title: 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 (K(i)) 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.
authors.affiliation: Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.
authors.forename: Jacob O
authors.initials: JO
authors.lastname: Agola
authors.forename: Lin
authors.initials: L
authors.lastname: Hong
authors.forename: Zurab
authors.initials: Z
authors.lastname: Surviladze
authors.forename: Oleg
authors.initials: O
authors.lastname: Ursu
authors.forename: Anna
authors.initials: A
authors.lastname: Waller
authors.forename: J Jacob
authors.initials: JJ
authors.lastname: Strouse
authors.forename: Denise S
authors.initials: DS
authors.lastname: Simpson
authors.forename: Chad E
authors.initials: CE
authors.lastname: Schroeder
authors.forename: Tudor I
authors.initials: TI
authors.lastname: Oprea
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Golden
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Aubé
authors.forename: Tione
authors.initials: T
authors.lastname: Buranda
authors.forename: Larry A
authors.initials: LA
authors.lastname: Sklar
authors.forename: Angela
authors.initials: A
authors.lastname: Wandinger-Ness
mesh_terms: D001667:Binding, Competitive; D002264:Carboxylic Acids / Q000737:chemistry / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology; D020691:rab GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D002264:Carboxylic Acids; D004791:Enzyme Inhibitors; D009711:Nucleotides; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; D006160:Guanosine Triphosphate; D020691:rab GTP-Binding Proteins