No abstract

Shp2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. It is involved in growth factorinduced activation of mitogen-activated protein (MAP) kinases Erk1 and Erk2 (Erk1/2) and has been implicated in the pathogenicity of the oncogenic bacterium Helicobacter pylori. Moreover, gain-of-function Shp2 mutations have been found in childhood leukemias and Noonan syndrome. Thus, small molecule Shp2 PTP inhibitors are much needed reagents for evaluation of Shp2 as a therapeutic target and for chemical biology studies of Shp2 function. By screening the National Cancer Institute (NCI) Diversity Set chemical library, we identified 8-hydroxy-7-(6-sulfonaphthalen-2-yl)diazenyl-quinoline-5-sulfonic acid (NSC-87877) as a potent Shp2 PTP inhibitor. Molecular modeling and site-directed mutagenesis studies suggested that NSC-87877 binds to the catalytic cleft of Shp2 PTP. NSC-87877 cross-inhibited Shp1 in vitro, but it was selective for Shp2 over other PTPs (PTP1B, HePTP, DEP1, CD45, and LAR). It is noteworthy that NSC-87877 inhibited epidermal growth factor (EGF)-induced activation of Shp2 PTP, Ras, and Erk1/2 in cell cultures but did not block EGF-induced Gab1 tyrosine phosphorylation or Gab1-Shp2 association. Furthermore, NSC-87877 inhibited Erk1/2 activation by a Gab1-Shp2 chimera but did not affect the Shp2-independent Erk1/2 activation by phorbol 12-myristate 13-acetate. These results identified NSC-87877 as the first PTP inhibitor capable of inhibiting Shp2 PTP in cell cultures without a detectable off-target effect. Our study also provides the first pharmacological evidence that Shp2 mediates EGF-induced Erk1/2 MAP kinase activation.

An analysis is presented of the similarities and differences in the bonding of CO to Ni(100), Ni(l 11), Co(0001), Fe(110), Cr(110), and Ti(0001). The primary interactions in every case are the expected forward and back donation, the mixing of CO 5 with surface dz2 and s states, and the mixing of CO 2:r with metal dXZi>.z. The latter interaction is dominant. Projections of the density of states and crystal orbital overlap population curves show these interactions clearly. As the metal changes from Ni to Ti, the effects of the rise of the Fermi level and the diffuseness of the d orbitals combine to put more and more electron density into 2 of CO. This is why CO dissociates on the earlier transition-metal surfaces. Our studies of surface coverage show little effect on the diatomic dissociation. There is some indication in the calculations that higher index surfaces should dissociate CO more readily than the lower index ones, but the effect is smaller than that of changing the metal.

A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X L , and Mcl-1, which bind to the BH3 ␣-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 ␣-helical mimetic BH3-M6 that binds to Bcl-X Apoptosis, a form of programmed cell death, is a highly conserved process in all multicellular organisms and is essential for embryonic development and adult tissue homeostasis. Deregulation of apoptosis contributes to several diseases including cancer (1). Apoptosis is primarily controlled by two major pathways, namely the death receptor (extrinsic) and the mitochondrial (intrinsic) pathways (2). The former is mediated by members of the tumor necrosis factor (TNF) 6 receptor superfamily, while the latter largely depends on multiple Bcl-2 family proteins, which affect the integrity of the mitochondrial outer membrane (MOM) (3). Both pathways converge on common cysteine proteases of the caspase family, which are responsible for the execution of apoptosis (4).The Bcl-2 family consists of anti-apoptotic and pro-apoptotic proteins. Anti-apoptotic proteins, such as Bcl-2, Bcl-X L , Bcl-w, Mcl-1, and Bfl-1 (Bcl-2A1) contain four Bcl-2 homology (BH) domains, while the pro-apoptotic members are divided into proteins with three BH domains BH1-BH3 (Bax, Bak, and Bok), and proteins with only a BH3 domain (e.g. Bim, Bad, Bik, Bmf, Bid, Noxa, and Puma) (5). Multi-domain proapoptotic proteins Bax and Bak are absolutely required for apoptosis (2). In response to cellular stress, they induce the release from mitochondria of apoptogenic factors such as cytochrome c, which then cooperate with APAF-1 to induce caspase-9 activation, followed by caspase-mediated apoptosis (6). BH3-only proteins act upstream of Bax and Bak and are important for the initiation of apoptosis. Importantly, the BH3 domain is essential for the killing function of pro-apoptotic proteins (7).An important feature of the Bcl-2 proteins is that they can homo-and heterodimerize, giving rise to three competing, but not necessarily exclusive models that could explain how the balance between pro-and anti-apoptotic proteins regulates apoptosis (7). For instance, upon receiving an apoptotic signal, BH3-only proteins directly or indirectly induce Bax and Bak activation and homo-oligomerization in the MOM, which is thought to be responsible for MOM permeabilization, resulting in the release of cytochrome c and the initiation of intrinsic apoptosis. However, activated Bax and Bak still can be kept in check by binding to anti-apoptotic Bcl-2 proteins (8 -10). X-ray diffraction and nuclear magnetic resonance (NMR) studies have shown that the amphipathic ␣-helices of pro-apoptotic proteins such as Bak or Bad BH3 domains fit into a hydrophobic pocket formed by the BH1, BH2, * This work was funded, in whole or in part, by National Institutes of Health P01 Grants CA118210 and GM69850.