| |
| The small GTPase cycles between the inactive GDP form and the activated GTP form, catalyzed by the upstream guanine exchange factors. The small molecule probes to their complex have proven to be a fruitful route for therapeutic intervention to prevent the over-activation of the small GTPase. The discovery of protein-protein interaction inhibitors, however, remains experimentally challenging. We have developed a highly automated NMR fragment screen equipped with a 96 well autosampler and custom-made automation scripts, in order to search for the interface blockers to the small GTPase RhoA and its upstream LARG. Our fragment library was first filtered for drug-like physiochemical properties and aqueous solubility as measured by the automated quantitative NMR. The compounds which passed the solubility criteria were then grouped into 10 compounds per tube, with minimal aromatic peak overlapping for better deconvolution. This streamlined screen identified seven hits, which were then interrogated by a secondary screen for those hits individually. One hit had a 66??M affinity (determined by HSQC titration) to RhoA, and binds in a novel binding cavity adjacent to the switch II region of RhoA. This hit is indeed a blocker to the LARG/RhoA complex formation, thus a potential ligand lead to inhibit RhoA activation, with its activity cross-validated by native gel electrophoresis and the titration of RhoA titrated to 15N LARG in the absence and presence the compound, respectively. |
|
| Keywords:Chemical biology, NMR Fragment Based Screening; protein-protein interaction inhibitor; Leukemia Associated Rho Guanine Exchange Factor; Quantitative NMR; NMR Automation; |
|
