Cryptococcus neoformans, and Aspergillus fumigatus, with particularly high potency against the multidrug-resistant pathogen Candida auris. Mechanistic analyses revealed rapid membrane and cell wall disruption accompanied by intracellular penetration, consistent with membrane-active antifungal activity. Importantly, experimental evolution assays demonstrated a markedly reduced capacity for resistance development in C. auris. FK20 inhibited biofilm formation and displayed substantial activity against mature, pre-formed biofilms, both alone and synergistically in combination with caspofungin. Finally, FK20 showed significant therapeutic efficacy in a murine model of systemic candidiasis. Collectively, these findings establish RPMs as a versatile antifungal platform with broad-spectrum activity, biofilm efficacy, and a low resistance footprint, highlighting their promise as a novel therapeutic strategy against drug-resistant fungal infections. IMPORTANCE The rising prevalence of invasive fungal infections, particularly among immunocompromised individuals, has become a critical public health concern. However, antifungal drug development has not kept pace with this growing need, and treatment options remain limited to a small number of drug classes. The emergence of multidrug-resistant fungal pathogens, such as Candida auris, further exacerbates this crisis by reducing the efficacy of existing therapeutics and increasing the risk of treatment failure. In this study, we evaluate the antifungal potential of FK20, a random peptide mixture (RPM) composed of L-phenylalanine and L-lysine. FK20 displays potent activity against C. auris and other clinically relevant Candida species, impairs biofilm formation, and exhibits synergy with caspofungin. Importantly, FK20 limits the emergence of resistance and demonstrates therapeutic efficacy in a murine model of systemic candidiasis. These findings establish RPMs as a promising new class of antifungals with broad-spectrum activity and clinical potential against drug-resistant fungal infections.