Yuval Blat

The Amyloid-β Rise and γ-Secretase Inhibitor Potency Depend on the Level of Substrate Expression

The amyloid-β (Aβ) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-β precursor protein (APP) through consecutive proteolytic cleavages by β-site APP-cleaving enzyme and γ-secretase. Unexpectedly γ-secretase inhibitors can increase the secretion of Aβ peptides under some circumstances. This “Aβ rise” phenomenon, the same inhibitor causing an increase in Aβ at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Aβ rise depends on the β-secretase-derived C-terminal fragment of APP (βCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Aβ, known as “p3,” formed by α-secretase cleavage, did not exhibit a rise. In addition to the Aβ rise, low βCTF or C99 expression decreased γ-secretase inhibitor potency. This “potency shift” may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, γ-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Aβ under conditions of low substrate expression. The Aβ rise was also observed in rat brain after dosing with the γ-secretase inhibitor BMS-299897. The Aβ rise and potency shift are therefore relevant factors in the development of γ-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Aβ rise, including the “incomplete processing” and endocytic models, are discussed.

Oligomerization of the Phosphatase CheZ Upon Interaction with the Phosphorylated Form of CheY THE SIGNAL PROTEIN OF BACTERIAL CHEMOTAXIS

Earlier studies have suggested that CheZ, the phosphatase of the signaling protein CheY in bacterial chemotaxis, may be in an oligomeric state both when bound to phosphorylated CheY (CheYP) (Blat, Y., and Eisenbach, M.(1994) Biochemistry 33, 902-906) or free (Stock, A., and Stock, J. B.(1987) J. Bacteriol. 169, 3301-3311). The purpose of the current study was to determine the oligomeric state of free CheZ and to investigate whether it changes upon binding to CheYP. By using either one of two different sets of cross-linking agents, free CheZ was found to be a dimer. The formation of the dimer was specific, as it was prevented by SDS which does not interfere with cross-linking mediated by random collisions. The dimeric form of CheZ was confirmed by sedimentation analysis, a cross-linking-free technique. In the presence of CheYP (but not in the presence of non-phosphorylated CheY), a high molecular size cross-linked complex (90-200 kDa) was formed, in which the CheZ:CheY ratio was 2:1. The size of the oligomeric complex was estimated by fluorescence depolarization to be 4-5-fold larger than the dimer, suggesting that its size is in the order of 200 kDa. These results indicate that CheZ oligomerizes upon interaction with CheYP. This phosphorylation-dependent oligomerization may be a mechanism for regulating CheZ activity.

Feedback activation of factor XI by thrombin does not occur in plasma

In this study, we tested the hypothesis that factor XI (FXI) activation occurs in plasma following activation of the extrinsic pathway by thrombin-mediated feedback activation. We used two different assays: (i) a direct measurement of activated FXI by ELISA and (ii) a functional assay that follows the activation of the coagulation cascade in the presence or absence of a FXI inhibiting antibody by monitoring thrombin activity. We failed to detect any FXI activation or functional contribution to the activation of the coagulation cascade in platelet poor or platelet-rich plasma, when activation was initiated by thrombin or tissue factor. Additionally, we found that, in the absence of a contact system inhibitor during blood draw, contact activation of FXI can mistakenly appear as thrombin- or tissue-factor-dependent activation. Thus, activation of FXI by thrombin in solution or on the surface of activated platelets does not appear to play a significant role in a plasma environment. These results call for reevaluation of the physiological role of the contact activation system in blood coagulation.

Mutants with Defective Phosphatase Activity Show No Phosphorylation-dependent Oligomerization of CheZ THE PHOSPHATASE OF BACTERIAL CHEMOTAXIS

CheZ is the phosphatase of CheY, the response regulator in bacterial chemotaxis. The mechanism by which the activity of CheZ is regulated is not known. We used cheZ mutants of Salmonella typhimurium, which had been isolated by Sockett et al. (Sockett, H., Yamaguchi, S., Kihara, M., Irikura, V. M., and Macnab, R. M.(1992) J. Bacteriol. 174, 793-806), for cloning the mutant cheZ genes, overexpressing and purifying their products. We then measured the phosphatase activity, binding to CheY and to phosphorylated CheY (CheYP), and CheYP-dependent oligomerization of the mutant CheZ proteins. While all the mutant proteins were defective in their phosphatase activity, they bound to CheY and CheYP as well as wild-type CheZ. However, unlike wild-type CheZ, all the four mutant proteins failed to oligomerize upon interaction with CheYP. On the basis of these and earlier results it is suggested that (i) oligomerization is required for the phosphatase activity of CheZ, (ii) the region defined by residues 141-145 plays an important role in mediating CheZ oligomerization and CheYP dephosphorylation but is not necessary for the binding to CheYP, (iii) the oligomerization and hence the phosphatase activity are regulated by the level of CheYP, and (iv) this regulation plays a role in the adaptation to chemotactic stimuli.

Tyrosine Kinase 2-mediated Signal Transduction in T Lymphocytes Is Blocked by Pharmacological Stabilization of Its Pseudokinase Domain

Background: Interleukin-23 mediates pathobiology in many autoimmune disorders. Results: A chemogenomics approach identified small molecule agents that block receptor-mediated activation or tyrosine kinase 2 (Tyk2) and downstream signaling. Compounds stabilize the pseudokinase domain of Tyk2. Conclusion: Small molecule ligands of the Tyk2 pseudokinase domain stabilize an autoinhibitory interaction with the catalytic domain. Significance: This work enables the discovery of selective therapeutics targeting Tyk2-dependent pathways critical in autoimmunity. Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified that bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor but not Tyk2-independent signaling and transcriptional cellular assays, including stimulation through the receptors for IL-2 (JAK1- and JAK3-dependent) and thrombopoietin (JAK2-dependent), demonstrating the high functional selectivity of this approach. A crystal structure of the pseudokinase domain liganded with a representative example showed the compound bound to a site analogous to the ATP-binding site in catalytic kinases with features consistent with high ligand selectivity. The results support a model where the pseudokinase domain regulates activation of the catalytic domain by forming receptor-regulated inhibitory interactions. Tyk2 pseudokinase stabilizers, therefore, represent a novel approach to the design of potent and selective agents for the treatment of autoimmunity.

Discovery of a Highly Selective JAK2 Inhibitor, BMS-911543, for the Treatment of Myeloproliferative Neoplasms

JAK2 kinase inhibitors are a promising new class of agents for the treatment of myeloproliferative neoplasms and have potential for the treatment of other diseases possessing a deregulated JAK2-STAT pathway. X-ray structure and ADME guided refinement of C-4 heterocycles to address metabolic liability present in dialkylthiazole 1 led to the discovery of a clinical candidate, BMS-911543 (11), with excellent kinome selectivity, in vivo PD activity, and safety profile.