Cell experiment:

To treat human and mouse B cells in vitro with the Rab7 inhibitor, CID-1067700 is diluted in DMSO and added to cell cultures to the final concentration of 40 μM. CID-1067700 or DMSO is added either at the time when B cell stimulation started, or 66 h after B cells are stimulated with LPS plus IL-4, TGF-β, anti-δ/dex and RA, for analysis of plasma cell survival[2].

Animal experiment:

Mice[2]For in vivo treatment, CID-1067700 dissolved in DMSO (stock concentration 40 mM, 16 mg/mL) is diluted with the solvent to the final volume of 50 μL and injected intraperitoneally (i.p.) once per week at the dose of 16 mg/kg body weight. C57, MRL/Faslpr/lpr and C57/Sle1Sle2Sle3 mice injected i. p. with the vehicle DMSO (50 μL). For survival studies and skin lesion analyses, MRL/Faslpr/lpr mice are treated with nil or CID-1067700 for 10 weeks and maintained until moribund (e.g., showing signs of severe loss of mobility, hunched back, piloerection, ruffled fur, dyspnea, gasping and weight loss), at which point they are euthanized[2].

CID-1067700 is the first competitive inhibitor of nucleotide binding by Ras-related GTPases [1].

The Ras superfamily of GTPases, which includes Arf, Rho, Ras and Rab GTPase subfamilies, regulate many cellular processes ranging from membrane trafficking to the control of cell proliferation. Alteration of small GTPase functions is a hallmark of genetic and sporadic human diseases, making GTPase family members attractive targets [1].

CID-1067700 is a competitive inhibitor of nucleotide binding by Ras-related GTPases. CID-1067700 exhibited EC50 values of 20-500 nM and at least 40% inhibitory activity against all tested GTPases. CID-1067700 significantly inhibited Rab GTPases (Rab2 and Rab7). CID 1067700 inhibited Rab7 binding of the BODIPY-linked nucleotides with EC50 values and Ki values of 11.22 ± 1.34 nM and 12.89 nM for BODIPY-GTP, and 20.96 ± 1.34 nM and 19.70 nM for BODIPY-GDP, respectively. The maximal inhibitory response was ≥97% for both nucleotides [1].

Reference:
[1]. Agola JO, Hong L, Surviladze Z, et al. A competitive nucleotide binding inhibitor: in vitro characterization of Rab7 GTPase inhibition. ACS Chem Biol. 2012 Jun 15;7(6):1095-108.