Comparative assessment of QM-based and MM-based models for prediction of protein-ligand binding affinity trends.

Methods which accurately predict proteinligand binding strengths are critical for drug discovery. In the last two decades, advances in chemical modelling have enabled steadily accelerating progress in the discovery and optimization of structure-based drug design. Most computational methods currently used in this context are based on molecular mechanics force fields that often have deficiencies in describing the quantum mechanical (QM) aspects of molecular binding.

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.

Discovery and Early Clinical Development of LY3202626, a Low-Dose, CNS-Penetrant BACE Inhibitor.

The beta-site APP cleaving enzyme 1, known as BACE1, has been a widely pursued Alzheimer's disease drug target owing to its critical role in the production of amyloid-beta. We have previously reported the clinical development of LY2811376 and LY2886721. LY2811376 advanced to Phase I before development was terminated due to nonclinical retinal toxicity.

Development of Dimethylisoxazole-Attached Imidazo[1,2-]pyridines as Potent and Selective CBP/P300 Inhibitors.

The use of epigenetic bromodomain inhibitors as anticancer therapeutics has transitioned from targeting bromodomain extraterminal domain (BET) proteins into targeting non-BET bromodomains. The two most relevant non-BET bromodomain oncology targets are cyclic AMP response element-binding protein (CBP) and E1A binding protein P300 (EP300). To explore the growing CBP/EP300 interest, we developed a highly efficient two-step synthetic route for dimethylisoxazole-attached imidazo[1,2-]pyridine scaffold-containing inhibitors.

Quantum Mechanical Investigation of Three-Dimensional Activity Cliffs Using the Molecules-in-Molecules Fragmentation-Based Method.

The concept of activity cliff (AC) (i.e., a small structural modification resulting in a substantial bioactivity change) is widely encountered in medicinal chemistry during compound design.

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.

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.

Preparation and biological evaluation of BACE1 inhibitors: Leveraging trans-cyclopropyl moieties as ligand efficient conformational constraints.

Inhibition of BACE1 has become an important strategy in the quest for disease modifying agents to slow the progression of Alzheimer's disease. We previously reported the fragment-based discovery of LY2811376, the first BACE1 inhibitor reported to demonstrate robust reduction of human CSF Aβ in a Phase I clinical trial. We also reported on the discovery of LY2886721, a potent BACE1 inhibitor that reached phase 2 clinical trials.

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.

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 (G) by catalyzing GDP-GTP exchange on its α subunit (Gα). To elucidate the determinants of G coupling and activation, we obtained cryo-EM structures of a fully functional, light-activated Rho-G complex in the presence and absence of a G-protein-stabilizing nanobody. The structures illustrate how G overcomes its low basal activity by engaging activated Rho in a conformation distinct from other GPCR-G-protein complexes.

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.

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.

[3 + 2] Cycloaddition and Cascade Radical Reactions for the Synthesis of Trifluoromethylated Tetrahydrobenzodiazepin-3-ones.

A reaction sequence involving three-component [3 + 2] cycloaddition of azomethine ylides followed by CuI-catalyzed cascade trifluoromethyl radical addition and cyclization is developed for diastereoselective synthesis of fused-tetrahydrobenzodiazepin-3-ones.

Idea2Data: Toward a New Paradigm for Drug Discovery.

Increasing the success rate and throughput of drug discovery will require efficiency improvements throughout the process that is currently used in the pharmaceutical community, including the crucial step of identifying hit compounds to act as drivers for subsequent optimization. Hit identification can be carried out through large compound collection screening and often involves the generation and testing of many hypotheses based on available knowledge. In practice, hypothesis generation can involve the selection of promising chemical structures from compound collections using predictive models built from previous screening/assay results.

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 (α) that is necessary to fully activate its effector enzyme, the cGMP phosphodiesterase (PDE).

Theoretical Study of Protein-Ligand Interactions Using the Molecules-in-Molecules Fragmentation-Based Method.

We have recently significantly expanded the applicability of our Molecules-in-Molecules (MIM) fragmentation method to large proteins by developing a three-layer model (MIM3) in which an accurate quantum-mechanical method is used in conjunction with a cost-effective, dispersion-corrected semiempirical model to overcome previous computational bottlenecks. In this work, we develop MIM3 as a structure-based drug design tool by application of the methodology for the accurate calculation of protein-ligand interaction energies. A systematic protocol is derived for the determination of the geometries of the protein-ligand complexes and to calculate their accurate interaction energies in the gas phase using MIM3.

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 (G). This results in the dissociation of G into its component α-GTP and βγ subunit complex. Structural information for the Rho*-G complex will be essential for understanding the molecular mechanism of visual photo-transduction.

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 [(2,4)-4-FGln, (2,4)-4-FGln] were previously developed for positron emission tomography. Label uptake into two tumor cell types was greater with [F](2,4)-4-FGln than with [F](2,4)-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 [(2,4)-4-(3-fluoropropyl)glutamine (FP-Gln)].

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 ( the "activation loop").

Discovery of (1S,2R,3S,4S,5R,6R)-2-Amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid Hydrochloride (LY3020371·HCl): A Potent, Metabotropic Glutamate 2/3 Receptor Antagonist with Antidepressant-Like Activity.

As part of our ongoing efforts to identify novel ligands for the metabotropic glutamate 2 and 3 (mGlu) receptors, we have incorporated substitution at the C3 and C4 positions of the (1S,2R,5R,6R)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid scaffold to generate mGlu antagonists.

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.

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.

Preparation and biological evaluation of conformationally constrained BACE1 inhibitors.

The BACE1 enzyme is a key target for Alzheimer's disease. During our BACE1 research efforts, fragment screening revealed that bicyclic thiazine 3 had low millimolar activity against BACE1. Analysis of the co-crystal structure of 3 suggested that potency could be increased through extension toward the S3 pocket and through conformational constraint of the thiazine core.

Fragment-based design of kinase inhibitors: a practical guide.

Fragment-based drug design has become an important strategy for drug design and development over the last decade. It has been used with particular success in the development of kinase inhibitors, which are one of the most widely explored classes of drug targets today. The application of fragment-based methods to discovering and optimizing kinase inhibitors can be a complicated and daunting task; however, a general process has emerged that has been highly fruitful.

The potent BACE1 inhibitor LY2886721 elicits robust central Aβ pharmacodynamic responses in mice, dogs, and humans.

BACE1 is a key protease controlling the formation of amyloid β, a peptide hypothesized to play a significant role in the pathogenesis of Alzheimer's disease (AD). Therefore, the development of potent and selective inhibitors of BACE1 has been a focus of many drug discovery efforts in academia and industry. Herein, we report the nonclinical and early clinical development of LY2886721, a BACE1 active site inhibitor that reached phase 2 clinical trials in AD.