Progettazione e sintesi di nuovi inibitori della proteina Racl e loro valutazione farmacologica nell'ambito della terapia cardiovascolare

Cardiovascular disease is a case-in-point of multifactorial pathology. Its occurrence is linked to genetic background, which means the involvement of a large number of genes coding for enzymes, receptors and carrier proteins of lipid metabolism, as well as clotting factors and adhesion molecules. Several aspects of the ‘life style', chiefly among them diet and exercise, as well as the interactions with further pathological conditions such as hypertension and diabetes mellitus also play a role. Due to its complexity, knowledge about cardiovascular disease is still incomplete, in spite of longstanding commitment in research and in spite of many if partial successful achievements.

The highest death toll from cardiovascular disease dates back to the early sixties, but after four decades this disease is still the most frequent cause of mortality and disability in industrialized countries and is becoming more and more commonplace in emerging countries.

The above considerations imply investigation should be continued while looking for breakthroughs both in basic and applied research. Specifically, it appears a must to make use of all novel biological insights in order to possibly develop new therapeutic approaches.

Along this line, Rho GTPases of the Ras superfamily are proteins involved in the regulation of multiple cell functions and have been implicated in the pathogenesis of cardiovascular diseases and cancer. The selective interaction of the different Rho GTPases with a variety of effectors determines the final outcome of their activation.

Several evidences have indicated that Rho proteins play a pivotal role in the pathogenesis of atherosclerosis, thus supporting Rho proteins as potential pharmacological targets for cardiovascular diseases. We have recently demonstrated that the activation of Rac1, but not RhoA, in human aortic smooth muscle cells induces the expression of matrix metalloproteinase 1 (MMP1) and MMP2, an event that may contribute to atherosclerotic plaque rupture. Emerging evidences are also pointing out the pivotal role of Rac1 in many other aspects of atherosclerotic plaque development, such as cell proliferation, endothelial cells permeability, inflammatory response, generation of reactive oxygen species, and regulation of nitric oxide synthase. Thus, a pharmacological inhibition of Rac1 may represent an important pharmacological tool to better understand the role of this protein in atherogenesis.

Recently, starting from the structure-function information on Rac1 and by using a computer based virtual screening of the National Cancer Institute database, Zheng et al have identified compound NSC23766 as a specific inhibitor of Rac1 activation. This compound is the only specific Rac1 inhibitor currently reported by the literature, but its use as a drug for the therapy of atherosclerosis may be prevented by insufficient potency. For those reasons, we recently undertook a study aiming to the identification of new Rac1 inhibitors through a virtual screening strategy. The study has been successful, and 5 new and rather potent inhibitors were identified.

However, those 5 hits need to be optimized in order to obtain a true lead compound, and this is the principal aim of this research project. Indeed, starting from those 5 hits, a similarity search will be performed aiming to identify new derivatives to be biologically tested, in order to dispose an adequate set for a Structure Activity Relationship (SAR) analysis and consequent hit-to-lead optimization.

The primary biological data will allow the derivation of a SAR model and the classification of the selected hits within a specific inhibitor class, characterized by a common scaffold and a certain range of substituents able to modulate the activity. Those information will be useful for a classical hit-to-lead optimization, but will also be used to derive specific Quantitative SAR (QSAR) models to be used for further ligand optimization, by themselves or in combination with molecular dockings and molecular dynamic simulations through a structure based ligand optimization approach.

In order to derive a predictive QSAR model that will be useful in future screening experiments, the activity values obtained from biological and pharmacological assays will be analysed using a tridimensional QSAR strategy. The 3D-QSAR model will be used to predict the activity of virtual compound libraries, automatically generated by varying the substitution pattern of the selected scaffolds. The compounds herein predicted as the most actives will be further optimized through a structure based procedure, such as an accurate molecular dockings, followed by molecular dynamic simulations and MM-PBSA calculations in order to obtain accurate information on the binding mode of the selected candidates. Those information will be used together with the 3D-QSAR models to guide the synthesis of a second generation of optimized Rac1 inhibitors. The synthesized compounds will be subjected to several in vitro biological assays in order to obtain a complete information about the pharmacological and toxicological profiles of the new compounds. Finally, the best drug candidates selected by in vitro testing will be subjected to an in vivo study by evaluating the potential antiatherosclerotic activity in a specific animal model, thus obtaining a rather complete description of the potentiality of the new compounds to be used for the treatment of atherosclerosis.