TY - JOUR

T1 - Discovery of MINC1, a GTPase-activating protein small molecule inhibitor, targeting MgcRacGAP

AU - van Adrichem, Arjan J

AU - Fagerholm, Annika

AU - Turunen, Laura

AU - Lehto, Anna

AU - Saarela, Jani

AU - Koskinen, Ari

AU - Repasky, Gretchen A

AU - Wennerberg, Krister

PY - 2015

Y1 - 2015

N2 - 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.

AB - 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.

KW - Animals

KW - Cell Cycle/drug effects

KW - Cell Line

KW - Cell Movement/drug effects

KW - Drug Discovery

KW - Enzyme Inhibitors/chemistry

KW - GTPase-Activating Proteins/antagonists & inhibitors

KW - HeLa Cells

KW - High-Throughput Screening Assays

KW - Humans

KW - Small Molecule Libraries/chemistry

M3 - Journal article

C2 - 25479424

VL - 18

SP - 3

EP - 17

JO - Combinatorial Chemistry & High Throughput Screening

JF - Combinatorial Chemistry & High Throughput Screening

SN - 1386-2073

IS - 1

ER -