Current Pharmaceutical Design - Volume 16, Issue 9, 2010

The production of new molecular entities endowed with salutary medicinal properties is a formidable challenge; synthetic molecules that can bind with high sequence specificity to a chosen target in a protein or gene sequence are of major interest in medicinal and biotechnological contexts. The general awareness of the importance of peptides in physiology and pathophysiology has markedly increased over the last few years. With progresses in the analysis of whole genomes, the knowledge base in gene sequence and expression data useful for protein and peptide analysis has drastically increased. The medical need for relevant biomarkers is enormous. Peptides have a number of advantages over small molecules in terms of specificity and affinity for targets, and over antibodies in terms of size. This is particularly true for the many types of cancers, but also for other diseases, e.g. type 2 diabetes or cardiac diseases, which also lack adequate diagnostic markers with high specificity and sensitivity. Imaging technologies for early detection of diseases, proteomic and peptidomic multiplex techniques have markedly evolved in recent years. Peptides can indeed be regarded as ideal agents (as “magic bullets”) for diagnostic and therapeutic applications because of their fast clearance, rapid tissue penetration, and low antigenicity, and also of their easy production, allowing innumerable biological applications. They can easily be engineered to improve their biological activities as well as their stability and their efficient delivery to specific targets. This third themed issue of Current Pharmaceutical Design, for which I have the honour to be Executive Guest Editor, addresses topical issues to some of these potential utilizations of peptide motifs for a variety of genetic and acquired diseases. Antimicrobial peptides are an essential part of innate immunity that evolved in most living organisms over 2.6 billion years to combat microbial challenges. These small peptides are multifunctional as effectors of innate immunity on skin and mucosal surfaces, representing a physical and chemical barrier against pathogen invasion, with a direct antimicrobial activity against various bacteria, viruses, fungi, and parasites, limiting pathogen growth in normal conditions. Moreover, they also possess additional roles in the regulation of adaptive immune responses, by recruiting or stimulating immune cells. Marie-Helene Metz-Boutigue, Peiman Shooshtarizadeh, Gilles Prevost, Youssef Haikel and Jean-François Chich [1] provide an up-to-date overview of the expression and the biological roles of the antimicrobial peptides found in the skin and gastrointestinal mucosa of the host in normal and pathological conditions. Despite recent advances in treatment modalities, cancer remains a major source of morbidity and mortality throughout the world. The development of novel anticancer treatments will be dependent on the ability to modulate cellular pathways that are aberrant in cancer cells as well as the capacity to refine the molecular diagnosis of the tumour and to create guided drugs able to target the tumour while sparing surrounding healthy tissues. This requires tumour-specific binding agents to probe the tumour cell surface phenotype and to customize treatment accordingly by conjugating the appropriate cell-targeting ligand to an anticancer drug. Kathlynn C. Brown [2] discusses the recent advances in the isolation of cancer-targeting peptides by unbiased biopanning methods and highlights the use of the isolated peptides in clinical applications. Membrane proteins account for a third of all proteins encoded in the human genome and protein-protein interactions play crucial roles in a number of biological processes, including viral self-assembly, cell proliferation, growth, differentiation, signal transduction, cell adhesion and programmed cell death. Studies of the structure and functions of transmembrane domains of membrane proteins are however difficult compared to the ones of water-soluble regions due to the lack of exogenous agents that can recognize them with high affinity and specificity. Tina X. Zhao, Alexander J. Martinko, Vy H. Le, Jing Zhao and Hang Yin [3] present the recent developments in using probing agents to specifically target transmembrane regions of proteins and study how they interact within their native environments as well as with nearby transmembrane segments.

Antimicrobial peptides are major components of the innate immune defence. They are well conserved along evolution, nontoxic and they ensure potent defences against a large number of pathogens. They act by direct killing of microorganisms and they possess additional roles in the regulation of adaptive immune responses, by recruting or stimulating immune cells. Skin and gut are positioned at the interface of internal milieu and external environment. They represent a physical and chemical barrier against pathogens invasion and the antimicrobial peptides limit pathogen growth in normal conditions. During infection or injury, some of these peptides are overexpressed and disrupt microbial membranes and/or stimulate immune cell recruitment, allowing to return to homeostasis or to increase inflammation. Antimicrobial peptides expression is altered in several diseases: α-defensins deficiency is related with Crohn's disease and in skin, cathelicidin LL-37 and β-defensin-2 are overexpressed in psoriasis, while in atopic dermatitis, their expression is decreased. The present review provides an up-to-date summary of the expression and the biological roles of the antimicrobial peptides found in the skin and gastrointestinal mucosa of the host, in normal and pathological conditions. The involvement of these natural antimicrobial peptides in inflammation, is also discussed.

Cancer has become the number one cause of death amongst Americans, killing approximately 1,600 people per day. Novel methods for early detection and the development of effective treatments are an eminent priority in medicine. For this reason, isolation of tumor-specific ligands is a growing area of research. Tumor-specific binding agents can be used to probe the tumor cell surface phenotype and customize treatment accordingly by conjugating the appropriate cell-targeting ligand to an anticancer drug. This refines the molecular diagnosis of the tumor and creates guided drugs that can target the tumor while sparing healthy tissues. Additionally, these targeting agents can be used as in vivo imaging agents that allow for earlier detection of tumors and micrometastasis. Phage display is a powerful technique for the isolation of peptides that bind to a particular target with high affinity and specificity. The biopanning of intact cancer cells or tumors in animals can be used to isolate peptides that bind to cancer-specific cell surface biomarkers. Over the past 10 years, unbiased biopanning of phage-displayed peptide libraries has generated a suite of cancer targeting peptidic ligands. This review discusses the recent advances in the isolation of cancer-targeting peptides by unbiased biopanning methods and highlights the use of the isolated peptides in clinical applications.

Membrane proteins account for approximately one third of all proteins in eukaryotic and prokaryotic cells. These proteins are critical in a diverse array of cellular functions. Despite their obvious importance, the effectiveness of research tools to study the structure and function of integral membrane proteins lags behind that of water-soluble proteins. This is due in part to the lack of probing agents that can specifically and selectively recognize these targets. This review focuses on methods developed to overcome the obstacles of studying membrane proteins. We describe TM protein properties as well as biophysical properties of amino acids within the membrane bilayer. We also summarize the known characteristics of membrane regions in their distinctive environments and generate a summary of current research approaches that succeed in probing interactions of TM proteins within their native setting. This allows further insight into protein-protein interactions in a hydrophobic environment as it pertains to drug development.

Toll-like receptors (TLRs) are a family of key proteins that permit mammals to detect microbes and endogenous molecules, which are present in body fluids, cell membranes and cytoplasm. They confer mechanisms to the host for maintaining homeostasis, activating innate immunity and inducing signals that lead to the activation of adaptive immunity. TLR signalling induces the expression of pro-inflammatory and anti-viral genes through different and intricate pathways. However, persistent signalling can be dangerous and all members of the TLR family are involved in the pathogenesis of acute and chronic inflammation, autoimmunity, allergy, cancer and aging. The pharmaceutical industry has begun intensive work developing novel immunotherapeutic approaches based on both activation and inhibition of TLR triggering. Further, clinical trials are pending to evaluate TLR agonists as novel vaccine adjuvants and for the treatment of infectious diseases, allergic diseases and asthma. Since systemic, metabolic and neuroendocrine changes are elicited by inflammation, TLR activity is susceptible of regulation by hormones and neuroendocrine factors. Neuroendocrine mediators are important players in modulating different phases of TLR regulation contributing to the endogenous control of homeostasis through local, regional and systemic routes. Vasoactive intestinal peptide (VIP) is an important signal molecule of the neuroendocrine-immune network that has recently emerged as a potential candidate for the treatment of inflammatory and autoimmune disorders by controlling innate and adaptive immunity. This review shows current advances in the understanding of TLR modulation by VIP that could contribute to the use of this natural peptide as a therapeutic tool.

The function of the Toll-like receptor (TLR) family members has been extensively studied in the recent decades. The TLR family is generally involved in the defense against microbial infections. TLRs are expressed mainly on macrophages and dendritic cells (DCs) and activate these cells upon ligand binding. The activation of TLRs basically initiates innate immune response, but can also induce adaptive immune response. TLRs have also been found on epithelial and tumor cells, but their role on tumor cells is still unclear. In some tumor types TLRs promote tumor proliferation and survival, while in others TLR2, -3 and -9 have been shown to be directly involved in apoptosis. Therefore, they seem to be promising candidates for the development of new, effective therapeutic options. It is however necessary to conduct comprehensive studies to assess the significance of these receptors in neoplastic cells. TLR ligands can also be used as immunostimulatory molecules to boost immune system in anticancer treatment. In this respect TLRs have been used in numerous preclinical and clinical studies. However, adjuvants can evoke distinct immune responses, either beneficial or deleterious in the neoplastic setting. Moreover, neoplastic processes may also subvert different signaling pathways and thereby advance cancer progression. From both points of view careful selection of adjuvants is a necessary prerequisite for cancer patients' treatment. Thus, TLRs have a dual role, when used as a target for immunostimulation, as well as when used directly to kill the cancer cell.

The autonomous nervous system of the gut is increasingly recognized as an important regulatory factor in intestinal permeability and immune cell activation. Neuropeptides released by neurons -or inflammatory cells- have emerged as neuro-immune modulators that can relay, for instance, stress-induced neuronal activity to immune processes. Such peptides can participate in processes reducing inflammatory responses, or augment resolution of inflammation. Neuropeptides and hormones such as vasoactive intestinal peptide, urocortin, ghrelin, and cortistatin have been shown to modulate the disease activity in a variety of experimental models of inflammatory and autoimmune disease via modulation of immune or neuronal cell activity. We review here the potential of neuropeptide receptor activation to modulate inflammatory diseases. We will highlight the role of neuropeptides in gastrointestinal (GI) physiology and immune regulation, and we will speculate on the therapeutic potential of peptides that bind G protein coupled receptors (GPCRs) in the management of inflammation in the GI tract.

Androgen receptor (AR) is a steroid hormone receptor that is activated by endogenous androgens, mainly testosterone and 5α- dihydrotestosterone (5α-DHT). AR is also an important drug target, and AR antagonists (antiandrogens) have been widely used for prostate cancer therapy. Antiandrogens currently available on the market are all small molecules that antagonize AR function via binding to the ligand binding domain (LBD). AR peptide antagonist has been proposed as a ‘mechanism-based’ approach to directly block AR function by interrupting AR-protein interactions from the surface of the receptor. Without targeting the rigid ligand binding pocket within LBD, peptide antagonists allow more flexibility in structure design, and are likely to provide more efficient and complete blockade of AR function as compared to small molecule antagonists. AR interacts with a variety of proteins, and the interaction may be mediated by different functional domains of the receptor. Although varieties of AR-protein interfaces might serve as the target for peptide antagonist, majority of ongoing research is still focusing on peptides that target the LBD, which is mainly due to the abundance of structural information revealed by crystal structures. This review gives an overview of the current research attempts to develop AR peptide antagonists, particularly peptide antagonists that target the LBD and N-terminal domain (NTD). The challenges and opportunities for future discovery and development of peptide antagonists are discussed as well.

Cyclic nucleotide phosphodiesterase (PDE), that is a multigenic enzyme superfamily ubiquitously distributed in mammalians, mainly contributes to intracellular signaling regulation. Its various isozymes specifically control in a spatio-temporal manner intracellular levels of cAMP and cGMP downstream receptor activation and nearby functional proteins. The PDE superfamily is constituted by 11 gene families (PDE1-PDE11), comprising 21 genes represented by more than 100 mRNA products due to alternative splicing. Among them, PDE3, PDE4 and PDE5 were viewed as therapeutic targets and therefore, due to the successful development of Viagra™ (sildenafil, potent selective PDE5 inhibitor), the knowledge in PDE field burst out with the help of academic/pharmaceutical collaborations. Organic medicinal chemistry, using crystallographic and docking approaches, has focused its search on the catalytic pocket of PDEs, leaving aside the development of variant subtype specific PDE inhibitors and activators. This review firstly describes the various properties of each PDE isozyme, focusing particularly on their regulatory domains, mainly located in the N-terminus. Thereafter, we review the possible peptidic regulations of PDE activity itself, then the PDE anchoring in macromolecular complexes and finally the direct interaction of PDE with some critical intracellular proteins, such as β-arrestin, immunophilin and proteins containing SH3-domain. Altogether, it appears that a peptidic approach would be helpful to study the intrinsic PDE regulation of each subfamily, and thereafter the PDE peptidic motifs implicated as well as PDE location in signaling cascades. Taking in account the various regulatory PDE domains could lead to design new peptides to conceive variant specific inhibitors as well as activators in a therapeutical goal.

Opioid receptors and opioid peptides constitute the endogenous opioid system. The most relevant function of the opioid system seems to be the inhibitory modulation of nociceptive information at supraspinal, spinal and peripheral sites, although it is also implicated in the modulation of many other processes in the body. Centrally acting plant opiates, such as morphine, are the most frequently used analgesics for the relief of severe pain, even though their undesired side-effects are serious limitation to their usefulness. Opioid peptides have the potential to be pharmaceutical agents for the treatment of pain, devoid of side-effects accompanying morphine. Unfortunately, peptides are generally hydrophilic compounds that will not enter the central nervous system via passive diffusion, due to the existence of the blood-brain barrier. Peptides are also easily degraded by proteolytic enzymes which further reduces their therapeutic value. Therefore, the design of peptide analogs based on the sequence of endogenous opioid peptides must be focused on increasing bioavailability and enhancing brain uptake.

Current pharmacologic treatments for inflammatory diseases are largely palliative rather than curative. Most of them result in nonspecific immunosuppression. This can be associated with disruption of natural and induced immunity with significant, sometimes dramatic, adverse effects. Among the novel strategies that are under development, tools that target specific molecular pathways and cells, and more precisely modulate the immune system to restore normal tolerance mechanisms are central. In these approaches, peptide therapeutics constitute a valuable class of therapeutic agents. They possess a number of intrinsic properties that are favorable for longterm treatments. They are also versatile components that can be modified to improve their capacities without affecting their bioactivity. Peptide-mediated immunotherapy has been evaluated in several appropriate experimental animal models. A few peptides are currently evaluated in clinical trials for the treatment of human chronic inflammatory diseases. In this review we describe a number of these emerging peptide therapeutics. We also discuss future challenges that, in addition to include selection of appropriate peptide drugs, also involve the optimization of peptide dosage and route of administration as well as the improvement of peptide stability for adequate bioavailability and specific targeting.

Viruses need to deliver their genomic information into the host cell lumen to establish productive infection. Enveloped viruses accomplish this task by fusing their membrane with a host cell membrane. Membrane fusion is facilitated by specialized viral membrane proteins, which mediate binding and entry into host cells. The architecture of the fusion machinery of envelope proteins can differ between viruses, and class I, II and III fusion systems have been described. However, the conformational rearrangements associated with membrane fusion are comparable and constitute attractive targets for intervention. The fusion apparatus of the human immunodeficiency virus (HIV) envelope protein (Env), a class I fusion protein, is located in the transmembrane unit gp41 of Env. The fusion machinery is activated by Env binding to CD4 and a chemokine coreceptor, and the structural rearrangements in gp41 associated with membrane fusion comprise the insertion of a fusion peptide into the target cell membrane and the formation of a stable six-helix bundle structure. These processes can be efficiently inhibited by peptides mimicking conserved functional elements in gp41. A prominent example for such peptides, termed fusion inhibitors, is the peptide T-20 (enfuvirtide, Fuzeon) which is used as salvage therapy of HIV/AIDS. Here, we will discuss how HIV mediates fusion with host cell membranes and how this process can be blocked by peptides targeting gp41. In addition, we will discuss peptide inhibitors of other class I viral fusion proteins.

The heart is a sophisticated endocrine gland synthesizing a family of peptide hormones by three different genes. These cardiac hormones are stored as 3 prohormones, i.e. atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) prohormones. Within the ANP prohormones are 4 peptide hormones, i.e. atrial natriuretic peptide, vessel dilator, kaliuretic peptide and long-acting natriuretic peptide (LANP) which decrease up to 97% of human pancreatic, breast, colon, prostate, kidney and ovarian carcinomas as well as small-cell and squamous cell lung cancer cells within 24 hours in cell culture. In vivo these 4 cardiac hormones eliminate up to 80% of human pancreatic adenocarcinomas, 2/3rds of human breast cancers, and up to 86% of human small-cell lung cancers in athymic mice. Their anticancer mechanism(s) target the Ras-MEK 1/2-ERK 1/2 kinase cascade in cancer cells. These 4 cardiac hormones inhibit up to 95% of the basal activity of Ras, 98% of the phosphorylation of MEK 1/2 and 97% of the activation of basal activity of ERK 1/2. They also completely block the activity of mitogens such as epidermal growth factor's ability to stimulate ERK. They do not inhibit the activity of ERK in healthy cells such as human fibroblasts. The final step in their anticancer mechanism of action is that they enter the nucleus as demonstrated by immunfluorescence to inhibit DNA synthesis within cancer cells.

Peptides, polypeptides, and proteins have been extensively studied for their various structural and functional roles in living organisms. However, breakthrough discoveries in the last decades identified some peptide-based matrices that posses the ability to traverse biological membranes, and many peptides, polypeptides and even complete proteins have been shown to have such properties. Hence, these matrices have been successfully used for the intracellular delivery of many therapeutic cargos including small molecules, proteins, peptides, oligonucleutides, plasmids and nanoparticles both in vitro and in vivo. Being neither toxic nor carcinogenic and meanwhile efficient in delivery, they are recognized as very promising vectors to overcome the shortcomings of the available technologies. The characteristics of these peptide-based matrices and their applications in drug delivery are here briefly illustrated together with current challenges and future prospects.