Kinase experiment:

The given cells are lysed with reporter lysis buffer and subjected to luciferase activity assay using luciferase assay system in a luminometer. Relative luciferase activity is normalized to protein content[2].

Cell experiment:

Cells are seeded in 96-well cell culture plates and treated the next day with the agents (including GGTI298 Trifluoroacetate). The viable cell number is determined using the sulforhodamine B assay[2].

Animal experiment:

The ileal loop experiment is performed in 6-8-week-old mice by a modifing rabbit ileal loop assay. Following gut sterilization, the animals are kept fasted for 24 h prior to surgery and fed only water ad libitum. Anesthesia is induced by a mixture of ketamine (35 mg/kg of body weight) and xylazine (5 mg/kg of body weight). A laparotomy is performed, and the experimental loops of 5-cm length are constricted at the terminal ileum by tying with non-absorbable silk. The following fluids are instilled in each loop by means of a tuberculin syringe fitting with a disposable needle through the ligated end of the loop: pure CT (1 μg; positive control), saline (negative control), CT (1 μg)+TRAM-34 (different concentrations in μM), CT (1 μg)+ H1152 (1 μM), and CT (1 μg)+GGTI298 Trifluoroacetate (different concentrations in μM), a specific inhibitor of Rap1A. The intestine is returned to the peritoneum, and the mice are sutured and returned to their cages. After 6 h, these animals are sacrificed by cervical dislocation, and the loops are excised[1].

GGTI298 Trifluoroacetate is a CAAZ peptidomimetic geranylgeranyltransferase I (GGTase I) inhibitor, which can inhibit Rap1A with IC50 of 3 uM; little effect on Ha-Ras with IC50 of >20 uM.

RhoA inhibitor (GGTI298 Trifluoroacetate) significantly reduces cAMP agonist-stimulated apical K+ conductance[1]. Knockdown of DR4 abolishes NF-κB activation, leading to sensitization of DR5-dependent apoptosis induced by the combination of GGTI298 Trifluoroacetate and TRAIL. GGTI298 Trifluoroacetate/TRAIL activates NF-κB and inhibits Akt. Knockdown of DR5, prevents GGTI298/TRAIL-induced IκBα and p-Akt reduction, suggesting that DR5 mediates reduction of IκBα and p-Akt induced by GGTI298/TRAIL. In contrast, DR4 knockdown further facilitates GGTI298/TRAIL-induced p-Akt reduction[2].

The vivo mouse ileal loop experiments show fluid accumulation is reduced in a dose-dependent manner by TRAM-34, GGTI298 Trifluoroacetate, or H1152 when inject together with cholera toxin into the loop[1].

References:
[1]. Sheikh IA, et al. The Epac1 signaling pathway regulates Cl- secretion via modulation of apical KCNN4c channels in diarrhea. J Biol Chem. 2013 Jul 12;288(28):20404-15.
[2]. Chen S, et al. Dissecting the roles of DR4, DR5 and c-FLIP in the regulation of geranylgeranyltransferase I inhibition-mediated augmentation of TRAIL-induced apoptosis. Mol Cancer. 2010 Jan 29;9:23.
[3]. McGuire TF, et al. Platelet-derived growth factor receptor tyrosine phosphorylation requires protein geranylgeranylation but not farnesylation. J Biol Chem. 1996 Nov 1;271(44):27402-7.