We describe a new serine protease inhibition motif in which binding is mediated by a cluster of very short hydrogen bonds (<2.3 Å) at the active site. This protease-inhibitor binding paradigm is ...observed at high resolution in a large set of crystal structures of trypsin, thrombin, and urokinase-type plasminogen activator (uPA) bound with a series of small molecule inhibitors (2-(2-phenol)indoles and 2-(2-phenol)benzimidazoles). In each complex there are eight enzyme-inhibitor or enzyme-water-inhibitor hydrogen bonds at the active site, three of which are very short. These short hydrogen bonds connect a triangle of oxygen atoms comprising OSer195γ, a water molecule co-bound in the oxyanion hole (H2Ooxy), and the phenolate oxygen atom of the inhibitor (O6′). Two of the other hydrogen bonds between the inhibitor and active site of the trypsin and uPA complexes become short in the thrombin counterparts, extending the three-centered short hydrogen-bonding array into a tetrahedral array of atoms (three oxygen and one nitrogen) involved in short hydrogen bonds. In the uPA complexes, the extensive hydrogen-bonding interactions at the active site prevent the inhibitor S1 amidine from forming direct hydrogen bonds with Asp189 because the S1 site is deeper in uPA than in trypsin or thrombin.
Ionization equilibria at the active site associated with inhibitor binding are probed through determination and comparison of structures over a wide range of pH (3.5 to 11.4) of thrombin complexes and of trypsin complexes in three different crystal forms. The high-pH trypsin-inhibitor structures suggest that His57 is protonated at pH values as high as 9.5. The pH-dependent inhibition of trypsin, thrombin, uPA and factor Xa by 2-(2-phenol)benzimidazole analogs in which the pKa of the phenol group is modulated is shown to be consistent with a binding process involving ionization of both the inhibitor and the enzyme. These data further suggest that the pKa of His57 of each protease in the unbound state in solution is about the same, ∼ 6.8. By comparing inhibition constants (Ki values), inhibitor solubilities, inhibitor conformational energies and corresponding structures of short and normal hydrogen bond-mediated complexes, we have estimated the contribution of the short hydrogen bond networks to inhibitor affinity (∼1.7 kcal/mol). The structures and Ki values associated with the short hydrogen-bonding motif are compared with those corresponding to an alternate, Zn2+-mediated inhibition motif at the active site. Structural differences among apo-enzymes, enzyme-inhibitor and enzyme-inhibitor-Zn2+ complexes are discussed in the context of affinity determinants, selectivity development, and structure-based inhibitor design.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Pyrimidine-based inhibitors of CaMKIIδ were identified. Through computational studies, a probable binding mode was identified leading to the design of ATP competitive inhibitors with improved ...potency, potential hinge interactions, and potent cellular activity.
Non-ATP competitive pyrimidine-based inhibitors of CaMKIIδ were identified. Computational studies were enlisted to predict the probable mode of binding. The results of the computational studies led to the design of ATP competitive inhibitors with optimized hinge interactions. Inhibitors of this class possessed improved enzyme and cellular activity compared to early leads.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
A new chip-based method to identify protein-protein interactions was developed using the guanine nucleotide exchange factor GRF2 and two interacting proteins, Ras and calmodulin, as model proteins. A ...generic immobilization strategy for FIAG-tagged bait proteins on a proteinrepellent streptavidin chip surface was implemented by presentation of an oriented anti-FUG antibody. A flow cell device, integrating different chip surfaces, was developed, and the interaction of immobilized GRF2 with the two analytes was verified by fluorescence assays. On-chip tryptic digest assays were then performed on the capture surface and analyzed by pLC-MS/MS. The interaction of GRF2 with calmodulin and Ras was demonstrated, and the lower limit of detection was determined. We also implemented an on-chip immunoprecipitation assay to identify GRF2-binding partners from complex protein mixtures. Cells overexpressing FIAG-GRF2 were lysed and then incubated with the anti-FIAG chip. In addition to detecting GRF2, we also identified calmodulin, demonstrating that this twhnique can successfully identify endogenous levels of proteins, bound to recombinant bait proteins. This chip-based method has the advantage that no subsequent gel separations of protein complexes prior to LC-MS analysis are required and is therefore amenable to miniaturized high-throughput determination of proteinprotein interactions. PERIODICAL ABSTRACT
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IJS, KILJ, NUK, PNG, UL, UM
Active site titration by a reversible tight-binding inhibitor normally depends on prior knowledge of the inhibition constant. Conversely, the determination of tight-binding inhibition constants ...normally requires prior knowledge of the active enzyme concentration. Often, neither of these quantities is known with sufficient accuracy. This paper describes experimental conditions under which both the enzyme active site concentration and the tight-binding inhibition constant can be determined simultaneously from a single dose-response curve. Representative experimental data are shown for the inhibition of human kallikrein.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Aryl-substituted maleimides were prepared and studied for their activity against CaMKII. Inhibitory activities ranged from 10
nM to >20
μM. Key predicted interactions with the kinase ATP site and ...hinge region were confirmed.
A family of aryl-substituted maleimides was prepared and studied for their activity against calmodulin dependant kinase. Inhibitory activities against the enzyme ranged from 10
nM to >20
μM and were dependant upon both the nature of the aryl group and the tether joining the basic amine to the indolyl maleimide core of the inhibitors. Key interactions with the kinase ATP site and hinge region, predicted by homology modeling, were confirmed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
A new chip-based method to identify protein−protein interactions was developed using the guanine nucleotide exchange factor GRF2 and two interacting proteins, Ras and calmodulin, as model proteins. A ...generic immobilization strategy for FLAG-tagged bait proteins on a protein-repellent streptavidin chip surface was implemented by presentation of an oriented anti-FLAG antibody. A flow cell device, integrating different chip surfaces, was developed, and the interaction of immobilized GRF2 with the two analytes was verified by fluorescence assays. On-chip tryptic digest assays were then performed on the capture surface and analyzed by μLC−MS/MS. The interaction of GRF2 with calmodulin and Ras was demonstrated, and the lower limit of detection was determined. We also implemented an on-chip immunoprecipitation assay to identify GRF2-binding partners from complex protein mixtures. Cells overexpressing FLAG-GRF2 were lysed and then incubated with the anti-FLAG chip. In addition to detecting GRF2, we also identified calmodulin, demonstrating that this technique can successfully identify endogenous levels of proteins, bound to recombinant bait proteins. This chip-based method has the advantage that no subsequent gel separations of protein complexes prior to LC−MS analysis are required and is therefore amenable to miniaturized high-throughput determination of protein−protein interactions.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
A new chip-based method to identify protein-protein interactions was developed using the guanine nucleotide exchange factor GRF2 and two interacting proteins, Ras and calmodulin, as model proteins. A ...generic immobilization strategy for FLAG-tagged bait proteins on a protein-repellent streptavidin chip surface was implemented by presentation of an oriented anti-FLAG antibody. A flow cell device, integrating different chip surfaces, was developed, and the interaction of immobilized GRF2 with the two analytes was verified by fluorescence assays. On-chip tryptic digest assays were then performed on the capture surface and analyzed by microLC-MS/MS. The interaction of GRF2 with calmodulin and Ras was demonstrated, and the lower limit of detection was determined. We also implemented an on-chip immunoprecipitation assay to identify GRF2-binding partners from complex protein mixtures. Cells overexpressing FLAG-GRF2 were lysed and then incubated with the anti-FLAG chip. In addition to detecting GRF2, we also identified calmodulin, demonstrating that this technique can successfully identify endogenous levels of proteins, bound to recombinant bait proteins. This chip-based method has the advantage that no subsequent gel separations of protein complexes prior to LC-MS analysis are required and is therefore amenable to miniaturized high-throughput determination of protein-protein interactions.
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IJS, KILJ, NUK, PNG, UL, UM
A family of aryl-substituted maleimides was prepared and studied for their activity against calmodulin dependant kinase. Inhibitory activities against the enzyme ranged from 10nM to >20microM and ...were dependant upon both the nature of the aryl group and the tether joining the basic amine to the indolyl maleimide core of the inhibitors. Key interactions with the kinase ATP site and hinge region, predicted by homology modeling, were confirmed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Non-ATP competitive pyrimidine-based inhibitors of CaMKIIdelta were identified. Computational studies were enlisted to predict the probable mode of binding. The results of the computational studies ...led to the design of ATP competitive inhibitors with optimized hinge interactions. Inhibitors of this class possessed improved enzyme and cellular activity compared to early leads.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
