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.

Figure 1. Identification of CID 1067700 as a pan GTPase inhibitor using high-throughput screening (HTS) on small GTPases

(a) Schematic diagram of the bead based assay used to measure BODIPY-linked guanosine triphosphate (GTP) binding by flow cytometry to glutathione-S-transferase (GST)–GTPase chimeras immobilized on GSH-beads. For HTS, beads of varying red fluorescence intensities were used as identifiers for individual protein-conjugated bead sets. The assay is sensitive to both increases and decreases in bead associated fluorescence and was used to identify both activators and inhibitors in a single screen. (b) Chemical structure of CID 1067700 an inhibitor of nucleotide binding with (c) Activity against multiple GTPases measured as residual nucleotide binding activity (BODIPY-GTP, 100 nM) in the presence of increasing concentrations of compound. (d) Nanomolar concentrations of CID 1067700 inhibit Rab7wt protein nucleotide binding; BODIPY-GTP (1 nM, filled squares) and BODIPY-GDP (0.4 nM, filled triangles). (e) Unlabeled nucleotides (GTP, GDP and GTP-γ-S) effectively competes BODIPY-GTP (100 nM) binding.

Figure 2. CID 1067700 competitively inhibits BODIPY-linked nucleotide binding by wildtype and mutant forms of Rab7

(a–b) CID 1067700 (100 nM) does not alter Rab7wt B_max for BODIPY-GTP, but does alter apparent EC₅₀ for BODIPY-GTP (filled squares); observed as a rightward shift of log plot of BODIPY-GTP binding by Rab7wt. (c–d) CID 1067700 (200 nM) does not alter Rab7wt B_max for BODIPY-GDP; but does alter apparent EC₅₀ for BODIPY-GDP (filled triangles); observed as rightward shift of log plot of BODIPY-GDP binding by Rab7wt. (e–f) CID 1067700 (100 nM) does not alter constitutively active Rab7Q67L mutant B_max for BODIPY-GTP, but does alter apparent EC₅₀ for BODIPY-GTP (filled squares); observed as a rightward shift of log plot of BODIPY-GTP binding by Rab7Q67L. (g–h) CID 1067700 (200 nM) does not alter Rab7T22N B_max for BODIPY-GDP; but does alter apparent EC₅₀ for BODIPY-GDP (filled triangles); observed as rightward shift of log plot of BODIPY-GDP binding by Rab7T22N. In all experiments, equilibrium binding reactions performed in 1% DMSO (final) served as the controls (open circles) and fluorescence attributed to non-specific nucleotide binding subtracted accordingly.

Figure 3. CID 1067700 has no effect on the rate of release of bound BODIPY-linked nucleotide by wild type Rab7 under equilibrium binding conditions

(a) Rab7 was pre-incubated with BODIPY-GTP (100 nM) for 2 h 15 min at 4°C, conditions that allow nucleotide binding to equilibrium. Dissociation assays were initiated by dilution +/− the addition of either CID 1067700 (10 μM) or unlabeled GDP (10 μM) and decrease in fluorescence due to nucleotide dissociation was measured in real time. (b) Two-phase exponential analysis of a, normalized by division of dilution only values (c). Rab7 was preincubated with BODIPY-GDP (40 nM) for 2 h 15 min at 4°C as for (a). Dissociation assays were initiated by dilution +/− CID 1067700 (10 μM) or unlabeled GDP (10 μM) and decrease in fluorescence due to nucleotide dissociation was measured in real time (d) Single phase exponential analysis of c, normalized by division of dilution only values (e) CID 1067700 exhibits fast binding kinetics and is reversible. Equal concentrations (140 nM) of Rab7 and CID 1067700 used against 100 nM BODIPY-GTP. CID 1067700 pre-incubated with Rab7 and either not washed (closed boxes) or washed (open blue triangles) and (f). Rab7 specifically binds guanine nucleotide moiety on BODIPY-GTP (100 nM, blue open boxes) without additional interaction with the BODIPY fluorescent moeity. 100 nM BODIPY dye was used in this control experiment and exhibited only background binding.

Figure 4. CID 1067700 docks optimally in the nucleotide binding pocket of Rab7 in the GTP-bound conformation

CID 1067700 docked in the nucleotide binding site of Rab7 in the (a–b) GDP-bound (PDB 1VG9) and (c–d) (GNP)- bound (PDB 1VG8) conformations. Molecular docking carried out using Fred docking software. Both GTP or GDP and CID 1067700 are shown simultaneously docked in the pocket for purposes of comparing their orientations in the pocket. Interaction maps for (e–f) Rab7-GDP vs. CID 1067700 and (g–h) Rab7-GNP vs. CID 1067700 illustrate differences in number and sites of interaction.

Figure 5. Structure activity relationships identify importance of linker and R-groups in inhibitory activity of CID 1067700

Blue and red dotted lines represent inhibitory dose response curves of the parent CID 1067700 compound against BODIPY-GTP (1 nM) and BODIPY-GDP (0.4 nM), respectively. (a) Nanomolar concentrations of CID 46916263 derivative with only single methyl replacement on the pyran group and intact carbamothioylamino linker inhibits Rab7wt protein nucleotide binding; BODIPY-GTP (1 nM, filled squares) and BODIPY-GDP (0.40 nM, filled triangles). (b) Nanomolar concentrations of CID 46916265 derivative with only alteration of the thiourea moiety of the carbamothioylamino linker inhibits Rab7wt protein nucleotide binding; BODIPY-GTP (1nM, filled squares) and BODIPY-GDP (0.4 nM, filled triangles) and (c) Nanomolar concentrations of CID 1251121 derivative with alteration of the carboxylic acid group does not inhibit nucleotide binding by Rab7wt protein.

Figure 6. General scheme used for synthesis of CID 1067700 analogs

Reagents and conditions: a) morpholine sulfur, EtOH, 50°C; b) PhCONCS or PhCONCO, THF, 50°C; c) TFA, CH₂Cl₂, rt.