In vitro assays to characterize inhibitors of the activation of small G proteins by their guanine nucleotide exchange factors.

摘要: Abstract Guanine nucleotide exchange factors (GEFs) are essential regulators of the spatiotemporal conditions small GTP‐binding protein (SMG) activation. Their cellular activities combine biochemical stimulation GDP/GTP exchange, which leads to active conformation SMG, detection upstream signals and, in some cases, interaction with downstream effectors. Inhibition GEF by molecules has become recently a very field, both for understanding biology tools chemistry and because GEFs emerging as therapeutic targets. The natural compound brefeldin A (BFA) was first inhibitor be characterized, several inhibitors SMG activation have since been discovered using variety screening methods. An step toward their use basic research or therapeutics is characterization mechanism inhibition. function according multistep mechanism, involving transient ternary (nucleotide‐bound) binary (nucleotide‐free) intermediates. This thereby offers many opportunities blockage, but thorough analysis necessary define inhibition steps reaction that affected inhibitor. Here, based on case study how BFA inhibits Arf Sec7 domains, we describe flowchart assays decipher SMGs GEFs.