RESUMO
In the last years, the development of new drugs in oncology has evolved notably. In particular, drug development has shifted from empirical screening of active cytotoxic compounds to molecularly targeted drugs blocking specific biologic pathways that drive cancer progression and metastasis. Using a rational design approach, our group has developed 1A-116 as a promising Rac1 inhibitor, with antitumoral and antimetastatic effects in several types of cancer. Rac1 is over activated in a wide range of tumor types and and it is one of the most studied proteins of the Rho GTPase family. Its role in actin cytoskeleton reorganization has effects on endocytosis, vesicular trafficking, cell cycle progression and cellular migration. In this context, the regulatory activity of Rac1 affects several key processes in the course of the cancer including invasion and metastasis. The purpose of this preclinical study was to focus on the mode of action of 1A-116, conducting an interdisciplinary approach with in silico bioinformatics tools and in vitro assays. Here, we demonstrate that the tryptophan 56 residue is necessary for the inhibitory effects of 1A-116 since this compound interferes with protein-protein interactions (PPI) of Rac1GTPase involving several GEF activators. 1A-116 is also able to inhibit the oncogenic Rac1P29S mutant protein, one of the oncogenic drivers found in sun-exposed melanoma. It also inhibits numerous Rac1-regulated cellular processes such as membrane ruffling and lamellipodia formation. These results deepen our knowledge of 1A-116 inhibition of Rac1 and its biological impact on cancer progression. They also represent a good example of how in silico analyses represent a valuable approach for drug development.
RESUMO
Rock proteins are Rho GTPase-dependent serine/ threonine kinases with crucial roles in F-actin dynamics and cell transformation. By analogy with other protein kinase families, it can be assumed that Rock proteins act, at least in part, through the regulation of gene expression events. However, with the exception of some singular transcriptional targets recently identified, the actual impact of these kinases on the overall cell transcriptome remains unknown. To address this issue, we have used a microarray approach to compare the transcriptomes of exponentially growing NIH3T3 cells that had been untreated or treated with Y27632, a well known specific inhibitor for Rock kinase activity. We show here that the Rock pathway promotes a weak impact on the fibroblast transcriptome, since its inhibition only results in changes in the expression of 2.3% of all the genes surveyed in the microarrays. Most Y27632-dependent genes are downregulated at moderate levels, indicating that the Rock pathway predominantly induces the upregulation of transcriptionally active genes. Although functionally diverse, a common functional leitmotiv of Y27632-dependent genes is the implication of their protein products in cytoskeletal-dependent processes. Taken together, these results indicate that Rock proteins can modify cytoskeletal dynamics by acting at post-transcriptional and transcriptional levels. In addition, they suggest that the main target of these serine/threonine kinases is the phosphoproteome and not the transcriptome.