Structural studies identify angiotensin II receptor blocker-like compounds as branched-chain ketoacid dehydrogenase kinase inhibitors.

The mammalian mitochondrial branched-chain ketoacid dehydrogenase (BCKD) complex is a multienzyme complex involved in the catabolism of branched-chain amino acids. BCKD is regulated by the BCKD kinase, or BCKDK, which binds to the E2 subunit of BCKD, phosphorylates its E1 subunit, and inhibits enzymatic activity. Inhibition of the BCKD complex results in increased levels of branched-chain amino acids and branched-chain ketoacids, and this buildup has been associated with heart failure, type 2 diabetes mellitus, and nonalcoholic fatty liver disease. To find BCKDK inhibitors for potential treatment of these diseases, we performed both NMR and virtual fragment screening and identified tetrazole-bearing fragments that bind BCKDK at multiple sites. Through structure-based virtual screening expanding from these fragments, the angiotensin receptor blocker class antihypertension drugs and angiotensin receptor blocker-like compounds were discovered to be potent BCKDK inhibitors, suggesting potential new avenues for heart failure treatment combining BCKDK inhibition and antihypertension.

Structural Insights into the Interleukin-17 Family Cytokines and Their Receptors.

The IL-17 family in humans consists of six distinct cytokines (IL-17A-F) that can interact with five IL-17 receptors (IL-17RA-E). The interaction between these cytokines and their receptors are critical in mediating host defenses while also making major contributions to inflammatory and autoimmune responses as demonstrated through both in vitro and in vivo experiments as well as human clinical trials. Inhibition of the IL-17A/IL-17RA interaction by monoclonal antibodies has also displayed remarkable efficacies in clinical trials against psoriasis and other autoimmune diseases. Recently, we and others reported the identification and characterization of both small-molecule and peptide IL-17A antagonists. These non-antibody IL-17A antagonists can effectively and selectively disrupt the IL-17A/IL-17RA complex and may provide alternative modalities to treat IL-17-related autoimmune and inflammatory diseases. This chapter summarizes the reported crystal structures of the IL-17 cytokines, their complexes with IL-17RA, and their complexes with both monoclonal antibodies as well as small-molecule and peptide antagonists.

Efficient Liver Targeting by Polyvalent Display of a Compact Ligand for the Asialoglycoprotein Receptor.

A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels-Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.

Crystal structure of a human IκB kinase ß asymmetric dimer.

Phosphorylation of inhibitor of nuclear transcription factor κB (IκB) by IκB kinase (IKK) triggers the degradation of IκB and migration of cytoplasmic κB to the nucleus where it promotes the transcription of its target genes. Activation of IKK is achieved by phosphorylation of its main subunit, IKKß, at the activation loop sites. Here, we report the 2.8 Å resolution crystal structure of human IKKß (hIKKß), which is partially phosphorylated and bound to the staurosporine analog K252a. The hIKKß protomer adopts a trimodular structure that closely resembles that from Xenopus laevis (xIKKß): an N-terminal kinase domain (KD), a central ubiquitin-like domain (ULD), and a C-terminal scaffold/dimerization domain (SDD). Although hIKKß and xIKKß utilize a similar dimerization mode, their overall geometries are distinct. In contrast to the structure resembling closed shears reported previously for xIKKß, hIKKß exists as an open asymmetric dimer in which the two KDs are further apart, with one in an active and the other in an inactive conformation. Dimer interactions are limited to the C-terminal six-helix bundle that acts as a hinge between the two subunits. The observed domain movements in the structures of IKKß may represent trans-phosphorylation steps that accompany IKKß activation.

Insights into mechanism of glucokinase activation: observation of multiple distinct protein conformations.

Human glucokinase (GK) is a principal regulating sensor of plasma glucose levels. Mutations that inactivate GK are linked to diabetes, and mutations that activate it are associated with hypoglycemia. Unique kinetic properties equip GK for its regulatory role: although it has weak basal affinity for glucose, positive cooperativity in its binding of glucose causes a rapid increase in catalytic activity when plasma glucose concentrations rise above euglycemic levels. In clinical trials, small molecule GK activators (GKAs) have been efficacious in lowering plasma glucose and enhancing glucose-stimulated insulin secretion, but they carry a risk of overly activating GK and causing hypoglycemia. The theoretical models proposed to date attribute the positive cooperativity of GK to the existence of distinct protein conformations that interconvert slowly and exhibit different affinities for glucose. Here we report the respective crystal structures of the catalytic complex of GK and of a GK-glucose complex in a wide open conformation. To assess conformations of GK in solution, we also carried out small angle x-ray scattering experiments. The results showed that glucose dose-dependently converts GK from an apo conformation to an active open conformation. Compared with wild type GK, activating mutants required notably lower concentrations of glucose to be converted to the active open conformation. GKAs decreased the level of glucose required for GK activation, and different compounds demonstrated distinct activation profiles. These results lead us to propose a modified mnemonic model to explain cooperativity in GK. Our findings may offer new approaches for designing GKAs with reduced hypoglycemic risk.

Biophysical and mechanistic insights into novel allosteric inhibitor of spleen tyrosine kinase.

Extracellular stimulation of the B cell receptor or mast cell FcεRI receptor activates a cascade of protein kinases, ultimately leading to antigenic or inflammation immune responses, respectively. Syk is a soluble kinase responsible for transmission of the receptor activation signal from the membrane to cytosolic targets. Control of Syk function is, therefore, critical to the human antigenic and inflammation immune response, and an inhibitor of Syk could provide therapy for autoimmune or inflammation diseases. We report here a novel allosteric Syk inhibitor, X1, that is noncompetitive against ATP (K(i) 4 ± 1 µM) and substrate peptide (K(i) 5 ± 1 µM), and competitive against activation of Syk by its upstream regulatory kinase LynB (K(i) 4 ± 1 µM). The inhibition mechanism was interrogated using a combination of structural, biophysical, and kinetic methods, which suggest the compound inhibits Syk by reinforcing the natural regulatory interactions between the SH2 and kinase domains. This novel mode of inhibition provides a new opportunity to improve the selectivity profile of Syk inhibitors for the development of safer drug candidates.

Crystal structures of cholesteryl ester transfer protein in complex with inhibitors.

Human plasma cholesteryl ester transfer protein (CETP) transports cholesteryl ester from the antiatherogenic high-density lipoproteins (HDL) to the proatherogenic low-density and very low-density lipoproteins (LDL and VLDL). Inhibition of CETP has been shown to raise human plasma HDL cholesterol (HDL-C) levels and is potentially a novel approach for the prevention of cardiovascular diseases. Here, we report the crystal structures of CETP in complex with torcetrapib, a CETP inhibitor that has been tested in phase 3 clinical trials, and compound 2, an analog from a structurally distinct inhibitor series. In both crystal structures, the inhibitors are buried deeply within the protein, shifting the bound cholesteryl ester in the N-terminal pocket of the long hydrophobic tunnel and displacing the phospholipid from that pocket. The lipids in the C-terminal pocket of the hydrophobic tunnel remain unchanged. The inhibitors are positioned near the narrowing neck of the hydrophobic tunnel of CETP and thus block the connection between the N- and C-terminal pockets. These structures illuminate the unusual inhibition mechanism of these compounds and support the tunnel mechanism for neutral lipid transfer by CETP. These highly lipophilic inhibitors bind mainly through extensive hydrophobic interactions with the protein and the shifted cholesteryl ester molecule. However, polar residues, such as Ser-230 and His-232, are also found in the inhibitor binding site. An enhanced understanding of the inhibitor binding site may provide opportunities to design novel CETP inhibitors possessing more drug-like physical properties, distinct modes of action, or alternative pharmacological profiles.

Pyrimidone-based series of glucokinase activators with alternative donor-acceptor motif.

Glucokinase activators are a class of experimental agents under investigation as a therapy for Type 2 diabetes mellitus. An X-ray crystal structure of a modestly potent agent revealed the potential to substitute the common heterocyclic amide donor-acceptor motif for a pyridone moiety. We have successfully demonstrated that both pyridone and pyrimidone heterocycles can be used as a potent donor-acceptor substituent. Several sub-micromolar analogs that possess the desired partial activator profile were synthesized and characterized. Unfortunately, the most potent activators suffered from sub-optimal pharmacokinetic properties. Nonetheless, these donor-acceptor motifs may find utility in other glucokinase activator series or beyond.

A covalent BTK ternary complex compatible with targeted protein degradation.

Targeted protein degradation using heterobifunctional chimeras holds the potential to expand target space and grow the druggable proteome. Most acutely, this provides an opportunity to target proteins that lack enzymatic activity or have otherwise proven intractable to small molecule inhibition. Limiting this potential, however, is the remaining need to develop a ligand for the target of interest. While a number of challenging proteins have been successfully targeted by covalent ligands, unless this modification affects form or function, it may lack the ability to drive a biological response. Bridging covalent ligand discovery with chimeric degrader design has emerged as a potential mechanism to advance both fields. In this work, we employ a set of biochemical and cellular tools to deconvolute the role of covalent modification in targeted protein degradation using Bruton's tyrosine kinase. Our results reveal that covalent target modification is fundamentally compatible with the protein degrader mechanism of action.

Structural basis of lipid-droplet localization of 17-beta-hydroxysteroid dehydrogenase 13.

Hydroxysteroid 17-beta-dehydrogenase 13 (HSD17B13) is a hepatic lipid droplet-associated enzyme that is upregulated in patients with non-alcoholic fatty liver disease. Recently, there have been several reports that predicted loss of function variants in HSD17B13 protect against the progression of steatosis to non-alcoholic steatohepatitis with fibrosis and hepatocellular carcinoma. Here we report crystal structures of full length HSD17B13 in complex with its NAD+ cofactor, and with lipid/detergent molecules and small molecule inhibitors from two distinct series in the ligand binding pocket. These structures provide insights into a mechanism for lipid droplet-associated proteins anchoring to membranes as well as a basis for HSD17B13 variants disrupting function. Two series of inhibitors interact with the active site residues and the bound cofactor similarly, yet they occupy different paths leading to the active site. These structures provide ideas for structure-based design of inhibitors that may be used in the treatment of liver disease.

Small molecule branched-chain ketoacid dehydrogenase kinase (BDK) inhibitors with opposing effects on BDK protein levels.

Branched chain amino acid (BCAA) catabolic impairments have been implicated in several diseases. Branched chain ketoacid dehydrogenase (BCKDH) controls the rate limiting step in BCAA degradation, the activity of which is inhibited by BCKDH kinase (BDK)-mediated phosphorylation. Screening efforts to discover BDK inhibitors led to identification of thiophene PF-07208254, which improved cardiometabolic endpoints in mice. Structure-activity relationship studies led to identification of a thiazole series of BDK inhibitors; however, these inhibitors did not improve metabolism in mice upon chronic administration. While the thiophenes demonstrated sustained branched chain ketoacid (BCKA) lowering and reduced BDK protein levels, the thiazoles increased BCKAs and BDK protein levels. Thiazoles increased BDK proximity to BCKDH-E2, whereas thiophenes reduced BDK proximity to BCKDH-E2, which may promote BDK degradation. Thus, we describe two BDK inhibitor series that possess differing attributes regarding BDK degradation or stabilization and provide a mechanistic understanding of the desirable features of an effective BDK inhibitor.

Affinity purification of a chimeric nicotinic acetylcholine receptor in the agonist and antagonist bound states.

Nicotinic acetylcholine receptors (nAChRs) form ligand-gated ion channels that mediate fast signal transmission at synapses. These receptors are members of a large family of pentameric ion channels that are of active medical interest. An expression system utilizing a chimerical construct of the N-terminal extracellular ligand binding domain of alpha7 type nAChR and the C-terminal transmembrane portion of 5HT3 type receptor resulted high level of expressions. Two ligand affinity chromatography purification methods for this receptor have been developed. One method relies on the covalent immobilization of a high affinity small molecule alpha7 nAChR agonist, (R)-5-(4-aminophenyl)-N-(quinuclidin-3-yl) furan-2-carboxamide, and the other uses mono biotinylated alpha-bungarotoxin, an antagonist, that forms a quasi-irreversible complex with alpha7 nAChR. Detergent solubilized alpha7/5HT(3) chimeric receptors were selectively retained on the affinity resins and could be eluted with free ligand or biotin. The proteins purified by both methods were characterized by gel electrophoresis, mass spectra, amino acid composition analysis, and N-terminal sequence determination. These analyses confirmed the isolation of a mature alpha7/5HT(3) receptor with the signal peptide removed. These results suggest a scalable path forward to generate multi-milligram amounts of purified complexes for additional studies including protein crystallization.

1-((3S,4S)-4-amino-1-(4-substituted-1,3,5-triazin-2-yl) pyrrolidin-3-yl)-5,5-difluoropiperidin-2-one inhibitors of DPP-4 for the treatment of type 2 diabetes.

A 3-amino-4-substituted pyrrolidine series of dipeptidyl peptidase IV (DPP-4) inhibitors was rapidly developed into a candidate series by identification of a polar valerolactam replacement for the lipophilic 2,4,5-trifluorophenyl pharmacophore. The addition of a gem-difluoro substituent to the lactam improved overall DPP-4 inhibition and an efficient asymmetric route to 3,4-diaminopyrrolidines was developed. Advanced profiling of a subset of analogs identified 5o with an acceptable human DPP-4 inhibition profile based on a rat PK/PD model and a projected human dose that was suitable for clinical development.

Understanding the impact of the P-loop conformation on kinase selectivity.

This work addresses the link between selectivity and an unusual, folded conformation for the P-loop observed initially for MAP4K4 and subsequently for other kinases. Statistical and computational analyses of our crystal structure database demonstrate that inhibitors that induce the P-loop folded conformation tend to be more selective, especially if they take advantage of this specific conformation by interacting more favorably with a conserved Tyr or Phe residue from the P-loop.

Structural basis for the catalytic mechanism of human phosphodiesterase 9.

The phosphodiesterases (PDEs) are metal ion-dependent enzymes that regulate cellular signaling by metabolic inactivation of the ubiquitous second messengers cAMP and cGMP. In this role, the PDEs are involved in many biological and metabolic processes and are proven targets of successful drugs for the treatments of a wide range of diseases. However, because of the rapidity of the hydrolysis reaction, an experimental knowledge of the enzymatic mechanisms of the PDEs at the atomic level is still lacking. Here, we report the structures of reaction intermediates accumulated at the reaction steady state in PDE9/crystal and preserved by freeze-trapping. These structures reveal the catalytic process of a PDE and explain the substrate specificity of PDE9 in an actual reaction and the cation requirements of PDEs in general.

(3,3-Difluoro-pyrrolidin-1-yl)-[(2S,4S)-(4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrrolidin-2-yl]-methanone: a potent, selective, orally active dipeptidyl peptidase IV inhibitor.

A series of 4-substituted proline amides was synthesized and evaluated as inhibitors of dipeptidyl pepdidase IV for the treatment of type 2 diabetes. (3,3-Difluoro-pyrrolidin-1-yl)-[(2S,4S)-(4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrrolidin-2-yl]-methanone (5) emerged as a potent (IC(50) = 13 nM) and selective compound, with high oral bioavailability in preclinical species and low plasma protein binding. Compound 5, PF-00734200, was selected for development as a potential new treatment for type 2 diabetes.

An Intracellular Allosteric Modulator Binding Pocket in SK2 Ion Channels Is Shared by Multiple Chemotypes.

Small conductance potassium (SK) ion channels define neuronal firing rates by conducting the after-hyperpolarization current. They are key targets in developing therapies where neuronal firing rates are dysfunctional, such as in epilepsy, Parkinson's, and amyotrophic lateral sclerosis (ALS). Here, we characterize a binding pocket situated at the intracellular interface of SK2 and calmodulin, which we show to be shared by multiple small-molecule chemotypes. Crystallization of this complex revealed that riluzole (approved for ALS) and an analog of the anti-ataxic agent (4-chloro-phenyl)-[2-(3,5-dimethyl-pyrazol-1-yl)-pyrimidin-4-yl]-amine (CyPPA) bind to and allosterically modulate via this site. Solution-state nuclear magnetic resonance demonstrates that riluzole, NS309, and CyPPA analogs bind at this bipartite pocket. We demonstrate, by patch-clamp electrophysiology, that both classes of ligand interact with overlapping but distinct residues within this pocket. These data define a clinically important site, laying the foundations for further studies of the mechanism of action of riluzole and related molecules.

2-Aminopyridine-Based Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Inhibitors: Assessment of Mechanism-Based Safety.

Studies have linked the serine-threonine kinase MAP4K4 to the regulation of a number of biological processes and/or diseases, including diabetes, cancer, inflammation, and angiogenesis. With a majority of the members of our lead series (e.g., 1) suffering from time-dependent inhibition (TDI) of CYP3A4, we sought design avenues that would eliminate this risk. One such approach arose from the observation that carboxylic acid-based intermediates employed in our discovery efforts retained high MAP4K4 inhibitory potency and were devoid of the TDI risk. The medicinal chemistry effort that led to the discovery of this central nervous system-impaired inhibitor together with its preclinical safety profile is described.

Lysosomal integral membrane protein-2 as a phospholipid receptor revealed by biophysical and cellular studies.

Lysosomal integral membrane protein-2 (LIMP-2/SCARB2) contributes to endosomal and lysosomal function. LIMP-2 deficiency is associated with neurological abnormalities and kidney failure and, as an acid glucocerebrosidase receptor, impacts Gaucher and Parkinson's diseases. Here we report a crystal structure of a LIMP-2 luminal domain dimer with bound cholesterol and phosphatidylcholine. Binding of these lipids alters LIMP-2 from functioning as a glucocerebrosidase-binding monomer toward a dimeric state that preferentially binds anionic phosphatidylserine over neutral phosphatidylcholine. In cellular uptake experiments, LIMP-2 facilitates transport of phospholipids into murine fibroblasts, with a strong substrate preference for phosphatidylserine. Taken together, these biophysical and cellular studies define the structural basis and functional importance of a form of LIMP-2 for lipid trafficking. We propose a model whereby switching between monomeric and dimeric forms allows LIMP-2 to engage distinct binding partners, a mechanism that may be shared by SR-BI and CD36, scavenger receptor proteins highly homologous to LIMP-2.

Identification and Profiling of a Selective and Brain Penetrant Radioligand for in Vivo Target Occupancy Measurement of Casein Kinase 1 (CK1) Inhibitors.

To enable the clinical development of our CNS casein kinase 1 delta/epsilon (CK1δ/ε) inhibitor project, we investigated the possibility of developing a CNS positron emission tomography (PET) radioligand. For this effort, we focused our design and synthesis efforts on the initial CK1δ/ε inhibitor HTS hits with the goal of identifying a compound that would fulfill a set of recommended PET ligand criteria. We identified [3H]PF-5236216 (9) as a tool ligand that meets most of the key CNS PET attributes including high CNS MPO PET desirability score and kinase selectivity, CNS penetration, and low nonspecific binding. We further used [3H]-9 to determine the binding affinity for PF-670462, a literature CK1δ/ε inhibitor tool compound. Lastly, [3H]-9 was used to measure in vivo target occupancy (TO) of PF-670462 in mouse and correlated TO with CK1δ/ε in vivo pharmacology (circadian rhythm modulation).