The discovery of new drugs is a long process and requires multidisciplinary research teams and an absolute scientific rigor. The discovery and development of a new drug usually takes between 7 and 15 years and has an associated average cost around 400 million euros. The process consists of several stages, with the initials where the contribution of basic research is fundamental. To the best of our references in Valencia has never found a drug or pharmaceutical companies are comparable to those of other regions with similar per capita income level as Madrid, Catalonia and the Basque Country. Can help to alleviate this situation and establish or create new initiatives in the Comunitat Valenciana of companies in this sector should be directed towards supporting and strengthening multidisciplinary teams whose research set the foundation for identifying new therapeutic targets and their modulators.

The last decade of research in cell biology has shown that protein-protein interactions, both between soluble proteins as membrane level, are a key mechanism for cellular regulation. The characterization of these interactions, together with the identification of modulators, offers new opportunities in biotechnology. In the present application intends to use modulators of protein-protein interactions of biological relevance for the discovery of new drug candidates. The proposal focuses on two fundamental biological processes, programmed cell death (apoptosis) and integrin-mediated interactions responsible for the formation of platelet aggregates causing thrombosis and tumor metastasis. The research project aims to use chemical biology techniques to modify / regulate cell signaling associated with these processes. The identification of modulators is performed from two complementary approaches. The use of in vitro and combinatorial chemistry of peptides and organic molecules of low molecular weight (SOM) and the identification of bioactive compounds in snake venoms.

The exploitation of SOM is part of human history, from use until the introduction of alkaloids in the twentieth century of antibiotics such as penicillin (natural) or ciprofloxacin (synthetic). The study of biological systems by chemical intervention was originally called 'chemical genetics' but more recently has adapted the broader term 'chemical biology'. It seeks the use of chemical tools to solve biological problems. By its very nature is multidisciplinary. Conceptually, the objective is based on the use of SOM and peptides to the understanding of cell biology at the molecular level and its future prospects in biotechnology has attracted interest from pharmaceutical companies. In recent decades, most drugs have been developed as enzyme inhibitors. However, today companies are exploring new possibilities. Among the potential candidates include inhibitors of protein-protein interactions. Many proteins, including enzymes, carry out their biological function through interaction with other proteins. Inhibition of these interactions is a form of modulation of the activity. This structural diversity and large number of possible interactions and specific offer an interesting panel of potential drug targets. The identification of peptides that inhibit SOM and interactions between proteins of high interest in biomedicine has confirmed the initial observations. Even very recently it has been proposed that inhibitors of protein-protein interactions can be useful to improve the so-called 'personalized medicine'.

Similarly, interest in the venoms of snakes for medicinal purposes is also part of human history. The venoms are complex mixtures of pharmacologically active molecules whose biological effects are also complex. The presumption of the curative powers of poisonous snakes is lost in the beginning of time and is based on widespread simplistic thinking: "He who carries the poison must take the antidote." Indigenous tribes worldwide have taken the remedies different body parts of snakes and modern science has shown that many poisonous snakes carry the cure for many diseases. Indeed, several hundred species of snakes are potentially dangerous to man in his house an arsenal of poison glands, chemical composition and biological action have been refined over millions of years of evolution that is both a deadly weapon for dam as a natural pharmacopoeia whose enormous biotechnological potential and is being actively explored clinically. Thus, platelet antiaggregatory Tirofiban (Aggrastat ®) and eptifibatide (Integrilin ®) are based on the RGD sequence of the disintegrin, potent inhibitors of platelet aggregation present in venoms of viper and pit vipers, and are in clinical use for prevention thromboembolic episodes. The ancrod serinoproteasas ®, Reptiles ® (or batroxobin ®) and Defibrase ®, isolated from the venom of Calloselasma rhodostoma, and Bothrops atrox Bothops moojeni respectively, have been approved for desfibrinantes therapies (elimination of fibrinogen / fibrin clotting) in order to alleviate the devastating effects of heart attack or stroke. Alfimeprase ® (Nuvelo-R & D), a metalloproteinase fibrinolytic derived recombinant fibrolasa the venom of Agkistrodon contortrix has completed phase II clinical trial, and Captopril Cinfa ® (Bristol-Myers), the first inhibitor of the enzyme converting angiotensin I produces relaxation of blood vessels and reduces blood pressure, based on the structure of the peptides bradykinin enhancers isolated from the venom of Bothrops jararaca. Likewise, the disintegrin contortrostatina, potent inhibitor of integrin v3, isolated from Agkistrodon contortrix contortrix venom, is being investigated by the group of Prof. Francis Markland (Univ. California) for their antiangiogenic potential. It is highly stimulating for the biomedical researcher that the same biological activities that make the poison into a lethal weapon for the natural prey of snakes could be used - properly managed, for the treatment of diseases. It is really fascinating to decipher the molecular mechanisms developed during evolution to enhance the toxic arsenal of snakes, convinced that such knowledge is needed to reverse the strategy in the laboratory of natural selection and transform toxins lethal drugs that can get save lives. In particular, this part of the project is based on the hypothesis that the detailed study of structure-function correlations of snake venom toxins, particularly those that inhibit angiogenesis by selectively blocking the integrin 11, represents a strategy for potential discovery of molecular tools for the visualization of tumor growth and eventually to halt tumor progression.

Apoptosis (programmed cell death) is a cellular process involved in cell turnover and immune system. Found evidence of deregulated apoptosis in neurodegenerative diseases and cancer. Moreover, apoptosis is responsible for cell death process observed in ischemia / reperfusion associated with transplantation. After activation by different apoptotic stimuli converge routes in the activation of proteases called caspases about. The so-called effector (caspase-3 and -7) are responsible for dismantling the cell, while the initiator caspases (-8, -9 and -10) are that activate the effector. One of the activation pathways of apoptosis involves the mitochondria (intrinsic pathway) that is used as the initiator caspase-9. The activation is mediated by the release of cytochrome c and other proapoptotic proteins from the mitochondrial intermembrane space. The formation of the apoptosome (multiprotein complex) is a key event in this pathway and consists of Apaf-1, cytochrome c, and procaspase-9. The main function is to activate the apoptosome caspase-9. To identify molecules mitigating the effects of excessive apoptosis in, for example, some neurodegenerative diseases, transplant rejection and ischemic processes, investigations have focused on the effector caspases. However, caspase inhibitors developed so far for various reasons, suffer from many problems of biodistribution and is widely believed to not exceed the controls required to be approved by regulatory agencies. Our hypothesis was based on consideration of pharmacological interest protein-protein interactions that lead to activation of the route. Specifically, we define the modulation of the formation of the apoptosome as drug target. In our laboratory, using different strategies have been identified first-generation inhibitors by binding to Apaf-1, inhibit the enzymatic activity of the apoptosome and decrease the apoptotic phenotype in cellular models of apoptosis. Moreover, in order to increase the viability of the inhibitors and their appropriate intracellular release polymers have been synthesized for the controlled release of the drug conjugate and heterocyclic molecules with an optimized pharmacological profile. These discoveries have also led to obtaining patent registration and awaken the interest of different groups of basic and applied research for our compounds. It has also signed a memorandum of transfer to the industry with a pharmaceutical company. This project aims at the molecular characterization of the binding site of inhibitors in Apaf-1 and study the consequences at the cellular level of the selective inhibition of apoptosome in various conditions. In particular, recent observations from our laboratory in collaboration with Dr. Guido Kroemer's laboratory (INSERM U848 and Institut Gustave Roussy, Villejuif, France) suggest that selective inhibition of Apaf-1, but not caspases, prevents the release of cytochrome c mitocondria15. This reinforces the strategy of Apaf-1 inhibition as a therapeutic target to prevent unwanted cell apoptosis but also, from the standpoint of basic research, raises new questions about the molecular mechanism of signaling process and may involve an activity-dependent Apaf-1 on the mitochondria. The release of cytochrome c from the mitochondrial intermembrane space is one of the cellular events has been proposed as the point of no return in the mechanism of programmed cell death. However, the soundness of this assertion has recently been questioned. In any case the release of cytochrome c involves the opening of the pore known as mitochondrial permeability transition (PTPM). The opening of PTPM is a complex process in which participants described family proteins Bcl-2. This family of proteins composing pro-and antiapoptotic members and their activity is regulated by an intricate balance based on protein-protein interactions involving the membrane. It has also proposed a possible interaction between Apaf-1 and pro-apoptotic family members Bcl-2. As part of this project is to analyze the consequences of this interaction and its influence on the protection of mitochondrial integrity observed for Apaf-1 inhibitors. Furthermore, in our opinion the role of the membrane is key. In this sense, it is noteworthy that in collaboration with the laboratory of Dr. Petra Schwille (Biophysics Group, BIOTEC, TU Dresden, Germany), we have shown that the interaction that the interaction between Bid, tBid and Bcl-xL is heavily regulated by the membrane. Specifically, a synthetic peptide derived from Bid is able to break the interaction between tBid and Bcl-xL in solution but not in the vicinity of the plasma membrane. These results suggest that interactions between pro-and antiapotósicas proteins are not of the same nature when analyzing only the soluble fragments that when these interactions are analyzed in model membranes using the complete protein including the transmembrane fragments. These observations, although novel in the field of family proteins Bcl-2, is unprecedented in the importance of selective interactions between proteins at the membrane and its importance in cell signaling pathways. Indeed, the increased number of membrane proteins that have been resolved three-dimensional structure has allowed a better understanding of how to carry out the folding and packaging of its fragments transmembrane (TM). Noteworthy are the proteins that have a single TM that direct and regulate protein-protein interactions of critical importance and are involved in signal transduction through the lipid bilayer. The TM has been implicated in dynamic conformational changes that regulate these signals. TM helices are involved, along with the soluble domains of proteins in the fine tuning of the conformational changes that initiate the signaling processes. These changes are propagated through the membrane-dependent oligomerization state and orientation of TM helices. An interesting example is constituted by integrins. These are modular signaling complexes recognize different ligands. 24 are known to heterodimer complex / compound combinations of 18 subunits and 8 subunits. Most integrins exist in equilibrium between an inactive state and an active state capable of binding its extracellular ligand. This project hypothesizes the existence of a dynamic equilibrium, similar to that described for integrins, including pro-and antiapoptotic proteins of the Bcl-2 in the membrane.

Interaction integrin / disintegrin. The interaction between cell surface integrins and extracellular matrix components is a key mechanism for controlling cell adhesion and migration. Signals transmitted from the extracellular region to the intracellular domains of members of the family of integrins modulate the expression of genes that regulate survival, proliferation and cell migration. The disintegrin were described in the laboratory of Stephan Niewiarowski (Temple University, Philadelphia, USA) in the late 1980's as potent inhibitors of platelet aggregation. It has subsequently been shown that the disintegrin family of panel has developed a limited, but selective, based on inhibition of integrin families b1 and b3. Thus, besides the reasons of platelet inhibition (RGD, KGD, WGD), also blocked with different affinity and enhances binding of other integrins to their natural ligands (ex. a5b1 to fibronectin, a8b1 to tenascin C, and avb1 and avb3 to vitronectin), have been characterized that possess disintegrin tripeptides active against other systems of integrin-ligand interaction. The reasons have MGD VGD and selectivity for integrin a5b1; the tripeptide MLD blocks the function of integrins α3β1, α4β1, α6β1, α7β1 and α9β1; and the KTS and RTS motifs selectively antagonize the binding of collagen I and IV to integrin a1b1. Mapping of active disintegrin tripeptide on the phylogenetic tree of the integrin chains (which confer specificity of ligand binding to heterodimers that have a common subunit), suggests an evolutionary adaptation of the disintegrin to the binding sites of ligands integrins.

Integrins target different disintegrin participating in various pathological processes: the integrin αIIbβ3 is responsible for the formation of platelet aggregates causing thrombosis and ischemic heart disease; integrin αvβ3 plays a role in tumor metastasis processes, and α4β1, α4β7 y α9β1 integrins participate in inflammation and autoimmune processes, integrins α1β1 and αvβ3 have been implicated in the mechanism of neovascularization (angiogenesis) of tumors. Antagonists of these integrins represents, therefore, potential therapeutic targets. The research group, described the production of recombinant disintegrin from Trimeresurus jerdostatina jerdonii. RTS has the tripeptide Jerdostatina manner with obtustatina (Vipera lebetina obtusa), viperistatina (Vipera palestinae) and lebestatina (Macrovipera lebetina Transmediterranea) the group of short disintegrin that selectively inhibit the integrin α1β1 in vitro and angiogenesis in vivo by preventing the participation of receptor in the mechanism of neovascularization (angiogenesis) that tumors require for obtaining nutrients, oxygen. needed for growth. Currently, the group led by Dr. J Calvete, which develops its research at the Institute of Biomedicine of Valencia, is generating conditional transgenic mouse models for the disintegrin jerdostatina with which to evaluate the antitumor effect of disintegrin in vivo . In addition, integrin α1β1 appears to play a role in biological processes such as psoriasis, asthma, allergies, organ rejection, and arterial restenosis after angioplasty. The effects of selective blockade of integrin α1β1 in these processes have not been studied in detail. This project intends to conduct such studies while they add new aspects on the study of conformational equilibrium in this type of membrane proteins. As mentioned above, the dynamic equilibrium of integrin receptor can be modulated by ligand binding (disintegrin) but also, as recently reported, the receptor interactions with cell signaling complexes can be modified by synthetic peptides.

Objectives

A.- Biology Chemistry protein-protein interactions in apoptosis.

A.1. Apoptosome. Characterization at the molecular level of the binding site of the inhibitors to Apaf-1. Biophysical techniques for structural characterization. Characterization of the effects of selective inhibition of the apoptosome in cells. Proteomic analysis. Analysis of the molecular mechanism of mitochondrial protection by Apaf-1.
A.2. Study of the dynamic balance of membrane protein pro-and antiapoptotic family Bcl-2.

B.- nteraction integrin / disintegrin.
Development of conditional transgenic mouse models for the disintegrin jerdostatina with which to evaluate the antitumor effect of disintegrin in vivo. Design, synthesis and biological evaluation of synthetic peptides that modulate the interaction between integrin and disintegrin jerdostatina 11. Establishment of the crystal structure of complex 11-jerdostatina.