Current Pharmaceutical Design - Volume 17, Issue 38, 2011

Broadly speaking, there are only two major structural classes of approved drugs, small molecules and protein therapeutics (also known as biologics). Small molecules typically show good stability and good pharmacological properties, but their intrinsic small size (≤100 atoms) endows them with only a modest overall surface area available to contact a protein target, which seriously limits their ability to effectively target large surfaces involved in protein-protein interactions. Protein-based therapeutics, on the other hand, have been shown to possess high specificity/selectivity and high affinity for protein targets. The use of therapeutic monoclonal antibodies to target extracellular protein receptors is just an example. Antibodies, however, suffer from clear limitations: they are expensive to produce, cannot be delivered orally, show low tissue penetration and can not target intracellular targets, among other issues. The potential problems associated with the use of antibody fragments have led to the exploration of alternative protein scaffolds as a source for novel protein-based therapeutics. However, the utility of protein-based therapeutics has been typically limited by their generally poor stability and limited bioavailability. In response to this challenge a number of technologies are starting to emerge to address these issues. Special attention has been recently given to the use of highly constrained peptides, also known as micro- or miniproteins, as extremely stable and versatile scaffolds for the production of high affinity ligands for specific protein capture and/or development of therapeutics. The present special issue covers the discovery, structure-activity and biomedical applications, which includes therapeutics and diagnostics, of novel highly-constrained peptidebased scaffolds. Conotoxins are small bioactive highly structured peptides isolated from the venom of marine cone snails (genus Conus) that have great potential for the development of novel peptide-based therapeutics. The conotoxin ω-MVIIA (Prialt®) received FDA approval in 2004 for the treatment of chronic pain. In Chapters one and two, Alewood and Craik, respectively, discuss the potential of α-conotoxins to develop novel peptide-based therapeutics. Defensins are also an important family of cationic and highly-constrained host defense peptides that are widely distributed in plants, fungi, and animals. In mammals, defensins exert potent antimicrobial and immunomodulatory activities linking the innate and adaptive immune defenses. These peptides play critical roles in health and disease, and defects in their production are usually associated with abnormal host responses to infection, chronic inflammatory diseases, and cancer. In Chapter three, Seveau reviews the biological activities of human and murine defensins in the host immune response and their potential to be used for developing novel antimicrobial and anti-inflammatory peptide-based therapeutics. Of particular interest is the recent discovery of -defensins, which are so far the only backbone cyclized peptides found in animals. -Defensins have very strong anti-inflammatory and anti-HIV activities, which makes them very attractive leads for the development of novel peptide-based drugs.....

Conotoxins are small bioactive highly structured peptides from the venom of marine cone snails (genus Conus). Over the past 50 million years these molluscs have developed a complex venom cocktail for each species that is comprised of 100-2000 distinct cysteine- rich peptides for prey capture and defence. This review focuses on an important and well-studied class of conotoxins, the α- conotoxins. These α-conotoxins are potent and selective antagonists of various subtypes of the nicotinic acetylcholine receptors (nAChRs). Key structure-activity relationship studies are presented to illustrate the common motifs, structural features and pharmacophores that define this interesting peptide class. Additionally, their synthesis, chemical modifications, the development of more selective and stable analogues and their therapeutic potential are discussed.

The peptides present in the venoms of marine snails are used by the snails to capture prey, but they have also attracted the interest of drug designers because of their potent activity against therapeutically important targets. These peptides are typically disulfiderich and target a wide range of ion channels, transporters and receptors with exquisite selectivity. In this article, we discuss structural and biological studies on several classes of conotoxins that have potential as drug leads for the treatment of pain. The chemical re-engineering of conotoxins via cyclization has been particularly valuable in improving their biopharmaceutical properties. An excellent example is the α-conotoxin Vc1.1, for which several cyclized analogs have been made. One of them was shown to be orally active in a rat pain model and this analog is currently undergoing pre-clinical development for the treatment of neuropathic pain. Several other α-conotoxins, including ImI, AuIB and MII, have proved amenable to cyclization and in all cases improvements in stability are obtained upon cyclization, suggesting that cyclization is a generally applicable approach to conotoxin stabilization. A variety of other chemical re-engineering approaches have also been used. Minor re-engineering of χ-conotoxin MrIa to convert its N-terminal residue to pyroglutamic acid proved particularly successful and the modified derivative, Xen2174, is currently in clinical trials for neuropathic pain.

Defensins are an important family of cationic and cysteine-rich host defense peptides that are widely distributed in plants, fungi, and animals. In mammals, defensins exert potent antimicrobial and immunomodulatory activities linking the innate and adaptive immune responses. These peptides play critical roles in health and disease as defects in their production are associated with abnormal host responses to infection, chronic inflammatory diseases, and cancer. There is much interest in elucidating the structure-function relation and modes of action of the defensins to better understand how these peptides kill microbes and regulate the host immune responses. Such knowledge is expected to help in the design of novel defensin-based therapeutics. This review focuses on the multifaceted antimicrobial and immunomodulatory activities of human and murine defensins.

Plant defensins are cationic peptides that are ubiquitous within the plant kingdom and belong to a large superfamily of antimicrobial peptides found in several organisms collectively called defensins. The primary structure of these peptides includes 45 to 54 amino acid residues with considerable sequence variation. At the level of three-dimensional structure, they are small and globular, composed of three anti-parallel β-sheets and one α-helix, which is highly conserved among these peptides. The three-dimensional structure is stabilized by four disulfide bridges formed by eight strictly conserved Cys residues. Two of these bridges compose the Cys-stabilized α-helix β-strand motif, which is found in other peptides with biological activities. Plant defensins present numerous biological activities, such as inhibiting protein synthesis, ion channel function and α-amylase and trypsin activity; impairing microbial, root hair and parasitic plant growth; mediating abiotic stress and Zn tolerance; altering ascorbic acid redox state; stimulating sweet taste sensation; serving as epigenetic factors; affecting self-incompatibility; and promoting male reproductive development. Some of these biological activities, such as microbial growth inhibition and sweet taste induction, coupled with a scaffold that provides these peptides with incredible physicochemical resistance to harsh environments and the potential for simple amino acid substitution, raise the opportunity to improve the function of defensins or introduce new activities, endowing these peptides with great biotechnological and medical significance. This review will cover the biological activities and roles of plant defensins and will focus on their application in the field of biotechnology.

Cyclotides are a unique and growing family of backbone cyclized peptides that also contain a cystine knot motif built from six conserved cysteine residues. This unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to thermal, chemical, and enzymatic degradation compared to other peptides of similar size. Aside from the conserved residues forming the cystine knot, cyclotides have been shown to have high variability in their sequences. Consisting of over 160 known members, cyclotides have many biological activities, ranging from anti-HIV, antimicrobial, hemolytic, and uterotonic capabilities; additionally, some cyclotides have been shown to have cell penetrating properties. Originally discovered and isolated from plants, cyclotides can also be produced synthetically and recombinantly. The high sequence variability, stability, and cell penetrating properties of cyclotides make them potential scaffolds to be used to graft known active peptides or engineer peptide-based drug design. The present review reports recent findings in the biological diversity and therapeutic potential of natural and engineered cyclotides.

This work is focused on SFTI-1, a member of the Bowman-Birk family of inhibitors. This 14 amino acid cyclic peptide exhibits several features i.e. compact rigidity, well-defined structure and small size that could result in a wide range of potential applications. Some examples of engineering of the specificity of this inhibitor along with structure - activity relationships will be discussed herein. Additionally, potential uses of STFI-1 and its analogs as pharmaceutical agents will be described.

Transpeptidation reactions result in the formation of new peptide bonds and this can occur between two separate peptides or within the one peptide. These reactions are catalyzed by enzymes and when the N- and C-terminus of the one peptide are joined it results in the formation of cyclic proteins. Cyclization via head-to-tail linkage of the termini of a peptide chain occurs in only a small percentage of proteins but gives the resultant cyclic proteins exceptional stability. The mechanisms are not well understood and this review documents what is known of the events that lead to cyclization. Gene encoded cyclic proteins are found in both prokaryotic and eukaryotic species. The prokaryotic circular proteins include the bacteriocins and pilins. The eukaryotic circular proteins in mammals include the - defensins and retrocyclins. Small cyclic proteins are also found in fungi and a large range of cyclic proteins are expressed in cyanobacteria. Three types of cyclic proteins have been found in plants, the small cyclic proteins of 5-12 amino acids, the cyclic proteins from sunflower which are made up of 12-14 amino acids, and the larger group known as cyclotides which contain 28-37 amino acids. Three classes of enzymes are able to catalyse transpeptidation reactions, these include the cysteine, serine and threonine proteases. However only cysteine and serine proteases have been documented to cyclize proteins. The cyclotides from Oldenlandia affinis, the plant in which cyclotides were first discovered, are processed by an asparaginyl endopeptidase which is a cysteine protease. These proteases cleave an amide bond and form an acyl enzyme intermediate before nucleophilic attack of the amine group of the N-terminal residue to form a peptide bond, resulting in a cyclic peptide.

Cystine knot miniproteins define a class of peptides in the size range of approximately 28-35 amino acid residues that often combine high chemical and biological stability with high potency and selectivity. They share a common structural motif that is defined by three intramolecular disulfide bonds that gives rise to a very stable scaffold. Members of this family cover a broad spectrum of natural bioactivities ranging from antimicrobial and antiviral activities to selective blockage or activation of ion channels, cell surface receptors and extracelluar proteases. In recent years, the spectrum of natural bioactivities of this class of miniproteins was further expanded by application of protein design and directed evolution technologies. Miniproteins have been developed that inhibit platelet aggregation, block asthma-related proteases, act as growth factor mimics or address human tumor targets. Recent reports on miniproteins binding to cancer specific targets indicate that these biomolecules due to their particularly high in vivo stability, high target affinity, good tissue distribution, and fast body clearance are very promising agents that can be endowed with important beneficial features for imaging and therapeutic applications. With the first cystine-knot miniprotein already marketed as an analgesic, more candidates can be expected to find their way into the clinic for diagnostic and therapeutic applications over next years

The knottins are extremely stable miniproteins present in many species and are able to perform various tasks. Owing to its small size and its amazing stability, the knottin structural domain is considered as an excellent scaffold for drug development. Several recent databases and web servers dedicated to or aware of knottins have appeared and are shortly described. Altogether they provide a valuable ensemble of data and of specific tools that greatly facilitate knottin-based studies. The essential structural features of the knottin scaffold, which heavily rest on the three knotted disulfide bridges for its stability, are reviewed. These include small but well-conserved secondary structures and hydrogen bonding networks, but also several further interactions that have been shown to be essential for stability and/or activity. Examples are supplementary disulfide bridges, side chain hydrogen bonds, or circularization. General structure prediction and modeling tools are not well fitted to knottins, and several specific tools have been developed. Specifically, methods to assign a disulfide connectivity pattern to small disulfide-rich sequences or to build accurate 3D models of knottins are available and are discussed in the review. Although more works are still needed to better understand sequence-structure-function relationships, recent studies strongly suggest that existing applications of knottins as drugs (i.e. painkillers), molecules for diagnosis, or insecticidal crop treatment should rapidly generalize and extend to other fields as well, e.g. as antimicrobials.

Crotamine, a low molecular weight cationic polypeptide from the venom of the South American rattlesnake Crotalus durissus terrificus is a natural cell-penetrating peptide with functional versatility. The presence of nine lysine residues and three disulfide bonds renders crotamine highly compact, stable and positively charged. Topologically, crotamine adopts an ancient β-defensin fold that is found in diverse families of endogenous and venom polypeptides dedicated to host defense. Crotamine is unique among several classes of bioactive peptides because it possesses both cell penetrating and antimicrobial activities and selective biological action toward some cell types at a given cell cycle phase. Because it can rapidly and efficiently translocate into actively proliferating cells, crotamine is being investigated for labeling highly replicating cells and for use as a chemotherapeutic adjuvant. Peptides derived from crotamine, nucleolar targeting peptides (NrTPs), have been designed and are being studied. NrTPs retain some crotamine properties, such as efficient cellular uptake and preferential nuclear localization whereas they improve upon other properties. For example, NrTPs are smaller than crotamine, show higher preferential nucleolar localization, and better facilitate ZIP-code localization of therapeutic proteins.

Surgical resection remains the primary component of cancer therapy. The precision required to successfully separate cancer tissue from normal tissue relies heavily on the surgeon's ability to delineate the tumor margins. Despite recent advances in surgical guidance and monitoring systems, intra-operative identification of these margins remains imprecise and directly influences patient prognosis. If the surgeon had improved tools to distinguish these margins, tumor progression and unacceptable morbidity could be avoided. In this article, we review the history of chlorotoxin and its tumor specificity and discuss the research currently being generated to target optical imaging agents to cancer tissue.

Inflammation and oxidative process are associated with inflammatory bowel disease (IBD). Regarding anti-inflammatory and antioxidant potentials, melatonin has been found beneficial in several experimental and clinical studies including inflammatory bowel disease (IBD). Our objective in this study is to review and evaluate all non-clinical and clinical studies on the efficacy of melatonin in IBD. All indexing databases were searched for ‘inflammatory bowel disease' and ‘melatonin’ keywords, without time limit up to May 2011. Three clinical trials and fifteen non-clinical studies are reviewed and analyzed. The majority of these studies indicate that melatonin has a positive impact on IBD with no or negligible side effects. Such results have been mostly explained through free radical scavenging and diminishing inflammation. It is yet crucial to determine the efficacy of melatonin in combination with other established drugs in more clinical trials, not only for further confirmation of its efficacy, but also to investigate its possible side effects in longer durations of therapy.

Accelerated atherosclerosis and microvascular complications are the leading causes of coronary heart disease, end-stage renal failure, acquired blindness and a variety of neuropathies, which could account for disabilities and high mortality rates in patients with diabetes. Glucagon-like peptide-1 (GLP-1) belongs to the incretin hormone family. L cells in the small intestine secrete GLP-1 in response to food intake. GLP-1 not only enhances glucose-evoked insulin release from pancreatic β-cells, but also suppresses glucagon secretion from pancreatic ??-cells. In addition, GLP-1 slows gastric emptying. Therefore, enhancement of GLP-1 secretion is a potential therapeutic target for the treatment of type 2 diabetes. Dipeptidyl peptidase-4 (DPP-4) is a responsible enzyme that mainly degrades GLP-1, and the half-life of circulating GLP-1 is very short. Recently, DPP-4 inhibitors and DPP-4-resistant GLP-1 receptor (GLP-1R) agonists have been developed and clinically used for the treatment of type 2 diabetes as a GLP-1-based medicine. GLP-1R is shown to exist in extra-pancreatic tissues such as vessels, kidney and heart, and could mediate the diverse biological actions of GLP-1 in a variety of tissues. So, in this paper, we review the pleiotropic effects of GLP-1-based therapies and its clinical utility in vascular complications in diabetes.