Structures of the Rhodopsin-Transducin Complex: Insights into G-Protein Activation.

Rhodopsin (Rho), a prototypical G-protein-coupled receptor (GPCR) in vertebrate vision, activates the G-protein transducin (GT) by catalyzing GDP-GTP exchange on its α subunit (GαT). To elucidate the determinants of GT coupling and activation, we obtained cryo-EM structures of a fully functional, light-activated Rho-GT complex in the presence and absence of a G-protein-stabilizing nanobody. The structures illustrate how GT overcomes its low basal activity by engaging activated Rho in a conformation distinct from other GPCR-G-protein complexes. Moreover, the nanobody-free structures reveal native conformations of G-protein components and capture three distinct conformers showing the GαT helical domain (αHD) contacting the Gßγ subunits. These findings uncover the molecular underpinnings of G-protein activation by visual rhodopsin and shed new light on the role played by Gßγ during receptor-catalyzed nucleotide exchange.

SIRT5 stabilizes mitochondrial glutaminase and supports breast cancer tumorigenesis.

The mitochondrial enzyme glutaminase (GLS) is frequently up-regulated during tumorigenesis and is being evaluated as a target for cancer therapy. GLS catalyzes the hydrolysis of glutamine to glutamate, which then supplies diverse metabolic pathways with carbon and/or nitrogen. Here, we report that SIRT5, a mitochondrial NAD+-dependent lysine deacylase, plays a key role in stabilizing GLS. In transformed cells, SIRT5 regulates glutamine metabolism by desuccinylating GLS and thereby protecting it from ubiquitin-mediated degradation. Moreover, we show that SIRT5 is up-regulated during cellular transformation and supports proliferation and tumorigenesis. Elevated SIRT5 expression in human breast tumors correlates with poor patient prognosis. These findings reveal a mechanism for increasing GLS expression in cancer cells and establish a role for SIRT5 in metabolic reprogramming and mammary tumorigenesis.

The activation loop and substrate-binding cleft of glutaminase C are allosterically coupled.

The glutaminase C (GAC) isoform of mitochondrial glutaminase is overexpressed in many cancer cells and therefore represents a potential therapeutic target. Understanding the regulation of GAC activity has been guided by the development of spectroscopic approaches that measure glutaminase activity in real time. Previously, we engineered a GAC protein (GAC(F327W)) in which a tryptophan residue is substituted for phenylalanine in an activation loop to explore the role of this loop in enzyme activity. We showed that the fluorescence emission of Trp-327 is enhanced in response to activator binding, but quenched by inhibitors of the BPTES class that bind to the GAC tetramer and contact the activation loop, thereby constraining it in an inactive conformation. In the present work, we took advantage of a tryptophan substitution at position 471, proximal to the GAC catalytic site, to examine the conformational coupling between the activation loop and the substrate-binding cleft, separated by ∼16 Å. Comparison of glutamine binding in the presence or absence of the BPTES analog CB-839 revealed a reciprocal relationship between the constraints imposed on the activation loop position and the affinity of GAC for substrate. Binding of the inhibitor weakened the affinity of GAC for glutamine, whereas activating anions such as Pi increased this affinity. These results indicate that the conformations of the activation loop and the substrate-binding cleft in GAC are allosterically coupled and that this coupling determines substrate affinity and enzymatic activity and explains the activities of CB-839, which is currently in clinical trials.

Gain-of-function screen of α-transducin identifies an essential phenylalanine residue necessary for full effector activation.

Two regions on the α subunits of heterotrimeric GTP-binding proteins (G-proteins), the Switch II/α2 helix (which changes conformation upon GDP-GTP exchange) and the α3 helix, have been shown to contain the binding sites for their effector proteins. However, how the binding of Gα subunits to their effector proteins is translated into the stimulation of effector activity is still poorly understood. Here, we took advantage of a reconstituted rhodopsin-coupled phototransduction system to address this question and identified a distinct surface and an essential residue on the α subunit of the G-protein transducin (αT) that is necessary to fully activate its effector enzyme, the cGMP phosphodiesterase (PDE). We started with a chimeric G-protein α subunit (αT*) comprising residues mainly from αT and a short stretch of residues from the Gi1 α subunit (αi1), which only weakly stimulates PDE activity. We then reinstated the αT residues by systematically replacing the corresponding αi1 residues within αT* with the aim of fully restoring PDE stimulatory activity. These experiments revealed that the αG/α4 loop and a phenylalanine residue at position 283 are essential for conferring the αT* subunit with full PDE stimulatory capability. We further demonstrated that this same region and amino acid within the α subunit of the Gs protein (αs) are necessary for full adenylyl cyclase activation. These findings highlight the importance of the αG/α4 loop and of an essential phenylalanine residue within this region on Gα subunits αT and αs as being pivotal for their selective and optimal stimulation of effector activity.

Conformational changes in the activation loop of mitochondrial glutaminase C: A direct fluorescence readout that distinguishes the binding of allosteric inhibitors from activators.

The first step in glutamine catabolism is catalysis by the mitochondrial enzyme glutaminase, with a specific isoform, glutaminase C (GAC), being highly expressed in cancer cells. GAC activation requires the formation of homotetramers, promoted by anionic allosteric activators such as inorganic phosphate. This leads to the proper orientation of a flexible loop proximal to the dimer-dimer interface that is essential for catalysis (i.e. the "activation loop"). A major class of allosteric inhibitors of GAC, with the prototype being bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and the related molecule CB-839, binds to the activation loop and induces the formation of an inactive tetramer (two inhibitors bound per active tetramer). Here we describe a direct readout for monitoring the dynamics of the activation loop of GAC in response to these allosteric inhibitors, as well as allosteric activators, through the substitution of phenylalanine at position 327 with tryptophan (F327W). The tryptophan fluorescence of the GAC(F327W) mutant undergoes a marked quenching upon the binding of BPTES or CB-839, yielding titration profiles that make it possible to measure the binding affinities of these inhibitors for the enzyme. Allosteric activators like phosphate induce the opposite effect (i.e. fluorescence enhancement). These results describe direct readouts for the binding of the BPTES class of allosteric inhibitors as well as for inorganic phosphate and related activators of GAC, which should facilitate screening for additional modulators of this important metabolic enzyme.

Isolation and structure-function characterization of a signaling-active rhodopsin-G protein complex.

The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (GT). This results in the dissociation of GT into its component αT-GTP and ß1γ1 subunit complex. Structural information for the Rho*-GT complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (GT*) comprising a GαT/Gαi1 chimera (αT*) and ß1γ1 The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to GαT* is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one GT*. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the ß2-adrenergic receptor-GS complex, including a flexible αT* helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex.

Mechanism by which a recently discovered allosteric inhibitor blocks glutamine metabolism in transformed cells.

The mitochondrial enzyme glutaminase C (GAC) catalyzes the hydrolysis of glutamine to glutamate plus ammonia, a key step in the metabolism of glutamine by cancer cells. Recently, we discovered a class of allosteric inhibitors of GAC that inhibit cancer cell growth without affecting their normal cellular counterparts, with the lead compound being the bromo-benzophenanthridinone 968. Here, we take advantage of mouse embryonic fibroblasts transformed by oncogenic Dbl, which hyperactivates Rho GTPases, together with (13)C-labeled glutamine and stable-isotope tracing methods, to establish that 968 selectively blocks the enhancement in glutaminolysis necessary for satisfying the glutamine addiction of cancer cells. We then determine how 968 inhibits the catalytic activity of GAC. First, we developed a FRET assay to examine the effects of 968 on the ability of GAC to undergo the dimer-to-tetramer transition necessary for enzyme activation. We next demonstrate how the fluorescence of a reporter group attached to GAC provides a direct read-out of the binding of 968 and related compounds to the enzyme. By combining these fluorescence assays with newly developed GAC mutants trapped in either the monomeric or dimeric state, we show that 968 has the highest affinity for monomeric GAC and that the dose-dependent binding of 968 to GAC monomers directly matches its dose-dependent inhibition of enzyme activity and cellular transformation. Together, these findings highlight the requirement of tetramer formation as the mechanism of GAC activation and shed new light on how a distinct class of allosteric GAC inhibitors impacts the metabolic program of transformed cells.

Mechanistic Basis of Glutaminase Activation: A KEY ENZYME THAT PROMOTES GLUTAMINE METABOLISM IN CANCER CELLS.

Glutamine-derived carbon becomes available for anabolic biosynthesis in cancer cells via the hydrolysis of glutamine to glutamate, as catalyzed by GAC, a splice variant of kidney-type glutaminase (GLS). Thus, there is significant interest in understanding the regulation of GAC activity, with the suggestion being that higher order oligomerization is required for its activation. We used x-ray crystallography, together with site-directed mutagenesis, to determine the minimal enzymatic unit capable of robust catalytic activity. Mutagenesis of the helical interface between the two pairs of dimers comprising a GAC tetramer yielded a non-active, GAC dimer whose x-ray structure displays a stationary loop ("activation loop") essential for coupling the binding of allosteric activators like inorganic phosphate to catalytic activity. Further mutagenesis that removed constraints on the activation loop yielded a constitutively active dimer, providing clues regarding how the activation loop communicates with the active site, as well as with a peptide segment that serves as a "lid" to close off the active site following substrate binding. Our studies show that the formation of large GAC oligomers is not a pre-requisite for full enzymatic activity. They also offer a mechanism by which the binding of activators like inorganic phosphate enables the activation loop to communicate with the active site to ensure maximal rates of catalysis, and promotes the opening of the lid to achieve optimal product release. Moreover, these findings provide new insights into how other regulatory events might induce GAC activation within cancer cells.

Fluorination at the 4 position alters the substrate behavior of L-glutamine and L-glutamate: Implications for positron emission tomography of neoplasias.

Two 4-fluoro-L-glutamine diastereoisomers [(2S,4R)-4-FGln, (2S,4S)-4-FGln] were previously developed for positron emission tomography. Label uptake into two tumor cell types was greater with [18F](2S,4R)-4-FGln than with [18F](2S,4S)-4-FGln. In the present work we investigated the enzymology of two diastereoisomers of 4-FGln, two diastereoisomers of 4-fluoroglutamate (4-FGlu) (potential metabolites of the 4-FGln diastereoisomers) and another fluoro-derivative of L-glutamine [(2S,4S)-4-(3-fluoropropyl)glutamine (FP-Gln)]. The two 4-FGlu diastereoisomers were found to be moderate-to-good substrates relative to L-glutamate of glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase. Additionally, alanine aminotransferase was shown to catalyze an unusual γ-elimination reaction with both 4-FGlu diastereoisomers. Both 4-FGlu diastereoisomers were shown to be poor substrates, but strong inhibitors of glutamine synthetase. Both 4-FGln diastereoisomers were shown to be poor substrates compared to L-glutamine of glutamine transaminase L and α-aminoadipate aminotransferase. However, (2S,4R)-4-FGln was found to be a poor substrate of glutamine transaminase K, whereas (2S,4S)-4-FGln was shown to be an excellent substrate. By contrast, FP-Gln was found to be a poor substrate of all enzymes examined. Evidently, substitution of H in position 4 by F in L-glutamine/L-glutamate has moderate-to-profound effects on enzyme-catalyzed reactions. The present results: 1) show that 4-FGln and 4-FGlu diastereoisomers may be useful for studying active site topology of glutamate- and glutamine-utilizing enzymes; 2) provide a framework for understanding possible metabolic transformations in tumors of 18F-labeled (2S,4R)-4-FGln, (2S,4S)-4-FGln, (2S,4R)-4-FGlu or (2S,4S)-4-FGlu; and 3) show that [18F]FP-Gln is likely to be much less metabolically active in vivo than are the [18F]4-FGln diastereoisomers.

Prenylation and membrane localization of Cdc42 are essential for activation by DOCK7.

The unconventional guanine nucleotide exchange factor (GEF) family comprising 11 DOCK180 related proteins is classified into four subfamilies, A through D, based on their relative GEF activity toward the closely related Rac and Cdc42 GTPases. DOCK proteins participate in the remodeling of the actin cytoskeleton and are key regulators of cell motility, phagocytosis, and adhesion. Here we show that the guanine nucleotide exchange domain of DOCK7, DHR2 (for DOCK homology region 2), is a potent GEF for prenylated Cdc42 and Rac1 in a model liposome system, demonstrating that the prenylation and membrane localization of Cdc42 or Rac1 are necessary for their activation by DOCK7. Additionally, we identify DOCK7 residues that confer GTPase GEF specificity. Finally, using our liposome reconstitution assay, we show that a more narrowly defined GEF domain of DHR2 (designated DHR2s) harbors an N-terminal site distinct from the GEF active site that binds preferentially to the active, GTP-bound forms of Cdc42 and Rac1 and thereby recruits free DHR2s from solution to the membrane surface. This recruitment results in a progressive increase in the effective concentration of DHR2s at the membrane surface that in turn provides for an accelerated rate of guanine nucleotide exchange on Cdc42. The positive cooperativity observed in our reconstituted system suggests that the action of DOCK7 in vivo may involve the coordinated integration of Cdc42/Rac signaling in the context of the membrane recruitment of a DOCK7 GEF complex.

C-terminal di-arginine motif of Cdc42 protein is essential for binding to phosphatidylinositol 4,5-bisphosphate-containing membranes and inducing cellular transformation.

Rho GTPases regulate a diverse range of processes that are dependent on their proper cellular localization. The membrane localization of these GTPases is due in large part to their carboxyl-terminal geranylgeranyl moiety. In addition, most of the Rho family members contain a cluster of positively charged residues (i.e. a "polybasic domain"), directly preceding their geranylgeranyl moiety, and it has been suggested that this domain serves to fine-tune their localization among different cellular membrane sites. Here, we have taken a closer look at the role of the polybasic domain of Cdc42 in its ability to bind to membranes and induce the transformation of fibroblasts. A FRET assay for the binding of Cdc42 to liposomes of defined composition showed that Cdc42 associates more strongly with liposomes containing phosphatidylinositol 4,5-bisphosphate (PIP(2)) when compared either with uncharged control membranes or with liposomes containing a charge-equivalent amount of phosphatidylserine. The carboxyl-terminal di-arginine motif (Arg-186 and Arg-187) was shown to play an essential role in the binding of Cdc42 to PIP(2)-containing membranes. We further showed that substitutions for the di-arginine motif, when introduced within a constitutively active ("fast cycling") Cdc42(F28L) background, had little effect on the ability of the activated Cdc42 mutant to induce microspikes/filopodia in NIH 3T3 cells, whereas they eliminated its ability to transform fibroblasts. Taken together, these findings suggest that the di-arginine motif within the carboxyl terminus of Cdc42 is necessary for this GTPase to bind at membrane sites containing PIP(2), where it can initiate signaling activities that are essential for the oncogenic transformation of cells.

A minimal Rac activation domain in the unconventional guanine nucleotide exchange factor Dock180.

Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by catalyzing the exchange of bound GDP for GTP, thereby resulting in downstream effector recognition. Two metazoan families of GEFs have been described: Dbl-GEF family members that share conserved Dbl homology (DH) and Pleckstrin homology (PH) domains and the more recently described Dock180 family members that share little sequence homology with the Dbl family and are characterized by conserved Dock homology regions 1 and 2 (DHR-1 and -2, respectively). While extensive characterization of the Dbl family has been performed, less is known about how Dock180 family members act as GEFs, with only a single X-ray structure having recently been reported for the Dock9-Cdc42 complex. To learn more about the mechanisms used by the founding member of the family, Dock180, to act as a Rac-specific GEF, we set out to identify and characterize its limit functional GEF domain. A C-terminal portion of the DHR-2 domain, composed of approximately 300 residues (designated as Dock180(DHR-2c)), is shown to be necessary and sufficient for robust Rac-specific GEF activity both in vitro and in vivo. We further show that Dock180(DHR-2c) binds to Rac in a manner distinct from that of Rac-GEFs of the Dbl family. Specifically, Ala(27) and Trp(56) of Rac appear to provide a bipartite binding site for the specific recognition of Dock180(DHR-2c), whereas for Dbl family Rac-GEFs, Trp(56) of Rac is the sole primary determinant of GEF specificity. On the basis of our findings, we are able to define the core of Dock180 responsible for its Rac-GEF activity as well as highlight key recognition sites that distinguish different Dock180 family members and determine their corresponding GTPase specificities.

KRAS-dependent cancer cells promote survival by producing exosomes enriched in Survivin.

Mutations in KRAS frequently occur in human cancer and are especially prevalent in pancreatic ductal adenocarcinoma (PDAC), where they have been shown to promote aggressive phenotypes. However, targeting this onco-protein has proven to be challenging, highlighting the need to further identify the various mechanisms used by KRAS to drive cancer progression. Here, we considered the role played by exosomes, a specific class of extracellular vesicles (EVs) derived from the endocytic cellular trafficking machinery, in mediating the ability of KRAS to promote cell survival. We found that exosomes isolated from the serum of PDAC patients, as well as from KRAS-transformed fibroblasts and pancreatic cancer cells, were all highly enriched in the cell survival protein Survivin. Exosomes containing Survivin, upon engaging serum-starved cells, strongly enhanced their survival. Moreover, they significantly compromised the effectiveness of the conventional chemotherapy drug paclitaxel, as well as a novel therapy that combines an ERK inhibitor with chloroquine, which is currently in clinical trials for PDAC. The survival benefits provided by oncogenic KRAS-derived exosomes were markedly reduced when depleted of Survivin using siRNA or upon treatment with the Survivin inhibitor YM155. Taken together, these findings demonstrate how KRAS mutations give rise to exosomes that provide a unique form of intercellular communication to promote cancer cell survival and therapy resistance, as well as raise interesting possibilities regarding their potential for serving as therapeutic targets and diagnostic markers for KRAS-dependent cancers.

Purification of the Rhodopsin-Transducin Complex for Structural Studies.

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors and are targets for over 30% of all drugs on the market. Structural information of GPCRs and more importantly that of the complex between GPCRs and their signaling partner heterotrimeric G proteins is of great importance. Here we present a method for the large-scale purification of the rhodopsin-transducin complex, the GPCR-G protein signaling complex in visual phototransduction, directly from their native retinal membrane using native proteins purified from bovine retinae. Formation of the complex on native membrane is orchestrated in part by the proper engagement of lipid-modified rhodopsin and transducin (i.e., palmitoylation of the rhodopsin C-terminus, myristoylation and farnesylation of the αT and γ1, respectively). The resulting complex is of high purity and stability and has proved suitable for further biophysical and structural studies. The methods described here should be applicable to other recombinantly expressed receptors from insect cells or mamalian cells by forming stable, functional complexes directly on purified cell membranes.

Reconstitution of the Rhodopsin-Transducin Complex into Lipid Nanodiscs.

Transmembrane proteins, such as G protein-coupled receptors (GPCR), require solubilization in detergents prior to purification. The recent development of novel detergents has allowed for the stabilization of GPCRs, which typically have a high degree of structural flexibility and are otherwise subject to denaturation. However, the detergent micelle environment is still very different from the native lipid membrane and the activity of GPCRs can be profoundly affected by interactions with annular lipid molecules. Moreover, GPCRs are often palmitoylated at their intracellular side, and a lipid bilayer environment would allow for proper orientation of these lipid modifications. Therefore, a reconstituted lipid bilayer environment would best mimic the physiological receptor microenvironment for biophysical studies of GPCRs and nanodiscs provide a methodology to address this aim. Nanodiscs are lipid bilayer discs stabilized by amphipathic membrane scaffolding proteins (MSP) where detergent-solubilized transmembrane proteins can be incorporated into them through a self-assembly process. Here we present a method for reconstituting the purified detergent-solubilized rhodopsin-transducin complex, the GPCR-G protein complex in visual phototransduction, into nanodiscs. The resulting complex incorporated into lipid nanodiscs can be used in biophysical studies including small-angle X-ray scattering and electron microscopy. This method is applicable to integral membrane proteins that mediate protein lipidation, including the zDHHC-family of S-acyltransferases and membrane-bound O-acyltransferases.

Liver-Type Glutaminase GLS2 Is a Druggable Metabolic Node in Luminal-Subtype Breast Cancer.

Efforts to target glutamine metabolism for cancer therapy have focused on the glutaminase isozyme GLS. The importance of the other isozyme, GLS2, in cancer has remained unclear, and it has been described as a tumor suppressor in some contexts. Here, we report that GLS2 is upregulated and essential in luminal-subtype breast tumors, which account for >70% of breast cancer incidence. We show that GLS2 expression is elevated by GATA3 in luminal-subtype cells but suppressed by promoter methylation in basal-subtype cells. Although luminal breast cancers resist GLS-selective inhibitors, we find that they can be targeted with a dual-GLS/GLS2 inhibitor. These results establish a critical role for GLS2 in mammary tumorigenesis and advance our understanding of how to target glutamine metabolism in cancer.

The oncogenic transcription factor c-Jun regulates glutaminase expression and sensitizes cells to glutaminase-targeted therapy.

Many transformed cells exhibit altered glucose metabolism and increased utilization of glutamine for anabolic and bioenergetic processes. These metabolic adaptations, which accompany tumorigenesis, are driven by oncogenic signals. Here we report that the transcription factor c-Jun, product of the proto-oncogene JUN, is a key regulator of mitochondrial glutaminase (GLS) levels. Activation of c-Jun downstream of oncogenic Rho GTPase signalling leads to elevated GLS gene expression and glutaminase activity. In human breast cancer cells, GLS protein levels and sensitivity to GLS inhibition correlate strongly with c-Jun levels. We show that c-Jun directly binds to the GLS promoter region, and is sufficient to increase gene expression. Furthermore, ectopic overexpression of c-Jun renders breast cancer cells dependent on GLS activity. These findings reveal a role for c-Jun as a driver of cancer cell metabolic reprogramming, and suggest that cancers overexpressing JUN may be especially sensitive to GLS-targeted therapies.

Rho GTPases and their roles in cancer metabolism.

Recently, the small molecule 968 was found to block the Rho GTPase-dependent growth of cancer cells in cell culture and mouse xenografts, and when the target of 968 was found to be the mitochondrial enzyme glutaminase (GLS1), it revealed a surprising link between Rho GTPases and mitochondrial glutamine metabolism. Signal transduction via the Rho GTPases, together with NF-κB, appears to elevate mitochondrial glutaminase activity in cancer cells, thereby helping cancer cells satisfy their altered metabolic demands. Here, we review what is known about the mechanism of glutaminase activation in cancer cells, compare the properties of two distinct glutaminase inhibitors, and discuss recent findings that shed new light on how glutamine metabolism might affect cancer progression.

A quantitative fluorometric approach for measuring the interaction of RhoGDI with membranes and Rho GTPases.

Tight regulation of Rho GTPase-signaling functions requires the proper localization of proteins to the membrane and cytosolic compartments, which can themselves undergo reconfiguration in response to signaling events. The importance of lipid-mediated membrane signal transduction continues to emerge as a critical event in many Rho GTPase-signaling pathways. Here we describe methods for the reconstitution of lipid-modified Rho GTPases with defined lipid vesicles and how this system can be used as a real-time assay for monitoring protein-membrane interactions.

Dibenzophenanthridines as inhibitors of glutaminase C and cancer cell proliferation.

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.