Publications by authors named "Xiayang Qiu"

35 Publications

Prefusion structure of human cytomegalovirus glycoprotein B and structural basis for membrane fusion.

Sci Adv 2021 Mar 5;7(10). Epub 2021 Mar 5.

Vaccine Research and Development, Pfizer Inc., Pearl River, NY 10965, USA.

Human cytomegalovirus (HCMV) causes congenital disease with long-term morbidity. HCMV glycoprotein B (gB) transitions irreversibly from a metastable prefusion to a stable postfusion conformation to fuse the viral envelope with a host cell membrane during entry. We stabilized prefusion gB on the virion with a fusion inhibitor and a chemical cross-linker, extracted and purified it, and then determined its structure to 3.6-Å resolution by electron cryomicroscopy. Our results revealed the structural rearrangements that mediate membrane fusion and details of the interactions among the fusion loops, the membrane-proximal region, transmembrane domain, and bound fusion inhibitor that stabilized gB in the prefusion state. The structure rationalizes known gB antigenic sites. By analogy to successful vaccine antigen engineering approaches for other viral pathogens, the high-resolution prefusion gB structure provides a basis to develop stabilized prefusion gB HCMV vaccine antigens.
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http://dx.doi.org/10.1126/sciadv.abf3178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935361PMC
March 2021

Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins.

Nat Commun 2020 03 27;11(1):1598. Epub 2020 Mar 27.

Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA.

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.
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http://dx.doi.org/10.1038/s41467-020-15363-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101349PMC
March 2020

Machine Learning for Prioritization of Thermostabilizing Mutations for G-Protein Coupled Receptors.

Biophys J 2019 12 24;117(11):2228-2239. Epub 2019 Oct 24.

Department of Computational and Quantitative Medicine, Beckman Research Institute of the City of Hope, Duarte, California. Electronic address:

Although the three-dimensional structures of G-protein coupled receptors (GPCRs), the largest superfamily of drug targets, have enabled structure-based drug design, there are no structures available for 87% of GPCRs. This is due to the stiff challenge in purifying the inherently flexible GPCRs. Identifying thermostabilized mutant GPCRs via systematic alanine scanning mutations has been a successful strategy in stabilizing GPCRs, but it remains a daunting task for each GPCR. We developed a computational method that combines sequence-, structure-, and dynamics-based molecular properties of GPCRs that recapitulate GPCR stability, with four different machine learning methods to predict thermostable mutations ahead of experiments. This method has been trained on thermostability data for 1231 mutants, the largest publicly available data set. A blind prediction for thermostable mutations of the complement factor C5a receptor 1 retrieved 36% of the thermostable mutants in the top 50 prioritized mutants compared to 3% in the first 50 attempts using systematic alanine scanning.
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http://dx.doi.org/10.1016/j.bpj.2019.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895739PMC
December 2019

Cryo-EM structures of the human glutamine transporter SLC1A5 (ASCT2) in the outward-facing conformation.

Elife 2019 10 3;8. Epub 2019 Oct 3.

Medicine Design, Pfizer Inc, Groton, United States.

Alanine-serine-cysteine transporter 2 (ASCT2, SLC1A5) is the primary transporter of glutamine in cancer cells and regulates the mTORC1 signaling pathway. The SLC1A5 function involves finely tuned orchestration of two domain movements that include the substrate-binding transport domain and the scaffold domain. Here, we present cryo-EM structures of human SLC1A5 and its complex with the substrate, L-glutamine in an outward-facing conformation. These structures reveal insights into the conformation of the critical ECL2a loop which connects the two domains, thus allowing rigid body movement of the transport domain throughout the transport cycle. Furthermore, the structures provide new insights into substrate recognition, which involves conformational changes in the HP2 loop. A putative cholesterol binding site was observed near the domain interface in the outward-facing state. Comparison with the previously determined inward-facing structure of SCL1A5 provides a basis for a more integrated understanding of substrate recognition and transport mechanism in the SLC1 family.
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http://dx.doi.org/10.7554/eLife.48120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800002PMC
October 2019

Delineating the role of cooperativity in the design of potent PROTACs for BTK.

Proc Natl Acad Sci U S A 2018 07 16;115(31):E7285-E7292. Epub 2018 Jul 16.

Discovery Sciences, Pfizer Worldwide Research and Development, Groton, CT 06340.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.
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http://dx.doi.org/10.1073/pnas.1803662115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6077745PMC
July 2018

Sites associated with Kalydeco binding on human Cystic Fibrosis Transmembrane Conductance Regulator revealed by Hydrogen/Deuterium Exchange.

Sci Rep 2018 03 16;8(1):4664. Epub 2018 Mar 16.

Structural & Molecular Sciences, Pfizer, 445 Eastern Point Road, Groton, Connecticut, 06340, United States.

Cystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Mutations associated with CF cause loss-of-function in CFTR leading to salt imbalance in epithelial tissues. Kalydeco (also called VX-770 or ivacaftor) was approved for CF treatment in 2012 but little is known regarding the compound's interactions with CFTR including the site of binding or mechanisms of action. In this study we use hydrogen/deuterium exchange (HDX) coupled with mass spectrometry to assess the conformational dynamics of a thermostabilized form of CFTR in apo and ligand-bound states. We observe HDX protection at a known binding site for AMPPNP and significant protection for several regions of CFTR in the presence of Kalydeco. The ligand-induced changes of CFTR in the presence of Kalydeco suggest a potential binding site.
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http://dx.doi.org/10.1038/s41598-018-22959-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856801PMC
March 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

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

Structural basis for allosteric, substrate-dependent stimulation of SIRT1 activity by resveratrol.

Genes Dev 2015 Jun;29(12):1316-25

National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China;

Sirtuins with an extended N-terminal domain (NTD), represented by yeast Sir2 and human SIRT1, harbor intrinsic mechanisms for regulation of their NAD-dependent deacetylase activities. Elucidation of the regulatory mechanisms is crucial for understanding the biological functions of sirtuins and development of potential therapeutics. In particular, SIRT1 has emerged as an attractive therapeutic target, and the search for SIRT1-activating compounds (STACs) has been actively pursued. However, the effectiveness of a class of reported STACs (represented by resveratrol) as direct SIRT1 activators is under debate due to the complication involving the use of fluorogenic substrates in in vitro assays. Future efforts of SIRT1-based therapeutics necessitate the dissection of the molecular mechanism of SIRT1 stimulation. We solved the structure of SIRT1 in complex with resveratrol and a 7-amino-4-methylcoumarin (AMC)-containing peptide. The structure reveals the presence of three resveratrol molecules, two of which mediate the interaction between the AMC peptide and the NTD of SIRT1. The two NTD-bound resveratrol molecules are principally responsible for promoting tighter binding between SIRT1 and the peptide and the stimulation of SIRT1 activity. The structural information provides valuable insights into regulation of SIRT1 activity and should benefit the development of authentic SIRT1 activators.
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http://dx.doi.org/10.1101/gad.265462.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4495401PMC
June 2015

HDL surface lipids mediate CETP binding as revealed by electron microscopy and molecular dynamics simulation.

Sci Rep 2015 Mar 4;5:8741. Epub 2015 Mar 4.

The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.

Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesterol esters (CE) from atheroprotective high-density lipoproteins (HDL) to atherogenic low-density lipoproteins (LDL). CETP inhibition has been regarded as a promising strategy for increasing HDL levels and subsequently reducing the risk of cardiovascular diseases (CVD). Although the crystal structure of CETP is known, little is known regarding how CETP binds to HDL. Here, we investigated how various HDL-like particles interact with CETP by electron microscopy and molecular dynamics simulations. Results showed that CETP binds to HDL via hydrophobic interactions rather than protein-protein interactions. The HDL surface lipid curvature generates a hydrophobic environment, leading to CETP hydrophobic distal end interaction. This interaction is independent of other HDL components, such as apolipoproteins, cholesteryl esters and triglycerides. Thus, disrupting these hydrophobic interactions could be a new therapeutic strategy for attenuating the interaction of CETP with HDL.
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http://dx.doi.org/10.1038/srep08741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4348656PMC
March 2015

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

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

Structure 2014 Aug 24;22(8):1161-1172. Epub 2014 Jul 24.

Worldwide Research and Development, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA. Electronic address:

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 α1β1γ1 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 α2β1γ1 structure and provide insight into the distinct molecular mechanisms of AMPK regulation by AMP and A769662.
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http://dx.doi.org/10.1016/j.str.2014.06.009DOI Listing
August 2014

A continuous and direct assay to monitor leucine-rich repeat kinase 2 activity.

Anal Biochem 2014 Apr 23;450:63-9. Epub 2014 Jan 23.

Structural Biology and Biophysics Group, Center for Chemistry Innovation and Excellence, Pfizer Worldwide Medicinal Chemistry, Groton, CT 06340, USA.

Leucine-rich repeat kinase 2 (LRRK2) is a multi-domain enzyme displaying activities of GTP hydrolase and protein threonine/serine kinase in separate domains. Mutations in both catalytic domains have been linked to the onset of Parkinson's disease, which triggered high interest in this enzyme as a potential target for drug development, particularly focusing on inhibition of the kinase activity. However, available activity assays are discontinuous, involving either radioactivity detection or coupling with antibodies. Here we describe a continuous and direct assay for LRRK2 kinase activity, combining a reported peptide sequence optimized for LRRK2 binding and an established strategy for fluorescence emission on magnesium ion chelation by phosphorylated peptides carrying an artificial amino acid. The assay was employed to evaluate apparent steady-state parameters for the wild type and two mutant forms of LRRK2 associated with Parkinson's disease as well as to probe the effects of GTP, GDP, and autophosphorylation on the kinase activity of the enzyme. Staurosporine was evaluated as an inhibitor of the wild-type enzyme. It is expected that this assay will aid in mechanistic investigations of LRRK2.
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http://dx.doi.org/10.1016/j.ab.2014.01.007DOI Listing
April 2014

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

Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein.

Nat Chem Biol 2012 Feb 19;8(4):342-9. Epub 2012 Feb 19.

Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.
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http://dx.doi.org/10.1038/nchembio.796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3792710PMC
February 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

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

Structural and biophysical insight into cholesteryl ester-transfer protein.

Biochem Soc Trans 2011 Aug;39(4):1000-5

Pfizer Research and Development, Eastern Point Road, Groton, CT 06430, USA.

CETP (cholesteryl ester-transfer protein) is essential for neutral lipid transfer between HDL (high-density lipoprotein) and LDL (low-density lipoprotein) and plays a critical role in the reverse cholesterol transfer pathway. In clinical trials, CETP inhibitors increase HDL levels and reduce LDL levels, and therefore may be used as a potential treatment for atherosclerosis. In this review, we cover the analysis of CETP structure and provide insights into CETP-mediated lipid transfer based on a collection of structural and biophysical data.
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http://dx.doi.org/10.1042/BST0391000DOI Listing
August 2011

Escherichia coli expression, purification and characterization of functional full-length recombinant alpha2beta2gamma3 heterotrimeric complex of human AMP-activated protein kinase.

Protein Expr Purif 2010 Oct 6;73(2):189-97. Epub 2010 May 6.

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

AMP-activated protein kinase (AMPK) is an energy-sensing serine/threonine protein kinase that plays a central role in whole-body energy homeostasis. AMPK is a heterotrimeric enzyme with a catalytic (alpha) subunit and two regulatory (beta and gamma) subunits. The muscle-specific AMPK heterotrimeric complex (alpha2beta2gamma3) is involved in glucose and fat metabolism in skeletal muscle and therefore has emerged as an attractive target for drug development for diabetes and metabolic syndrome. To date, expression of recombinant full-length human AMPK alpha2beta2gamma3 has not been reported. Here we describe the expression, purification and biochemical characterization of functional full-length AMPK alpha2beta2gamma3 heterotrimeric complex using an Escherichia coli expression system. All three subunits of AMPK alpha2beta2gamma3 were transcribed as a single tricistronic transcript driven by the T7 RNA polymerase promoter, allowing spontaneous formation of the heterotrimeric complex in the bacterial cytosol. The self-assembled trimeric complex was purified from the cell lysate by nickel-ion chromatography using the hexahistidine tag fused exclusively at the N-terminus of the alpha 2 domain. The un-assembled beta 2 and gamma 3 domains were removed by extensive washing of the column. Further purification of the heterotrimer was performed using size exclusion chromatography. The final yield of the recombinant AMPK alpha2beta2gamma3 complex was 1.1mg/L culture in shaker flasks. The E. coli expressed enzyme was catalytically inactive after purification, but was activated in vitro by upstream kinases such as CaMKKbeta and LKB1. The kinase activity of activated AMPK alpha2beta2gamma3 complex was significantly enhanced by AMP (an allosteric activator) but not by thienopyridone A-769662, a known small molecule activator of AMPK. Mass spectrometric characterization of recombinant AMPK alpha2beta2gamma3 showed significant heterogeneity before and after activation that could potentially hamper crystallographic studies of this complex.
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http://dx.doi.org/10.1016/j.pep.2010.04.022DOI Listing
October 2010

SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.

J Biol Chem 2010 Mar 8;285(11):8340-51. Epub 2010 Jan 8.

Department of Cardiovascular, Pfizer Global Research and Development, Groton, Connecticut 06340, USA.

Sirtuins catalyze NAD(+)-dependent protein deacetylation and are critical regulators of transcription, apoptosis, metabolism, and aging. There are seven human sirtuins (SIRT1-7), and SIRT1 has been implicated as a key mediator of the pathways downstream of calorie restriction that have been shown to delay the onset and reduce the incidence of age-related diseases such as type 2 diabetes. Increasing SIRT1 activity, either by transgenic overexpression of the Sirt1 gene in mice or by pharmacological activation by small molecule activators resveratrol and SRT1720, has shown beneficial effects in rodent models of type 2 diabetes, indicating that SIRT1 may represent an attractive therapeutic target. Herein, we have assessed purported SIRT1 activators by employing biochemical assays utilizing native substrates, including a p53-derived peptide substrate lacking a fluorophore as well as the purified native full-length protein substrates p53 and acetyl-CoA synthetase1. SRT1720, its structurally related compounds SRT2183 and SRT1460, and resveratrol do not lead to apparent activation of SIRT1 with native peptide or full-length protein substrates, whereas they do activate SIRT1 with peptide substrate containing a covalently attached fluorophore. Employing NMR, surface plasmon resonance, and isothermal calorimetry techniques, we provide evidence that these compounds directly interact with fluorophore-containing peptide substrates. Furthermore, we demonstrate that SRT1720 neither lowers plasma glucose nor improves mitochondrial capacity in mice fed a high fat diet. SRT1720, SRT2183, SRT1460, and resveratrol exhibit multiple off-target activities against receptors, enzymes, transporters, and ion channels. Taken together, we conclude that SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.
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http://dx.doi.org/10.1074/jbc.M109.088682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832984PMC
March 2010

Assessment of cholesteryl ester transfer protein inhibitors for interaction with proteins involved in the immune response to infection.

J Lipid Res 2010 May 21;51(5):967-74. Epub 2009 Oct 21.

Department of Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA.

The CETP inhibitor, torcetrapib, was prematurely terminated from phase 3 clinical trials due to an increase in cardiovascular and noncardiovascular mortality. Because nearly half of the latter deaths involved patients with infection, we have tested torcetrapib and other CETPIs to see if they interfere with lipopolysaccharide binding protein (LBP) or bactericidal/permeability increasing protein (BPI). No effect of these potent CETPIs on LPS binding to either protein was detected. Purified CETP itself bound weakly to LPS with a Kd >or= 25 microM compared with 0.8 and 0.5 nM for LBP and BPI, respectively, and this binding was not blocked by torcetrapib. In whole blood, LPS induced tumor necrosis factor-alpha normally in the presence of torcetrapib. Furthermore, LPS had no effect on CETP activity. We conclude that the sepsis-related mortality of the ILLUMINATE trial was unlikely due to a direct effect of torcetrapib on LBP or BPI function, nor to inhibition of an interaction of CETP with LPS. Instead, we speculate that the negative outcome seen for patients with infections might be related to the changes in plasma lipoprotein composition and metabolism, or alternatively to the known off-target effects of torcetrapib, such as aldosterone elevation, which may have aggravated the effects of sepsis.
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http://dx.doi.org/10.1194/jlr.M002295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2853464PMC
May 2010

(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.

Bioorg Med Chem Lett 2009 Apr 13;19(7):1991-5. Epub 2009 Feb 13.

Pfizer Global Research & Development, Groton/New London Laboratories, Pfizer Inc, Groton, CT 06340, United States.

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.
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http://dx.doi.org/10.1016/j.bmcl.2009.02.041DOI Listing
April 2009

Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia.

Nat Struct Mol Biol 2007 May 15;14(5):413-9. Epub 2007 Apr 15.

Pfizer Inc., Eastern Point Road, Groton, Connecticut 06430, USA.

Proprotein convertase subtilisin kexin type 9 (PCSK9) lowers the abundance of surface low-density lipoprotein (LDL) receptor through an undefined mechanism. The structure of human PCSK9 shows the subtilisin-like catalytic site blocked by the prodomain in a noncovalent complex and inaccessible to exogenous ligands, and that the C-terminal domain has a novel fold. Biosensor studies show that PCSK9 binds the extracellular domain of LDL receptor with K(d) = 170 nM at the neutral pH of plasma, but with a K(d) as low as 1 nM at the acidic pH of endosomes. The D374Y gain-of-function mutant, associated with hypercholesterolemia and early-onset cardiovascular disease, binds the receptor 25 times more tightly than wild-type PCSK9 at neutral pH and remains exclusively in a high-affinity complex at the acidic pH. PCSK9 may diminish LDL receptors by a mechanism that requires direct binding but not necessarily receptor proteolysis.
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http://dx.doi.org/10.1038/nsmb1235DOI Listing
May 2007

Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules.

Nat Struct Mol Biol 2007 Feb 21;14(2):106-13. Epub 2007 Jan 21.

Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06430, USA.

Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.2-A resolution, revealing a 60-A-long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic phosphatidylcholine at each end. The two tunnel openings are large enough to allow lipid access, which is aided by a flexible helix and possibly also by a mobile flap. The curvature of the concave surface of CETP matches the radius of curvature of HDL particles, and potential conformational changes may occur to accommodate larger lipoprotein particles. Point mutations blocking the middle of the tunnel abolish lipid-transfer activities, suggesting that neutral lipids pass through this continuous tunnel.
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http://dx.doi.org/10.1038/nsmb1197DOI Listing
February 2007

Expression, purification, crystallization and structure of human adipocyte lipid-binding protein (aP2).

Acta Crystallogr Sect F Struct Biol Cryst Commun 2006 Nov 25;62(Pt 11):1058-60. Epub 2006 Oct 25.

Exploratory Medicinal Sciences, Pfizer Global Research and Development Groton Laboratories, Eastern Point Road, Groton, CT 06340, USA.

Human adipocyte lipid-binding protein (aP2) belongs to a family of intracellular lipid-binding proteins involved in the transport and storage of lipids. Here, the crystal structure of human aP2 with a bound palmitate is described at 1.5 A resolution. Unlike the known crystal structure of murine aP2 in complex with palmitate, this structure shows that the fatty acid is in a folded conformation and that the loop containing Phe57 acts as a lid to regulate ligand binding by excluding solvent exposure to the central binding cavity.
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http://dx.doi.org/10.1107/S1744309106038656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2225221PMC
November 2006

cis-2,5-dicyanopyrrolidine inhibitors of dipeptidyl peptidase IV: synthesis and in vitro, in vivo, and X-ray crystallographic characterization.

J Med Chem 2006 Jun;49(11):3068-76

Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06340, USA.

Inhibitors of the glucagon-like peptide-1 (GLP-1) degrading enzyme dipeptidyl peptidase IV (DPP-IV) have been shown to be effective treatments for type 2 diabetes in animal models and in human subjects. A novel series of cis-2,5-dicyanopyrrolidine alpha-amino amides were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-IV) for the treatment of type 2 diabetes. 1-({[1-(Hydroxymethyl)cyclopentyl]amino}acetyl)pyrrolidine-2,5-cis-dicarbonitrile (1c) is an achiral, slow-binding (time-dependent) inhibitor of DPP-IV that is selective for DPP-IV over other DPP isozymes and proline specific serine proteases, and which has oral bioavailability in preclinical species and in vivo efficacy in animal models. The mode of binding of the cis-2,5-dicyanopyrrolidine moiety was determined by X-ray crystallography. The hydrochloride salt of 1c was further profiled for development as a potential new treatment for type 2 diabetes.
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http://dx.doi.org/10.1021/jm0600085DOI Listing
June 2006

Crystal structure of the herpes simplex virus 1 DNA polymerase.

J Biol Chem 2006 Jun 24;281(26):18193-200. Epub 2006 Apr 24.

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

Herpesviruses are the second leading cause of human viral diseases. Herpes Simplex Virus types 1 and 2 and Varicella-zoster virus produce neurotropic infections such as cutaneous and genital herpes, chickenpox, and shingles. Infections of a lymphotropic nature are caused by cytomegalovirus, HSV-6, HSV-7, and Epstein-Barr virus producing lymphoma, carcinoma, and congenital abnormalities. Yet another series of serious health problems are posed by infections in immunocompromised individuals. Common therapies for herpes viral infections employ nucleoside analogs, such as Acyclovir, and target the viral DNA polymerase, essential for viral DNA replication. Although clinically useful, this class of drugs exhibits a narrow antiviral spectrum, and resistance to these agents is an emerging problem for disease management. A better understanding of herpes virus replication will help the development of new safe and effective broad spectrum anti-herpetic drugs that fill an unmet need. Here, we present the first crystal structure of a herpesvirus polymerase, the Herpes Simplex Virus type 1 DNA polymerase, at 2.7 A resolution. The structural similarity of this polymerase to other alpha polymerases has allowed us to construct high confidence models of a replication complex of the polymerase and of Acyclovir as a DNA chain terminator. We propose a novel inhibition mechanism in which a representative of a series of non-nucleosidic viral polymerase inhibitors, the 4-oxo-dihydroquinolines, binds at the polymerase active site interacting non-covalently with both the polymerase and the DNA duplex.
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http://dx.doi.org/10.1074/jbc.M602414200DOI Listing
June 2006

Crystal structure and substrate specificity of the beta-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus.

Protein Sci 2005 Aug 29;14(8):2087-94. Epub 2005 Jun 29.

GlaxoSmithKline, King of Prussia, Pennsylvania 19406, USA.

beta-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 A resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rank order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- > acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.
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http://dx.doi.org/10.1110/ps.051501605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2279320PMC
August 2005

Cholesteryl ester transfer protein variants have differential stability but uniform inhibition by torcetrapib.

J Biol Chem 2005 Apr 28;280(15):14918-22. Epub 2005 Jan 28.

Department of Discovery Pharmacogenomics, Pfizer Global Research and Development, Groton, Connecticut 06340, USA.

Cholesteryl ester transfer protein (CETP) is an important modulator of high density lipoprotein cholesterol in humans and thus considered to be a therapeutic target for preventing cardiovascular disease. The gene encoding CETP has been shown to be highly variable, with multiple single nucleotide polymorphisms responsible for altering both its transcription and sequence. Examining nine missense variants of CETP, we found some had significant associations with CETP mass and high density lipoprotein cholesterol levels. Two variants, Pro-373 and Gln-451, appear to be more stable in vivo, an observation mirrored by partial proteolysis studies performed in vitro. Because these naturally occurring variant proteins are potentially present in clinical populations that will be treated with CETP inhibitors, all commonly occurring haplotypes were tested to determine whether the proteins they encode could be inhibited by torcetrapib, a compound currently in clinical trials in combination with atorvastatin. Torcetrapib behaved similarly with all variants, with no significant differences in inhibition.
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http://dx.doi.org/10.1074/jbc.M500523200DOI Listing
April 2005