title: Automated NMR fragment based screening identified a novel interface blocker to the LARG/RhoA complex.
abstract: The small GTPase cycles between the inactive GDP form and the activated GTP form, catalyzed by the upstream guanine exchange factors. The modulation of such process by small molecules has been proven to be a fruitful route for therapeutic intervention to prevent the over-activation of the small GTPase. The fragment based approach emerging in the past decade has demonstrated its paramount potential in the discovery of inhibitors targeting such novel and challenging protein-protein interactions. The details regarding the procedure of NMR fragment screening from scratch have been rarely disclosed comprehensively, thus restricts its wider applications. To achieve a consistent screening applicable to a number of targets, we developed a highly automated protocol to cover every aspect of NMR fragment screening as possible, including the construction of small but diverse libray, determination of the aqueous solubility by NMR, grouping compounds with mutual dispersity to a cocktail, and the automated processing and visualization of the ligand based screening spectra. We exemplified our streamlined screening in RhoA alone and the complex of the small GTPase RhoA and its upstream guanine exchange factor LARG. Two hits were confirmed from the primary screening in cocktail and secondary screening over individual hits for LARG/RhoA complex, while one of them was also identified from the screening for RhoA alone. HSQC titration of the two hits over RhoA and LARG alone, respectively, identified one compound binding to RhoA.GDP at a 0.11 mM affinity, and perturbed the residues at the switch II region of RhoA. This hit blocked the formation of the LARG/RhoA complex, validated by the native gel electrophoresis, and the titration of RhoA to ¹⁵N labeled LARG in the absence and presence the compound, respectively. It therefore provides us a starting point toward a more potent inhibitor to RhoA activation catalyzed by LARG.
authors: Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Science, University of Science and Technology of China, Hefei, Anhui, China.
authors: Jia
authors: J
authors: Gao
authors: Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Science, University of Science and Technology of China, Hefei, Anhui, China.
authors: Rongsheng
authors: R
authors: Ma
authors: Pfizer Worldwide Research and Development, San Diego, California, United States of America.
authors: Wei
authors: W
authors: Wang
authors: Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Science, University of Science and Technology of China, Hefei, Anhui, China.
authors: Na
authors: N
authors: Wang
authors: Advanced Chemistry Development Inc., Toronto, Ontario, Canada.
authors: Ryan
authors: R
authors: Sasaki
authors: Advanced Chemistry Development Inc., Toronto, Ontario, Canada.
authors: David
authors: D
authors: Snyderman
authors: Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Science, University of Science and Technology of China, Hefei, Anhui, China.
authors: Jihui
authors: J
authors: Wu
authors: Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Science, University of Science and Technology of China, Hefei, Anhui, China.
authors: Ke
authors: K
authors: Ruan
mesh_terms: D004353:Drug Evaluation, Preclinical / Q000379:methods; D006801:Humans; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D060066:Protein Interaction Maps / Q000187:drug effects; D017434:Protein Structure, Tertiary; D064067:Rho Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D014867:Water / Q000737:chemistry; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: C577222:ARHGEF12 protein, human; D064067:Rho Guanine Nucleotide Exchange Factors; D014867:Water; D020742:rhoA GTP-Binding Protein