Current Pharmaceutical Design - Volume 17, Issue 25, 2011

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 the advancements 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. Novel therapeutic peptides currently derived from active pre-existing peptides or from high-throughput screening and are optimized following a rational drug design approach. Molecules of interest have to prove their ability to influence the disease outcome in animal models and must respond to a set of criteria based on toxicity studies, ease of administration, cost of their synthesis and logistic for clinical use to validate it; as a good candidate in a therapeutic perspective. 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 fifth themed issue of Current Pharmaceutical Design, for which I have the honor to be the Executive Guest Editor, addresses topical issues to some of these potential utilizations of peptide motifs for a variety of genetic and acquired diseases. Alzheimer's disease, the most common form of age-related neurodegenerative disorder, is characterized by the presence of extracellular senile plaques, neurofibrillary tangles and neuronal loss in the brain. Senile plaques are composed of aggregations of small peptides called amyloid β (Aβ). Aβ is produced during normal cell metabolism through proteolytic processing of the amyloid precursor protein, expressed constitutively by many cell types throughout life. Recent studies have shown that Aβ is neurotoxic and that this neurotoxicity is related to its aggregation state into insoluble β-sheet fibrils. Erik Portelius, Niklas Mattsson, Ulf Andreasson, Kaj Blennow and Henrik Zetterberg [1] present an overview of the many aspects of Aβ and its isoforms with special focus on their potential role as diagnostic and theragnostic markers. The major constituent of neurofibrillary tangles is Tau, a microtubule-associated protein, whose increased hyperphosphorylation is known to cause memory impairments. In the amyloid cascade hypothesis, toxic concentrations of Aβ would trigger changes in Tau and consequent neurofibrillary tangle formation. Natalia Shiryaev, Regina Pickman, Eliezer Giladi, and Illana Gozes [2] examine the protecting effects of chronic daily administration of short peptide snippets against deficits in spatial memory and the underlying tauopathy. Endothelins (ETs) are peptides produced primarily in the endothelium and playing a key role in vascular homeostasis. The ET system consists of three ET isoforms (ET-1, -2 and -3) and two G-protein-coupled receptors, ET(A) and ET(B). In the cardiovascular system, ETs, particularly ET-1, are expressed in smooth muscle cells, cardiomyocytes, fibroblasts, and notably in vascular endothelial cells, and many of the cardiovascular complications associated with aging and cardiovascular risk factors (including e.g. hypertension, atherosclerosis or fibrosis) appear initially attributable, at least in part, to endothelial dysfunction. Martin Houde, Julie Labonté and Pedro D'Orléans-Juste [3] summarize the knowledge to date and future perspectives related to the use of peptide antagonists targeting endothelin receptors in physiological and pathological settings. Neuropeptides are peptides released by neurons to communicate with each other by acting on cell surface receptors, particularly G protein-coupled receptors (GPCRs) to control a wide spectrum of physiological functions. Genomic sequencing efforts have yielded a large number of cDNA sequences that potentially encode novel candidate peptide precursors, as well as hundreds of GPCRs with no known cognate ligands, termed “orphan GPCRs”, that became the roots of reverse pharmacology, in which receptors are attempted to be matched to potential transmitters. Yan Zhang, Zhiwei Wang, Gregory Scott Parks and Olivier Civelli [4] retrace the history of the orphan GPCRs and the discoveries of their endogenous ligands and discuss the difficulties that the search for new ligands is presently encountering and the challenges facing neuropeptide discovery....

The last decades have witnessed an explosion in studies of the role of amyloid-β (Aβ) in the progress of the neurodegenerative disorder Alzheimer´s disease (AD) and it is now widely accepted that Aβ is related to the pathogenesis of AD. For example, studies have shown that Aβ is neurotoxic and that the neurotoxicity of Aβ is related to its aggregation state. The concentration of the 42 amino acid form of Aβ (Aβ1-42) is reduced in the cerebrospinal fluid (CSF) from AD patients, which is believed to reflect the AD pathology with plaques in the brain acting as sinks. Less well investigated, however, is the ability of other Aβ isoforms to distinguish AD patients from controls and to identify treatment effects in clinical trials. Recently, novel C-truncated forms of Aβ (Aβ1-14, Aβ1-15, and Aβ1-16) were identified in human CSF. The presence of these small peptides is consistent with a catabolic amyloid precursor protein cleavage pathway by β- followed by α-secretase. It has been shown that Aβ1-14, Aβ1-15, and Aβ1-16 increase dose-dependently in response to γ-secretase inhibitor treatment while Aβ1-42 levels are unchanged. Here, we review the many aspects of Aβ and its isoforms with special focus on their potential role as diagnostic and theragnostic markers.

Activity-dependent neuroprotective protein (ADNP) is essential for brain formation and partial deficiency in ADNP results in cognitive deficits coupled with tauopathy and neuronal cell death. Our previous results indicated that a peptide snippet from ADNP, NAPVSIPQ (NAP, generic name, davunetide) can restore in part ADNP deficiencies. NAP interacts with tubulin and this interaction is displaced by the NAP related peptide that is derived from activity-dependent neurotrophic factor (ADNF), SALLRSIPA (SAL) and its all D-amino acid peptide derivative (D-SAL, also known as AL-309). Both NAP and D-SAL were shown to protect neurons against amyloid beta toxicity however the mechanism of protection is still under investigation. In addition, NAP protects against tau hyperphosphorylation associated with ADNP deficiency, in vivo. To investigate whether the mechanism of in vitro neuroprotection relates to the in vivo protection against tauopathy and to draw potential additional parallelism between NAP and D-SAL, we asked if: 1]NAP and D-SAL protect against amyloid beta related tau hyperphosphorylation in vitro; and 2] D-SAL protects against haploinsufficiency in ADNP, inhibiting tauopathy in vivo. Assessment of NAP and D-SAL neuroprotection in primary cortical neuro-glial cultures treated with amyloid beta showed that both peptides reduced toxin-related neuronal damage and protected against tau hyperphosphorylation. In vivo, chronic DSAL administration protected against tau hyperphosphorylation associated with ADNP deficiency (ADNP+/- mice), showing for the first time protection against deficits in odor discrimination and in social recognition. These studies associate neuroprotection in vivo and in vitro and provide a broad base for future drug development based on NAP and D-SAL against multiple neurodegenerative conditions.

As for other peptides such as bradykinin, neurokinins and angiotensins, peptide antagonists for endothelin-1 (ET-1) have been early on developed towards the pharmacological characterization of both ETA and ETB receptors. Interestingly, unlike the previously mentioned three peptides, receptors for ET-1 were cloned and purified prior to the report of ETA and ETB receptor antagonists such as BQ-123 and BQ-788. The availability of such pharmacological tools and the use of molecular approaches have certainly fast-tracked the development of non-peptide ET receptor antagonists for clinical applications. Albeit rapid degradation by gastric enzymes and short halflife in plasma of peptide receptor antagonists limit their use in clinical settings, those molecules have been of importance in the identification of mediators and modulators of ET-1 induced properties in vitro and in vivo, as described further in this review. Peptide antagonists acting selectively or, with equivalent affinities against ETA and ETB receptors were reported prior to the advent of clinically relevant non-peptide blockers such as Bosentan. Confounding mechanisms involving, for example, the endogenous modulators nitric oxide and prostacyclin as well as allosteric interactions between ET receptor types, have also been clarified with the use of peptide antagonists for endothelins. Finally, peptide antagonists were also used to identify the precise pharmacology of ET-1 precursors such as big-endothelin-1 and ET-1 (1-31). The present review will thus attempt to summarize the knowledge to date and future perspectives related to use of peptide antagonists targeting endothelin receptors in physiological and pathological settings.

Neuropeptides control a wide spectrum of physiological functions. They are central to our understanding of brain functions. They exert their actions by interacting with specific G protein-coupled receptors. We however have not found all the neuropeptides that exist in organisms. The search for novel neuropeptides is thus of great interest as it will lead to a better understanding of brain function and disorders. In this review, we will discuss the historical as well as the current approaches to neuropeptide discovery, with a particular emphasis on the orphan GPCR-based strategies. We will also discuss two novel peptides, neuropeptide S and neuromedin S, as examples of the impact of neuropeptide discovery on our understanding of brain functions. Finally, the challenges facing neuropeptide discovery will be discussed.

Activation of the estrogen receptor alpha (ERα) is of prime importance for the development of hormone-dependent breast cancers. Hence, drugs able to impede the emergence of an active folding of ERα have been used for a long time as a first line therapeutic strategy. Aromatase inhibitors that block estradiol synthesis and / or antiestrogens that compete with hormone binding to the receptor are routinely prescribed. Unfortunately, emergence of tumor resistance almost invariably results from currently used antihormonal approaches. One may anticipate that a “multi-target” strategy affecting key regulatory domains distinct from ligand binding pocket of ERα may help to circumvent this problem. To reach this goal, the synthesis of peptides that may specifically inhibit intra- or inter-molecular interactions has been proposed. This paper describes functional motifs potentially suitable for the design of such antagonists. Activity of available peptidic and non-peptidic mimics of these motifs is also reviewed.

A fair amount of data indicates that bradykinin and lysyl-bradykinin exert arterial, cardiac and renal effects which afford protection against organ damage in diseases, especially in the settings of ischemia or diabetes. The concept of kinins acting as therapeutic agents is supported by the wide use of angiotensin I-converting enzyme (ACE) inhibitors. These inhibitors indeed potentiate kinin action by inhibiting kinin degradation. Experimental evidence strongly suggests that the cardiac and renal effects of ACE inhibitors are due, at least in part, to kinins. Angiotensin AT1 receptor antagonists act also partly through kinins. This paper reviews available evidence supporting a role for kinins in the therapeutic effect of current drugs. It then discusses the opportunity to develop new drugs based on kinin action. Direct activation of the kinin B2 receptor by pharmacological agonists might provide higher therapeutic benefit than existing kinin- potentiating drugs. Possible occurrence of side effects is however a concern.

Bioactive biomaterials are desirable as tissue engineering scaffolds by virtue of their capability to mimic the natural environment of the extracellular matrix. Bioactive biomaterials have been achieved by incorporating synthetic short peptide sequences into suitable materials either by surface modification or by bulk incorporation. The goal is to enhance cell attachment and other basic functions. Bioactive peptides can be obtained from biological or chemically synthesized sources, increasing their specific cellular responses for tissue growth and development. Compared to using an entire growth factor in regenerative therapy, these peptides demonstrate potential advantages such as overcoming possible immunogenicity, being less susceptible to degradation, and producing fewer tumor-related side effects. Biomaterial scaffolds modified with peptides can provide biological ligands for cell-scaffold interactions that promote cell attachment, proliferation, and differentiation. Peptide-based biomaterial scaffolds can be fabricated to form two- and three-dimensional structures. This review discusses cell-binding, biominerailization inducing peptides, and receptor-binding peptides for bone regeneration. This review also addresses issues related to peptide immobilization as well as potential complications that may develop as a result of using these versatile bioactive peptides. The development of self-assembled peptide amphiphiles with the goal of generating new threedimensional scaffolds for tissue engineering is also summarized.

We have employed computer-based molecular modeling approaches to design peptides from the ras-p21 and p53 proteins that either induce tumor cell reversion to the untransformed phenotype or induce tumor cell necrosis without affecting normal cells. For rasp21, we have computed and superimposed the average low energy structures for the wild-type protein and oncogenic forms of this protein and found that specific domains change conformation in the oncogenic proteins. We have synthesized peptides corresponding to these and found that ras peptides, 35-47 (PNC-7) and 96-110 (PNC-2), block oncogenic ras-p21-induced oocyte maturation but have no effect on insulin-induced oocyte maturation that requires activation of endogenous wild-type ras-p21. These results show signal transduction pathway differences between oncogenic and activated wild-type ras-p21. Both peptides, attached to a membrane-penetrating peptide (membrane residency peptide or MRP), either induce phenotypic reversion to the untransformed phenotype or tumor cell necrosis of several ras-transformed cell lines, but have no effect on the growth of normal cells. Using other computational methods, we have designed two peptides, PNC-27 and 28, containing HDM-2-protein-binding domain sequences from p53 linked on their C-termini to the MRP that induce pore formation in the membranes of a wide range of cancer cells but not any normal cells tested. This is due to the expression of HDM-2 in the cancer cell membrane that does not occur in normal cells. These peptides eradicate a highly malignant tumor in nude mice with no apparent side effects. Both ras and p53 peptides show promise as anti-tumor agents in humans.

The first step in drug absorption is the passage of drug molecules across epithelial cell sheets. Epithelial cell sheets are pivotal for the maintenance of homeostasis in the body by acting as a biological barrier that separates the inside of the body from the outside environment. Intercellular space between the adjacent epithelial cells is tightly sealed by tight junctions (TJs), which prevent solutes from freely moving across the epithelial cell sheets. Modulation of the TJ barrier has been a potent strategy for drug absorption. Absorption enhancers have been investigated since the 1980s, and sodium caprate is clinically used as an absorption enhancer. However, the biochemical constituents and structures of TJs were not elucidated until 1993. Occludin, a tetra-transmembrane protein, was identified to be a structural component of TJs in 1993. Claudin, another tetra-transmembrane protein, was identified as a structural and functional component of TJs in 1998. Modulation of occludin- or claudin-barrier is novel methods to enhance drug absorption. Recently, synthetic TJbinding peptides, a kinase of claudin and peptide fragments of toxins have been developed. In the present review, we summarize the recent progress in TJ-modulating peptides and discuss their potencies.

Neurotrophic factors were originally identified based on their ability to prevent naturally occurring cell death in the developing nervous system. Many of these proteins also promote survival after injury or protect neurons in toxin-disease models in animals. In addition to neuroprotective effects, these factors exert trophic effects on neurons, stimulating increases in neuronal metabolism, cell size, and process outgrowth. These properties underlie expectations for neurorestoration, in which growth of new axons and synapses could lead to functional improvement, which is of great interest for those patients who are already significantly disabled by disease. Preclinical and clinical data suggest that subcutaneous or intravenous administration of neurotrophic factors may be effective for the treatment of peripheral nervous system diseases. However, even though these proteins are natural products, they do present specific problems when used as therapeutic agents. They cannot be given orally, present uncertain pharmacokinetic behavior, and large-scale production is labor and costintensive. Neurotrophic factor treatment of central nervous system diseases presents an even more complex scenario, since they are not able to cross the blood-brain barrier and must be given intracerebrally. Although there is an active search for alternative delivery strategies, for central nervous system diseases in particular the advantages of small molecule mimetics over proteins are evident. Small organic molecules can be modified to penetrate freely into the brain parenchyma and can be designed for oral administration. There are several possible approaches for replacing neurotrophic proteins with small molecule mimetics. For therapeutic use in the peripheral nervous system, neurotrophic proteins could be replaced by active peptide fragments with receptor binding properties similar to the full-length protein, but improved pharmacokinetic properties and lower production costs. In principle, it should be possible to replace the entire protein or fully active peptide fragment by a non-peptidic molecule binding to the same receptor site. It may be possible to regulate neurotrophic factor receptor activity by allosterically-acting molecules which influence the functional efficacy of the receptors. Other strategies include intracellular effector-targeting approaches, which are based on knowledge of signaling pathways involved in neuronal cell survival and demise, and which can be agonized or antagonized to promote neuroprotection. This chapter will begin with a brief overview on the biology neurotrophic proteins, followed with a description of strategies taken towards the development of small molecule mimetics for neurotrophic factors and the emerging drug candidates. The latter will encompass both receptor-directed as well as intracellular signalling approaches.

Antimicrobial peptides include a diverse array of both natural and synthetic molecules varying greatly in size, charge, hydrophobicity and secondary-structural features. Although better known as antibacterial agents, many peptides have demonstrated activity against the malarial parasite Plasmodium either in its vertebrate blood stages or mosquito stages or both. The antimalarial peptides reviewed here consist of (i) cationic, amphipathic ‘host-defence’ peptides including some (e.g. defensins and cecropins) that are naturally produced by mosquitos, (ii) other membrane-active peptide antibiotics such as gramicidins, (iii) hydrophobic peptides, most notably cyclosporins, (iv) thiopeptides, such as thiostrepton, and (v) some other naturally occurring or synthetic peptides. Many of these peptides affect membrane integrity and some are selective for parasite membranes over those of the host, while others are thought to have more specific intracellular targets. The mechanisms of action of the majority of antimalarial peptides are however either uncertain or totally unknown. Very few of these agents have been tested in rodent malaria models and none has undergone significant pre-clinical or clinical development for malaria. Issues such as metabolic lability, high cost, and a lack of information about systemic toxicity are likely to be serious obstacles to further development of peptides as antimalarial drugs. On the other hand, they offer potential advantages, including the possibility of being much less prone to resistance than the drugs in current use. An alternative to conventional chemotherapy, namely the release of malaria-refractory, transgenic mosquitos overproducing antimalarial peptides, has already passed the ‘proof of concept’ stage.

Peptoids are a developing class of peptide-like oligomers originally invented for drug discovery in the early 1990s. While peptides hold great promise for therapeutic applications, current development of peptide-based pharmaceuticals is hindered by their potential for misfolding and aggregation, and particularly, for rapid in vivo degradation post-administration. Researchers have investigated alternative peptide-like constructs that may be able to circumvent such complications. Peptoids comprise a peptide-based backbone and Nsubstituted glycines for side chain residues, resulting in complete protease-resistance. Synthesis of peptoid sequences up to 50 units in length allows for controlled sequence composition and incorporation of diverse side chain chemistries. Though the landscape of peptoid structure is not clearly defined, secondary, tertiary, loop, turn, and random structures have been identified. As protease-resistant isomers of peptides, peptoids are being developed as versatile molecular tools in biochemistry and biophysics, and are becoming attractive candidates for therapeutic and diagnostic applications. Peptoids have thus far demonstrated bioactivity as protein mimics and as replacements for small molecule drugs. In this review, we discuss the most recent advances in peptoid research on the therapeutic front in the last few years, including in vitro and in vivo studies in the fields of lung surfactant therapy, antimicrobial agents, diagnostics, and cancer. We particularly focus on the biophysical activity of lipid-associated peptoids and their potential therapeutic applications.