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
SN - 1386-2073
VL - 18
SP - 3
EP - 17
JO - Combinatorial Chemistry and High Throughput Screening
JF - Combinatorial Chemistry and High Throughput Screening
IS - 1
ER -
