Methodological development and applications of cyclic cysteine-rich peptides for drug design
Date of Issue2014
School of Biological Sciences
Drug Discovery Centre
My thesis focuses on the development of novel synthetic strategies for preparing the cyclic cysteine-rich peptides and applying the newly developed strategies for preparing bioactive cyclic cysteine-rich peptides with potential therapeutic applications. Cysteine-rich peptides are mini-proteins (2-8 kDa) with two to five intramolecular disulfide bonds. These disulfide bonds provide conformational constraints to enhance the structural stability. Cysteine-rich peptides occur ubiquitously in all organisms and they exhibit a broad range of bioactivities including antimicrobial, neurotoxic, enzyme inhibitory and hormonal functions. Interestingly, certain plant cysteine-rich peptides such as cyclotides possess an end-to-end cyclic backbone that gives an additional constraint and exceptional stability against heat and enzyme degradation. Cyclic cysteine-rich peptides have been exploited for designing and engineering orally active peptidyl therapeutics by grafting bioactive fragments into their scaffolds. A challenge in developing such engineered cyclic cysteine-rich peptides is developing an efficient and practical approach for their synthesis, and in particular, for the formation of cyclic backbones and oxidative folding of the multiple disulfide bonds. My synthetic strategy employed Cys-thioester ligation (native chemical ligation) for preparing cyclic peptides. It involved the formation of a new peptide bond between an N-terminal cysteine and a C-terminal thioester to afford a cyclic backbone. To prepare thioesters, I developed two novel 9-fluorenylmethoxycarbonyl (Fmoc)-compatible thioester surrogates using readily available starting materials including N-methylated cysteine and thioethylbutylamide. These surrogates were incorporated as a C-terminal thioethylamido moiety of a peptide by solid-phase synthesis. Under acidic conditions, they transform into a thioester via an intramolecular N-S acyl shift reaction that is facilitated by the cis-conformation of the tertiary amide bond. In the presence of the intramolecular thiols of a cysteine-rich peptide, the C-terminal thioester is rapidly transformed into thiolactones via transthioesterification and cyclization accelerated by a thia zip mechanism. A novel oxidative folding strategy was developed in organic system to form multiple disulfide bonds of cysteine-rich peptides within one hour. It overcame the limitations of conventional aqueous folding methods that required high dilution and long duration for completion. A combination of the newly developed cyclization and the organic folding strategy in a one-pot manner successfully afforded a shorter reaction time and an improved yield than the conventional methods. They could be used as a simple and high-throughput synthetic platform to prepare cyclic cysteine-rich peptides. For designing cyclic peptides, I studied synthetic cyclic ω-conotoxin MVII analogs as models to demonstrate the consequence of engineering a linear peptide to form a cyclic peptide. The end-to-end cyclization of the linear created a functional neo-epitope that exhibited antimicrobial activities against bacteria and fungi. The results suggested that backbone cyclization could create new functions on a bioactive cysteine-rich peptide to expand its versatility for drug development. Collectively, my thesis works provide a new synthetic strategy for preparing and engineering cysteine-rich peptide-based biologics through backbone cyclization.