Dibenzophenanthridines as inhibitors of glutaminase C and cancer cell proliferation - PubMed (original) (raw)

Dibenzophenanthridines as inhibitors of glutaminase C and cancer cell proliferation

William P Katt et al. Mol Cancer Ther. 2012 Jun.

Abstract

One hallmark of cancer cells is their adaptation to rely upon an altered metabolic scheme that includes changes in the glycolytic pathway, known as the Warburg effect, and elevated glutamine metabolism. Glutaminase, a mitochondrial enzyme, plays a key role in the metabolism of glutamine in cancer cells, and its inhibition could significantly impact malignant transformation. The small molecule 968, a dibenzophenanthridine, was recently shown to inhibit recombinantly expressed glutaminase C, to block the proliferation and anchorage-independent colony formation of human cancer cells in culture, and to inhibit tumor formation in mouse xenograft models. Here, we examine the structure-activity relationship that leads to 968-based inhibition of glutaminase and cancer cell proliferation, focusing upon a "hot-spot" ring previously identified as critical to 968 activity. We find that the hot-spot ring must be substituted with a large, nonplanar functionality (e.g., a t-butyl group) to bestow activity to the series, leading us to a model whereby the molecule binds glutaminase at a previously undescribed allosteric site. We conduct docking studies to locate potential 968-binding sites and proceed to test a specific set of docking solutions via site-directed mutagenesis. We verify the results from our initial assay of 968 and its analogues by cellular studies using MDA-MB-231 breast cancer cells.

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Conflict of interest statement

Conflict of Interest: None.

Figures

Figure 1

Derivatives of 968 exhibit dose-dependent behavior when assaying recombinant GAC activity. This was the case even for the less potent compound 27. GAC (50 nM) was initially exposed to glutamine (21 mM) and inhibitor, followed by the addition of inorganic phosphate (136 mM). The solution was incubated for 10 minutes, and then glutamate turnover was measured. Curves were fit with a fixed slope inhibitor model in Sigma Plot. IC50 values were determined to be 9.3 μM (968), 7.6 μM (5), 15.1 μM (17) and 47.4 μM (27).

Figure 2

Structures of 968 and GAC. A, 968, energy minimized with the MMFF94 force field. Carbon atoms are shown in grey, hydrogen in white, nitrogen in blue, oxygen in red, and bromine in darker red. The ‘hot-spot’ ring projects to the upper right. B, The x-ray crystal structure for the human GAC dimer (3CZD). Glutamine-binding pockets are highlighted in blue. The proposed 968-binding pocket is highlighted in red. The N- and C-termini are labeled as indicated. C, Docked pose of 968 (blue) in the concave surface region formed at the dimerization interface of two GAC monomers (green and brown). This image is a 90° rotation of B, along the horizontal axis. D, An interaction diagram, showing close contacts between the interface formed by two GAC monomers (denoted A and B) and 968, as predicted by docking.

Figure 3

Inhibitor effects upon MDA-MB-231 cell proliferation. MDA-MB-231 cells were treated with DMSO (negative control) or the indicated compound (10 μM) over 6 days.

Figure 4

Inhibition data for representative compounds tested vs. MDA-MB-231 cells (10 μM compound, blue) and vs. recombinant GAC (25 μM compound, red) via the protocols described. Briefly, MDA-MB-231 cells were treated with DMSO or the indicated compound (10 μM) over 6 days. Reported inhibition was calculated based on cells counted on day 6, relative to a DMSO control run in that time course. Recombinant GAC (50 nM) was exposed to glutamine (21 mM) and inhibitor, followed by the addition of inorganic phosphate (136 mM). The solution was incubated for 10 minutes, at which time glutamate turnover was measured.

Figure 5

Proposed model of GAC activity, showing GAC monomers (green squares) in different configurations. From an inactive conformation (upper left), 968 can be added to form an inhibited complex which cannot be significantly activated via addition of inorganic phosphate (lower left). Inorganic phosphate can be added to the inactive protein to achieve an active tetrameric complex (upper right). From this state, addition of 968 does not affect inhibition (lower right).

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Dibenzophenanthridines as inhibitors of glutaminase C and cancer cell proliferation - PubMed (original) (raw)