Abstract
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.
Highlights
Rho GTPases are molecular switches that cycle between two conformational states: an inactive GDP-bound form and an active GTP-bound form
We showed that 1A-116 was able to inhibit related C3 botulinum toxin substrate 1 (Rac1)-guanine nucleotide exchange factors (GEFs) interactions reducing Rac1 activation levels and showing anti-proliferative effects on different cancer cell lines (Figure 1B; Cardama et al, 2014a,b; Cabrera et al, 2017; Gonzalez et al, 2017) but not in COS-1 cells used in the luciferase assays (Figure 1C)
To identify which interactions between Rac1 and different GEFs were inhibited by 1A-116, we evaluated the effect of the 1A116 inhibitor in the serum response element (SRE) activity elicited by a number of activated versions of Rac1 GEFs when ectopically expressed in COS1 cells
Summary
Rho GTPases are molecular switches that cycle between two conformational states: an inactive GDP-bound form and an active GTP-bound form. This cycle is highly regulated by guanine nucleotide exchange factors (GEFs), which catalyze nucleotide exchange and mediate Rho GTPase activation, and GTPase-activating proteins (GAPs), which stimulate GTP hydrolysis to return the Pharmacodynamics of 1A116 Rac Inhibitor. Rho GTPases are readily activated by different stimuli that activate a wide variety of cell-surface receptors, including receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs), cytokine receptors, integrins and cadherins (Bustelo, 2018). These stimulated receptors promote the exchange of GDP for GTP on Rho proteins, mainly by GEF activation. Some of the most well described GEFs include Tiam, Dbl, Vav family, P-Rex, Dock-180 (Vigil et al, 2010)
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