Crystal structure of human lysosomal acid lipase and its implications in cholesteryl ester storage disease.

Lysosomal acid lipase (LAL) is a serine hydrolase that hydrolyzes cholesteryl ester (CE) and TGs delivered to the lysosomes into free cholesterol and fatty acids. LAL deficiency due to mutations in the LAL gene (LIPA) results in accumulation of TGs and cholesterol esters in various tissues of the body leading to pathological conditions such as Wolman's disease and CE storage disease (CESD). Here, we present the first crystal structure of recombinant human LAL (HLAL) to 2.6 Å resolution in its closed form. The crystal structure was enabled by mutating three of the six potential glycosylation sites. The overall structure of HLAL closely resembles that of the evolutionarily related human gastric lipase (HGL). It consists of a core domain belonging to the classical α/ß hydrolase-fold family with a classical catalytic triad (Ser-153, His-353, Asp-324), an oxyanion hole, and a "cap" domain, which regulates substrate entry to the catalytic site. Most significant structural differences between HLAL and HGL exist at the lid region. Deletion of the short helix, 238NLCFLLC244, at the lid region implied a possible role in regulating the highly hydrophobic substrate binding site from self-oligomerization during interfacial activation. We also performed molecular dynamic simulations of dog gastric lipase (lid-open form) and HLAL to gain insights and speculated a possible role of the human mutant, H274Y, leading to CESD.

Structural Basis for Achieving GSK-3ß Inhibition with High Potency, Selectivity, and Brain Exposure for Positron Emission Tomography Imaging and Drug Discovery.

Using structure-guided design, several cell based assays, and microdosed positron emission tomography (PET) imaging, we identified a series of highly potent, selective, and brain-penetrant oxazole-4-carboxamide-based inhibitors of glycogen synthase kinase-3 (GSK-3). An isotopologue of our first-generation lead, [3H]PF-367, demonstrates selective and specific target engagement in vitro, irrespective of the activation state. We discovered substantial ubiquitous GSK-3-specific radioligand binding in Tg2576 Alzheimer's disease (AD), suggesting application for these compounds in AD diagnosis and identified [11C]OCM-44 as our lead GSK-3 radiotracer, with optimized brain uptake by PET imaging in nonhuman primates. GSK-3ß-isozyme selectivity was assessed to reveal OCM-51, the most potent (IC50 = 0.030 nM) and selective (>10-fold GSK-3ß/GSK-3α) GSK-3ß inhibitor known to date. Inhibition of CRMP2T514 and tau phosphorylation, as well as favorable therapeutic window against WNT/ß-catenin signaling activation, was observed in cells.

Discovery of 3-Cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide: A Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor C2 Inverse Agonist.

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.

Acyl Glucuronide Metabolites of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1 H-indole-3-carboxylic Acid (PF-06409577) and Related Indole-3-carboxylic Acid Derivatives are Direct Activators of Adenosine Monophosphate-Activated Protein Kinase (AMPK).

Studies on indole-3-carboxylic acid derivatives as direct activators of human adenosine monophosphate-activated protein kinase (AMPK) α1ß1γ1 isoform have culminated in the identification of PF-06409577 (1), PF-06885249 (2), and PF-06679142 (3) as potential clinical candidates. Compounds 1-3 are primarily cleared in animals and humans via glucuronidation. Herein, we describe the biosynthetic preparation, purification, and structural characterization of the glucuronide conjugates of 1-3. Spectral characterization of the purified glucuronides M1, M2, and M3 indicated that they were acyl glucuronide derivatives. In vitro pharmacological evaluation revealed that all three acyl glucuronides retained selective activation of ß1-containing AMPK isoforms. Inhibition of de novo lipogenesis with representative parent carboxylic acids and their respective acyl glucuronide conjugates in human hepatocytes demonstrated their propensity to activate cellular AMPK. Cocrystallization of the AMPK α1ß1γ1 isoform with 1-3 and M1-M3 provided molecular insights into the structural basis for AMPK activation by the glucuronide conjugates.

Optimization of Metabolic and Renal Clearance in a Series of Indole Acid Direct Activators of 5'-Adenosine Monophosphate-Activated Protein Kinase (AMPK).

Optimization of the pharmacokinetic (PK) properties of a series of activators of adenosine monophosphate-activated protein kinase (AMPK) is described. Derivatives of the previously described 5-aryl-indole-3-carboxylic acid clinical candidate (1) were examined with the goal of reducing glucuronidation rate and minimizing renal excretion. Compounds 10 (PF-06679142) and 14 (PF-06685249) exhibited robust activation of AMPK in rat kidneys as well as desirable oral absorption, low plasma clearance, and negligible renal clearance in preclinical species. A correlation of in vivo renal clearance in rats with in vitro uptake by human and rat renal organic anion transporters (human OAT/rat Oat) was identified. Variation of polar functional groups was critical to mitigate active renal clearance mediated by the Oat3 transporter. Modification of either the 6-chloroindole core to a 4,6-difluoroindole or the 5-phenyl substituent to a substituted 5-(3-pyridyl) group provided improved metabolic stability while minimizing propensity for active transport by OAT3.

Biophysical Interactions of Direct AMPK Activators.

Protein-ligand interactions can be evaluated by a number of different biophysical methods. Here we describe some of the experimental methods that we have used to generate AMPK protein reagents and characterize its interactions with direct synthetic activators. Recombinant heterotrimeric AMPK complexes were generated using standard molecular biology methods by expression either in insect cells via infection with three different viruses or more routinely in Escherichia coli with a tricistronic expression vector. Hydrogen/deuterium exchange (HDX) coupled with mass spectrometry was used to probe protein conformational changes and potential binding sites of activators on AMPK. X-ray crystallographic studies were carried out on crystals of AMPK with bound ligands to reveal detailed molecular interactions formed by AMPK activators at near-atomic resolution. In order to gain insights into the mechanism of enzyme activation and to probe the effects of AMPK activators on kinetic parameters such as Michaelis-Menten constant (K m ) or maximal reaction velocity (V max), we performed classical enzyme kinetic studies using radioactive 33P-ATP-based filter assay. Equilibrium dissociation constants (K D ) and on and off rates of ligand binding were obtained by application of surface plasmon resonance (SPR) technique.

Activation of Skeletal Muscle AMPK Promotes Glucose Disposal and Glucose Lowering in Non-human Primates and Mice.

The AMP-activated protein kinase (AMPK) is a potential therapeutic target for metabolic diseases based on its reported actions in the liver and skeletal muscle. We evaluated two distinct direct activators of AMPK: a non-selective activator of all AMPK complexes, PF-739, and an activator selective for AMPK ß1-containing complexes, PF-249. In cells and animals, both compounds were effective at activating AMPK in hepatocytes, but only PF-739 was capable of activating AMPK in skeletal muscle. In diabetic mice, PF-739, but not PF-249, caused a rapid lowering of plasma glucose levels that was diminished in the absence of skeletal muscle, but not liver, AMPK heterotrimers and was the result of an increase in systemic glucose disposal with no impact on hepatic glucose production. Studies of PF-739 in cynomolgus monkeys confirmed translation of the glucose lowering and established activation of AMPK in skeletal muscle as a potential therapeutic approach to treat diabetic patients.

Selective Activation of AMPK ß1-Containing Isoforms Improves Kidney Function in a Rat Model of Diabetic Nephropathy.

Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the ß1 subunit. After confirming that human and rodent kidney predominately express AMPK ß1, we explore the effects of pharmacological activation of AMPK in the ZSF1 rat model of diabetic nephropathy. Chronic administration of these direct activators elevates the phosphorylation of AMPK in the kidney, without impacting blood glucose levels, and reduces the progression of proteinuria to a greater degree than the current standard of care, angiotensin-converting enzyme inhibitor ramipril. Further analyses of urine biomarkers and kidney tissue gene expression reveal AMPK activation leads to the modulation of multiple pathways implicated in kidney injury, including cellular hypertrophy, fibrosis, and oxidative stress. These results support the need for further investigation into the potential beneficial effects of AMPK activation in kidney disease.

Development of a high-throughput crystal structure-determination platform for JAK1 using a novel metal-chelator soaking system.

Crystals of phosphorylated JAK1 kinase domain were initially generated in complex with nucleotide (ADP) and magnesium. The tightly bound Mg2+-ADP at the ATP-binding site proved recalcitrant to ligand displacement. Addition of a molar excess of EDTA helped to dislodge the divalent metal ion, promoting the release of ADP and allowing facile exchange with ATP-competitive small-molecule ligands. Many kinases require the presence of a stabilizing ligand in the ATP site for crystallization. This procedure could be useful for developing co-crystallization systems with an exchangeable ligand to enable structure-based drug design of other protein kinases.

Structure and Regulation of AMPK.

AMP-activated protein kinase is a family of heterotrimeric serine/threonine protein kinases that come in twelve different flavors. They serve an essential function in all eukaryotes of conserving cellular energy levels. AMPK complexes are regulated by changes in cellular AMP:ATP or ADP:ATP ratios and by a number of neutraceuticals and some of the widely-used diabetes medications such as metformin and thiazolinonediones. Moreover, biochemical activities of AMPK are tightly regulated by phosphorylation or dephosphorylation by upstream kinases and phosphatases respectively. Efforts are underway in many pharmaceutical companies to discover direct AMPK activators for the treatment of cardiovascular and metabolic diseases such as diabetes, non-alcoholic steatohepatitis (NASH) and diabetic nephropathy. Many advances have been made in the AMPK structural biology arena over the last few years that are beginning to provide detailed molecular insights into the overall topology of these fascinating enzymes and how binding of small molecules elicit subtle conformational changes leading to their activation and protection from dephosphorylation. In the brief review below on AMPK structure and function, we have focused on the recent crystallographic results especially on specific molecular interactions of direct synthetic AMPK activators which lead to biased activation of a sub-family of AMPK isoforms.

Recent progress in the identification of adenosine monophosphate-activated protein kinase (AMPK) activators.

Adenosine monophosphate-activated protein kinase (AMPK), a serine/threonine heterotrimeric protein kinase, is a critical regulator of cellular and whole body energy homeostasis. There are twelve known AMPK isoforms that are differentially expressed in tissues and species. Dysregulation of AMPK signaling is associated with a multitude of human pathologies. Hence isoform-selective activators of AMPK are actively being sought for the treatment of cardiovascular and metabolic diseases. The present review summarizes the status of direct AMPK activators from the patent and published literature.

Discovery and Preclinical Characterization of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic Acid (PF-06409577), a Direct Activator of Adenosine Monophosphate-activated Protein Kinase (AMPK), for the Potential Treatment of Diabetic Nephropathy.

Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound. Optimization of the core and aryl appendage improved oral absorption and culminated in the identification of indole acid, PF-06409577 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.

Discovery of a Highly Selective Glycogen Synthase Kinase-3 Inhibitor (PF-04802367) That Modulates Tau Phosphorylation in the Brain: Translation for PET Neuroimaging.

Glycogen synthase kinase-3 (GSK-3) regulates multiple cellular processes in diabetes, oncology, and neurology. N-(3-(1H-1,2,4-triazol-1-yl)propyl)-5-(3-chloro-4-methoxyphenyl)oxazole-4-carboxamide (PF-04802367 or PF-367) has been identified as a highly potent inhibitor, which is among the most selective antagonists of GSK-3 to date. Its efficacy was demonstrated in modulation of tau phosphorylation in vitro and in vivo. Whereas the kinetics of PF-367 binding in brain tissues are too fast for an effective therapeutic agent, the pharmacokinetic profile of PF-367 is ideal for discovery of radiopharmaceuticals for GSK-3 in the central nervous system. A (11) C-isotopologue of PF-367 was synthesized and preliminary PET imaging studies in non-human primates confirmed that we have overcome the two major obstacles for imaging GSK-3, namely, reasonable brain permeability and displaceable binding.

Probing the enzyme kinetics, allosteric modulation and activation of α1- and α2-subunit-containing AMP-activated protein kinase (AMPK) heterotrimeric complexes by pharmacological and physiological activators.

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that serves as a pleotropic regulator of whole body energy homoeostasis. AMPK exists as a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (ß and γ), each present as multiple isoforms. In the present study, we compared the enzyme kinetics and allosteric modulation of six recombinant AMPK isoforms, α1ß1γ1, α1ß2γ1, α1ß2γ3, α2ß1γ1, α2ß2γ1 and α2ß2γ3 using known activators, A769662 and AMP. The α1-containing complexes exhibited higher specific activities and lower Km values for a widely used peptide substrate (SAMS) compared with α2-complexes. Surface plasmon resonance (SPR)-based direct binding measurements revealed biphasic binding modes with two distinct equilibrium binding constants for AMP, ADP and ATP across all isoforms tested. The α2-complexes were ∼25-fold more sensitive than α1-complexes to dephosphorylation of a critical threonine on their activation loop (pThr(172/174)). However, α2-complexes were more readily activated by AMP than α1-complexes. Compared with ß1-containing heterotrimers, ß2-containing AMPK isoforms are less sensitive to activation by A769662, a synthetic activator. These data demonstrate that ligand induced activation of AMPK isoforms may vary significantly based on their AMPK subunit composition. Our studies provide insights for the design of isoform-selective AMPK activators for the treatment of metabolic diseases.

Expression and functional characterization of human lysosomal acid lipase gene (LIPA) mutation responsible for cholesteryl ester storage disease (CESD) phenotype.

Lysosomal acid lipase (LAL) is a serine hydrolase which hydrolyzes cholesteryl ester and triglycerides delivered to the lysosomes into free cholesterol and free fatty acids. Mutations in the LAL gene (LIPA) result in accumulation of triglycerides and cholesterol esters in various tissues of the body, leading to pathological conditions such as Wolman's disease (WD) and cholesteryl ester storage disease (CESD). CESD patients homozygous for His295Tyr (H295Y) mutation have less than 5% of normal LAL activity. To shed light on the molecular basis for this loss-of-function phenotype, we have generated the recombinant H295Y enzyme and studied its biophysical and biochemical properties. No significant differences were observed in the expression levels or glycosylation patterns between the mutant and the wild type LAL. However, the H295Y mutant displayed only residual enzymatic activity (<5%) compared to the wild type. While wild type LAL is mostly a monomer at pH 5.0, the vast majority H295Y exists as a high molecular soluble aggregate. Besides, the H295Y mutant has a 20°C lower melting temperature compared to the wild type. Transient expression studies in WD fibroblasts showed that mutation of His295 to other amino acids resulted in a significant loss of enzymatic activity. A homology model of LAL revealed that His295 is located on an α-helix of the cap domain and could be important for tethering it to its core domain. The observed loss-of-function phenotype in CESD patients might arise from a combination of protein destabilization and the shift to a non-functional soluble aggregate.

Discovery and preclinical profiling of 3-[4-(morpholin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]benzonitrile (PF-06447475), a highly potent, selective, brain penetrant, and in vivo active LRRK2 kinase inhibitor.

Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson's disease (PD) by genome-wide association studies (GWAS). The most common LRRK2 mutation, G2019S, which is relatively rare in the total population, gives rise to increased kinase activity. As such, LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the discovery and optimization of a novel series of potent LRRK2 inhibitors, focusing on improving kinome selectivity using a surrogate crystallography approach. This resulted in the identification of 14 (PF-06447475), a highly potent, brain penetrant and selective LRRK2 inhibitor which has been further profiled in in vivo safety and pharmacodynamic studies.

Kinase domain inhibition of leucine rich repeat kinase 2 (LRRK2) using a [1,2,4]triazolo[4,3-b]pyridazine scaffold.

Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson's disease (PD). The most common mutant, G2019S, increases kinase activity, thus LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the structure, potential ligand-protein binding interactions, and pharmacological profiling of potent and highly selective kinase inhibitors based on a triazolopyridazine chemical scaffold.

Structural basis for AMPK activation: natural and synthetic ligands regulate kinase activity from opposite poles by different molecular mechanisms.

AMP-activated protein kinase (AMPK) is a principal metabolic regulator affecting growth and response to cellular stress. Comprised of catalytic and regulatory subunits, each present in multiple forms, AMPK is best described as a family of related enzymes. In recent years, AMPK has emerged as a desirable target for modulation of numerous diseases, yet clinical therapies remain elusive. Challenges result, in part, from an incomplete understanding of the structure and function of full-length heterotrimeric complexes. In this work, we provide the full-length structure of the widely expressed α1ß1γ1 isoform of mammalian AMPK, along with detailed kinetic and biophysical characterization. We characterize binding of the broadly studied synthetic activator A769662 and its analogs. Our studies follow on the heels of the recent disclosure of the α2ß1γ1 structure and provide insight into the distinct molecular mechanisms of AMPK regulation by AMP and A769662.

Selectively targeting an inactive conformation of interleukin-2-inducible T-cell kinase by allosteric inhibitors.

ITK (interleukin-2-inducible T-cell kinase) is a critical component of signal transduction in T-cells and has a well-validated role in their proliferation, cytokine release and chemotaxis. ITK is an attractive target for the treatment of T-cell-mediated inflammatory diseases. In the present study we describe the discovery of kinase inhibitors that preferentially bind to an allosteric pocket of ITK. The novel ITK allosteric site was characterized by NMR, surface plasmon resonance, isothermal titration calorimetry, enzymology and X-ray crystallography. Initial screening hits bound to both the allosteric pocket and the ATP site. Successful lead optimization was achieved by improving the contribution of the allosteric component to the overall inhibition. NMR competition experiments demonstrated that the dual-site binders showed higher affinity for the allosteric site compared with the ATP site. Moreover, an optimized inhibitor displayed non-competitive inhibition with respect to ATP as shown by steady-state enzyme kinetics. The activity of the isolated kinase domain and auto-activation of the full-length enzyme were inhibited with similar potency. However, inhibition of the activated full-length enzyme was weaker, presumably because the allosteric site is altered when ITK becomes activated. An optimized lead showed exquisite kinome selectivity and is efficacious in human whole blood and proximal cell-based assays.

Activation of AMP-activated protein kinase revealed by hydrogen/deuterium exchange mass spectrometry.

AMP-activated protein kinase (AMPK) monitors cellular energy, regulates genes involved in ATP synthesis and consumption, and is allosterically activated by nucleotides and synthetic ligands. Analysis of the intact enzyme with hydrogen/deuterium exchange mass spectrometry reveals conformational perturbations of AMPK in response to binding of nucleotides, cyclodextrin, and a synthetic small molecule activator, A769662. Results from this analysis clearly show that binding of AMP leads to conformational changes primarily in the γ subunit of AMPK and subtle changes in the α and ß subunits. In contrast, A769662 causes profound conformational changes in the glycogen binding module of the ß subunit and in the kinase domain of the α subunit, suggesting that the molecular binding site of the latter resides between the α and ß subunits. The distinct short- and long-range perturbations induced upon binding of AMP and A769662 suggest fundamentally different molecular mechanisms for activation of AMPK by these two ligands.