The signaling mechanisms leading to the formation of neutrophil extracellular traps (NETs), relevant in infections, sepsis and autoimmune diseases, are poorly understood. Neutrophils are not amenable to studies with conventional genetic techniques. Using a new chemical genetic analysis we show that the Raf-MEK-ERK pathway is involved in NET formation through activation of NADPH oxidase and upregulation of antiapoptotic proteins. We identify potential targets for drugs addressing NET-associated diseases.

Guanine nucleotide exchange factors (GEFs) activate Ras proteins by stimulating the exchange of GTP for GDP in a multistep mechanism which involves binary and ternary complexes between Ras, guanine nucleotide, and GEF. We present fluorescence measurements to define the kinetic constants that characterize the interactions between Ras, GEF, and nucleotides, similar to the characterization of the action of RCC1 on Ran [Klebe et al. (1995) Biochemistry 34, 12543-12552]. The dissociation constant for the binary complex between nucleotide-free Ras and the catalytic domain of mouse Cdc25, Cdc25(Mm285), was 4.6 nM, i.e., a 500-fold lower affinity than the Ras.GDP interaction. The affinities defining the ternary complex Ras. nucleotide.Cdc25(Mm285) are several orders of magnitude lower. The maximum acceleration by Cdc25(Mm285) of the GDP dissociation from Ras was more than 10(5)-fold. Kinetic measurements of the association of nucleotide to nucleotide-free Ras and to the binary complex Ras. Cdc25(Mm285) show that these reactions are practically identical: a fast binding step is followed by a reaction of the first order which becomes rate limiting at high nucleotide concentrations. The second reaction is thought to be a conformational change from a low- to a high-affinity nucleotide binding conformation in Ras. Taking into consideration all experimental data, the reverse isomerization reaction from a high- to a low-affinity binding conformation in the ternary complex Ras. GDP.Cdc25(Mm285) is postulated to be the rate-limiting step of the GEF-catalyzed exchange. Furthermore, we demonstrate that the disruption of the Mg2+-binding site is not the only factor in the mechanism of GEF-catalyzed nucleotide exchange on Ras.

Myosin binding protein C is a protein of the myosin filaments of striated muscle which is expressed in isoforms specific for cardiac and skeletal muscle. The cardiac isoform is phosphorylated rapidly upon adrenergic stimulation of myocardium by cAMP-dependent protein kinase, and together with the phosphorylation of troponin-I and phospholamban contributes to the positive inotropy that results from adrenergic stimulation of the heart. Cardiac myosin binding protein C is phosphorylated by cAMP-dependent protein kinase on three sites in a myosin binding protein C specific N-terminal domain which binds to myosin-S2. This interaction with myosin close to the motor domain is likely to mediate the regulatory function of the protein.Cardiac myosin binding protein C is a common target gene of familial hypertrophic cardiomyopathy and most mutations encode N-terminal subfragments of myosin binding protein C. The understanding of the signalling interactions of the N-terminal region is therefore important for understanding the pathophysiology of myosin binding protein C associated cardiomyopathy. We demonstrate here by cosedimentation assays and isothermal titration calorimetry that the myosin-S2 binding properties of the myosin binding protein C motif are abolished by cAMP-dependent protein kinase-mediated trisphosphorylation, decreasing the S2 affinity from a K d of W W5 W WM to undetectable levels. We show that the slow and fast skeletal muscle isoforms are no cAMP-dependent protein kinase substrates and that the S2 interaction of these myosin binding protein C isoforms is therefore constitutively on. The regulation of cardiac contractility by myosin binding protein C therefore appears to be a`brake-off' mechanism that will free a specific subset of myosin heads from sterical constraints imposed by the binding to the myosin binding protein C motif.z 1999 Federation of European Biochemical Societies.

The interaction of Ran, a Ras-related nuclear GTP-binding protein, with its guanine nucleotide exchange factor RCC1 has been studied by equilibrium and transient kinetic measurements using fluorescent nucleotides. The four-step mechanism of catalyzed nucleotide exchange involves the formation of ternary complexes consisting of Ran, RCC1, and GXP as well as a nucleotide-free dimeric Ran.RCC1 complex. This model is sufficient to describe all experimental data obtained, so that no additional reaction steps must be assumed. All the rate and equilibrium constants for the four-step mechanism have been determined either experimentally or from a simultaneous theoretical fit to all experimental data sets. The affinities of RCC1 to Ran.GDP and Ran.GTP are similar (1.3 x 10(5) and 1.8 x 10(5) M-1, respectively) and are high enough to allow formation of the ternary complex under appropriate concentration conditions. In the absence of excess nucleotide and at low Ran concentrations, GDP (or GTP) can be efficiently displaced by excess RCC1 and the ternary complex can be produced. The affinities of both nucleotides (GDP or GTP) to Ran in the corresponding ternary complexes are reduced by orders of magnitude in comparison with the respective binary complexes. The reduction of affinity of both nucleotides in the ternary complexes leads to a dramatic increase in the dissociation rate constants by similar orders of magnitude (from 1.5 x 10(-5) s(-1) to 21 s(-1) for GDP) and thus to facilitated nucleotide exchange. The quantitative results of the kinetic analysis suggest that the exchange reaction does not per se favor the formation of the Ran.GTP complex, but rather accelerates the formation of the equilibrium dictated by the relative affinities of Ran for GDP/GTP and the respective concentrations of the nucleotide in the cell. The extent of Ran.GTP formation in vivo can be calculated using the constants derived.

Hill coefficients (n H ) derived from four parameter logistic fits to dose-response curves were compared to calculated realistic reaction schemes and related to experimental data: (1) Hill coefficients may give information on the number of interacting sites but cannot distinguish between competitive, non-competitive or ortho-, iso-, or allosteric mechanisms. (2) For enzymatic dose-inhibition curves, Hill coefficients smaller than one do not indicate anticooperative binding but show that at least one ternary complex has enzymatic activity. (3) Hill coefficients different from one are proof for multiple ligand binding. The large variations of reported Hill coefficients corresponds to multiple allosteric binding, where induced conformational changes cause loss of the active conformation. Such a denaturation mechanism is in stark contrast to the desired specificity of drugs. The discussion is open.

No abstract

Protein phosphatases have very recently emerged as important targets for chemical biology and medicinal chemistry research, and new phosphatase inhibitor classes are in high demand. The underlying frameworks of natural products represent the evolutionarily selected fractions of chemical space explored by nature so far and meet the criteria of relevance to nature and biological prevalidation most crucial to inhibitor development. We refer to synthesis efforts and compound collection development based on these criteria as biology-oriented synthesis. For the discovery of phosphatase inhibitor classes by means of this approach, four natural product-derived or -inspired medium-sized compound collections were synthesized and investigated for inhibition of the tyrosine phosphatases VE-PTP, Shp-2, PTP1B, MptpA, and MptpB and the dual-specificity phosphatases Cdc25A and VHR. The screen yielded four unprecedented and selective phosphatase inhibitor classes for four phosphatases with high hit rates. For VE-PTP and MptpB the first inhibitors were discovered. These results demonstrate that biology-oriented synthesis is an efficient approach to the discovery of new compound classes for medicinal chemistry and chemical biology research that opens up new opportunities for the study of phosphatases, which may lead to the development of new drug candidates.chemical biology ͉ medicinal chemistry ͉ phosphatase inhibition P rotein phosphatases are key regulators of innumerable biological processes (1, 2). Small-molecule modulators of phosphatase activity have proven to be powerful tools for the study of the chemical biology of these enzymes (3), and, in particular, protein tyrosine phosphatases (PTPs) (4, 5) and dual-specificity phosphatases (6) have recently moved into the focus of a growing number of drug discovery programs, for instance in diabetes and anticancer research. However, although important progress has been made, the development of potent and selective phosphatase inhibitors is still in its early stages, and structurally new phosphatase inhibitor classes are in high demand.Relevance to nature is one of the most important criteria to be met by compound classes for chemical biology and medicinal chemistry research. The underlying frameworks of natural products (NPs) provide evolutionarily selected chemical structures encoding the properties required for binding to proteins, and their structural scaffolds represent the biologically relevant and prevalidated fractions of chemical space explored by nature so far (7-9). Consequently, it is to be expected that compound collections designed on the basis of NP structure will be enriched in biochemical and biological activity. Based on this reasoning, we have introduced a structural classification of natural products (SCONP) in a tree-like arrangement as an idea-and hypothesis-generating tool for the design and synthesis of compound collections (8). It permits the selection of library scaffolds based on relevance to and prevalidation by nature. We refer to synthesis efforts base...

IgE antibodies bind the high affinity IgE Fc receptor (FcεRI), found primarily on mast cells and basophils, and trigger inflammatory cascades of the allergic response1,2. Inhibitors of IgE:FcεRI binding have been identified and an anti-IgE therapeutic antibody (omalizumab) is used to treat severe allergic asthma3,4. However, preformed IgE:FcεRI complexes that prime cells prior to allergen exposure dissociate extremely slowly5 and cannot be disrupted by strictly competitive inhibitors. IgE-Fc conformational flexibility indicated that inhibition could be mediated by allosteric or other non-classical mechanisms6–8. Here we demonstrate that an engineered protein inhibitor, DARPin E2_799–11, acts through a non-classical inhibition mechanism, not only blocking IgE:FcεRI interactions, but actively stimulating the dissociation of preformed ligand-receptor complexes. The structure of the E2_79:IgE-Fc3-4 complex predicts the presence of two non-equivalent E2_79 sites in the asymmetric IgE:FcεRI complex, with Site 1 distant from the receptor and Site 2 exhibiting partial steric overlap. While the structure is suggestive of an allosteric inhibition mechanism, mutational studies and quantitative kinetic modeling indicate that E2_79 acts through a facilitated dissociation mechanism at Site 2 alone. These results demonstrate that high affinity IgE:FcεRI complexes can be actively dissociated to block the allergic response and suggest that protein:protein complexes may be more generally amenable to active disruption by macromolecular inhibitors.