Current Medicinal Chemistry (v.24, #17)

Foreword by Atta-ur-Rahman (1721-1721).

Editorial: Bioactive Proteins and Peptides. by Vadim Ivanov (1722-1723).

A Therapeutic Potential of Animal β-hairpin Antimicrobial Peptides by Pavel V. Panteleev, Sergey V. Balandin, Vadim T. Ivanov, Tatiana V. Ovchinnikova (1724-1746).
Endogenous antimicrobial peptides (AMPs) are evolutionary ancient molecular factors of innate immunity that play the key role in host defense. Because of the low resistance rate, AMPs have caught extensive attention as possible alternatives to conventional antibiotics. Over the last years, it has become evident that biological functions of AMPs are beyond direct killing of microbial cells. This review focuses on a relatively small family of animal host defense peptides with the β-hairpin structure stabilized by disulfide bridges. Their small size, rigid structure, stability to proteases, and plethora of biological functions, including antibacterial, antifungal, antiviral, anticancer, endotoxin-binding, metabolism- and immune- modulating activities, make natural β-hairpin AMPs an attractive molecular basis for drug design.

Prothymosin Alpha: An Alarmin and More... by Pinelopi Samara, Chrysoula-Evangelia Karachaliou, Kyriaki Ioannou, Nikos E. Papaioannou, Ioannis F. Voutsas, Christos Zikos, Ioannis Pirmettis, Minas Papadopoulos, Hubert Kalbacher, Evangelia Livaniou, Ourania E. Tsitsilonis, Wolfgang Voelter (1747-1760).
Background/Objective: Prothymosin alpha (proTα) is a ubiquitous polypeptide first isolated by Haritos in 1984, whose role still remains partly elusive. We know that proTα acts both, intracellularly, as an anti-apoptotic and proliferation mediator, and extracellularly, as a biologic response modifier mediating immune responses similarly to molecules termed as 'alarmins'. Our research team pioneered the elucidation of the mechanisms underlying the observed activities of proTα.

Results: We were the first to demonstrate that proTα levels increase during normal and abnormal cell proliferation. We showed that proTα acts pleiotropically, inducing immunomodulatory effects on immune cell populations. We revealed that the immunoreactive region of proTα is the carboxyterminal decapeptide proTα(100-109) and both molecules stimulate innate immune responses, signaling through Toll-like receptors (TLRs), specifically TLR-4. We reported that proTα and proTα(100-109) bind on the surface of human neutrophils on sites involving TLR-4, and cell activation is complemented by cytoplasmic calcium ion influx. Further, we showed that proTα and proTα(100-109) act as adjuvants upstream of lymphocyte stimulation and, in the presence of antigen, promote the expansion of antigen-reactive effectors. Most recently, we reported that proTα(100-109) may accumulate in experimentally inflamed sites and can serve as a surrogate biomarker in severe bacterial infections, proposing that extracellular release of proTα or proTα(100- 109) alerts the immune system during conditions of danger.

Conclusion: We, therefore, suggest that proTα, and likely proTα(100-109), act as alarmins, being important immune mediators as well as biomarkers, and could eventually become targets for new therapeutic/diagnostic approaches in immune-related diseases like cancer, inflammation, and sepsis.

Peptides Against Autoimmune Neurodegeneration by Alexey Stepanov, Yakov Lomakin, Alexander Gabibov, Alexey Belogurov (1761-1771).
The mammalian immune system is a nearly perfect defensive system polished by a hundred million years of evolution. Unique flexibility and adaptivity have created a virtually impenetrable barrier to numerous exogenous pathogens that are assaulting us every moment. Unfortunately, triggers that remain mostly enigmatic will sometimes persuade the immune system to retarget against self-antigens. This civil war remains underway, showing no mercy and taking no captives, eventually leading to irreversible pathological changes in the human body. Research that has emerged during the last two decades has given us hope that we may have a chance to overcome autoimmune diseases using a variety of techniques to 'reset' the immune system. In this report, we summarize recent advances in utilizing short polypeptides - mostly fragments of autoantigens - in the treatment of autoimmune neurodegeneration.

Plant Pathogenesis-Related Proteins PR-10 and PR-14 as Components of Innate Immunity System and Ubiquitous Allergens by Ekaterina I. Finkina, Daria N. Melnikova, Ivan V. Bogdanov, Tatiana V. Ovchinnikova (1772-1787).
Pathogenesis-related (PR) proteins are components of innate immunity system in plants. They play an important role in plant defense against pathogens. Lipid transfer proteins (LTPs) and Bet v 1 homologs comprise of two separate families of PR-proteins. Both LTPs (PR-14) and Bet v 1 homologs (PR-10) are multifunctional small proteins involving in plant response to abiotic and biotic stress conditions. The representatives of these PR-protein families do not show any sequence similarity but have other common biochemical features such as low molecular masses, the presence of hydrophobic cavities, ligand binding properties, and antimicrobial activities. Besides, many members of PR-10 and PR-14 families are ubiquitous plant panallergens which are able to cause sensitization of human immune system and crossreactive allergic reactions to plant food and pollen. This review is aimed at comparative analysis of structure-functional and allergenic properties of the PR-10 and PR-14 families, as well as prospects for their medicinal application.

NMR-based Drug Development and Improvement Against Malignant Melanoma – Implications for the MIA Protein Family by Oliver Arnolds, Xueyin Zhong, King Tuo Yip, Miriam Schöpel, Bastian Kohl, Stefanie Pütz, Raid Abdel-Jalil, Raphael Stoll (1788-1796).
The Melanoma Inhibitory Activity (MIA) protein is strongly expressed and secreted by malignant melanoma cells and was shown to promote melanoma development and invasion. The MIA protein was the first extracellular protein shown to adopt an Src homology 3 (SH3) domain-like fold in solution that can bind to fibronectin type III domains. Together with MIA, the homologous proteins OTOR (or FDP), MIA-2, and TANGO (or MIA-3) constitute a protein family of non-cytosolic and – except for fulllength TANGO and TANGO1-like (TALI) – extracellular SH3-domain containing proteins. Members of this protein family modulate collagen maturation and export, cartilage development, cell attachment in the extracellular matrix, and melanoma metastasis. These proteins may thus serve as promising targets for drug development against malignant melanoma.

For the last twenty years, NMR spectroscopy has become a powerful technique in medicinal chemistry. While traditional high throughput screenings only report on the activity or affinity of low molecular weight compounds, NMR spectroscopy does not only relate to the structure of those compounds with their activity, but it can also unravel structural information on the ligand binding site on the protein at atomic resolution. Based on the molecular details of the interaction between the ligand and its target protein, the binding affinities of initial fragment hits can be further improved more efficiently in order to generate lead structures that exhibit significant therapeutic effects. The NMR-based approach promises to greatly contribute to the quest for low molecular weight compounds that ultimately could yield drugs to treat skin-related diseases such as malignant melanoma more effectively.

Synthetic Peptide Drugs for Targeting Skin Cancer: Malignant Melanoma and Melanotic Lesions by Alex N. Eberle, Bhimsen Rout, Mei Bigliardi Qi, Paul L. Bigliardi (1797-1826).
Background: Peptides play decisive roles in the skin, ranging from host defense responses to various forms of neuroendocrine regulation of cell and organelle function. Synthetic peptides conjugated to radionuclides or photosensitizers may serve to identify and treat skin tumors and their metastatic forms in other organs of the body. In the introductory part of this review, the role and interplay of the different peptides in the skin are briefly summarized, including their potential application for the management of frequently occurring skin cancers. Special emphasis is given to different targeting options for the treatment of melanoma and melanotic lesions.

Radionuclide Targeting: α-Melanocyte-stimulating hormone (α-MSH) is the most prominent peptide for targeting of melanoma tumors via the G protein-coupled melanocortin-1 receptor that is (over-)expressed by melanoma cells and melanocytes. More than 100 different linear and cyclic analogs of α-MSH containing chelators for 111In, 67/68Ga, 64Cu, 90Y, 212Pb, 99mTc, 188Re were synthesized and examined with experimental animals and in a few clinical studies. Linear Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys-NH2 (NAP-amide) and Re-cyclized Cys- Cys-Glu-His-D-Phe-Arg-Trp-Cys-Arg-Pro-Val-NH2 (Re[Arg11]CCMSH) containing different chelators at the N- or C-terminus served as lead compounds for peptide drugs with further optimized characteristics. Alternatively, melanoma may be targeted with radiopeptides that bind to melanin granules occurring extracellularly in these tumors.

Photosensitizer targeting: A more recent approach is the application of photosensitizers attached to the MSH molecule for targeted photodynamic therapy using LED or coherent laser light that specifically activates the photosensitizer. Experimental studies have demonstrated the feasibility of this approach as a more gentle and convenient alternative compared to radionuclides.

Divergent Roles of IRS (Insulin Receptor Substrate) 1 and 2 in Liver and Skeletal Muscle by Sabine Sarah Eckstein, Cora Weigert, Rainer Lehmann (1827-1852).
IRS1 and IRS2 are the most important representatives of the IRS protein family and critical nodes in insulin/IGF1-signaling. Although they are quite similar in their structural and functional features they show tissue-specific differences. In this review, we outline the functions of IRS1 and IRS2 in skeletal muscle and liver with regard to their importance for metabolism, growth and differentiation. Mechanisms contributing to IRS1 and IRS2 dysregulation in disease states as well as consequences thereof are discussed. IRS1 plays the dominant role in skeletal muscle. It is crucial for normal growth and differentiation of myofibers, insulin-dependent glucose uptake and glycogen synthesis. The presence of IRS2 in skeletal muscle is negligible for insulin-induced glucose uptake and the general role of IRS2 in muscle is still not fully understood. In liver IRS1 and IRS2 are important to mediate insulindependent regulation of glucose and lipid metabolism and complement each other in the diurnal regulation thereof. IRS1 in the liver is more important for signaling in the late refeeding period, whereas IRS2 signaling is mostly dominating in the period directly after food intake and during fasting. Importantly, the expression level of IRS1 and IRS2 is different within the liver lobule, which could be an explanation for the phenomenon of selective insulin resistance. Dysregulated muscular or hepatic abundance and/or phosphorylation status of IRS1 and IRS2 are important factors in the pathogenesis of insulin resistance, type 2 diabetes and muscle wasting.

During the last years, the interest in combining the features of metal-containing molecules with biomolecules, particularly peptides, has been increased. Large series of new innovative organometallic compounds, as well as potent coordination complexes have been designed and, especially in medicinal chemistry, the library of bioactive compounds was excessively expanded by the introduction of metal complexes. The research foci are divers and not limited to e.g. the development of therapeutics with anti-proliferative or antibacterial activity, or the design of novel biosensors useful for pharmaceutical applications. By introduction of a metal centre, new attributes might be added that could help to overcome the problems of difficult to treat diseases, as well as to combat issues with arising drug resistances. However, the application of a number of very potent metal complexes is restricted owing to their poor water-solubility, air-stability and only poor uptake when in contact with cells. In this context, one possibility to optimize promising lead structures is to couple them to bioactive peptides. Within this review, an overview on metal complex-peptide conjugates used for drug design and future pharmaceutical application is presented.

Linear Peptides in Intracellular Applications by Cristiane R. Zuconelli, Roland Brock, Merel J.W. Adjobo-Hermans (1862-1873).
To this point, efforts to develop therapeutic peptides for intracellular applications were guided by the perception that unmodified linear peptides are highly unstable and therefore structural modifications are required to reduce proteolytic breakdown. Largely, this concept is a consequence of the fact that most research on intracellular peptides hitherto has focused on peptide degradation in the context of antigen processing, rather than on peptide stability. Interestingly, inside cells, endogenous peptides lacking any chemical modifications to enhance stability escape degradation to the point that they may even modulate intracellular signaling pathways. In addition, many unmodified synthetic peptides designed to interfere with intracellular signaling, following introduction into cells, have the expected activity demonstrating that biologically relevant concentrations can be reached. This review provides an overview of results and techniques relating to the exploration and application of linear, unmodified peptides. After an introduction to intracellular peptide turnover, the review mentions examples for synthetic peptides as modulators of intracellular signaling, introduces endogenous peptides with bioactivity, techniques to measure peptide stability, and peptide delivery. Future experiments should elucidate the rules needed to predict promising peptide candidates.

Snake Venom: From Deadly Toxins to Life-saving Therapeutics by Humera Waheed, Syed F. Moin, M. I. Choudhary (1874-1891).
Snakes are fascinating creatures and have been residents of this planet well before ancient humans dwelled the earth. Venomous snakes have been a figure of fear, and cause notable mortality throughout the world. The venom constitutes families of proteins and peptides with various isoforms that make it a cocktail of diverse molecules. These biomolecules are responsible for the disturbance in fundamental physiological systems of the envenomed victim, leading to morbidity which can lead to death if left untreated. Researchers have turned these life-threatening toxins into life-saving therapeutics via technological advancements. Since the development of captopril, the first drug that was derived from bradykininpotentiating peptide of Bothrops jararaca, to the disintegrins that have potent activity against certain types of cancers, snake venom components have shown great potential for the development of lead compounds for new drugs. There is a continuous development of new drugs from snake venom for coagulopathy and hemostasis to anti-cancer agents. In this review, we have focused on different snake venom proteins / peptides derived drugs that are in clinical use or in developmental stages till to date. Also, some commonly used snake venom derived diagnostic tools along with the recent updates in this exciting field are discussed.

Protein Profile Analysis of Two Australian Snake Venoms by One- Dimensional Gel Electrophoresis and MS/MS Experiments by Dessislava Georgieva, Diana Hildebrand, Rodrigo Simas, Monika A. Coronado, Marcel Kwiatkowski, Hartmut Schlüter, Raghuvir Arni, Patrick Spencer, Christian Betzel (1892-1908).
The Pseudechis colletti and Pseudechis butleri venoms were analyzed by 1-D gel electrophoresis, followed by mass spectrometric analysis of tryptic peptides obtained from the protein bands. Both venoms contain highly potent pharmacologically active components, which were assigned to the following protein families: basic and acidic phospholipases A2 (PLA2s), L-amino acid oxidases (LAAOs), P-III metalloproteinases (P-III SVMPs), 5'- nucleotidases (5'-NTDs), cysteine-rich secretory proteins (CRISPs), venom nerve growth factors (VNGFs) and post-synaptic neurotoxins. Considerable predominance of PLA2s over other toxins is a characteristic feature of both venoms. The major differences in the venom compositions are the higher concentration of SVMPs and CRISPs in the P. butleri venom, as well as the presence of post-synaptic neurotoxins. Furthermore, the analysis revealed a high concentration of proteins with myotoxic, coagulopathic and apoptotic activities. PLA2s are responsible for the myotoxic and anticoagulant effects observed in patients after envenomation (4). The other protein families, encountered in the two venoms, probably contribute to the major symptoms described for these venoms. These results explain the observed clinical effects of the black snake envenomation. The analyzed venoms contain group P-III metalloproteinases of medical importance with the potency to be used for diagnostic purposes of von Willebrand factor (vWF) disease, for regulation of vWF in thrombosis and haemostasis, for studying the function of the complement system in host defense and in the pathogenesis of diseases. Comparison of venomic data showed similarities in the major venom components of snakes from the genus Pseudechis, resulting in common clinical effects of envenomation, and demonstrating close relationships between venom toxins of Elapidae snakes.