Enzyme-bound Substrate Research Articles

High Resolution 31P Field Cycling NMR Reveals Unsuspected Features of Enzyme-Substrate-Cofactor Dynamics.

The dynamic interactions of enzymes and substrates underpins catalysis, yet few techniques can interrogate the dynamics of protein-bound ligands. Here we describe the use of field cycling NMR relaxometry to measure the dynamics of enzyme-bound substrates and cofactors in catalytically competent complexes of GMP reductase. These studies reveal new binding modes unanticipated by x-ray crystal structures and reaction-specific dynamic networks. Importantly, this work demonstrates that distal interactions not usually considered part of the reaction coordinate can play an active role in catalysis. The commercialization of shuttling apparatus will make field cycling relaxometry more accessible and expand its use to additional nuclei, promising more intriguing findings to come.

Molecular Dynamics-Derived Descriptor Informs the Impact of Mutation on the Catalytic Turnover Number in Lactonase Across Substrates.

Molecular dynamics simulations have been extensively employed to reveal the roles of protein dynamics in mediating enzyme catalysis. However, simulation-derived predictive descriptors that inform the impacts of mutations on catalytic turnover numbers remain largely unexplored. In this work, we report the identification of molecular modeling-derived descriptors to predict mutation effect on the turnover number of lactonase SsoPox with both native and non-native substrates. The study consists of 10 enzyme-substrate complexes resulting from a combination of five enzyme variants with two substrates. For each complex, we derived 15 descriptors from molecular dynamics simulations and applied principal component analysis to rank the predictive capability of the descriptors. A top-ranked descriptor was identified, which is the solvent-accessible surface area (SASA) ratio of the substrate to the active site pocket. A uniform volcano-shaped plot was observed in the distribution of experimental activation free energy against the SASA ratio. To achieve efficient lactonase hydrolysis, a non-native substrate-bound enzyme variant needs to involve a similar range of the SASA ratio to the native substrate-bound wild-type enzyme. The descriptor reflects how well the enzyme active site pocket accommodates a substrate for reaction, which has the potential of guiding optimization of enzyme reaction turnover for non-native chemical transformations.

Computational Analysis of Histone Deacetylase 10 Mechanism by the ONIOM Method: A Complementary Approach to X-ray and Kinetics Studies.

Histone deacetylase 10 (HDAC 10) catalyzes deacetylation of N8-acetylspermidine into spermidine in the cytosolic region of eukaryotic cells. Inhibition of HDAC 10 has clinical importance in certain types of cancers. Recently, X-ray crystal structures corresponding to the substrate-bound, tetrahedral intermediate-bound, and product-bound enzymes have been resolved using variant forms of humanized HDAC 10. Based on these structures, it was proposed that Y307 residue polarizes the carbonyl of the acetyl group in N8-acetylspermidine together with a zinc atom, which is coordinated by D174, H176, D267, and an H2O molecule. The H2O molecule undergoes nucleophilic addition to the carbonyl carbon of N8-acetylspermidine to form the tetrahedral intermediate. During this process, it is suggested that H136 acts as a general base to deprotonate the H2O molecule. It is further proposed that the protonation of the amide N atom of the tetrahedral intermediate by H137 causes the deacetylation forming the final products, spermidine and acetate ion. In this study, computational models based on the ONIOM method were employed to study the proposed mechanism for the two steps of the deacetylation process based on the crystal structure of the substrate-bound enzyme. The energy profiles of each step as well as the roles of the active site residues were investigated for the catalysis. The calculated activation barrier is in good agreement with the reported kcat value.

Unlocking Asymmetric Michael Additions in an Archetypical Class I Aldolase by Directed Evolution.

Class I aldolases catalyze asymmetric aldol addition reactions and have found extensive application in the biocatalytic synthesis of chiral β-hydroxy-carbonyl compounds. However, the usefulness of these powerful enzymes for application in other C–C bond-forming reactions remains thus far unexplored. The redesign of class I aldolases to expand their catalytic repertoire to include non-native carboligation reactions therefore continues to be a major challenge. Here, we report the successful redesign of 2-deoxy-d-ribose-5-phosphate aldolase (DERA) from Escherichia coli, an archetypical class I aldolase, to proficiently catalyze enantioselective Michael additions of nitromethane to α,β-unsaturated aldehydes to yield various pharmaceutically relevant chiral synthons. After 11 rounds of directed evolution, the redesigned DERA enzyme (DERA-MA) carried 12 amino-acid substitutions and had an impressive 190-fold enhancement in catalytic activity compared to the wildtype enzyme. The high catalytic efficiency of DERA-MA for this abiological reaction makes it a proficient “Michaelase” with potential for biocatalytic application. Crystallographic analysis provides a structural context for the evolved activity. Whereas an aldolase acts naturally by activating the enzyme-bound substrate as a nucleophile (enamine-based mechanism), DERA-MA instead acts by activating the enzyme-bound substrate as an electrophile (iminium-based mechanism). This work demonstrates the power of directed evolution to expand the reaction scope of natural aldolases to include asymmetric Michael addition reactions and presents opportunities to explore iminium catalysis with DERA-derived catalysts inspired by developments in the organocatalysis field.

Computational Analysis of the Nicotine Oxidoreductase Mechanism by the ONIOM Method.

Nicotine oxidoreductase (NicA2) is a monoamine oxidase (MAO)-based flavoenzyme that catalyzes the oxidation of S-nicotine into N-methylmyosmine. Due to its nanomolar binding affinity toward nicotine, it is seen as an ideal candidate for the treatment of nicotine addiction. Based on the crystal structure of the substrate-bound enzyme, hydrophobic interactions mainly govern the binding of the substrate in the active site through Trp108, Trp364, Trp427, and Leu217 residues. In addition, Tyr308 forms H-bonding with the pyridyl nitrogen of the substrate. Experimental and computational studies support the hydride transfer mechanism for MAO-based enzymes. In this mechanism, a hydride ion transfers from the substrate to the flavin cofactor. In this study, computational models involving the ONIOM method were formulated to study the hydride transfer mechanism based on the crystal structure of the enzyme–substrate complex. The geometry and energetics of the hydride transfer mechanism were analyzed, and the roles of active site residues were highlighted.

Exifone Is a Potent HDAC1 Activator with Neuroprotective Activity in Human Neuronal Models of Neurodegeneration.

Genomic instability caused by a deficiency in the DNA damage response and repair has been linked to age-related cognitive decline and neurodegenerative diseases. Preventing genomic instability that ultimately leads to neuronal death may provide a broadly effective strategy to protect against multiple potential genotoxic stressors. Recently, the zinc-dependent class I histone deacetylase (HDAC1) has been identified as a critical factor for protecting neurons from deleterious effects of DNA damage in Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Translating these observations to a novel neuroprotective therapy for AD, ALS, and FTD may be advanced by the identification of small molecules capable of increasing the deacetylase activity of HDAC1 selectively over other structurally similar HDACs. Here, we demonstrate that exifone, a drug previously shown to be effective in treating cognitive deficits associated with AD and Parkinson's disease, the molecular mechanism of which has remained poorly understood, potently activates the deacetylase activity of HDAC1. We show that exifone acts as a mixed, nonessential activator of HDAC1 that is capable of binding to both free and substrate-bound enzyme, resulting in an increased relative maximal rate of HDAC1-catalyzed deacetylation. Exifone can directly bind to HDAC1 based upon biolayer interferometry assays with kinetic and selectivity profiling, suggesting that HDAC1 is preferentially targeted compared to other class I HDACs and the kinase CDK5, which have also been implicated in neurodegeneration. Consistent with a mechanism of deacetylase activation intracellularly, the treatment of human induced pluripotent stem cell (iPSC)-derived neuronal cells resulted in globally decreased histone acetylation. Moreover, exifone treatment was neuroprotective in a tauopathy patient iPSC-derived neuronal model subject to oxidative stress. Taken together, these findings reveal exifone as a potent activator of HDAC1-mediated deacetylation, thereby offering a lead for novel therapeutic development aiming to protect genomic integrity in the context of neurodegeneration and aging.

Effector role of cytochrome P450 reductase for androstenedione binding to human aromatase

Cytochromes P450 constitute a large superfamily of monooxygenases involved in many metabolic pathways. Most of them are not self-sufficient and need a reductase protein to provide the electrons necessary for catalysis. It was shown that the redox partner plays a role in the modulation of the structure and function of some bacterial P450 enzymes.Here, the effect of NADPH-cytochrome reductase (CPR) on human aromatase (Aro) is studied for what concerns its role in substrate binding. Pre-steady-state kinetic experiments indicate that both the substrate binding rates and the percentage of spin shift detected for aromatase are increased when CPR is present. Moreover, aromatase binds the substrate through a conformational selection mechanism, suggesting a possible effector role of CPR. The thermodynamic parameters for the formation of the CPR-Aro complex were studied by isothermal titration calorimetry. The dissociation constant of the complex formation is 4.5 folds lower for substrate-free compared to the substrate-bound enzyme. The enthalpy change observed when the CPR-Aro complex forms in the absence of the substrate are higher than in its presence, indicating that more interactions are formed/broken in the former case.Taken together, our data confirm that CPR has a role in promoting aromatase conformation optimal for substrate binding.

Enzyme-Substrate-Cofactor Dynamical Networks Revealed by High-Resolution Field Cycling Relaxometry.

The remarkable power and specificity of enzyme catalysis rely on the dynamic alignment of the enzyme, substrates, and cofactors, yet the role of dynamics has usually been approached from the perspective of the protein. We have been using an underappreciated NMR technique, subtesla high-resolution field cycling 31P NMR relaxometry, to investigate the dynamics of the enzyme-bound substrates and cofactor on guanosine-5'-monophosphate reductase (GMPR). GMPR forms two dead end, yet catalytically competent, complexes that mimic distinct steps in the catalytic cycle: E·IMP·NADP+ undergoes a partial hydride transfer reaction, while E·GMP·NADP+ undergoes a partial deamination reaction. A different cofactor conformation is required for each partial reaction. Here we report the effects of mutations designed to perturb cofactor conformation and ammonia binding with the goal of identifying the structural features that contribute to the distinct dynamic signatures of the hydride transfer and deamination complexes. These experiments suggest that Asp129 is a central cog in a dynamic network required for both hydride transfer and deamination. In contrast, Lys77 modulates the conformation and mobility of substrates and cofactors in a reaction-specific manner. Thr105 and Tyr318 are part of a deamination-specific dynamic network that includes the 2'-OH of GMP. These residues have comparatively little effect on the dynamic properties of the hydride transfer complex. These results further illustrate the potential of high-resolution field cycling NMR relaxometry for the investigation of ligand dynamics. In addition, exchange experiments indicate that NH3/NH4+ has a high affinity for the deamination complex but a low affinity for the hydride transfer complex, suggesting that the movement of ammonia may gate the cofactor conformational change. Collectively, these experiments reinforce the view that the enzyme, substrates, and cofactor are linked in intricate, reaction-specific, dynamic networks and demonstrate that distal portions of the substrates and cofactors are critical features in these networks.

Crystal Structures of L-DOPA Dioxygenase from Streptomyces sclerotialus.

Extradiol dioxygenases are essential biocatalysts for breaking down catechols. The vicinal oxygen chelate (VOC) superfamily contains a large number of extradiol dioxygenases, most of which are found as part of catabolic pathways degrading a variety of natural and human-made aromatic rings. The l-3,4-dihydroxyphenylalanine (L-DOPA) extradiol dioxygenases compose a multitude of pathways that produce various antibacterial or antitumor natural products. The structural features of these dioxygenases are anticipated to be distinct from those of other VOC extradiol dioxygenases. Herein, we identified a new L-DOPA dioxygenase from the thermophilic bacterium Streptomyces sclerotialus (SsDDO) through a sequence and genome context analysis. The activity of SsDDO was kinetically characterized with L-DOPA using an ultraviolet-visible spectrophotometer and an oxygen electrode. The optimal temperature of the assay was 55 °C, at which the Km and kcat of SsDDO were 110 ± 10 μM and 2.0 ± 0.1 s-1, respectively. We determined the de novo crystal structures of SsDDO in the ligand-free form and as a substrate-bound complex, refined to 1.99 and 2.31 Å resolution, respectively. These structures reveal that SsDDO possesses a form IV arrangement of βαβββ modules, the first characterization of this assembly from among the VOC/type I extradiol dioxygenase protein family. Electron paramagnetic resonance spectra of Fe-NO adducts for the resting and substrate-bound enzyme were obtained. This work contributes to our understanding of a growing class of topologically distinct VOC dioxygenases, and the obtained structural features will improve our understanding of the extradiol cleavage reaction within the VOC superfamily.

Enzyme Architecture: Amino Acid Side-Chains That Function To Optimize the Basicity of the Active Site Glutamate of Triosephosphate Isomerase.

We report pH rate profiles for kcat and Km for the isomerization reaction of glyceraldehyde 3-phosphate catalyzed by wildtype triosephosphate isomerase (TIM) from three organisms and by ten mutants of TIM; and, for Ki for inhibition of this reaction by phosphoglycolate trianion (I3–). The pH profiles for Ki show that the binding of I3– to TIM (E) to form EH·I3– is accompanied by uptake of a proton by the carboxylate side-chain of E165, whose function is to abstract a proton from substrate. The complexes for several mutants exist mainly as E–·I3– at high pH, in which cases the pH profiles define the pKa for deprotonation of EH·I3–. The linear free energy correlation, with slope of 0.73 (r2 = 0.96), between kcat/Km for TIM-catalyzed isomerization and the disassociation constant of PGA trianion for TIM shows that EH·I3– and the transition state are stabilized by similar interactions with the protein catalyst. Values of pKa = 10–10.5 were estimated for deprotonation of EH·I3– for wildtype TIM. This pKa decreases to as low as 6.3 for the severely crippled Y208F mutant. There is a correlation between the effect of several mutations on kcat/Km and on pKa for EH·I3–. The results support a model where the strong basicity of E165 at the complex to the enediolate reaction intermediate is promoted by side-chains from Y208 and S211, which serve to clamp loop 6 over the substrate; I170, which assists in the creation of a hydrophobic environment for E165; and P166, which functions in driving the carboxylate side-chain of E165 toward enzyme-bound substrate.

Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target

Protein dynamics manifested through structural flexibility play a central role in the function of biological molecules. Here we explore the substrate-mediated change in protein flexibility of an antibiotic target enzyme, Clostridium botulinum dihydrodipicolinate synthase. We demonstrate that the substrate, pyruvate, stabilizes the more active dimer-of-dimers or tetrameric form. Surprisingly, there is little difference between the crystal structures of apo and substrate-bound enzyme, suggesting protein dynamics may be important. Neutron and small-angle X-ray scattering experiments were used to probe substrate-induced dynamics on the sub-second timescale, but no significant changes were observed. We therefore developed a simple technique, coined protein dynamics-mass spectrometry (ProD-MS), which enables measurement of time-dependent alkylation of cysteine residues. ProD-MS together with X-ray crystallography and analytical ultracentrifugation analyses indicates that pyruvate locks the conformation of the dimer that promotes docking to the more active tetrameric form, offering insight into ligand-mediated stabilization of multimeric enzymes.

Substituent Effects on Carbon Acidity in Aqueous Solution and at Enzyme Active Sites.

Methods are described for the determination of pKas for weak carbon acids in water. The application of these methods to the determination of the pKas for a variety of carbon acids including nitriles, imidazolium cations, amino acids, peptides and their derivatives and, α-iminium cations is presented. The substituent effects on the acidity of these different classes of carbon acids are discussed; and, the relevance of these results to catalysis of the deprotonation of amino acids by enzymes and by pyridoxal 5'-phosphate is reviewed. The procedure for estimating the pKa of uridine 5'-phosphate for C-6 deprotonation at the active site of orotidine 5'-phosphate decarboxylase is described, and the effect of a 5-F substituent on carbon acidity of the enzyme-bound substrate is discussed.

Substrate and Cofactor Dynamics on Guanosine Monophosphate Reductase Probed by High Resolution Field Cycling 31P NMR Relaxometry

Guanosine-5'-monophosphate reductase (GMPR) catalyzes the reduction of GMP to IMP and ammonia with concomitant oxidation of NADPH. Here we investigated the structure and dynamics of enzyme-bound substrates and cofactors by measuring 31P relaxation rates over a large magnetic field range using high resolution field cycling NMR relaxometry. Surprisingly, these experiments reveal differences in the low field relaxation profiles for the monophosphate of GMP compared with IMP in their respective NADP+ complexes. These complexes undergo partial reactions that mimic different steps in the overall catalytic cycle. The relaxation profiles indicate that the substrate monophosphates have distinct interactions in E·IMP·NADP+ and E·GMP·NADP+ complexes. These findings were not anticipated by x-ray crystal structures, which show identical interactions for the monophosphates of GMP and IMP in several inert complexes. In addition, the motion of the cofactor is enhanced in the E·GMP·NADP+ complex. Last, the motions of the substrate and cofactor are coordinately regulated; the cofactor has faster local motions than GMP in the deamination complex but is more constrained than IMP in that complex, leading to hydride transfer. These results show that field cycling can be used to investigate the dynamics of protein-bound ligands and provide new insights into how portions of the substrate remote from the site of chemical transformation promote catalysis.

Control of the Position of Oxygen Delivery in Soybean Lipoxygenase-1 by Amino Acid Side Chains within a Gas Migration Channel

Understanding gas migration pathways is critical to unraveling structure-function relationships in enzymes that process gaseous substrates such as O2, H2, and N2 This work investigates the role of a defined pathway for O2 in regulating the peroxidation of linoleic acid by soybean lipoxygenase 1. Computational and mutagenesis studies provide strong support for a dominant delivery channel that shuttles molecular oxygen to a specific region of the active site, thereby ensuring the regio- and stereospecificity of product. Analysis of reaction kinetics and product distribution in channel mutants also reveals a plasticity to the gas migration pathway. The findings show that a single site mutation (I553W) limits oxygen accessibility to the active site, greatly increasing the fraction of substrate that reacts with oxygen free in solution. They also show how a neighboring site mutation (L496W) can result in a redirection of oxygen toward an alternate position of the substrate, changing the regio- and stereospecificity of peroxidation. The present data indicate that modest changes in a protein scaffold may modulate the access of small gaseous molecules to enzyme-bound substrates.

Tyr217 and His213 are important for substrate binding and hydroxylation of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1.

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that contains FAD as a redox-active cofactor. The enzyme catalyzes para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on the enzyme crystal structure, residue His213 is located close to the hydroxyl moiety, whereas Tyr217 is close to the carboxylate group of 3HB. Y217A and Y217S did not show any perturbation of flavin absorption upon addition of 3HB, whereas Y217F has a Kd value for 3HB binding of 7.5 mm, which is ~ 50-fold larger than that found for wild-type enzyme. The results clearly indicate that Tyr217 is necessary for substrate binding. All His213 variants can bind to 3HB with similar affinity as the wild-type enzyme and form C4a-hydroperoxy intermediate. H213S, H213D and H213E produce 2,5-DHB with yields of 28 ± 5%, 52 ± 7% and 92 ± 6%, respectively, whereas H213A cannot catalyze hydroxylation. The results indicate that the interaction between the hydroxyl group of 3HB and residue 213 is important for substrate hydroxylation. Interestingly, the hydroxylation rate constant of H213E (35 s(-1) ) is similar to that of wild-type enzyme (36 s(-1) ) and this variant has an efficiency of hydroxylation (92 ± 6%) similar to the wild-type enzyme (86 ± 2%). Difference spectra of enzyme-bound substrate suggest that 3HB binds to H213E in the phenolic form. The results indicate that His213 and Glu213 in H213E may act as a catalytic base to initiate the substrate deprotonation and facilitate the electrophilic aromatic substitution of 3HB.

Active Role of the Substrate During Catalysis by the Therapeutic Enzyme L‐Asparaginase II

Bacterial type II L‐Asparaginases (ASPII) have been used for over four decades to treat acute lymphoblastic leukemia, yet a full reaction mechanism remains unknown. ASPII enzymes catalyze the deamidation of both asparagine (Asn) and glutamine (Gln), which results in the formation of aspartate (Asp) and glutamate (Glu) respectively, and the by‐product ammonia. Proposed ASPII mechanisms to date have yet to explain the absolute requirement of a substrate α‐carboxyl group, and clearly identify the role of the catalytic threonines T12 and T89. Here, we study the reaction mechanism of asparagine degradation by ASPII through ab initio molecular dynamics (MD) simulations. We select a reduced system from the substrate‐bound enzyme obtained by classical MD, and explore different initial reaction pathways by driving the system in steered simulations. Our results show that direct nucleophilic attack by T12 produces a highly unstable substrate‐enzyme intermediate, as the stabilization provided by the nearby protons (i.e., the “oxyanion hole”) is insufficient to sustain the high energy state. We find that the substrate‐enzyme intermediate can be stabilized by first protonating the substrate's amide oxygen through the K162‐T89 proton bridge. Furthermore, the α‐carboxyl of the substrate acts as a proton acceptor for the hydroxyl sidechain of T12 during nucleophilic attack. We conclude by showing that a complete deamidation mechanism may require a sequence of several nucleophilic attacks by both T12 and T89, with K162 playing a critical role as a proton buffer during the course of the reaction.

The Pseudomonas aeruginosa antimetabolite L -2-amino-4-methoxy-trans-3-butenoic acid (AMB) is made from glutamate and two alanine residues via a thiotemplate-linked tripeptide precursor.

The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a non-proteinogenic amino acid which is toxic for prokaryotes and eukaryotes. Production of AMB requires a five-gene cluster encoding a putative LysE-type transporter (AmbA), two non-ribosomal peptide synthetases (AmbB and AmbE), and two iron(II)/α-ketoglutarate-dependent oxygenases (AmbC and AmbD). Bioinformatics analysis predicts one thiolation (T) domain for AmbB and two T domains (T1 and T2) for AmbE, suggesting that AMB is generated by a processing step from a precursor tripeptide assembled on a thiotemplate. Using a combination of ATP-PPi exchange assays, aminoacylation assays, and mass spectrometry-based analysis of enzyme-bound substrates and pathway intermediates, the AmbB substrate was identified to be L-alanine (L-Ala), while the T1 and T2 domains of AmbE were loaded with L-glutamate (L-Glu) and L-Ala, respectively. Loading of L-Ala at T2 of AmbE occurred only in the presence of AmbB, indicative of a trans loading mechanism. In vitro assays performed with AmbB and AmbE revealed the dipeptide L-Glu-L-Ala at T1 and the tripeptide L-Ala-L-Glu-L-Ala attached at T2. When AmbC and AmbD were included in the assay, these peptides were no longer detected. Instead, an L-Ala-AMB-L-Ala tripeptide was found at T2. These data are in agreement with a biosynthetic model in which L-Glu is converted into AMB by the action of AmbC, AmbD, and tailoring domains of AmbE. The importance of the flanking L-Ala residues in the precursor tripeptide is discussed.

Active Role of the Substrate during Catalysis by the Therapeutic Enzyme L-Asparaginase II

Bacterial type II L-Asparaginases (ASPII) have been used for over four decades to treat acute lymphoblastic leukemia, yet a full reaction mechanism remains unknown. ASPII enzymes catalyze the deamidation of both asparagine (Asn) and glutamine (Gln), which results in the formation of aspartate (Asp) and glutamate (Glu) respectively, and the by-product ammonia. Proposed ASPII mechanisms to date have yet to explain the absolute requirement of a substrate α-carboxyl group, and clearly identify the role of the catalytic threonines T12 and T89. Here, we study the reaction mechanism of asparagine degradation by ASPII through ab initio molecular dynamics (MD) simulations. We selected a reduced system from the substrate-bound enzyme obtained by classical MD, and explore different initial reaction pathways by driving the system in steered simulations. Our results show that direct nucleophilic attack by T12 produces a highly unstable substrate-enzyme intermediate, as the stabilization provided by the nearby protons (i.e., the “oxyanion hole”) is insufficient to sustain the high energy state. We find that the substrate-enzyme intermediate can be stabilized by first protonating the substrate's amide oxygen through the K162-T89 proton bridge. Furthermore, the α-carboxyl of the substrate acts as a proton acceptor for the hydroxyl sidechain of T12 during nucleophilic attack. We conclude by showing that a complete deamidation mechanism may require a sequence of several nucleophilic attacks by both T12 and T89, with K162 playing a critical role as a proton buffer during the course of the reaction.

Abstract 4746: Selective inhibition of mutant IDH1 via small molecule binding to the dimer interface

Abstract Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitric acid to α-ketoglutaric acid (αKG). Point mutations in IDH1 and IDH2 confer a neomorphic enzymatic activity: the reduction of αKG to D-2-hydroxyglutaric acid (2HG), which acts as an oncometabolite by inducing hypermethylation of histones and DNA. IDH1 mutations (predominantly R132H) have been found in 50-80% of grade II gliomas. To find potential IDH1-targeted therapeutics, we conducted a full small-molecule HTS and hit validation campaign using recombinant R132H IDH1. This campaign identified Compound 1, which belongs to a phenol-azole series, as a potent and selective inhibitor of mutant IDH1. We investigated the mode of inhibition of Compound 1 and a published IDH1 mutant inhibitor with a different chemical scaffold (Compound 2). Steady-state kinetics and biophysical studies showed that both inhibitors reversibly interact with both free and substrate-bound enzyme at an allosteric site. A crystal structure of Compound 1 complexed with R132H demonstrates binding of the inhibitor at the dimer interface. A competitive binding study showed that Compound 2 competes with Compound 1, suggesting that Compound 2 also binds at the dimer interface. This is contrary to a previously published statement that Compound 2 is a competitive inhibitor with respect to αKG. In cancer cell lines engineered to produce high levels of 2HG, both inhibitors penetrate cells efficiently and inhibit 2HG production with minimal cell toxicity. This study indicates that the dimer interface pocket is a druggable binding site for IDH1 inhibitors, and suggests that Compound 1 is a promising new starting point for future structure-based drug discovery efforts. Citation Format: Gejing Deng, Stuart Licht, Junqing Shen, Ming Yin, Jessica McManus, Patricia Gee, Tim He, Giang Gao, Bailin Zhang, Magali Mathieu, Alexey Rak, Olivier Bedel, Chaomei Shi, Stefan Gross, Dietmar Hoffmann, Eamonn Rooney, Aurelie Vassort, Walter Englaro, Yi Li, Dmitri Wiederschain, Vinod Patel, Francisco Adrian, Hong Cheng. Selective inhibition of mutant IDH1 via small molecule binding to the dimer interface. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4746. doi:10.1158/1538-7445.AM2014-4746

Abstract 683: Sensitizing tumors to pro-oxidant therapy by inhibiting gamma-glutamyl transpeptidase

Abstract We have reported that 73% of ovarian tumors express the cell surface enzyme gamma-glutamyl transpeptidase (GGT). Clinical studies have shown a poorer prognosis for ovarian tumors that are GGT-positive compared to GGT-negative tumors. GGT-positive tumors are able to use the glutathione in the interstitial fluid as an additional source of cysteine, the limiting component of intracellular glutathione synthesis in tumors. The concentration of cysteine/cystine in standard tissue culture media is more than 3 times the concentration in interstitial fluid. In this study we show that in studies of cultured cells, the high concentration of cysteine in tissue culture media obscured the role of GGT expression in drug resistance. However, culturing human ovarian tumor cells in tissue culture media containing physiological concentrations of cystine and glutathione demonstrated that the supply of cysteine/cystine regulates intracellular glutathione concentrations. In these studies, inhibition of GGT blocked the use of extracellular glutathione as a source of cysteine and sensitized the tumors to cisplatin, carboplatin and melphalan. Inhibiting GGT in patients would also inhibit GGT in the kidney resulting in the excretion of glutathione in the urine and depletion of cysteine from serum and interstitial fluid. As a result, inhibition of GGT in patients would sensitize both GGT-positive and GGT-negative tumors to pro-oxidant chemotherapy. Clinical application of this therapeutic strategy is limited by the fact that most inhibitors of GGT are glutamate analogs and too toxic for use in humans. We discovered a non-glutamate analog that is an uncompetitive inhibitor of GGT, and are developing more potent, non-toxic GGT inhibitors. We recently published the crystal structure of human GGT. We have now also solved the structure of the substrate-bound enzyme, the form of the enzyme to which our uncompetitive inhibitors bind. Using structure-based drug design and our physiologically relevant tissue culture system we are designing new GGT inhibitors and evaluating them for their ability to sensitize tumors to chemotherapy regimens used in the clinic. Supported by an Institutional Development Award (IDeA) from the National Institutes of Health under grant P20GM103640. Citation Format: Marie H. Hanigan, Nancy Wakeham, Stephanie Wickham, Simon S. Terzyan. Sensitizing tumors to pro-oxidant therapy by inhibiting gamma-glutamyl transpeptidase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 683. doi:10.1158/1538-7445.AM2014-683