Publications by authors named "Shenping Liu"

39 Publications

The RESOLUTE consortium: unlocking SLC transporters for drug discovery.

Authors:
Giulio Superti-Furga Daniel Lackner Tabea Wiedmer Alvaro Ingles-Prieto Barbara Barbosa Enrico Girardi Ulrich Goldmann Bettina Gürtl Kristaps Klavins Christoph Klimek Sabrina Lindinger Eva Liñeiro-Retes André C Müller Svenja Onstein Gregor Redinger Daniela Reil Vitaly Sedlyarov Gernot Wolf Matthew Crawford Robert Everley David Hepworth Shenping Liu Stephen Noell Mary Piotrowski Robert Stanton Hui Zhang Salvatore Corallino Andrea Faedo Maria Insidioso Giovanna Maresca Loredana Redaelli Francesca Sassone Lia Scarabottolo Michela Stucchi Paola Tarroni Sara Tremolada Helena Batoulis Andreas Becker Eckhard Bender Yung-Ning Chang Alexander Ehrmann Anke Müller-Fahrnow Vera Pütter Diana Zindel Bradford Hamilton Martin Lenter Diana Santacruz Coralie Viollet Charles Whitehurst Kai Johnsson Philipp Leippe Birgit Baumgarten Lena Chang Yvonne Ibig Martin Pfeifer Jürgen Reinhardt Julian Schönbett Paul Selzer Klaus Seuwen Charles Bettembourg Bruno Biton Jörg Czech Hélène de Foucauld Michel Didier Thomas Licher Vincent Mikol Antje Pommereau Frédéric Puech Veeranagouda Yaligara Aled Edwards Brandon J Bongers Laura H Heitman Ad P IJzerman Huub J Sijben Gerard J P van Westen Justine Grixti Douglas B Kell Farah Mughal Neil Swainston Marina Wright-Muelas Tina Bohstedt Nicola Burgess-Brown Liz Carpenter Katharina Dürr Jesper Hansen Andreea Scacioc Giulia Banci Claire Colas Daniela Digles Gerhard Ecker Barbara Füzi Viktoria Gamsjäger Melanie Grandits Riccardo Martini Florentina Troger Patrick Altermatt Cédric Doucerain Franz Dürrenberger Vania Manolova Anna-Lena Steck Hanna Sundström Maria Wilhelm Claire M Steppan

Nat Rev Drug Discov 2020 07;19(7):429-430

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http://dx.doi.org/10.1038/d41573-020-00056-6DOI Listing
July 2020

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

Authors:
Shenping Liu

Adv Exp Med Biol 2019 ;1172:97-117

Discovery Sciences, Pfizer Inc., Eastern Point Road, Groton, CT, 06340, USA.

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.
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http://dx.doi.org/10.1007/978-981-13-9367-9_5DOI Listing
October 2019

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

Structure 2018 04 22;26(4):533-544.e3. Epub 2018 Mar 22.

Pfizer Neuroscience and Pain Research Unit, Granta Park, Great Abington, Cambridge CB21 6GS, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.str.2018.02.017DOI Listing
April 2018

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

J Med Chem 2018 04 3;61(7):3114-3125. Epub 2018 Apr 3.

Pfizer Worldwide Research & Development , Cambridge , Massachusetts 02139 , United States.

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.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00152DOI Listing
April 2018

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

Nat Commun 2017 12 4;8(1):1908. Epub 2017 Dec 4.

Neuroscience Research Unit, Pfizer Worldwide R&D, 610 Main Street, Cambridge, MA, 02139, USA.

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.
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http://dx.doi.org/10.1038/s41467-017-02044-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712522PMC
December 2017

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

ACS Chem Neurosci 2017 09 30;8(9):1995-2004. Epub 2017 Jun 30.

Chemical Research and Development, Pharmaceutical Sciences, Pfizer Worldwide Research and Development , 558 Eastern Point Rd, Groton, Connecticut 06340, United States.

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 [H]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 [H]-9 to determine the binding affinity for PF-670462, a literature CK1δ/ε inhibitor tool compound. Lastly, [H]-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).
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http://dx.doi.org/10.1021/acschemneuro.7b00155DOI Listing
September 2017

Structure-Based Design of Highly Selective Inhibitors of the CREB Binding Protein Bromodomain.

J Med Chem 2017 07 12;60(13):5349-5363. Epub 2017 May 12.

Medicine Design, Pfizer , 610 Main Street, Cambridge Massachusetts 02139, United States.

Chemical probes are required for preclinical target validation to interrogate novel biological targets and pathways. Selective inhibitors of the CREB binding protein (CREBBP)/EP300 bromodomains are required to facilitate the elucidation of biology associated with these important epigenetic targets. Medicinal chemistry optimization that paid particular attention to physiochemical properties delivered chemical probes with desirable potency, selectivity, and permeability attributes. An important feature of the optimization process was the successful application of rational structure-based drug design to address bromodomain selectivity issues (particularly against the structurally related BRD4 protein).
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http://dx.doi.org/10.1021/acs.jmedchem.6b01839DOI Listing
July 2017

Design of Potent mRNA Decapping Scavenger Enzyme (DcpS) Inhibitors with Improved Physicochemical Properties To Investigate the Mechanism of Therapeutic Benefit in Spinal Muscular Atrophy (SMA).

J Med Chem 2017 04 16;60(7):3094-3108. Epub 2017 Mar 16.

Medicine Design and ‡Rare Disease Research Unit, #Pharmacokinetics and Drug Metabolism, Pfizer , 610 Main Street, Cambridge, Massachusetts 02139, United States.

The C-5 substituted 2,4-diaminoquinazoline RG3039 (compound 1), a member of a chemical series that was identified and optimized using an SMN2 promoter screen, prolongs survival and improves motor function in a mouse model of spinal muscular atrophy (SMA). It is a potent inhibitor of the mRNA decapping scavenger enzyme (DcpS), but the mechanism whereby DcpS inhibition leads to therapeutic benefit is unclear. Compound 1 is a dibasic lipophilic molecule that is predicted to accumulate in lysosomes. To understand if the in vivo efficacy is due to DcpS inhibition or other effects resulting from the physicochemical properties of the chemotype, we undertook structure based molecular design to identify DcpS inhibitors with improved physicochemical properties. Herein we describe the design, synthesis, and in vitro pharmacological characterization of these DcpS inhibitors along with the in vivo mouse CNS PK profile of PF-DcpSi (compound 24), one of the analogs found to be efficacious in SMA mouse model.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00124DOI Listing
April 2017

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

J Am Chem Soc 2017 03 23;139(9):3528-3536. Epub 2017 Feb 23.

Pfizer Medicine Design , Eastern Point Road, Groton, Connecticut 06340, United States.

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.
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http://dx.doi.org/10.1021/jacs.6b12964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6991140PMC
March 2017

Binding site elucidation and structure guided design of macrocyclic IL-17A antagonists.

Sci Rep 2016 08 16;6:30859. Epub 2016 Aug 16.

Worldwide Medicinal Chemistry, Pfizer Worldwide R&D, 610 Main Street, Cambridge, MA 02139, USA.

Interleukin-17A (IL-17A) is a principal driver of multiple inflammatory and immune disorders. Antibodies that neutralize IL-17A or its receptor (IL-17RA) deliver efficacy in autoimmune diseases, but no small-molecule IL-17A antagonists have yet progressed into clinical trials. Investigation of a series of linear peptide ligands to IL-17A and characterization of their binding site has enabled the design of novel macrocyclic ligands that are themselves potent IL-17A antagonists.
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http://dx.doi.org/10.1038/srep30859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4985813PMC
August 2016

Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide.

Sci Rep 2016 05 17;6:26071. Epub 2016 May 17.

Pfizer Worldwide Research and Development, San Diego, CA 92121, USA.

IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. The N-terminal portions of HAP form a β-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. This mode of inhibition suggests opportunities for developing peptide antagonists against this challenging target.
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http://dx.doi.org/10.1038/srep26071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869123PMC
May 2016

Transcriptional Profiling of a Selective CREB Binding Protein Bromodomain Inhibitor Highlights Therapeutic Opportunities.

Chem Biol 2015 Dec 3;22(12):1588-96. Epub 2015 Dec 3.

Worldwide Medicinal Chemistry, Pfizer, 610 Main Street, Cambridge, MA 02139, USA. Electronic address:

Bromodomains are involved in transcriptional regulation through the recognition of acetyl lysine modifications on diverse proteins. Selective pharmacological modulators of bromodomains are lacking, although the largely hydrophobic nature of the pocket makes these modules attractive targets for small-molecule inhibitors. This work describes the structure-based design of a highly selective inhibitor of the CREB binding protein (CBP) bromodomain and its use in cell-based transcriptional profiling experiments. The inhibitor downregulated a number of inflammatory genes in macrophages that were not affected by a selective BET bromodomain inhibitor. In addition, the CBP bromodomain inhibitor modulated the mRNA level of the regulator of G-protein signaling 4 (RGS4) gene in neurons, suggesting a potential therapeutic opportunity for CBP inhibitors in the treatment of neurological disorders.
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http://dx.doi.org/10.1016/j.chembiol.2015.10.013DOI Listing
December 2015

Discovery of an in Vivo Tool to Establish Proof-of-Concept for MAP4K4-Based Antidiabetic Treatment.

ACS Med Chem Lett 2015 Nov 6;6(11):1128-33. Epub 2015 Oct 6.

Worldwide Medicinal Chemistry, Cardiovascular and Metabolic Research Unit, External Research Solutions, Primary Pharmacology Group, and Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research & Development , 610 Main Street, Cambridge, Massachusetts 02139, United States.

Recent studies in adipose tissue, pancreas, muscle, and macrophages suggest that MAP4K4, a serine/threonine protein kinase may be a viable target for antidiabetic drugs. As part of the evaluation of MAP4K4 as a novel antidiabetic target, a tool compound, 16 (PF-6260933) and a lead 17 possessing excellent kinome selectivity and suitable properties were delivered to establish proof of concept in vivo. The medicinal chemistry effort that led to the discovery of these lead compounds is described herein together with in vivo pharmacokinetic properties and activity in a model of insulin resistance.
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http://dx.doi.org/10.1021/acsmedchemlett.5b00215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4645242PMC
November 2015

Rational targeting of active-site tyrosine residues using sulfonyl fluoride probes.

ACS Chem Biol 2015 Apr 29;10(4):1094-8. Epub 2015 Jan 29.

†Worldwide Medicinal Chemistry and ‡Rare Disease Research Unit, Pfizer, 610 Main Street, Cambridge, Massachusetts 02139, United States.

This work describes the first rational targeting of tyrosine residues in a protein binding site by small-molecule covalent probes. Specific tyrosine residues in the active site of the mRNA-decapping scavenger enzyme DcpS were modified using reactive sulfonyl fluoride covalent inhibitors. Structure-based molecular design was used to create an alkyne-tagged probe bearing the sulfonyl fluoride warhead, thus enabling the efficient capture of the protein from a complex proteome. Use of the probe in competition experiments with a diaminoquinazoline DcpS inhibitor permitted the quantification of intracellular target occupancy. As a result, diaminoquinazoline upregulators of survival motor neuron protein that are used for the treatment of spinal muscular atrophy were confirmed as inhibitors of DcpS in human primary cells. This work illustrates the utility of sulfonyl fluoride probes designed to react with specific tyrosine residues of a protein and augments the chemical biology toolkit by these probes uses in target validation and molecular pharmacology.
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http://dx.doi.org/10.1021/cb5009475DOI Listing
April 2015

Casein kinase 1δ/ε inhibitor PF-5006739 attenuates opioid drug-seeking behavior.

ACS Chem Neurosci 2014 Dec 28;5(12):1253-65. Epub 2014 Oct 28.

Pfizer Worldwide Research and Development , 610 Main Street, Cambridge, Massachusetts 02139, United States.

Casein kinase 1 delta (CK1δ) and casein kinase 1 epsilon (CK1ε) inhibitors are potential therapeutic agents for a range of psychiatric disorders. The feasibility of developing a CNS kinase inhibitor has been limited by an inability to identify safe brain-penetrant compounds with high kinome selectivity. Guided by structure-based drug design, potent and selective CK1δ/ε inhibitors have now been identified that address this gap, through the design and synthesis of novel 4-[4-(4-fluorophenyl)-1-(piperidin-4-yl)-1H-imidazol-5-yl]pyrimidin-2-amine derivatives. PF-5006739 (6) possesses a desirable profile, with low nanomolar in vitro potency for CK1δ/ε (IC50 = 3.9 and 17.0 nM, respectively) and high kinome selectivity. In vivo, 6 demonstrated robust centrally mediated circadian rhythm phase-delaying effects in both nocturnal and diurnal animal models. Further, 6 dose-dependently attenuated opioid drug-seeking behavior in a rodent operant reinstatement model in animals trained to self-administer fentanyl. Collectively, our data supports further development of 6 as a promising candidate to test the hypothesis of CK1δ/ε inhibition in treating multiple indications in the clinic.
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http://dx.doi.org/10.1021/cn500201xDOI Listing
December 2014

Discovery and structural characterization of an allosteric inhibitor of bacterial cis-prenyltransferase.

Protein Sci 2015 Jan 6;24(1):20-6. Epub 2014 Nov 6.

Worldwide Research and Development, Pfizer Inc, Groton, Connecticut, 06340.

Undecaprenyl pyrophosphate synthase (UPPs) is an essential enzyme in a key bacterial cell wall synthesis pathway. It catalyzes the consecutive condensations of isopentenyl pyrophosphate (IPP) groups on to a trans-farnesyl pyrophosphate (FPP) to produce a C55 isoprenoid, undecaprenyl pyrophosphate (UPP). Here we report the discovery and co-crystal structures of a drug-like UPPs inhibitor in complex with Streptococcus pneumoniae UPPs, with and without substrate FPP, at resolutions of 2.2 and 2.1 Å, respectively. The UPPs inhibitor has a low molecular weight (355 Da), but displays potent inhibition of UPP synthesis in vitro (IC50 50 nM) that translates into excellent whole cell antimicrobial activity against pathogenic strains of Streptococcal species (MIC90 0.4 µg mL(-1) ). Interestingly, the inhibitor does not compete with the substrates but rather binds at a site adjacent to the FPP binding site and interacts with the tail of the substrate. Based on the structures, an allosteric inhibition mechanism of UPPs is proposed for this inhibitor. This inhibition mechanism is supported by biochemical and biophysical experiments, and provides a basis for the development of novel antibiotics targeting Streptococcus pneumoniae.
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http://dx.doi.org/10.1002/pro.2579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4282408PMC
January 2015

Design and synthesis of truncated EGF-A peptides that restore LDL-R recycling in the presence of PCSK9 in vitro.

Chem Biol 2014 Feb 16;21(2):284-94. Epub 2014 Jan 16.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, 4072 QLD, Australia. Electronic address:

Disrupting the binding interaction between proprotein convertase (PCSK9) and the epidermal growth factor-like domain A (EGF-A domain) in the low-density lipoprotein receptor (LDL-R) is a promising strategy to promote LDL-R recycling and thereby lower circulating cholesterol levels. In this study, truncated 26 amino acid EGF-A analogs were designed and synthesized, and their structures were analyzed in solution and in complex with PCSK9. The most potent peptide had an increased binding affinity for PCSK9 (KD = 0.6 μM) compared with wild-type EGF-A (KD = 1.2 μM), and the ability to increase LDL-R recycling in the presence of PCSK9 in a cell-based assay.
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http://dx.doi.org/10.1016/j.chembiol.2013.11.014DOI Listing
February 2014

Ligand-protein interactions of selective casein kinase 1δ inhibitors.

J Med Chem 2013 Sep 28;56(17):6819-28. Epub 2013 Aug 28.

Pfizer Worldwide Research and Development , 700 Main Street, Cambridge, Massachusetts 02139, United States.

Casein kinase 1δ (CK1δ) and 1ε (CK1ε) are believed to be necessary enzymes for the regulation of circadian rhythms in all mammals. On the basis of our previously published work demonstrating a CK1ε-preferring compound to be an ineffective circadian clock modulator, we have synthesized a series of pyrazole-substitued pyridine inhibitors, selective for the CK1δ isoform. Additionally, using structure-based drug design, we have been able to exploit differences in the hinge region between CK1δ and p38 to find selective inhibitors that have minimal p38 activity. The SAR, brain exposure, and the effect of these inhibitors on mouse circadian rhythms are described. The in vivo evaluation of these inhibitors demonstrates that selective inhibition of CK1δ at sufficient central exposure levels is capable of modulating circadian rhythms.
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http://dx.doi.org/10.1021/jm4006324DOI Listing
September 2013

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

Bioorg Med Chem Lett 2013 Aug 20;23(16):4571-8. Epub 2013 Jun 20.

Cardiovascular, Metabolic, and Endocrine Diseases Chemistry, Pfizer Worldwide Research and Development, 620 Memorial Dr, Cambridge, MA 02139, USA.

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.
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http://dx.doi.org/10.1016/j.bmcl.2013.06.036DOI Listing
August 2013

Crystal structure of a human IκB kinase β asymmetric dimer.

J Biol Chem 2013 Aug 21;288(31):22758-67. Epub 2013 Jun 21.

Structural Biology and Biophysics Group, Pfizer Worldwide Research, Groton, Connecticut 06340, USA.

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, IKKβ, at the activation loop sites. Here, we report the 2.8 Å resolution crystal structure of human IKKβ (hIKKβ), which is partially phosphorylated and bound to the staurosporine analog K252a. The hIKKβ protomer adopts a trimodular structure that closely resembles that from Xenopus laevis (xIKKβ): an N-terminal kinase domain (KD), a central ubiquitin-like domain (ULD), and a C-terminal scaffold/dimerization domain (SDD). Although hIKKβ and xIKKβ utilize a similar dimerization mode, their overall geometries are distinct. In contrast to the structure resembling closed shears reported previously for xIKKβ, hIKKβ 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 IKKβ may represent trans-phosphorylation steps that accompany IKKβ activation.
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http://dx.doi.org/10.1074/jbc.M113.482596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829360PMC
August 2013

Crystal structures of interleukin 17A and its complex with IL-17 receptor A.

Nat Commun 2013 ;4:1888

Structural Biology and Biophysics Group, Pfizer Groton Laboratories, Eastern Point Road, Groton, Connecticut 06340, USA.

The constituent polypeptides of the interleukin-17 family form six different homodimeric cytokines (IL-17A-F) and the heterodimeric IL-17A/F. Their interactions with IL-17 receptors A-E (IL-17RA-E) mediate host defenses while also contributing to inflammatory and autoimmune responses. IL-17A and IL-17F both preferentially engage a receptor complex containing one molecule of IL-17RA and one molecule of IL-17RC. More generally, IL-17RA appears to be a shared receptor that pairs with other members of its family to allow signaling of different IL-17 cytokines. Here we report crystal structures of homodimeric IL-17A and its complex with IL-17RA. Binding to IL-17RA at one side of the IL-17A molecule induces a conformational change in the second, symmetry-related receptor site of IL-17A. This change favors, and is sufficient to account for, the selection of a different receptor polypeptide to complete the cytokine-receptor complex. The structural results are supported by biophysical studies with IL-17A variants produced by site-directed mutagenesis.
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http://dx.doi.org/10.1038/ncomms2880DOI Listing
December 2013

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

J Biol Chem 2012 Oct 7;287(44):37321-9. Epub 2012 Sep 7.

Department of Structural Biology & Biophysics, Pfizer Groton Laboratories, Groton, Connecticut 06340, USA.

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.
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http://dx.doi.org/10.1074/jbc.M112.380063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3481329PMC
October 2012

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

J Biol Chem 2012 Apr 1;287(17):13598-610. Epub 2012 Feb 1.

Structural Biology and Biophysics, Pfizer Groton Laboratories, Groton, Connecticut 06340, USA.

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.
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http://dx.doi.org/10.1074/jbc.M111.274126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340189PMC
April 2012

Pyridone methylsulfone hydroxamate LpxC inhibitors for the treatment of serious gram-negative infections.

J Med Chem 2012 Feb 8;55(4):1662-70. Epub 2012 Feb 8.

Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, 445 Eastern Point Road, Groton, Connecticut 06340, United States.

The synthesis and biological activity of a new series of LpxC inhibitors represented by pyridone methylsulfone hydroxamate 2a is presented. Members of this series have improved solubility and free fraction when compared to compounds in the previously described biphenyl methylsulfone hydroxamate series, and they maintain superior Gram-negative antibacterial activity to comparator agents.
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http://dx.doi.org/10.1021/jm2014875DOI Listing
February 2012

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

J Biol Chem 2012 Mar 4;287(10):7717-27. Epub 2012 Jan 4.

Structural Biology and Biophysics Group, Pfizer, Groton, Connecticut 06340, USA.

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.
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http://dx.doi.org/10.1074/jbc.M111.311993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3293529PMC
March 2012

Discovery of (S)-6-(3-cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-yl)propanamido)nicotinic acid as a hepatoselective glucokinase activator clinical candidate for treating type 2 diabetes mellitus.

J Med Chem 2012 Feb 24;55(3):1318-33. Epub 2012 Jan 24.

Cambridge Laboratories, Pfizer Worldwide Research & Development, 620 Memorial Drive, Cambridge, Massachusetts 02139, United States.

Glucokinase is a key regulator of glucose homeostasis, and small molecule allosteric activators of this enzyme represent a promising opportunity for the treatment of type 2 diabetes. Systemically acting glucokinase activators (liver and pancreas) have been reported to be efficacious but in many cases present hypoglycaemia risk due to activation of the enzyme at low glucose levels in the pancreas, leading to inappropriately excessive insulin secretion. It was therefore postulated that a liver selective activator may offer effective glycemic control with reduced hypoglycemia risk. Herein, we report structure-activity studies on a carboxylic acid containing series of glucokinase activators with preferential activity in hepatocytes versus pancreatic β-cells. These activators were designed to have low passive permeability thereby minimizing distribution into extrahepatic tissues; concurrently, they were also optimized as substrates for active liver uptake via members of the organic anion transporting polypeptide (OATP) family. These studies lead to the identification of 19 as a potent glucokinase activator with a greater than 50-fold liver-to-pancreas ratio of tissue distribution in rodent and non-rodent species. In preclinical diabetic animals, 19 was found to robustly lower fasting and postprandial glucose with no hypoglycemia, leading to its selection as a clinical development candidate for treating type 2 diabetes.
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http://dx.doi.org/10.1021/jm2014887DOI Listing
February 2012

Potent inhibitors of LpxC for the treatment of Gram-negative infections.

J Med Chem 2012 Jan 11;55(2):914-23. Epub 2012 Jan 11.

Worldwide Medicinal Chemistry, Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06340, United States.

In this paper, we present the synthesis and SAR as well as selectivity, pharmacokinetic, and infection model data for representative analogues of a novel series of potent antibacterial LpxC inhibitors represented by hydroxamic acid.
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http://dx.doi.org/10.1021/jm2014748DOI Listing
January 2012

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

Protein Expr Purif 2011 Sep 2;79(1):102-10. Epub 2011 Jun 2.

Pfizer Inc., Pfizer Global Research and Development, Groton, CT 06340, United 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.
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http://dx.doi.org/10.1016/j.pep.2011.05.019DOI Listing
September 2011

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

J Chem Inf Model 2011 Jun 24;51(6):1199-204. Epub 2011 May 24.

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.
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http://dx.doi.org/10.1021/ci200153cDOI Listing
June 2011

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.

Bioorg Med Chem Lett 2011 Mar 31;21(6):1810-4. Epub 2011 Jan 31.

Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340, USA.

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.
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http://dx.doi.org/10.1016/j.bmcl.2011.01.055DOI Listing
March 2011