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1.
ACS Omega ; 6(29): 18635-18650, 2021 Jul 27.
Article in English | MEDLINE | ID: mdl-34337203

ABSTRACT

Here, we described the design, by fragment merging and multiparameter optimization, of selective MMP-13 inhibitors that display an appropriate balance of potency and physicochemical properties to qualify as tool compounds suitable for in vivo testing. Optimization of potency was guided by structure-based insights, specifically to replace an ester moiety and introduce polar directional hydrogen bonding interactions in the core of the molecule. By introducing polar enthalpic interactions in this series of inhibitors, the overall beneficial physicochemical properties were maintained. These physicochemical properties translated to excellent drug-like properties beyond potency. In a murine model of rheumatoid arthritis, treatment of mice with selective inhibitors of MMP-13 resulted in a statistically significant reduction in the mean arthritic score vs control when dosed over a 14 day period.

3.
J Med Chem ; 56(11): 4465-81, 2013 Jun 13.
Article in English | MEDLINE | ID: mdl-23659209

ABSTRACT

Chymase plays an important and diverse role in the homeostasis of a number of cardiovascular processes. Herein, we describe the identification of potent, selective chymase inhibitors, developed using fragment-based, structure-guided linking and optimization techniques. High-concentration biophysical screening methods followed by high-throughput crystallography identified an oxindole fragment bound to the S1 pocket of the protein exhibiting a novel interaction pattern hitherto not observed in chymase inhibitors. X-ray crystallographic structures were used to guide the elaboration/linking of the fragment, ultimately leading to a potent inhibitor that was >100-fold selective over cathepsin G and that mitigated a number of liabilities associated with poor physicochemical properties of the series it was derived from.


Subject(s)
Benzimidazoles/chemistry , Cardiovascular Agents/chemistry , Chymases/antagonists & inhibitors , Serine Proteinase Inhibitors/chemistry , Benzimidazoles/chemical synthesis , Benzimidazoles/metabolism , Cardiovascular Agents/chemical synthesis , Cardiovascular Agents/metabolism , Catalytic Domain , Chymases/chemistry , Crystallography, X-Ray , Humans , In Vitro Techniques , Microsomes, Liver/metabolism , Models, Molecular , Molecular Structure , Protein Binding , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/metabolism , Structure-Activity Relationship
4.
J Med Chem ; 54(23): 8174-87, 2011 Dec 08.
Article in English | MEDLINE | ID: mdl-22017539

ABSTRACT

Matrix metalloproteases (MMPs) play an important role in cartilage homeostasis under both normal and inflamed disease states and, thus, have become attractive targets for the treatment of arthritic diseases. Herein, we describe the identification of a potent, selective MMP-13 inhibitor, developed using fragment-based structure-guided lead identification and optimization techniques. Virtual screening methods identified a novel, indole-based MMP-13 inhibitor that bound into the S1' pocket of the protein exhibiting a novel interaction pattern hitherto not observed in MMP-13 inhibitors. X-ray crystallographic structures were used to guide the elaboration of the fragment, ultimately leading to a potent inhibitor that was >100-fold selective over nine other MMP isoforms tested.


Subject(s)
Indoles/chemical synthesis , Matrix Metalloproteinase Inhibitors , Crystallography, X-Ray , Humans , Indoles/chemistry , Matrix Metalloproteinase 13/chemistry , Models, Molecular , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Structure-Activity Relationship
5.
Bioorg Med Chem Lett ; 19(18): 5321-4, 2009 Sep 15.
Article in English | MEDLINE | ID: mdl-19692239

ABSTRACT

Discovery and optimization of potency and selectivity of a non-Zn-chelating MMP-13 inhibitor with the aid of protein co-crystal structural information is reported. This inhibitor was observed to have a binding mode distinct from previously published MMP-13 inhibitors. Potency and selectivity were improved by extending the hit structure out from the active site into the S1' pocket.


Subject(s)
Chelating Agents/pharmacology , Matrix Metalloproteinase 13/metabolism , Matrix Metalloproteinase Inhibitors , Protease Inhibitors/pharmacology , Catalytic Domain , Chelating Agents/chemistry , Matrix Metalloproteinase 13/chemistry , Models, Molecular , Protease Inhibitors/chemistry , Protein Binding , Structure-Activity Relationship
6.
J Med Chem ; 52(7): 1814-27, 2009 Apr 09.
Article in English | MEDLINE | ID: mdl-19256503

ABSTRACT

A series of inhibitors of Pim-2 kinase identified by high-throughput screening is described. Details of the hit validation and lead generation process and structure-activity relationship (SAR) studies are presented. Disclosure of an unconventional binding mode for 1, as revealed by X-ray crystallography using the highly homologous Pim-1 protein, is also presented, and observed binding features are shown to correlate with the Pim-2 SAR. While highly selective within the kinase family, the series shows similar potency for both Pim-1 and Pim-2, which was expected on the basis of homology, but unusual in light of reports in the literature documenting a bias for Pim-1. A rationale for these observations based on Pim-1 and Pim-2 K(M(ATP)) values is suggested. Some interesting cross reactivity with casein kinase-2 was also identified, and structural features which may contribute to the association are discussed.


Subject(s)
Azepines/chemistry , Models, Molecular , Phenylpropionates/chemistry , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Proto-Oncogene Proteins c-pim-1/chemistry , Azepines/chemical synthesis , Binding Sites , Casein Kinase II/antagonists & inhibitors , Casein Kinase II/chemistry , Crystallography, X-Ray , Phenylpropionates/chemical synthesis , Stereoisomerism , Structure-Activity Relationship
7.
Proc Natl Acad Sci U S A ; 104(15): 6353-8, 2007 Apr 10.
Article in English | MEDLINE | ID: mdl-17395714

ABSTRACT

p38 MAPK and MAPK-activated protein kinase 2 (MK2) are key components of signaling pathways leading to many cellular responses, notably the proinflammatory cytokine production. The physical association of p38alpha isoform and MK2 is believed to be physiologically important for this signaling. We report the 2.7-A resolution crystal structure of the unphosphorylated complex between p38alpha and MK2. These protein kinases bind "head-to-head," present their respective active sites on approximately the same side of the heterodimer, and form extensive intermolecular interactions. Among these interactions, the MK2 Ile-366-Ala-390, which includes the bipartite nuclear localization signal, binds to the p38alpha-docking region. This binding supports the involvement of noncatalytic regions to the tight binding of the MK2:p38alpha binary assembly. The MK2 residues 345-365, containing the nuclear export signal, block access to the p38alpha active site. Some regulatory phosphorylation regions of both protein kinases engage in multiple interactions with one another in this complex. This structure gives new insights into the regulation of the protein kinases p38alpha and MK2, aids in the better understanding of their known cellular and biochemical studies, and provides a basis for understanding other regulatory protein-protein interactions involving signal transduction proteins.


Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Models, Molecular , Multiprotein Complexes/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/immunology , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cloning, Molecular , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Mice , Multiprotein Complexes/chemistry , Phosphorylation , Protein Binding , Protein Conformation , Protein Serine-Threonine Kinases/chemistry , X-Ray Diffraction , p38 Mitogen-Activated Protein Kinases/chemistry
8.
J Med Chem ; 49(16): 5013-7, 2006 Aug 10.
Article in English | MEDLINE | ID: mdl-16884313

ABSTRACT

The NMR structure is presented for compound 1 (BMS-480404) (Ki = 33 (+/-2) nM) bound to keratinocyte fatty acid-binding protein. This article describes interactions between a high affinity drug-like compound and a member of the fatty acid-binding protein family. A benzyl group ortho to the mandelic acid in 1 occupies an area of the protein that fatty acids do not normally contact. Similar to that in the kFABP-palmitic acid structure, the acid moiety in 1 is proximal to R129 and Y131. Computational modeling indicates that the acid moiety in 1 interacts indirectly via a modeled water molecule to R109.


Subject(s)
Fatty Acid-Binding Proteins/antagonists & inhibitors , Fatty Acid-Binding Proteins/chemistry , Keratinocytes/metabolism , Binding Sites , Computer Simulation , Fatty Acid-Binding Proteins/metabolism , Humans , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Structure
9.
Protein Sci ; 14(6): 1472-84, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15929997

ABSTRACT

CFE88 is a conserved essential gene product from Streptococcus pneumoniae. This 227-residue protein has minimal sequence similarity to proteins of known 3D structure. Sequence alignment models and computational protein threading studies suggest that CFE88 is a methyltransferase. Characterization of the conformation and function of CFE88 has been performed by using several techniques. Backbone atom and limited side-chain atom NMR resonance assignments have been obtained. The data indicate that CFE88 has two domains: an N-terminal domain with 163 residues and a C-terminal domain with 64 residues. The C-terminal domain is primarily helical, while the N-terminal domain has a mixed helical/extended (Rossmann) fold. By aligning the experimentally observed elements of secondary structure, an initial unrefined model of CFE88 has been constructed based on the X-ray structure of ErmC' methyltransferase (Protein Data Bank entry 1QAN). NMR and biophysical studies demonstrate binding of S-adenosyl-L-homocysteine (SAH) to CFE88; these interactions have been localized by NMR to the predicted active site in the N-terminal domain. Mutants that target this predicted active site (H26W, E46R, and E46W) have been constructed and characterized. Overall, our results both indicate that CFE88 is a methyltransferase and further suggest that the methyltransferase activity is essential for bacterial survival.


Subject(s)
Bacterial Proteins/chemistry , Methyltransferases/chemistry , Streptococcus pneumoniae/enzymology , Structural Homology, Protein , Amino Acid Sequence , Molecular Sequence Data , Protein Structure, Tertiary
10.
Article in English | MEDLINE | ID: mdl-16508102

ABSTRACT

Pim kinases, including Pim-1, Pim-2 and Pim-3, belong to a distinctive serine/threonine protein-kinase family. They are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Their kinase domains are highly homologous to one another, but share low sequence identity to other kinases. Specifically, there are two proline residues in the conserved hinge-region sequence ERPXPX separated by a residue that is non-conserved among Pim kinases. Full-length human Pim-1 kinase (1-313) was cloned and expressed in Escherichia coli as a GST-fusion protein and truncated to Pim-1 (14-313) by thrombin digestion during purification. The Pim-1 (14-313) protein was purified to high homogeneity and monodispersity. This protein preparation yielded small crystals in the initial screening and large crystals after optimization. The large crystals of apo Pim-1 enzyme diffracted to 2.1 A resolution and belong to space group P6(5), with unit-cell parameters a = b = 95.9, c = 80.0 A, beta = 120 degrees and one molecule per asymmetric unit.


Subject(s)
Proto-Oncogene Proteins c-pim-1/chemistry , Proto-Oncogene Proteins c-pim-1/isolation & purification , Apoenzymes/chemistry , Base Sequence , Cloning, Molecular , Crystallization , Crystallography, X-Ray , DNA Primers , Humans , Protein Conformation , Proto-Oncogene Proteins c-pim-1/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Restriction Mapping , Transfection
11.
J Biol Chem ; 280(7): 6130-7, 2005 Feb 18.
Article in English | MEDLINE | ID: mdl-15525646

ABSTRACT

Pim-1 kinase is a member of a distinct class of serine/threonine kinases consisting of Pim-1, Pim-2, and Pim-3. Pim kinases are highly homologous to one another and share a unique consensus hinge region sequence, ER-PXPX, with its two proline residues separated by a non-conserved residue, but they (Pim kinases) have <30% sequence identity with other kinases. Pim-1 has been implicated in both cytokine-induced signal transduction and the development of lymphoid malignancies. We have determined the crystal structures of apo Pim-1 kinase and its AMP-PNP (5'-adenylyl-beta,gamma-imidodiphosphate) complex to 2.1-angstroms resolutions. The structures reveal the following. 1) The kinase adopts a constitutively active conformation, and extensive hydrophobic and hydrogen bond interactions between the activation loop and the catalytic loop might be the structural basis for maintaining such a conformation. 2) The hinge region has a novel architecture and hydrogen-bonding pattern, which not only expand the ATP pocket but also serve to establish unambiguously the alignment of the Pim-1 hinge region with that of other kinases. 3) The binding mode of AMP-PNP to Pim-1 kinase is unique and does not involve a critical hinge region hydrogen bond interaction. Analysis of the reported Pim-1 kinase-domain structures leads to a hypothesis as to how Pim kinase activity might be regulated in vivo.


Subject(s)
Adenylyl Imidodiphosphate/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Adenylyl Imidodiphosphate/chemistry , Amino Acid Sequence , Apoproteins/chemistry , Apoproteins/metabolism , Catalytic Domain , Crystallography, X-Ray , Enzyme Activation , Humans , Models, Molecular , Protein Binding , Protein Conformation , Protein Folding , Proto-Oncogene Proteins c-pim-1 , Structure-Activity Relationship
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