Small-molecule inhibitors binding to protein kinases. Part I: exceptions from the traditional pharmacophore approach of type I inhibition.

BACKGROUND: Protein kinases are essential enzymes propagating cellular signal transduction processes and consequently have emerged as central targets for drug discovery against a wide range of diseases with a strong historical focus on oncological disorders. A large number of high-resolution crystal structures of various ATP-competitive inhibitors in complex with their target protein kinases have been determined and present a wealth of detailed information about binding modes, inhibition mechanisms and associated structure-activity relationships of target-bound small molecules. OBJECTIVE: In this first part of a two-part review, exceptions to the type I binding mode of kinase inhibitors that follow the traditional pharmacophore model are discussed, highlighting unexpected structural features. METHODS: The scope of this review covers published crystal structures of protein kinases in complex with various ligands. RESULTS: Structural parameters of both inhibitors and kinases contribute to the complexity of designing kinase inhibitors. The continued study of high-resolution structures of ligand-enzyme complexes in combination with a more dynamic understanding of accessible conformational states of the target proteins, supported by detailed kinetic studies, will in the long-term help in developing new low-molecular weight kinase inhibitors.

Small-molecule inhibitors binding to protein kinase. Part II: the novel pharmacophore approach of type II and type III inhibition.

BACKGROUND: Protein kinases are essential enzymes propagating cellular signal transduction processes and consequently emerged as central targets for drug discovery against a wide range of diseases with a strong historical focus on oncological disorders. Several high-resolution crystal structures of various ATP-competitive inhibitors in complex with their target protein kinases have been determined and represent a wealth of detailed information about binding modes, inhibition mechanisms, and associated structure- activity relationships of target-bound small molecules. OBJECTIVE: In this second part of a two-part review, we discuss the binding mode of inhibitors that target protein kinases in their inactive state. METHODS: The scope of this review covers inhibitors for which crystal structures in complex with their respective kinases in the inactive state are available. RESULTS: Structural parameters of both inhibitors and kinases contribute to the complexity of designing kinase inhibitors. Kinase inhibitors that target the inactive state of a kinase have become a novel rule in the design of highly active and selective compounds. The combination of high-resolution structures of ligand-enzyme complexes with especially detailed kinetic studies will in the long-term help to develop new low-molecular weight type II inhibitors.

Mass spectrometric analysis of nitric oxide-modified caspase-3.

Caspases are a family of cysteine proteases activated during apoptosis. Modification of caspases by nitric oxide and its relevance during apoptosis is currently a controversial subject. In this study we analyzed the S-nitrosated form of caspase-3 at a molecular level. By using electrospray ionization-mass spectrometry, we detected poly-S-nitrosation of caspase-3 with an average of about 2 molecules of NO bound per enzyme. Although NO treatment completely inhibited enzyme activity, S-nitrosation was not restricted to the active site cysteine. Rather, we detected multiple relative mass increases of 30 +/- 1 Da in both the p12 and p17 subunits of caspase-3, corresponding to single to triple S-nitrosation. The stability of these S-nitrosations differed in physiologically relevant concentrations of 5 mM glutathione. Whereas all S-nitroso bonds in the p12 subunit were cleaved with release of NO and partial formation of protein-mixed disulfides with glutathione, a single S-nitrosation in the p17 subunit remained stable. Since this S-nitrosation was not observed in a mutant form of caspase-3 lacking the active site cysteine, we conclude that NO nitrosates the active site cysteine of caspase-3 and that this modification is notably inert to fast trans-nitrosation with glutathione. Furthermore, we provide evidence that treatment of caspase-3 with NO can lead to mixed disulfide formation with glutathione, demonstrating the oxidative character of NO.

Inhibition of caspase-3 by S-nitrosation and oxidation caused by nitric oxide.

Apoptotic signaling cascades converge in the activation of caspases (interleukin-1beta converting enzyme like proteases). Treatment of the human promyelocytic leukaemia cell line U937 with actinomycin D resulted in the activation of caspase-3 also known as CPP32. Protease activity was measured in cytosolic extracts by fluorometric analysis of the time-dependent cleavage of acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-AMC), a caspase-3 substrate. Caspase activity was inhibited by thiol modifying agents such as N-ethylmaleimide or iodoacetamide and NO donors such as S-nitrosoglutathione (GSNO), BF4NO, and spermine-NO. NO-mediated enzyme inhibition was fully reversible upon the addition of DTT (dithiothreitol). NO. itself was not primarily responsible for downregulation of caspase-3, as we found no correlation between rates of NO* release and the magnitude of enzyme inhibition. It is likely that S-nitrosation accounts for enzyme inhibition by various NO donors. SIN-1 and peroxynitrite were inhibitory as well. In this case, however, enzyme activity was not restored upon DTT addition, suggesting oxidation as an additional thiol modification mechanism. Our studies provide evidence that caspases are targeted by NO via S-nitrosation and oxidation of critical thiol groups.

Interindividual variability of isometric endurance with regard to the endurance performance limit for static work.

In literature there are doubts concerning the 15% MVC (maximal voluntary contraction) level and invariability of isometric endurance, as described by ROHMERT (1962) as a "biologische Konstante". Therefore, 3 experimental sets using different levels of relative loads from approximately 2.9% (individual weight of forearm) to 50% MVC and different holding times (up to 1 hr) were performed concerning elbow flexion and knee extension. Measurements of the times until exhaustion and muscular fatigue (EMG) underlined the great variability of the holding times and showed that the mean endurance performance limit for isometric exercise lies neither at the 15% MVC level nor can it be used as a "biologische Konstante". The muscle fibre composition is discussed as the main reason for the observed variability.