An Antivirulence Approach for Preventing Cryptococcus neoformans from Crossing the Blood-Brain Barrier via Novel Natural Product Inhibitors of a Fungal Metalloprotease

Abstract

Cryptococcus neoformans (Cn) is the leading cause of fungal meningitis, a deadly disease with limited therapeutic options. Dissemination to the central nervous system hinges on the ability of Cn to breach the blood-brain barrier (BBB) and is considered an attribute of Cn virulence. Targeting virulence instead of growth for antifungal drug development has not been fully exploited despite the benefits of this approach. Mpr1 is a secreted fungal metalloprotease not required for fungal growth, but rather, it functions as a virulence factor by facilitating Cn migration across the BBB. This central role for Mpr1, its extracellular location, and lack of expression in mammalian cells make Mpr1 a high-value target for an antivirulence approach aimed at developing therapeutics for cryptococcal meningitis. To test this notion, we devised a large-scale screen to identify compounds that prohibited Cn from crossing the BBB by selectively blocking Mpr1 proteolytic activity, without inhibiting the growth of Cn. A phytochemical natural product-derived library was screened to identify new molecular scaffolds of prototypes unique to a Cn microecosystem. Of the 240 pure natural products examined, 3 lead compounds, abietic acid, diosgenin, and lupinine inhibited Mpr1 proteolytic activity with 50% inhibitory concentration (IC50) values of <10 μM, displayed little to no mammalian cell toxicity, and did not affect Cn growth. Notably, the lead compounds blocked Cn from crossing the BBB, without damaging the barrier integrity, suggesting the bioactive molecules had no off-target effects. We propose that these new drug scaffolds are promising candidates for the development of antivirulence therapy against cryptococcal meningitis. IMPORTANCE Fungal infections like cryptococcal meningitis are difficult to resolve because of the limited therapies available. The small arsenal of antifungal drugs reflect the difficulty in finding available targets in fungi because like mammalian cells, fungi are eukaryotes. The limited efficacy, toxicity, and rising resistance of antifungals contribute to the high morbidity and mortality of fungal infections and further underscore the dire but unmet need for new antifungal drugs. The traditional approach in antifungal drug development has been to target fungal growth, but an attractive alternative is to target mechanisms of pathogenesis. An important attribute of Cryptococcus neoformans (Cn) pathogenesis is its ability to enter the central nervous system. Here, we describe a large-scale screen that identified three natural products that prevented Cn from crossing the blood-brain barrier by inhibiting the virulence factor Mpr1 without affecting the growth of Cn. We propose that compounds identified here could be further developed as antivirulence therapy that would be administered preemptively or serve as a prophylactic in patients at high risk for developing cryptococcal meningitis.

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

“…Several natural protease inhibitors exert anti-virulence effects by targeting extracellular proteases, impairing nutritional and/or growth functions [30][31][32], or hindering virulence mechanisms, such as tissue invasion (Figure 1) [33]. 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].…”

“…Lastly, Lupinine and Diosgenin are two plant derived compounds that possess antifungal properties against C. neoformans [33]. These compounds inhibit a secreted metallopeptidase relevant in brain invasion by cryptococcal cells causing meningoencephalitis, CnMpr-1 (Inhibitory concentration [IC 50 ] 5.025 µM and 9.659 µM, respectively) [68].…”

“…Lupinine is a quinolizidine alkaloid found primarily within flowering plants of the Lupinus genus [69], whereas diosgenin is a plant steroidal sapogenin isolated from dietary fenugreek (Trigonella foenum-graecum) seeds [70]. Interestingly, these compounds impair fungal crossing of the blood-brain barrier without detrimental effects to the host [33]. Diosgenin also inhibits matrix metalloproteinases (e.g., MMP-2 and MMP-9) involved in matrix integrity or cell migration [71][72][73][74][75].…”

Identification of Disease-Associated Cryptococcal Proteins Reactive With Serum IgG From Cryptococcal Meningitis Patients

“…This was demonstrated using an mpr1D C. neoformans strain, with mpr1 being the serotype D homologue of the elastinolytic metalloprotease identified in our study (88,89). Extracellular elastinolytic metalloprotease is an especially interesting target protein for development of anti-cryptococcal treatment options, as inhibition of Mpr1 by natural product inhibitors prevented cryptococcal cells from crossing the blood brain barrier in an in vitro transwell model (90). Extracellular elastinolytic metalloproteinase contains a secretory signal peptide making it also an attractive candidate for vaccine development.…”

“…Several proteins are implicated in cryptococcal virulence or fungal metabolism and survival and could therefore be targeted by anti-fungal agents. One disease-associated protein identified in our screen, extracellular elastinolytic protease, was already successfully inhibited by natural products in an in vitro transwell model, decreasing cryptococcal virulence (90). Therefore, targeting other virulence-associated proteins using similar approaches could be beneficial.…”