A Competitive Nucleotide Binding Inhibitor: In Vitro Characterization of Rab7 GTPase Inhibition

Abstract

Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (Ki) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase as well as serving as a model for other small molecular weight GTPase inhibitors.

Ethanol exposure inhibits hepatocyte lipophagy by inactivating the small guanosine triphosphatase Rab7

“…Briefly, hepatocytes suspended in Williams’ E media were seeded onto collagen‐coated six‐well plates with or without coverslips. After 2 hours at 37°C, cells were washed with phosphate‐buffered saline (PBS) and incubated for 4 hours in either Williams’ E media with 5% fetal bovine serum or in the nutrient‐free buffer Krebs‐Ringer‐4‐(2‐hydroxyethyl)‐1‐piperazine ethanesulfonic acid (HEPES) (KRH) either with or without 80 µM CID1067700 (Rab7‐specific inhibitor) 20, 27. Following the incubation period, cells were collected and the pellets reconstituted in PBS.…”

Direct Modulation of Small GTPase Activity and Function

“…However,s everal high-throughput screens (HTSs) identified compounds interfering with GTPase nucleotide binding in vitro. [49][50][51] By using ab ead-based flow cytometry assay [52] to test 300 000 compounds,CID1067700 (1)was identified as adirect competitor for nucleotide binding (Figure 3a). [49] This compound impairs nucleotide binding by several small GTPases with low nanomolar inhibitory constants (K i = 12.9-19.7 nm) in vitro.I na ni dentical setup,C ID29950007 (ML141, 2)w as identified as aC dc42-selective noncompetitive allosteric inhibitor (Figure 3a).…”

“…[49][50][51] By using ab ead-based flow cytometry assay [52] to test 300 000 compounds,CID1067700 (1)was identified as adirect competitor for nucleotide binding (Figure 3a). [49] This compound impairs nucleotide binding by several small GTPases with low nanomolar inhibitory constants (K i = 12.9-19.7 nm) in vitro.I na ni dentical setup,C ID29950007 (ML141, 2)w as identified as aC dc42-selective noncompetitive allosteric inhibitor (Figure 3a). [50] In this case,t he binding of 2 locks Cdc42 in an inactive conformation, thereby inducing nucleotide release and inhibition of Cdc42-mediated cellular functions such as filopodia formation or cell migration (Figure 3b).…”

Tiyoüreler, açiltiyoüreler ve 4-tiyazolidinonların sentezi, karakterizasyonu ve antikanser ve antiviral etkilerinin değerlendirilmesi

“…Acyl thiourea compounds, cambinol and tenovin-1 are small molecule inhibitors of the NAD + -dependent family of protein deacetylases that is one of the targets in the treatment of cancer and neurodegenerative diseases (13,14). In the other research, CID 1067700 carrying acyl thiourea structure has been reported as an inhibitor of nucleotide binding by Ras-related GTPases (15). In addition, N-substituted phenylthioureas have been found that have antitumoral effects against skin cancer (16,17).…”