Re-emerging Aspartic Protease Targets: Examining Cryptococcus neoformans Major Aspartyl Peptidase 1 as a Target for Antifungal Drug Discovery

Abstract

Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.

From Naturally-Sourced Protease Inhibitors to New Treatments for Fungal Infections

“…For instance, secreted aspartic proteases (SAPs), are involved in several virulence processes, including tissue invasion, growth, and immune system evasion among the important human fungal pathogens, Candida albicans and C. neoformans [34,35]. Additionally, SAPs have been assessed as antifungal targets using protease inhibitors with promising results for further exploration [36][37][38]. Other important anti-virulence mechanisms include cell wall disruption or membrane pore formation initiated by protease inhibitors to deregulate ion flow and/or membrane disruption to cause leakage of internal cellular components, affecting cell viability [30,31].…”

“…ATBI binds within the active site of the HIV-1 protease (competitive inhibition), leading to inactivation of the enzyme, and thereby suggesting pharmaceutical potential as a drug for the treatment of AIDS. As described above, compounds such as HIV-1-protease inhibitors (e.g., indinavir or ritonavir) possess antifungal properties [36][37][38]99], highlighting the need for more research using ATBI against human fungal pathogens, such as Candida spp. and C. neoformans.…”

“…As identified here, important areas for further exploration include the search for natural compounds that mimic current synthetic compounds with anti-HIV activity. For example, the beneficial effects of anti-HIV protease inhibitors on the incidence of disease and the subsequent outcome of opportunistic fungal infections, such as candidiasis [36,37,98] and cryptococcosis [38,99,100,107,108]. This includes the off-target effects of anti-HIV aspartic protease inhibitors (e.g., saquinavir, indinavir and ritonavir) against hydrolytic enzymes (e.g., SAPs in C. albicans), which correspond with reduced fungal infections in HIVinfected patients [36,37,98,109].…”

The Possible Role of Microbial Proteases in Facilitating SARS-CoV-2 Brain Invasion

“…They display a domain organisation that has a large pro-domain with an activation locus, a catalytic domain with a zinc-binding site, and a C-terminal hemopexin domain [64,70]. Kryštůfek et al [71] reported that homology modelling predicted that the May1 adopts a renin fold, which is a predominantly β-sheet conformation characteristic of the aspartyl proteinase family. The substrate-binding cleft and the active site are at the junction of two structurally similar domains of approximately equal size.…”

“…The substrate-binding cleft and the active site are at the junction of two structurally similar domains of approximately equal size. Moreover, the catalytic residues are located centrally in the cleft with the carboxyl side chains and surrounding main chain scaffolding related by an approximate twofold interdomain axis [71]. The cryptococcal serine-based protease(s) are, at present, unnamed [72].…”