Structural basis for selective inhibition of Src family kinases by PP1.

BACKGROUND: Small-molecule inhibitors that can target individual kinases are powerful tools for use in signal transduction research. It is difficult to find such compounds because of the enormous number of protein kinases and the highly conserved nature of their catalytic domains. Recently, a novel, potent, Src family selective tyrosine kinase inhibitor was reported (PP1). Here, we study the structural basis for this inhibitor's specificity for Src family kinases. RESULTS: A single residue corresponding to Ile338 (v-Src numbering; Thr338 in c-Src) in Src family tyrosine kinases largely controls PP1's ability to inhibit protein kinases. Mutation of Ile338 to a larger residue such as methionine or phenylalanine in v-Src makes this inhibitor less potent. Conversely, mutation of Ile338 to alanine or glycine increases PP1's potency. PP1 can inhibit Ser/Thr kinases if the residue corresponding to Ile338 in v-Src is mutated to glycine. We have accurately predicted several non-Src family kinases that are moderately (IC(50) approximately 1 microM) inhibited by PP1, including c-Abl and the MAP kinase p38. CONCLUSIONS: Our mutagenesis studies of the ATP-binding site in both tyrosine kinases and Ser/Thr kinases explain why PP1 is a specific inhibitor of Src family tyrosine kinases. Determination of the structural basis of inhibitor specificity will aid in the design of more potent and more selective protein kinase inhibitors. The ability to desensitize a particular kinase to PP1 inhibition of residue 338 or conversely to sensitize a kinase to PP1 inhibition by mutation should provide a useful basis for chemical genetic studies of kinase signal transduction.     

Structural basis for cyclophellitol inhibition of a beta-glucosidase

The structural basis for beta-glucosidase inhibition by cyclophellitol is demonstrated using X-ray crystallography, enzyme kinetics and mass spectrometry.     

Structural basis for the broad-spectrum inhibition of metallo-beta-lactamases by thiols

The development of broad-spectrum metallo-beta-lactamase (MBL) inhibitors is challenging due to structural diversity and differences in metal utilisation by these enzymes. Analysis of structural data, followed by non-denturing mass spectrometric analyses, identified thiols proposed to inhibit representative MBLs from all three sub-classes: B1, B2 and B3. Solution analyses led to the identification of broad spectrum inhibitors, including potent inhibitors of the CphA MBL (Aeromonas hydrophila). Structural studies revealed that, as observed for other B1 and B3 MBLs, inhibition of the L1 MBL thiols involves metal chelation. Evidence is reported that this is not the case for inhibition of the CphA enzyme by some thiols; the crystal structure of the CphA-Zn-inhibitor complex reveals a binding mode in which the thiol does not interact with the zinc. The structural data enabled the design and the production of further more potent inhibitors. Overall the results suggest that the development of reasonably broad-spectrum MBL inhibitors should be possible.     

Structural basis for selective inhibition of trypanosomatid glyceraldehyde-3-phosphate dehydrogenase: molecular docking and 3D QSAR studies

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is as an attractive target for the development of novel antitrypanosomatid agents. In the present work, comparative molecular field analysis and comparative molecular similarity index analysis were conducted on a large series of selective inhibitors of trypanosomatid GAPDH. Four statistically significant models were obtained ( r2 > 0.90 and q2 > 0.70), indicating their predictive ability for untested compounds. The models were then used to predict the potency of an external test set, and the predicted values were in good agreement with the experimental results. Molecular modeling studies provided further insight into the structural basis for selective inhibition of trypanosomatid GAPDH.     

Collisionally assisted, highly selective laser isotope separation of carbon-13

We have further developed our recently reported two-laser technique for highly selective molecular isotope separation of carbon-13 [Boyarkin, Kowalczyk, and Rizzo, J. Chem. Phys. 118, 93 (2003)] with the objective of increasing the yield. An essential feature of this approach in its original conception is the significant increase of isotopic selectivity that occurs through collisions during the time between the overtone preexcitation laser pulse and the multiphoton dissociation pulse. We demonstrate here that under certain conditions, this collisional enhancement of the selectivity works equally well when the two pulses are overlapped in time, allowing the overall isotopic selectivity of the process to remain high while achieving a significant increase in the absolute dissociation yield. We also find that proper shaping of the CO2 laser dissociation pulse makes the fluence required for dissociation sufficiently low to allow irradiation of a large reaction volume by unfocused laser beams. Together, these factors may make this laser isotope separation scheme competitive with existing separation methods.     

Unconventional, highly selective CO2 adsorption in zeolite SSZ-13

Low-pressure adsorption of carbon dioxide and nitrogen was studied in both acidic and copper-exchanged forms of SSZ-13, a zeolite containing an 8-ring window. Under ideal conditions for industrial separations of CO(2) from N(2), the ideal adsorbed solution theory selectivity is >70 in each compound. For low gas coverage, the isosteric heat of adsorption for CO(2) was found to be 33.1 and 34.0 kJ/mol for Cu- and H-SSZ-13, respectively. From in situ neutron powder diffraction measurements, we ascribe the CO(2) over N(2) selectivity to differences in binding sites for the two gases, where the primary CO(2) binding site is located in the center of the 8-membered-ring pore window. This CO(2) binding mode, which has important implications for use of zeolites in separations, has not been observed before and is rationalized and discussed relative to the high selectivity for CO(2) over N(2) in SSZ-13 and other zeolites containing 8-ring windows.     

Structural basis for thrombin activation of a protease-activated receptor: inhibition of intramolecular liganding

Protease-activated G protein-coupled receptors (PAR1-4) are tethered-ligand receptors that are activated by proteolytic cleavage of the extracellular domain (exodomain) of the receptor. PAR1, the prototypic member of the PAR family, is the high-affinity thrombin receptor of platelets and vascular endothelium and plays a critical role in blood coagulation, thrombosis, and inflammation. Here, we describe the solution structure of the thrombin-cleaved exodomain of PAR1. The side chains of a hydrophobic hirudin-like (Hir) sequence and adjacent anionic motif project into solution. Docking of the exodomain Hir sequence to exosite I of thrombin reveals that the tethered ligand in the cleaved exodomain bends away from thrombin, leaving its active site available to another large macromolecular substrate. The N-terminal ligand is longer than anticipated and forms an intramolecular complex with a region located in the C terminus of the exodomain. Mutational analysis confirmed that this C-terminal region is a ligand binding site for both intra- and intermolecular ligands. A lipidated-ligand binding site peptide was found to be an effective inhibitor of thrombin-induced platelet aggregation.     

Structural basis for inhibition of protein tyrosine phosphatases by Keggin compounds phosphomolybdate and phosphotungstate

Protein-tyrosine phosphatases (PTPs) constitute a family of receptor-like, and cytoplasmic enzymes, which catalyze the dephosphorylation of phosphotyrosine residues in a variety of receptors and signaling molecules. Together with protein tyrosine kinases (PTKs), PTPs are critically involved in regulating many cellular signaling processes. In this study, diverse compounds were screened for PTP inhibition and selectively screened for inhibitors with the end product inhibition properties. Among phosphate analogues and their derivatives for PTP inhibition, Keggin compounds phosphomolybdate (PM) and phosphotungstate (PT) strongly inhibited both PTP-1B and SHP-1, with K(i) values of 0.06-1.2 micromM in the presence of EDTA. Unlike the vanadium compounds, inhibition potencies of PM and PT were not significantly affected by EDTA. PM and PT were potent, competitive inhibitors for PTPs, but relatively poor inhibitors of Ser/Thr phosphatase. Interestingly, PM and PT did not inhibit alkaline phosphatase at all. The crystal structure of PTP-1B in complex with PM, at 2.0 A resolution, reveals that MoO(3), derived from PM by hydrolysis, binds at the active site. The molybdenium atom of the inhibitor is coordinated with six ligands: three oxo-ligands, two apical water molecules and a S atom of the catalytic cysteine residue. In support of the crystallographic finding, we observed that molybdenium oxides (MoO(3), MoO(2), and MoO(2)Cl(2)) inhibited PTP-1B with IC(50) in the range 5-15 micromM.     

A highly selective method for the synthesis of phenylacetaldehyde

Phenylacetaldehyde is synthesized in high yield by isomerization of styrene oxide and by dehydratation of styrene glycol in gas phase using H-ZSM-5 zeolite, Silicalite and silica gels as catalysts.     

A highly selective colorimetric sulfide assay based on the inhibition of the peroxidase-like activity of copper nanoclusters

The authors report that sulfide ions are capable of inhibiting the peroxidase-like activity of copper nanoclusters (CuNCs). The catalytic activity of CuNCs toward the oxidation of the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine by H₂O₂ is remarkably decreased in the presence of sulfide. Based on this finding, a colorimetric assay was developed for the rapid determination of sulfide. Best operated at a wavelength of 652 nm, it has a 0.5 μM detection limit. The method is highly selective and has been successfully applied to the quantification of sulfide in environmental water samples.

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Graphical abstract The catalytic activity of CuNCs toward the oxidation of 3,3′,5,5′-tetramethylbenzidine by H₂O₂ is remarkably decreased in the presence of sulfide ions. This finding has been applied to design a method for colorimetric quantification of sulfide ions in environmental samples.

     

Coordination of the 13C-NMR. spectrum of rifamycin S on the basis of selective uncoupling of protons

¹³C-NMR. spectra of rifamycin-S and some of its derivatives have been measured. A unambigous assignment of the signals, due to the hydrogen bearing ¹³C atoms, was possible with heteronuclear double resonance experiments. On the other hand the resonances of the quarternary ¹³C atoms could be ascribed only tentatively. ¹³C-NMR. spectroscopy gives a more complete picture of the rifamycins, especially about the naphthoquinone chromophore, than previously obtained by ¹H-NMR.     

Structural determinants of CDK4 inhibition and design of selective ATP competitive inhibitors

A number of selective inhibitors of the CDK4/cyclin D1 complex have been reported recently. Due to the absence of an experimental CDK4 structure, the ligand and protein determinants contributing to CDK4 selectivity are poorly understood at present. Here, we report the use of computational methods to elucidate the characteristics of selectivity and to derive the structural basis for specific, high-affinity binding of inhibitors to the CDK4 active site. From these data, the hypothesis emerged that appropriate incorporation of an ionizable function into a CDK2 inhibitor results in more favorable binding to CDK4. This knowledge was applied to the design of compounds in the otherwise CDK2-selective 2-anilino-4-(thiazol-5-yl)pyrimidine pharmacophore that are potent and highly selective ATP antagonists of CDK4/cyclin D1. The findings of this study also have significant implications in the design of CDK4 mimic structures based on CDK2.     

Structural basis for bending of organic crystals

Bending is observed in organic crystals when the packing is anisotropic in such a way that strong and weak interaction patterns occur in nearly perpendicular directions.     

A highly sensitive and selective chemosensor for cyanide

A highly sensitive and selective cyanide chemosensor based on fused indoline and benzooxazine fragment was reported with fast response. The detection of cyanide was performed via the nucleophilic attack of cyanide anion on the oxazine. (1)H NMR and MS studies confirmed the cleavage of C-O bond of oxazine and binding of cyanide to the spiro center of oxazine. The specific reaction results in high selectivity for cyanide ion. Addition of cyanide anion to the oxazine in MeCN/H(2)O solution results in a loss in absorbance at 343 nm and an increase in new absorbance at 411 nm, thus resulting in obvious color changes. Cyanide can be detected down to 1 microM levels in a fast response of less than 30s with no interference of other anionic species. The cyanide detection method should have potential application in a variety of settings requiring rapid and accurate analysis of cyanide anion for drinking and fresh water.     

A highly selective fluorescent chemosensor for lead ions

A simple fluorescent chemosensor 1 exhibits a 40-fold fluorescence enhancement upon addition of lead ion and yet is insensitive to most of univalent and divalent metal ions. The high affinity of 1 toward Pb2+ presumably results from the cooperative binding behaviors of two potential metal binding sites: the dicarbonyl and the 15-monoazacrown-5 ether.