Filament formation drives catalysis by glutaminase enzymes important in cancer progression.

The glutaminase enzymes GAC and GLS2 catalyze the hydrolysis of glutamine to glutamate, satisfying the 'glutamine addiction' of cancer cells. They are the targets of anti-cancer drugs; however, their mechanisms of activation and catalytic activity have been unclear. Here we demonstrate that the ability of GAC and GLS2 to form filaments is directly coupled to their catalytic activity and present their cryo-EM structures which provide a view of the conformational states essential for catalysis. Filament formation guides an 'activation loop' to assume a specific conformation that works together with a 'lid' to close over the active site and position glutamine for nucleophilic attack by an essential serine. Our findings highlight how ankyrin repeats on GLS2 regulate enzymatic activity, while allosteric activators stabilize, and clinically relevant inhibitors block, filament formation that enables glutaminases to catalyze glutaminolysis and support cancer progression.

Alone and together: current approaches to targeting glutaminase enzymes as part of anti-cancer therapies.

Metabolic reprogramming is a major hallmark of malignant transformation in cancer, and part of the so-called Warburg effect, in which the upregulation of glutamine catabolism plays a major role. The glutaminase enzymes convert glutamine to glutamate, which initiates this pathway. Inhibition of different forms of glutaminase (KGA, GAC, or LGA) demonstrated potential as an emerging anti-cancer therapeutic strategy. The regulation of these enzymes, and the molecular basis for their inhibition, have been the focus of much recent research. This review will explore the recent progress in understanding the molecular basis for activation and inhibition of different forms of glutaminase, as well as the recent focus on combination therapies of glutaminase inhibitors with other anti-cancer drugs.

Many strategies exist to inhibit cancer progression, from chemotherapy to more targeted therapies that exploit differences between tumors and healthy tissue. One such targeted strategy involves inhibition of the enzyme glutaminase, which converts glutamine obtained from the bloodstream into nutrients that fuel tumor growth. Research into glutaminase is ongoing, with regulation of the enzyme, and novel molecular approaches to inhibit its activity, being key focus areas. Here, we review recent progress on targeting glutaminase enzymes for anti-cancer therapy, including several approaches in which glutaminase inhibitors are combined with inhibitors of other cancer-relevant targets, to increase the overall effectiveness of the treatment.

Filament formation drives catalysis by glutaminase enzymes important in cancer progression.

The glutaminase enzymes GAC and GLS2 catalyze the hydrolysis of glutamine to glutamate, satisfying the 'glutamine addiction' of cancer cells. They are the targets of anti-cancer drugs; however, their mechanisms of activation and catalytic activity have been unclear. Here we demonstrate that the ability of GAC and GLS2 to form filaments is directly coupled to their catalytic activity and present their cryo-EM structures which provide an unprecedented view of the conformational states essential for catalysis. Filament formation guides an 'activation loop' to assume a specific conformation that works together with a 'lid' to close over the active site and position glutamine for nucleophilic attack by an essential serine. Our findings highlight how ankyrin repeats on GLS2 regulate enzymatic activity, while allosteric activators stabilize, and clinically relevant inhibitors block, filament formation that enables glutaminases to catalyze glutaminolysis and support cancer progression.

High-resolution structures of mitochondrial glutaminase C tetramers indicate conformational changes upon phosphate binding.

The mitochondrial enzyme glutaminase C (GAC) is upregulated in many cancer cells to catalyze the first step in glutamine metabolism, the hydrolysis of glutamine to glutamate. The dependence of cancer cells on this transformed metabolic pathway highlights GAC as a potentially important therapeutic target. GAC acquires maximal catalytic activity upon binding to anionic activators such as inorganic phosphate. To delineate the mechanism of GAC activation, we used the tryptophan substitution of tyrosine 466 in the catalytic site of the enzyme as a fluorescent reporter for glutamine binding in the presence and absence of phosphate. We show that in the absence of phosphate, glutamine binding to the Y466W GAC tetramer exhibits positive cooperativity. A high-resolution X-ray structure of tetrameric Y466W GAC bound to glutamine suggests that cooperativity in substrate binding is coupled to tyrosine 249, located at the edge of the catalytic site (i.e., the "lid"), adopting two distinct conformations. In one dimer within the GAC tetramer, the lids are open and glutamine binds weakly, whereas, in the adjoining dimer, the lids are closed over the substrates, resulting in higher affinity interactions. When crystallized in the presence of glutamine and phosphate, all four subunits of the Y466W GAC tetramer exhibited bound glutamine with closed lids. Glutamine can bind with high affinity to each subunit, which subsequently undergo simultaneous catalysis. These findings explain how the regulated transitioning of GAC between different conformational states ensures that maximal catalytic activity is reached in cancer cells only when an allosteric activator is available.

New insights into the molecular mechanisms of glutaminase C inhibitors in cancer cells using serial room temperature crystallography.

Cancer cells frequently exhibit uncoupling of the glycolytic pathway from the TCA cycle (i.e., the "Warburg effect") and as a result, often become dependent on their ability to increase glutamine catabolism. The mitochondrial enzyme Glutaminase C (GAC) helps to satisfy this 'glutamine addiction' of cancer cells by catalyzing the hydrolysis of glutamine to glutamate, which is then converted to the TCA-cycle intermediate α-ketoglutarate. This makes GAC an intriguing drug target and spurred the molecules derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (the so-called BPTES class of allosteric GAC inhibitors), including CB-839, which is currently in clinical trials. However, none of the drugs targeting GAC are yet approved for cancer treatment and their mechanism of action is not well understood. Here, we shed new light on the underlying basis for the differential potencies exhibited by members of the BPTES/CB-839 family of compounds, which could not previously be explained with standard cryo-cooled X-ray crystal structures of GAC bound to CB-839 or its analogs. Using an emerging technique known as serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors with significantly different potencies. We also developed a computational model to further elucidate the molecular basis of differential inhibitor potency. We then corroborated the results from our modeling efforts using recently established fluorescence assays that directly read out inhibitor binding to GAC. Together, these findings should aid in future design of more potent GAC inhibitors with better clinical outlook.

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.

Fluorescent Sensing of a Nerve Agent Simulant with Dual Emission over Wide pH Range in Aqueous Solution.

A new 1,8-naphthalimide-based fluorescent probe for the detection of diethyl cyanophosphonate, a very common nerve agent simulant, is designed, synthesized, and characterized fully. The probe shows around 50-fold enhancement of fluorescence intensity over other nerve agent simulants. Importantly, the probe is able to work under aqueous conditions in a wide pH range. Two reactive groups, the oxime and the phenol, allow a dual emission with different kinetic reactions. The reaction of diethyl cyanophosphonate with the oxime group occurs in advance; the resulting time response of the fluorescence enhancement is observed within approximately 30 s. After the oxime reaction, then phenol also undergoes a substitution reaction with diethyl cyanophosphonate, resulting in a blue emission. The real application of this new probe is demonstrated through the use of silica plate assays for the detection of diethyl cyanophosphonate in both gas and liquid phases through dual emission channels.

Bioinorganic Chemistry of the Alkali Metal Ions.

The common Group 1 alkali metals are indeed ubiquitous on earth, in the oceans and in biological systems. In this introductory chapter, concepts involving aqueous chemistry and aspects of general coordination chemistry and oxygen atom donor chemistry are introduced. Also, there are nuclear isotopes of importance. A general discussion of Group 1 begins from the prevalence of the ions, and from a comparison of their ionic radii and ionization energies. While oxygen and water molecule binding have the most relevance to biology and in forming a detailed understanding between the elements, there is a wide range of basic chemistry that is potentially important, especially with respect to biological chelation and synthetic multi-dentate ligand design. The elements are widely distributed in life forms, in the terrestrial environment and in the oceans. The details about the workings in animal, as well as plant life are presented in this volume. Important biometallic aspects of human health and medicine are introduced as well. Seeing as the elements are widely present in biology, various particular endogenous molecules and enzymatic systems can be studied. Sodium and potassium are by far the most important and central elements for consideration. Aspects of lithium, rubidium, cesium and francium chemistry are also included; they help in making important comparisons related to the coordination chemistry of Na(+) and K(+). Physical methods are also introduced.