Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII

Abstract

Conotoxins (CTXs), with their exquisite specificity and potency, have recently created much excitement as drug leads. However, like most peptides, their beneficial activities may potentially be undermined by susceptibility to proteolysis in vivo. By cyclizing the ␣-CTX MII by using a range of linkers, we have engineered peptides that preserve their full activity but have greatly improved resistance to proteolytic degradation. The cyclic MII analogue containing a seven-residue linker joining the N and C termini was as active and selective as the native peptide for native and recombinant neuronal nicotinic acetylcholine receptor subtypes present in bovine chromaffin cells and expressed in Xenopus oocytes, respectively. Furthermore, its resistance to proteolysis against a specific protease and in human plasma was significantly improved. More generally, to our knowledge, this report is the first on the cyclization of disulfide-rich toxins. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.conotoxins ͉ drug delivery ͉ molecular engineering V enoms from marine snails of the Conus genus comprise a myriad of peptides called conotoxins (CTXs) for the rapid immobilization of prey (1, 2). These 12-to 30-aa peptides target membrane receptors with exquisite selectivity and potency and have become invaluable neurophysiological probes and drug leads. Recently, the CTX ziconotide (MVIIA) was approved for use in the treatment of severe chronic pain by the FDA, and other CTXs have entered clinical trials as treatments for pain (3,4). In addition, CTXs have played a critical role in dissecting the molecular mechanisms of ion channel and transporter functions in the nervous system (2). One family of CTXs, the ␣-CTXs, consists of members that antagonize the nicotinic acetylcholine receptors (nAChRs). Ranging in size from 12 to 19 residues, ␣-CTXs are the smallest of all of the CTXs, yet this family is the most widely distributed among Conus venoms (5).Despite their exciting applications, many peptide toxins are susceptible to enzymatic degradation by proteases. This characteristic may limit the therapeutic applications of CTXs, and, hence, methods that provide improvements in biological half-life are valuable. Cyclization has been used in the past as a strategy in the pharmaceutical industry for stabilizing and locking the conformation of small peptides (6). Similarly, microorganisms are known to produce cyclized peptides, such as cyclosporin A, which is now in widespread use as an immunosuppressant. Such a strategy has not been applied in the past to disulfide-rich proteins, but with the recent discovery of the cyclotide family of macrocyclic miniproteins (7), it is clear that the approach can be applied to disulfide-rich toxins to produce additional stabilization with the potential to dramatically increase the therapeutic potential of these molecules when limited by poor in vivo stability.This stu...

Sortase-catalyzed transformations that improve the properties of cytokines

“…Cyclic proteins are also resistant to exoproteolytic attack (25,26), a feature that may enhance utility of any therapeutic proteins exposed to exoproteases, for example upon receptor mediated internalization. In addition, cyclization of proteins and peptides has been shown to improve potency, stability, and oral bioavailability (27)(28)(29)(30).…”

Developmental Biology of Neoplastic Growth

“…They are structurally related to other peptide families of pore-forming toxins such as mellitin and have a tendency to aggregate both in solution and in lipidic membrane and to create voltage-gated ion channels (Lazarovici et al 1986). The pore-forming activity is highly dependent on the ability on both the peptide to form α helices and an intramolecular interaction between the N-and C-termini of the peptide suggesting that cyclisation might play an important role in this process, as suggested for a number of other neurotoxins (Clark et al 2005). A molecular view of membrane inserted pardaxin was recently obtained by NMR, which highlighted the importance of non-covalent interactions in promoting a stable secondary structure (Porcelli et al 2004).…”

“…The prospect of discovering novel potent antinociceptive drugs has promoted investigations into neurotransmitter release from c-fibres in the spinal cord. One of the most challenging features of the use of peptides as drugs is their high sensitivity to protease degradation -a problem that can be overcome by strategies such as tail to tail cyclisation (Clark et al 2005). …”

Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A

“…As well as being valuable scaffolds, naturally occurring cyclic peptides, such as cyclotides and SFTI-1, have inspired the concept of using artificial head-to-tail backbone cyclization as a stabilizing strategy for peptides and proteins of therapeutic interest (3,6). For example, Clark et al (3) showed that cyclization of ␣-conotoxin Vc1.1, a potential neuropathic pain treatment, endowed it with oral activity.…”

“…Several classes of cyclic peptides, including the sunflower trypsin inhibitors and cyclotides, have now been discovered in a variety of plant species (5). In addition to the plant-derived cyclic peptides, disulfide-rich peptides isolated from cone snails, including ␣-conotoxins, have successfully been cyclized (3,6) and are showing promise as potential treatments for neuropathic pain (3).…”