Targeting PfUCHL3 Using Chemically Constrained Peptides

Abstract

PfUCHL3 is a highly conserved, dual specificity deubiquitinating and deNeddylating enzyme acting within the ubiquitin proteasome system (UPS) of Plasmodium falciparum, the deadliest and most prominent Plasmodium species responsible for the transmission of malaria to humans. Malaria kills on average 450,000 people per year across 90 different countries, and there is at present no broadly effective vaccine against the disease. PfUCHL3 is essential to P. falciparum cell viability, and through the use of the RaPID peptide discovery system (Yamagishi Y et al, 2011), 10 novel, non-natural, cyclic peptides were selected for against PfUCHL3. The peptides all display tight dissociation constants for PfUCHL3 in the low nanomolar range (6-35 nM) and 4 of the 10 peptides inhibit the activity of PfUCHL3 in cleaving its ubiquitin substrate in vitro. The peptides all have selectivity for PfUCHL3 over HsUCHL3, the human homologue of the enzyme, and through NMR analysis it was shown that all 4 of the inhibitory peptides bind to the substrate recognition region of PfUCHL3. Thus, the peptides act by interfering with ubiquitin’s ability to bind to PfUCHL3 and represent the first reported inhibitory peptides for PfUCHL3. Through the use of thermal shift assays the inhibitory peptide’s were shown to stabilise the proteins structure upon binding and increase the melting temperature. Work is currently underway to determine the cell permeability properties of the peptides and their ability to kill Plasmodium parasites in vivo with the long-term aim of taking these peptides forward for the generation of a novel class of anti-malarial therapeutics. (Yamagishi Y et al, 2011) - Yamagishi Y, Shoji I, Miyagawa S, Kawakami T, Katoh T, Goto Y, Suga H. Natural product-like macrocyclic N-methyl-peptide inhibitors against a ubiquitin ligase uncovered from a ribosome-expressed de novo library. Chem Biol. Dec 23;18(12):1562-70. 2011