Send Orders for Reprints to reprints@benthamscience.ae Combinatorial Chemistry & High Throughput Screening, 2015, 18, 3-17 3 1875-5402/15 $58.00+.00 © 2015 Bentham Science Publishers Discovery of MINC1, a GTPase-Activating Protein Small Molecule Inhibitor, Targeting MgcRacGAP Arjan J. van Adrichem 1 , Annika Fagerholm 2 , Laura Turunen 1 , Anna Lehto 1 , Jani Saarela 1 , Ari Koskinen 2 , Gretchen A. Repasky 1 and Krister Wennerberg *,1,3 1 Institute for Molecular Medicine Finland, University of Helsinki, Helsinki, Finland 2 Laboratory of Organic Chemistry, Department of Chemistry, School of Chemical Technology, Aalto University, Espoo, Finland 3 Drug Discovery Division, Southern Research Institute, Birmingham, AL, USA Abstract: The Rho family of Ras superfamily small GTPases regulates a broad range of biological processes such as migration, differentiation, cell growth and cell survival. Therefore, the availability of small molecule modulators as tool compounds could greatly enhance research on these proteins and their biological function. To this end, we designed a biochemical, high throughput screening assay with complementary follow-up assays to identify small molecule compounds inhibiting MgcRacGAP, a Rho family GTPase activating protein involved in cytokinesis and transcriptionally upregulated in many cancers. We first performed an in-house screen of 20,480 compounds, and later we tested the assay against 342,046 compounds from the NIH Molecular Libraries Small Molecule Repository. Primary screening hit rates were about 1% with the majority of those affecting the primary readout, an enzyme-coupled GDP detection assay. After orthogonal and counter screens, we identified two hits with high selectivity towards MgcRacGAP, compared with other RhoGAPs, and potencies in the low micromolar range. The most promising hit, termed MINC1, was then examined with cell-based testing where it was observed to induce an increased rate of cytokinetic failure and multinucleation in addition to other cell division defects, suggesting that it may act as an MgcRacGAP inhibitor also in cells. Keywords: Biochemical assays, cytokinesis, HTS, MgcRacGAP, Rac1, small molecule inhibitor. INTRODUCTION Rho GTPases are regulated molecular binary switches that cycle between active, GTP-loaded and inactive, GDP- loaded states. This protein family controls a wide array of cellular functions such as cell proliferation, apoptosis and cell morphology in health and disease [1, 2], and therefore are interesting targets for pharmacological inhibition. Small molecule modulators could have many advantages over conventional molecular biology tools such as mutant proteins and RNA interference (RNAi), which, although having provided global insight into cell biological function, seemingly led to controversial results in the Rho GTPase research field [3, 4]. Small molecule inhibitors can easily be applied in a dose-dependent, temporal or domain-specific manner, inhibiting one particular function, whereas RNAi methods completely remove the protein, disrupting possible protein-protein interactions that are mediated through domains other than the one of interest. Therefore, knockdown of proteins that have multiple functions often leads to different phenotypes observed, based on different levels of depletion. Introduction of mutant proteins might *Address correspondence to this author at the Institute for Molecular Medicine Finland FIMM, Nordic EMBL Partnership for Molecular Medicine, Biomedicum Helsinki 2U, P.O. Box 20 (Tukholmankatu 8), FI- 00014, University of Helsinki, Finland; Tel: +358 50 415 4900; E-mail: krister.wennerberg@fimm.fi specifically target a function of interest leaving the other functions untouched; however, controlled temporal regulation is not easily achieved and non-physiological concentrations of the mutant proteins can distort the delicate balance needed for normal cellular behavior. For the last couple of decades, protein kinases have been the most important target class in cancer drug discovery, and the majority of inhibitors achieve their activity by competing for binding with the phosphate donor substrate ATP. Like protein kinases, Rho GTPases are nucleotide-binding enzymes; therefore, it is conceivable that they could be inhibited by nucleotide-competitive small molecules. However, unlike protein kinases, which have moderate binding affinities toward ATP, Rho GTPases and most other small GTPases have very high affinity toward guanine nucleotides making them difficult to target with nucleotide- competitive small molecule inhibitors [5]. Furthermore, many of the Rho GTPases are involved in multiple cellular processes and general inhibition of a Rho GTPase may therefore have nonspecific effects. Instead, by targeting Rho protein regulators, the biological effect is expected to be more selective toward specific physiological and pathological processes. Illustrating this principle, for the 20 mammalian Rho GTPases there are more than 80 guanine nucleotide exchange factors (GEFs) that facilitate guanine nucleotide release and more than 60 GTPase activating proteins (GAPs) that stimulate the intrinsic GTPase activity [6, 7].