Characterization of the interactions of potent allosteric inhibitors with glutaminase C, a key enzyme in cancer cell glutamine metabolism

Abstract

Altered glycolytic flux in cancer cells (the "Warburg effect") causes their proliferation to rely upon elevated glutamine metabolism ("glutamine addiction"). This requirement is met by the overexpression of glutaminase C (GAC), which catalyzes the first step in glutamine metabolism and therefore represents a potential therapeutic target. The small molecule CB-839 was reported to be more potent than other allosteric GAC inhibitors, including the parent compound bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl (BPTES), and is in clinical trials. Recently, we described the synthesis of BPTES analogs having distinct saturated heterocyclic cores as a replacement for the flexible chain moiety, with improved microsomal stability relative to CB-839 and BPTES. Here, we show that one of these new compounds, UPGL00004, like CB-839, more potently inhibits the enzymatic activity of GAC, compared with BPTES. We also compare the abilities of UPGL00004, CB-839, and BPTES to directly bind to recombinant GAC and demonstrate that UPGL00004 has a similar binding affinity as CB-839 for GAC. We also show that UPGL00004 potently inhibits the growth of triple-negative breast cancer cells, as well as tumor growth when combined with the anti-vascular endothelial growth factor antibody bevacizumab. Finally, we compare the X-ray crystal structures for UPGL00004 and CB-839 bound to GAC, verifying that UPGL00004 occupies the same binding site as CB-839 or BPTES and that all three inhibitors regulate the enzymatic activity of GAC via a similar allosteric mechanism. These results provide insights regarding the potency of these inhibitors that will be useful in designing novel small-molecules that target a key enzyme in cancer cell metabolism.

Keywords: BPTES; CB-839; anticancer drug; cancer; crystallography; glutaminase; metabolism.

Figure 1.

Chemical structures and IC₅₀ values for inhibitors of GAC. *, IC₅₀ value reported by Shukla et al. (12); **, IC₅₀ value reported by Gross et al. (13); ***, IC₅₀ value determined here.

Figure 2.

Comparison of the effects of UPGL00004 and BPTES to inhibit and directly bind to recombinant GAC. A, BPTES (black circles) and UPGL00004 (white circles) were used to treat 50 nm recombinant GAC, and then enzymatic activity was assayed in the presence of inorganic phosphate, as described under “Experimental procedures.” B, changes in the fluorescence emission of the GAC(F327W) mutant provides a read-out for the direct binding of inhibitors to purified GAC. The addition of increasing concentrations of BPTES (black circles), CB-839 (gray triangles), and UPGL00004 (white circles) quenches the tryptophan fluorescence of 100 nm GAC(F327W). The fluorescence quenching curves were fit to a bimolecular interaction equation, giving K_D values of 70 ± 5, 26 ± 5, and 27 ± 2 nm, for BPTES, CB-839, and UPGL00004, respectively. Error bars for all panels represent the mean ± S.D. of three independent experiments.

Figure 3.

Comparison of the effects of UPGL00004, BPTES, and CB-839 on the growth of breast cancer cells. A, BPTES, CB-839, and UPGL00004 were added to MDA-MB-231 cells in the indicated amounts and their effects on cell proliferation were assayed. B, HS578T, MDA-MB-231, TSE, and MDA-MB-453 cells were treated in serum-free media for 14 h with BPTES, CB-839, or UPGL00004 (UPGL4). The media was then collected, and the amount of ammonia generated was determined for each sample. Error bars for panels A and B represent the mean ± S.D. of three independent experiments. C, plots showing mean tumor volumes (mm³) as a function of time for mice containing triple-negative HCI-002 grafts, which were treated with the different drug combinations. When tumors reached ∼3 mm in diameter, IP injections with vehicle alone (control) (black diamonds), bevacizumab (2.5 mg/kg weight of the animal) (white triangles), UPGL00004 (1 mg/kg weight of the animal) (white circles), or the two drugs together (gray squares), were injected every other day for 4 weeks. The tumor volumes for the bevacizumab plus UPGL00004 (n = 6), versus treatment with bevacizumab (n = 6), UPGL00004 (n = 6), or DMSO alone, were statistically significant. Error bars represent the S.E. for this experiment.

Figure 4.

X-ray crystal structures of UPGL00004 and CB-839 complexed to GAC. Shown are GAC crystallized with: A, UPGL00004 (PDB code 5WJ6); B, CB-839 (PDB code 5HL1); C, BPTES (PDB code 4JKT); and D, no drug (PDB code 5D3O). Each inhibitor is shown in an enlarged inset for A–C. B-factor coloring ranges from dark blue (low B factor, well resolved atoms) to bright red (high B factor, poorly resolved residues). The activation loop (indicated by the orange arrow for one subunit in each structure) generally has the highest B factors for the entire structure, although this decreases when an inhibitor is present. The location of the catalytic site for one monomer within each GAC complex is indicated with a red arrow.

Figure 5.

X-ray overlays of UPGL00004, BPTES, and CB-839. A, the X-ray structure for GAC complexed to UPGL00004 (yellow) overlaid with the structure (PDB code 4JKT) for GAC complexed to BPTES (blue). The important interacting residues from chain A (green) and chain D (light blue) are shown. B, the X-ray structure for GAC complexed to CB-839 (orange) overlaid with the structure (PDB code 4JKT) for GAC complexed to BPTES (blue). Critical interacting residues from chain A (green) and chain D (light blue) are shown.

Figure 6.

Hydrogen bonds formed between UPGL00004 and CB-839, BPTES, and GAC. Each ligand binds primarily to two chains (colored green and cyan if reported here, or magenta and peach if previously reported). Hydrogen bonds that fulfill the Berndt criteria are shown as dotted lines. Hydrogen bonds to Tyr-394 (A or B chain) are not shown for any molecule, as the bond is obstructed by the chosen pose. A, UPGL00004 (yellow) bound to GAC in crystal structure 5WJ6. B, CB-839 (orange) bound to the A and D chains of GAC in crystal structure 5HL1. C, CB-839 bound primarily to the B and C chains of GAC in crystal structure 5HL1. A single (D) chain residue, Arg-317, also provides a hydrogen bond to this ligand. The double-dashed line shows a putative hydrogen bond that may form if the pyridine ring rotates 180°. D, BPTES bound to GAC in crystal structure 4JKT. E, BPTES bound to GAC in crystal structure 3UO9.