Discovery and Characterization of a Rapidly Fungicidal and Minimally Toxic Peptoid against Cryptococcus neoformans

Abstract

A limited number of antifungals are available to treat infections caused by fungal pathogens such as Cryptococcus neoformans and Candida albicans. Current clinical antifungals are generally toxic, and increasing resistance to these therapies is being observed, necessitating new, effective, and safe antifungals. Peptoids, or N-substituted glycines, have shown promise as antimicrobial agents against bacteria, fungi, and parasites. Herein we report the discovery and characterization of an antifungal peptoid termed RMG8-8. This compound was originally discovered from a combinatorial peptoid library using the Peptoid Library Agar Diffusion assay to screen against C. albicans. Though the efficacy of RMG8-8 against C. albicans was modest (25 μg/mL), the efficacy against C. neoformans was excellent (1.56 μg/mL). Cytotoxicity against a panel of cell lines proved RMG8-8 to be minimally toxic, with selectivity ratios ranging from 34 to 121. Additional studies were carried out to determine the pharmacological importance of each peptoid monomer in RMG8-8, characterize the killing kinetics of this compound against C. neoformans (t 1/2 = 6.5 min), and evaluate plasma protein binding and proteolytic stability. Finally, a liposomal lysis assay suggested that RMG8-8 likely exerts fungal killing through membrane permeabilization, the generally accepted mechanism of action for most antimicrobial peptides and peptoids.

Synthesis and Characterization of Derivatives of the Antifungal Peptoid RMG8-8

“…42 Further characterization indicated that RMG8-8 killed fungi rapidly (t 1/2 = 6.5 min), was proteolytically stable, and likely exerted antifungal activity through membrane disruption. 35 The natural next step after discovery of any promising lead compound is structure modification to improve biological activity. Structure−activity relationship (SAR) studies in peptoids utilize iterative design to modify structures and can be helpful in determining the pharmacological significance of each peptoid monomer.…”

“…36 The sarcosine scan for RMG8-8 was completed shortly after discovery and initial characterization, revealing the lipophilic tail of RMG8-8 to be most pharmacologically important, followed by the cyclohexyl groups, followed by the cationic moieties, which were primarily responsible for mitigating cytotoxicity. 35 Here, we report the attempted optimization of RMG8-8 through an iterative SAR study against C. neoformans. A three-round modular SAR study yielded 25 different compounds for analysis containing various lipophilic tails, aliphatic and aromatic substitutions of the cyclohexyl groups, and trimethylation of the cationic amino side chains.…”

“…N-substituted oligoglycines, or peptoids, place the side chain on the nitrogen of the amide backbone instead of on the α-carbon. Due to this unique structure, peptoids are not recognized by proteases and have better in vivo stability than their peptide counterparts, while demonstrating low toxicity both in vitro and in vivo. − Since the first demonstration of peptoid antimicrobial activity over 20 years ago, antimicrobial peptoids have been developed against numerous bacteria, fungi, parasites, and viruses. , Promising antimicrobial peptoids have been developed with activity against the ESKAPE bacteria, ,,− Mycobacterium tuberculosis, fungi, including Cryptococcus neoformans, Candida albicans, and Candida auris, ,− and viruses, including HSV-1 and SARS-CoV-2 …”

“…Design, synthesis, and screening of potentially effective antimicrobial peptoids is a tedious endeavor, and so, combinatorial libraries and high-throughput screening methods are essential for quick identification. The Peptoid Library Agar Diffusion (PLAD) assay has been developed to interrogate combinatorial peptoid libraries to identify compounds with promising antimicrobial activity. ,, One particular PLAD screening against C. albicans led to the discovery of a peptoid with moderate activity against C.…”

“…albicans, termed RMG8-8 (Figure ). With a minimum inhibitory concentration (MIC) of 25 μg/mL and low mammalian cytotoxicity against human liver cells (TD 50 = 189 μg/mL), RMG8-8 was characterized further. While only moderately active against C.…”