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1. Introduction and Background Rho GTPases are a family of small GTP-binding proteins functionally and structurally related to Ras protein. They act as molecular switches cycling between inactive GDP-bound and active GTP-bound forms. The most representative members of this family are Rho, Rac and Cdc24 which in an active form can interact with specific effectors that mediate their effects on the cell1. Over the last years many information about the participation of these Rho GTPases in cellular processes associated to malignant transformation has been accumulated. They play an important role controlling cell cytoskeleton organization, migration, transcription, and proliferation which makes them one of the most important molecular target towards the development of new generation oncological drugs. Particularly, Rac 1, is one of the most studied proteins of this family. It is over expressed and rarely mutated in many kinds of tumors2,3. Like the other members of the Rho GTPases family, Rac1 activation is mediated through its interaction with Guanine Nucleotide Exchange Factors (GEFs) and It was proposed that the tryptophan 56 is essential for this interaction4. In this work we present the identification of a Hit structure through docking based virtual screening targeting Rac1/GEFs interaction, the rational design and synthesis of novel analogues with potential antitumor activity and the selection of a new lead compound based on in-vitro and in-vivo biological essays for further investigation. Synthesis and development of novel small molecules targeting Rac1 signaling as new potential leads for antitumor agentsCiarlantini, M.1; Gonzalez, N2; Cardama, G. A.2; Defelipe, L.3; Turjanski, A.;3 Alonso, D. F.2; Gomez, D. E.2; Lorenzano Menna, P.2 and Comin, M. J.11Centro Investigación y Desarrollo en Química, Instituto Nacional de Tecnología Industrial, Buenos Aires, Argentina; 2Laboratorio de Oncología Molecular, Universidad Nacional de Quilmes, Buenos Aires, Argentina; 3INQUIMAE-CONICET, FCEyN, Universidad de Buenos Aires, Buenos Aires, Argentina. Docking based Virtual Screening Virtual screening is a computational technique widely used to predict the affinity of a large database of compounds to a particular target and select the most promising ones. With the aim to identify compounds with high affinity and specificity for Rac1 activation site, a docking-based virtual screening was conducted. Using the crystalline structure of Rac1 (1,38 Å, PDB ID 1MH1), more than 200.000 drug-like compounds obtained from ZINC database5 were challenged to interact with the surface containing Trp56. After visual inspection of the top 100 scored compounds, a group of 12 selected candidates were acquired from commercial sources and tested in-vitro. From this small set of chemicals with high docking affinities the compound ZINC69391 showed significant antiproliferative effect on different cancer cell lines. Therefore, it was selected as the leader compound and a detailed preclinical characterization was carried on. Synthesis and evaluation of novel analogues A B C A-ring B-ring Ser71 % Control Scheme 2. Synthetic pathways developed towards ZINC69391 analogues and the first group of synthesized analogues. Docking of 1A-116 predicted a higher affinity than other analogues for Rac1 activation site, it also showed an important antiproliferative effect on F3II, GL26, LN229 and MCF7 cells. For these reasons, it was selected as the lead compound to carry on with the investigation. This new lead compound showed in-vitro a 10-fold reduction of IC50 compared to ZINC69391, and antimetastatic effect in-vivo with an 8 times lower daily dose than the leader. (Figure 2). Trp56 D Asp57 A B 3 mg/day 25 mg/day Figure 1. A. Docking of ZINC69391 with Rac1 activation site. It presents an aromatic pi interaction between pyrimidine moisture and Trp56 , and two double hydrogen bonds from guanidine moisture to Asp57 and Ser71. B. Immunoprecipitation essay shows inhibition of Rac1-Tiam1 GEF interaction. C. Antiproliferative effect of ZINC69391 on different cancer cell lines. D. Antimetastatic effect on in-vivo syngenic model, ** means P< 0.01 for mann-whitney test. Design of ZINC69391 derivatives Employing ZINC69391 as lead structure, a series of disubstituted guanidines with structural variations of the aromatic rings were synthesized. For that purpose, the molecule was divided in three main portions and the retrosynthetic analysis led to two different cyanamide synthons for each ring variation that could be prepared from accessible starting materials as aniline, cyanoguanidine and acetylacetone. Figure 2. A. Antiproliferative effect of 1A-116 on F3II cells compared to ZINC69391, IC50 is 10 times lower. B. Comparative antimetastatic effect on animal syngenic model. Conclusion ZINC69391, a novel Rac1 inhibitor was identified by docking-based virtual library screening. This compound affected Rac1 activation in-vitro and in-vivo. We further designed and synthesized 1A-116, a more potent Rac1 inhibitor analogue that showed an enhanced antiproliferative activity and a similar in-vivo response as its parental compound, using a significantly lower dose. Due to the importance of Rac1 activity to cancer cell migration and invasion, the novel inhibitors presented here are promising candidates for further development. We are currently working on the synthesis of new promising analogues with higher predicted docking affinities than the new lead compound. Concentration (µM) Acknowledgments This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Quilmes, Agencia de Promoción Científica y Tecnológica and INTI. • References • 1- Etienne-Manneville, S.; Hall, A. Nature, 2002, 420(6916), 629. • 2- Fritz, G.; Just, I.; Kaina, B. Int J Cancer, 1999, 81(5), 682. • 3- Fritz, G.; Brachetti, C.; Bahlmann, F.; Schmidt, M.; Kaina, B. Br J Cancer, 2002, 87(6), 635 • 4- Wu, X.; Ramachandran, S.; Lin, M.C.; Cerione, R.A.; Erickson, J.W. Biochemistry.2011, 50(6), 1070 • 5- Irwin, J.J. & Shoichet, B.K. J ChemInf Model. 2005, 45(1), 177 Scheme 1. Retrosynthetic analysis for the preparation of A-ring and B-ring analogues of ZINC69391 lead compound from accesible commercially available materials.