Final Report Summary - CYCLOTIDE MECHANISM (Elucidating the mechanism of action of cyclotides: ultra-stable proteins from plants) Project context and objectivesPeptides have promising pharmaceutical applications, but their application is limited by their poor stability and bioavailability. Cyclotides are a large family of plant peptides that incorporate a unique knotted macrocyclic structure that confers exceptional resistance to thermal, chemical and enzymatic degradation. In addition, several intrinsic activities, including anti-microbial, anti-cancer, anti-fouling and insecticidal activities, have been reported for cyclotides. Such properties make cyclotides very advantageous, relative to conventional linear peptides, and have inspired applications in drug design for pharmaceutical and agricultural applications. To fully explore the usefulness of the cyclotides, understanding how they exert their biological effects is crucial for a more effective and rational drug design and is the basis of this project. Briefly, this project aims to provide for the first time a chemical understanding of the biological activities of cyclotides. Experimental evidences suggest that these actions are mediated by peptide-membrane interactions. Therefore, the main goal of this project is to explore the interaction of a selected set of cyclotides with model membranes and correlate them with biological assays to elucidate their mechanism of action.Peptides belonging to the subfamilies Möbius and bracelet were tested (Table 1). Model membranes of different lipid composition were used and the results compared with naturally occurring cell membranes of distinct compositions (i.e. red blood cells, bacteria and human immunodeficiency virus (HIV) particles). Fluorescence spectroscopy, UV-vis spectroscopy, surface plasmon resonance and confocal microscopy have been used to analyse the peptides.To further explore the importance of the cell membrane in the activity of the cyclotides, the prototypic cyclotide kalata B1 (kB1) was compared with synthetic analogues. Inactive and more active mutants of kB1, but also a mirror image enantiomer D-kalata B1 and D-kalata B2 were included in the study.Based on biophysical studies and bioassays with native cyclotides and kB1 analogues it is possible to conclude that the biological activity of cyclotides is dependent on their affinity for the membrane, which is governed by the lipid composition of the target cell membrane (Figure 1). Of relevance, no evidence for a protein receptor involved in the activity of cyclotides was found (Figure 2). Interestingly, the activity of cyclotides requires specific interactions with phospholipids containing phosphatidylethanolamine (PE) headgroups, but is further modulated by nonspecific peptide-lipid hydrophobic interactions, in which a fine selection was found across the family. Negatively-charged phospholipids do not favour membrane affinity. Overall, the results show that cyclotide activity is correlated with membrane binding and that both the sequence and the cyclic knotted structure of cyclotides are important for membrane targeting. Although with similar membrane selectivity, different cyclotides have different effects on the membrane stability, which suggests distinct mechanisms of action.In addition, we have shown that the anti-HIV activity of cyclotides is the result of their ability to target and disrupt the membranes of HIV particles. Inactivating HIV through a lipid-targeting mechanism is unlikely to result in the emergence of cyclotide resistant strains of HIV, as replacement of membrane lipids is virtually impossible for the virus.In addition we have shown kB1 has cell-penetrating properties, although this has not been tested yet with other cyclotides belonging to the Mobius and bracelet subfamilies, such finding opens up the possibility of using cyclotides as a scaffold with functional epitopes for intracellular targets.A growing number of peptides are showing promise in pharmaceutical applications. Despite this promise, peptides have several drawbacks that limit their broad application, including their inactivation in serum and sensitivity to enzymatic degradation. The potential use of cyclotides as a scaffold for therapeutic purposes has emerged not only because of their remarkable stability and potential to overcome enzymatic degradation normally associated with peptides, but also due to their plasticity and tolerance for substitution. Understanding the structural features important for binding to cell membranes will assist in designing cyclotides with enhanced bioactivity and bioavailability and lower toxicity.Beside the possibility of using cyclotides as a template to introduce foreign sequences into them, cyclotides are now known to have cell-penetrating properties which increases their value as template for drug design with a biological function but also as a vector that can be readily taken up by cells with more bioavailability than linear cell-penetrating peptides (CPPs) that suffer from low stability.In addition, the anti-HIV activity of cyclotides can also be pursued. Cyclotides are promising leads in HIV therapy that can destroy the virus particle at non cytotoxic concentrations, and have potential to be used as a topical microbicide to prevent the sexual transmission of HIV. The use of topical microbicides has the advantage of delivering the drugs locally and therefore minimising the potential for systemic toxicity. There is an urgent need for female-controlled HIV prevention strategies such as microbicide gels. Nevertheless, the formulation of anti-HIV gels is often complicated, as the drug needs to be compatible with the excipients and stable for long periods of time and at the high temperatures often encountered in African countries. With a long half-life, thermal resistant and high efficacy against HIV, cyclotides are promising anti-HIV active pharmaceutical agents for microbicide gels.
