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Kenneth M Comess,
Chaohong Sun,
Cele Abad-Zapatero,
Eric R Goedken,
Rebecca J Gum,
David W Borhani,
Maria Argiriadi,
Duncan R Groebe,
Yong Jia,
Jill E Clampit, [......],
Zhili Xin,
Gang Liu, Elizabeth H Fry,
Vincent Stoll,
Teresa I Ng,
David Banach,
Doug Marcotte,
David J Burns,
David J Calderwood,
Philip J Hajduk
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ABSTRACT: Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38α (involved in the formation of TNFα and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional (1)H/(13)C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38α both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.
ACS Chemical Biology 01/2011; 6(3):234-44. · 6.45 Impact Factor
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Thomas D Penning,
Gui-Dong Zhu,
Jianchun Gong,
Sheela Thomas,
Viraj B Gandhi,
Xuesong Liu,
Yan Shi,
Vered Klinghofer,
Eric F Johnson,
Chang H Park, [......],
Fritz G Buchanan,
Gail T Bukofzer,
Luis E Rodriguez,
Velitchka Bontcheva-Diaz,
Jennifer J Bouska,
Donald J Osterling,
Amanda M Olson,
Kennan C Marsh,
Yan Luo,
Vincent L Giranda
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ABSTRACT: We have developed a series of phenylpyrrolidine- and phenylpiperidine-substituted benzimidazole carboxamide poly(ADP-ribose) polymerase (PARP) inhibitors with excellent PARP enzyme potency as well as single-digit nanomolar cellular potency. These efforts led to the identification of (S)-2-(2-fluoro-4-(pyrrolidin-2-yl)phenyl)-1H-benzimidazole-4-carboxamide (22b, A-966492). Compound 22b displayed excellent potency against the PARP-1 enzyme with a K(i) of 1 nM and an EC(50) of 1 nM in a whole cell assay. In addition, 22b is orally bioavailable across multiple species, crosses the blood-brain barrier, and appears to distribute into tumor tissue. It also demonstrated good in vivo efficacy in a B16F10 subcutaneous murine melanoma model in combination with temozolomide and in an MX-1 breast cancer xenograft model both as a single agent and in combination with carboplatin.
Journal of Medicinal Chemistry 03/2010; 53(8):3142-53. · 4.80 Impact Factor
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ABSTRACT: Ionic liquids exhibit a variety of properties that make them attractive solvents for biomaterials. Given the potential for productive interaction between ionic liquids and biological macromolecules, we investigated the use of ionic liquids as precipitating agents and additives for protein crystallization for six model proteins (lysozyme, catalase, myoglobin, trypsin, glucose isomerase, and xylanase). The ionic liquids produced changes in crystal morphology and mediated significant increases in crystal size in some cases. Crystals grown using ionic liquids as precipitating agents or as additives provided X-ray diffraction resolution similar to or better than that obtained without ionic liquids. Based upon the experiments performed with model proteins, the ionic liquids were used as additives for the crystallization of the poorly diffracting monoclonal antibody 106.3 Fab in complex with the B-type natriuretic peptide (5-13). The ionic liquids improved the crystallization behavior and provided improved diffraction resulting in the determination of the structure. Ionic liquids should be considered as useful additives for the crystallization of other proteins.
08/2009;
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Julie Miyashiro,
Keith W Woods,
Chang H Park,
Xuesong Liu,
Yan Shi,
Eric F Johnson,
Jennifer J Bouska,
Amanda M Olson,
Yan Luo, Elizabeth H Fry,
Vincent L Giranda,
Thomas D Penning
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ABSTRACT: Based on screening hit 1, a series of tricyclic quinoxalinones have been designed and evaluated for inhibition of PARP-1. Substitutions at the 7- and 8-positions of the quinoxalinone ring led to a number of compounds with good enzymatic and cellular potency. The tricyclic quinoxalinone class is sensitive to modifications of both the amine substituent and the tricyclic core. The synthesis and structure-activity relationship studies are presented.
Bioorganic & medicinal chemistry letters 07/2009; 19(15):4050-4. · 2.65 Impact Factor
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ABSTRACT: B-type natriuretic peptide (BNP) is a naturally secreted regulatory hormone that influences blood pressure and vascular water retention in human physiology. The plasma BNP concentration is a clinically recognized biomarker for various cardiovascular diseases. Quantitative detection of BNP can be achieved in immunoassays using the high-affinity monoclonal IgG1 antibody 106.3, which binds an epitope spanning residues 5-13 of the mature bioactive peptide. To understand the structural basis of this molecular recognition, we crystallized the Fab fragment complexed with the peptide epitope and determined the three-dimensional structure by X-ray diffraction to 2.1 A resolution. The structure reveals the detailed interactions that five of the complementarity-determining regions make with the partially folded peptide. Thermodynamic measurements using fluorescence spectroscopy suggest that the interaction is enthalpy driven, with an overall change in free energy of binding, DeltaG = -54 kJ/mol, at room temperature. The parameters are interpreted on the basis of the structural information. The kinetics of binding suggest a diffusion-limited mechanism, whereby the peptide easily adopts a bound conformation upon interaction with the antibody. Moreover, comparative analysis with alanine-scanning results of the epitope explains the basis of selectivity for BNP over other related natriuretic peptides.
Proteins Structure Function and Bioinformatics 02/2009; 76(3):536-47. · 3.39 Impact Factor
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Thomas D Penning,
Gui-Dong Zhu,
Viraj B Gandhi,
Jianchun Gong,
Sheela Thomas,
Wilfried Lubisch,
Roland Grandel,
Wolfgang Wernet,
Chang H Park, Elizabeth H Fry, [......],
Eric F Johnson,
Cherrie K Donawho,
David J Frost,
Velitchka Bontcheva-Diaz,
Jennifer J Bouska,
Amanda M Olson,
Kennan C Marsh,
Yan Luo,
Saul H Rosenberg,
Vincent L Giranda
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ABSTRACT: We have developed a series of cyclic amine-containing benzimidazole carboxamide poly(ADP-ribose)polymerase (PARP) inhibitors, with good PARP-1 enzyme potency, as well as cellular potency. These efforts led to the identification of a lead preclinical candidate, 10b, 2-(1-propylpiperidin-4-yl)-1H-benzimidazole-4-carboxamide (A-620223). 10b displayed very good potency against both the PARP-1 enzyme with a K(i) of 8nM and in a whole cell assay with an EC(50) of 3nM. 10b is aqueous soluble, orally bioavailable across multiple species, and demonstrated good in vivo efficacy in a B16F10 subcutaneous murine melanoma model in combination with temozolomide (TMZ) and in an MX-1 breast xenograph model in combination with cisplatin.
Bioorganic & medicinal chemistry 06/2008; 16(14):6965-75. · 2.82 Impact Factor
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Mei Liu,
Sanyi Wang,
Jill E Clampit,
Rebecca J Gum,
Deanna L Haasch,
Cristina M Rondinone,
James M Trevillyan,
Cele Abad-Zapatero, Elizabeth H Fry,
Hing L Sham,
Gang Liu
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ABSTRACT: A new series of 4-anilinopyrimidines has been synthesized and evaluated as JNK1 inhibitors. SAR studies led to the discovery of potent JNK1 inhibitors with good enzymatic activity as well as cellular potency represented by compound 2b. Kinase selectivity profile and the crystal structure of 2b are also described.
Bioorganic & Medicinal Chemistry Letters 03/2007; 17(3):668-72. · 2.55 Impact Factor
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Hongyu Zhao,
Michael D Serby,
Zhili Xin,
Bruce G Szczepankiewicz,
Mei Liu,
Christi Kosogof,
Bo Liu,
Lissa T J Nelson,
Eric F Johnson,
Sanyi Wang,
Terry Pederson,
Rebecca J Gum,
Jill E Clampit,
Deanna L Haasch,
Cele Abad-Zapatero, Elizabeth H Fry,
Cristina Rondinone,
James M Trevillyan,
Hing L Sham,
Gang Liu
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ABSTRACT: C-Jun NH2 terminal kinases (JNKs) are important cell signaling enzymes. JNK1 plays a central role in linking obesity and insulin resistance. JNK2 and JNK3 may be involved in inflammatory and neurological disorders, respectively. Small-molecule JNK inhibitors could be valuable tools to study the therapeutic benefits of inhibiting these enzymes and as leads for potential drugs targeting JNKs. In this report, we disclose a series of potent and highly selective JNK inhibitors with good pharmacokinetic profiles.
Journal of Medicinal Chemistry 08/2006; 49(15):4455-8. · 5.25 Impact Factor
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Bruce G Szczepankiewicz,
Christi Kosogof,
Lissa T J Nelson,
Gang Liu,
Bo Liu,
Hongyu Zhao,
Michael D Serby,
Zhili Xin,
Mei Liu,
Rebecca J Gum, [......],
Edward T Olejniczak,
Chaohong Sun,
Sarah A Dorwin,
Kristi Haskins,
Cele Abad-Zapatero, Elizabeth H Fry,
Charles W Hutchins,
Hing L Sham,
Cristina M Rondinone,
James M Trevillyan
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ABSTRACT: The c-Jun N-terminal kinases (JNK-1, -2, and -3) are members of the mitogen activated protein (MAP) kinase family of enzymes. They are activated in response to certain cytokines, as well as by cellular stresses including chemotoxins, peroxides, and irradiation. They have been implicated in the pathology of a variety of different diseases with an inflammatory component including asthma, stroke, Alzheimer's disease, and type 2 diabetes mellitus. In this work, high-throughput screening identified a JNK inhibitor with an excellent kinase selectivity profile. Using X-ray crystallography and biochemical screening to guide our lead optimization, we prepared compounds with inhibitory potencies in the low-double-digit nanomolar range, activity in whole cells, and pharmacokinetics suitable for in vivo use. The new compounds were over 1,000-fold selective for JNK-1 and -2 over other MAP kinases including ERK2, p38alpha, and p38delta and showed little inhibitory activity against a panel of 74 kinases.
Journal of Medicinal Chemistry 07/2006; 49(12):3563-80. · 5.25 Impact Factor
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Kenton L Longenecker,
Kent D Stewart,
David J Madar,
Clarissa G Jakob, Elizabeth H Fry,
Sherwin Wilk,
Chun W Lin,
Stephen J Ballaron,
Michael A Stashko,
Thomas H Lubben,
Hong Yong,
Daisy Pireh,
Zhonghua Pei,
Fatima Basha,
Paul E Wiedeman,
Thomas W von Geldern,
James M Trevillyan,
Vincent S Stoll
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ABSTRACT: Dipeptidyl peptidase IV (DPP-IV) belongs to a family of serine peptidases, and due to its indirect regulatory role in plasma glucose modulation, DPP-IV has become an attractive pharmaceutical target for diabetes therapy. DPP-IV inactivates the glucagon-like peptide (GLP-1) and several other naturally produced bioactive peptides that contain preferentially a proline or alanine residue in the second amino acid sequence position by cleaving the N-terminal dipeptide. To elucidate the details of the active site for structure-based drug design, we crystallized a natural source preparation of DPP-IV isolated from rat kidney and determined its three-dimensional structure using X-ray diffraction techniques. With a high degree of similarity to structures of human DPP-IV, the active site architecture provides important details for the design of inhibitory compounds, and structures of inhibitor-protein complexes offer detailed insight into three-dimensional structure-activity relationships that include a conformational change of Tyr548. Such accommodation is exemplified by the response to chemical substitution on 2-cyanopyrrolidine inhibitors at the 5 position, which conveys inhibitory selectivity for DPP-IV over closely related homologues. A similar conformational change is also observed in the complex with an unrelated synthetic inhibitor containing a xanthine core that is also selective for DPP-IV. These results suggest the conformational flexibility of Tyr548 is unique among protein family members and may be utilized in drug design to achieve peptidase selectivity.
Biochemistry 07/2006; 45(24):7474-82. · 3.42 Impact Factor
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Zhonghua Pei,
Xiaofeng Li,
Kenton Longenecker,
Thomas W von Geldern,
Paul E Wiedeman,
Thomas H Lubben,
Bradley A Zinker,
Kent Stewart,
Stephen J Ballaron,
Michael A Stashko, [......],
Todd S McDermott,
Lakshmi Bhagavatula,
Michael G Fickes,
Daisy Pireh,
Larry R Solomon,
Marc R Lake,
Rohinton Edalji, Elizabeth H Fry,
Hing L Sham,
James M Trevillyan
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ABSTRACT: A series of (5-substituted pyrrolidinyl-2-carbonyl)-2-cyanopyrrolidine (C5-Pro-Pro) analogues was discovered as dipeptidyl peptidase IV (DPPIV) inhibitors as a potential treatment of diabetes and obesity. X-ray crystallography data show that these inhibitors bind to the catalytic site of DPPIV with the cyano group forming a covalent bond with the serine residue of DPPIV. The C5-substituents make various interactions with the enzyme and affect potency, chemical stability, selectivity, and PK properties of the inhibitors. Optimized analogues are extremely potent with subnanomolar K(i)'s, are chemically stable, show very little potency decrease in the presence of plasma, and exhibit more than 1,000-fold selectivity against related peptidases. The best compounds also possess good PK and are efficacious in lowering blood glucose in an oral glucose tolerance test in ZDF rats.
Journal of Medicinal Chemistry 07/2006; 49(12):3520-35. · 5.25 Impact Factor
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Mei Liu,
Zhili Xin,
Jill E Clampit,
Sanyi Wang,
Rebecca J Gum,
Deanna L Haasch,
James M Trevillyan,
Cele Abad-Zapatero, Elizabeth H Fry,
Hing L Sham,
Gang Liu
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ABSTRACT: A novel class of 1,9-dihydro-9-hydroxypyrazolo[3,4-b]quinolin-4-ones as c-Jun-N-terminal kinase (JNK) inhibitors is described. These compounds were synthesized via the condensation of 2-nitrobenzaldehydes and hydroxypyrazoles. The structure-activity relationships (SAR) and kinase selectivity profile of the inhibitors are also discussed. Compound 16 was identified as a potent JNK inhibitor with good cellular potency.
Bioorganic & Medicinal Chemistry Letters 06/2006; 16(10):2590-4. · 2.55 Impact Factor
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ABSTRACT: We have identified benzoxazole benzenesulfonamide 1 as a novel allosteric inhibitor of fructose-1,6-bisphosphatase (FBPase-1). X-ray crystallographic and biological studies of 1 indicate a distinct binding mode that recapitulates features of several previously reported FBPase-1 inhibitor classes.
Bioorganic & Medicinal Chemistry Letters 05/2006; 16(7):1811-5. · 2.55 Impact Factor
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ABSTRACT: A series of novel benzoxazole benzenesulfonamides was synthesized as inhibitors of fructose-1,6-bisphosphatase (FBPase-1). Extensive SAR studies led to a potent inhibitor, 53, with an IC(50) of 0.57microM. Compound 17 exhibited excellent bioavailability and a good pharmacokinetic profile in rats.
Bioorganic & Medicinal Chemistry Letters 05/2006; 16(7):1807-10. · 2.55 Impact Factor
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Geoffrey F Stamper,
Kenton L Longenecker, Elizabeth H Fry,
Clarissa G Jakob,
Alan S Florjancic,
Yu-Gui Gu,
David D Anderson,
Curt S Cooper,
Tianyuan Zhang,
Richard F Clark,
Yingna Cia,
Candace L Black-Schaefer,
J Owen McCall,
Claude G Lerner,
Philip J Hajduk,
Bruce A Beutel,
Vincent S Stoll
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ABSTRACT: The D-Ala-D-Ala adding enzyme (MurF) from Streptococcus pneumoniae catalyzes the ATP-dependent formation of the UDP-MurNAc-pentapeptide, a critical component of the bacterial cell wall. MurF is a potential target for antibacterial design because it is unique to bacteria and performs an essential non-redundant function in the bacterial cell. The recent discovery and subsequent cocrystal structure determination of MurF in complex with a new class of inhibitors served as a catalyst to begin a medicinal chemistry program aimed at improving their potency. We report here a multidisciplinary approach to this effort that allowed for rapid generation of cocrystal structures, thereby providing the crystallographic information critical for driving the inhibitor optimization process. This effort resulted in the discovery of low-nanomolar inhibitors of this bacterial enzyme.
Chemical Biology & Drug Design 02/2006; 67(1):58-65. · 2.28 Impact Factor
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Kenton L Longenecker,
Geoffrey F Stamper,
Philip J Hajduk, Elizabeth H Fry,
Clarissa G Jakob,
John E Harlan,
Rohinton Edalji,
Diane M Bartley,
Karl A Walter,
Larry R Solomon,
Thomas F Holzman,
Yu Gui Gu,
Claude G Lerner,
Bruce A Beutel,
Vincent S Stoll
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ABSTRACT: In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF-inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target.
Protein Science 01/2006; 14(12):3039-47. · 2.80 Impact Factor
