Fig. 2.

KRA-533 directly binds to and activates KRAS in an in vitro cell-free system. (a) Thermal shift melting curve of purified KRAS proteins (WT, G12C, G12D or G13D) incubated with increasing concentrations of KRA-533. Melting temperature (Tm) values of DMSO Ctrl and 15 μM of KRA-533 are shown. *P < 0.05 by 2-tailed t test. (b) 600 nM Purified KRAS protein (WT, G12C, G12D or G13D) was incubated with 11 μM [γ-³⁵S] GTPγS, purified GFP (RASGRP1, 180 nM) and GAP (RASA1, 180 nM) at 25 °C in the presence or absence of increasing concentrations of KRA-533. The radioactivity remaining on the protein after intensive washing was quantified by liquid scintillation. Error bars represent ± SD. **P < 0.01 by 2-tailed t test