Small molecule peptidomimetics containing a novel phosphotyrosine bioisostere inhibit protein tyrosine phosphatase 1B and augment insulin action.

Protein tyrosine phosphatase 1B (PTP1B) attenuates insulin signaling by catalyzing dephosphorylation of insulin receptors (IR) and is an attractive target of potential new drugs for treating the insulin resistance that is central to type II diabetes. Several analogues of cholecystokinin(26)(-)(33) (CCK-8) were found to be surprisingly potent inhibitors of PTP1B, and a common N-terminal tripeptide, N-acetyl-Asp-Tyr(SO(3)H)-Nle-, was shown to be necessary and sufficient for inhibition. This tripeptide was modified to reduce size and peptide character, and to replace the metabolically unstable sulfotyrosyl group. This led to the discovery of a novel phosphotyrosine bioisostere, 2-carboxymethoxybenzoic acid, and to analogues that were >100-fold more potent than the CCK-8 analogues and >10-fold selective for PTP1B over two other PTP enzymes (LAR and SHP-2), a dual specificity phosphatase (cdc25b), and a serine/threonine phosphatase (calcineurin). These inhibitors disrupted the binding of PTP1B to activated IR in vitro and prevented the loss of tyrosine kinase (IRTK) activity that accompanied PTP1B-catalyzed dephosphorylation of IR. Introduction of these poorly cell permeant inhibitors into insulin-treated cells by microinjection (oocytes) or by esterification to more lipophilic proinhibitors (3T3-L1 adipocytes and L6 myocytes) resulted in increased potency, but not efficacy, of insulin. In some instances, PTP1B inhibitors were insulin-mimetic, suggesting that in unstimulated cells PTP1B may suppress basal IRTK activity. X-ray crystallography of PTP1B-inhibitor complexes revealed that binding of an inhibitor incorporating phenyl-O-malonic acid as a phosphotyrosine bioisostere occurred with the mobile WPD loop in the open conformation, while a closely related inhibitor with a 2-carboxymethoxybenzoic acid bioisostere bound with the WPD loop closed, perhaps accounting for its superior potency. These CCK-derived peptidomimetic inhibitors of PTP1B represent a novel template for further development of potent, selective inhibitors, and their cell activity further justifies the selection of PTP1B as a therapeutic target.

Crystallization and preliminary X-ray diffraction studies of a new crystal form of human secretory type IIA phospholipase A2.

Human synovial type IIA phospholipase A(2) (sPLA(2)-IIA) has been implicated in the pathogenesis of a number of inflammatory diseases and is a target for the development of therapeutically useful inhibitors. Biochemical evidence suggests a novel mechanism of inhibition for a series of peptide inhibitors originally derived from the primary sequence of the protein. On co-incubation with one of these inhibitors, single crystals of a hitherto unreported crystallographic form of sPLA2-IIA suitable for diffraction analysis were obtained. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 140.8, b = 38.9, c = 109.1 A, beta = 125.1 degrees, and diffraction at 2.4 A resolution has been observed.

Periplasmic chaperone recognition motif of subunits mediates quaternary interactions in the pilus.

The class of proteins collectively known as periplasmic immunoglobulin-like chaperones play an essential role in the assembly of a diverse set of adhesive organelles used by pathogenic strains of Gram-negative bacteria. Herein, we present a combination of genetic and structural data that sheds new light on chaperone-subunit and subunit-subunit interactions in the prototypical P pilus system, and provides new insights into how PapD controls pilus biogenesis. New crystallographic data of PapD with the C-terminal fragment of a subunit suggest a mechanism for how periplasmic chaperones mediate the extraction of pilus subunits from the inner membrane, a prerequisite step for subunit folding. In addition, the conserved N- and C-terminal regions of pilus subunits are shown to participate in the quaternary interactions of the mature pilus following their uncapping by the chaperone. By coupling the folding of subunit proteins to the capping of their nascent assembly surfaces, periplasmic chaperones are thereby able to protect pilus subunits from premature oligomerization until their delivery to the outer membrane assembly site.

Structural basis of pilus subunit recognition by the PapD chaperone.

The assembly of different types of virulence-associated surface fibers called pili in Gram-negative bacteria requires periplasmic chaperones. PapD is the prototype member of the periplasmic chaperone family, and the structural basis of its interactions with pilus subunits was investigated. Peptides corresponding to the carboxyl terminus of pilus subunits bound PapD and blocked the ability of PapD to bind to the pilus adhesin PapG in vitro. The crystal structure of PapD complexed to the PapG carboxyl-terminal peptide was determined to 3.0 A resolution. The peptide bound in an extended conformation with its carboxyl terminus anchored in the interdomain cleft of the chaperone via hydrogen bonds to invariant chaperone residues Arg8 and Lys112. Main chain hydrogen bonds and contacts between hydrophobic residues in the peptide and the chaperone stabilized the complex and may play a role in determining binding specificity. Site-directed mutations in Arg8 and Lys112 abolished the ability of PapD to bind pilus subunits and mediate pilus assembly in vivo, an indication that the PapD-peptide crystal structure is a reflection of at least part of the PapD-subunit interaction.

Successful outcome after Nd : YAG laser separation of chorioangiopagus-twins under sonoendoscopic control.

Endoscopic laser coagulation of the chorioangiopagus was successfully performed at 25 weeks' gestation in a pregnancy presenting with acute polyhydramnios due to twin transfusion syndrome. Under local anesthesia, a rigid fetoscope housed in a 2.7 mm diameter cannula was introduced transabdominally into the amniotic cavity and the communicating vessels were coagulated by Nd : YAG laser through a fiber that was passed down the side-arm of the cannula. There was rapid normalization of urine output in both fetuses and resolution of the oligo- and polyhydramnios in the donor and recipient twins, respectively. Furthermore, there was normalization in both fetal circulations, as assessed by Doppler velocimetry, and there was gradual resolution of the hydrops in the recipient. Two healthy babies were born 9 weeks after the procedure.

The structure of mouse L1210 dihydrofolate reductase-drug complexes and the construction of a model of human enzyme.

The structure of mouse L1210 dihydrofolate reductase (DHFR) complexed with NADPH and trimethoprim has been refined at 2.0 A resolution. The analogous complex with NADPH and methotrexate has been refined at 2.5 A resolution. These structures reveal for the first time details of drug interactions with a mammalian DHFR, which are compared with those observed from previous X-ray investigations of DHFR/inhibitor complexes. The refined L1210 structure has been used as the basis for the construction of a model of the human enzyme. There are only twenty-one sequence differences between mouse L1210 and human DHFRs, and all but two of these are located close to the molecular surface: a strong indication that the active sites are essentially identical in these two mammalian enzymes.

Purification and preliminary characterization of phosphoglycerate mutase from Schizosaccharomyces pombe.

Phosphoglycerate mutase could be purified to over 95% homogeneity by a single step procedure involving elution from Cibacron Blue-Sepharose by a pulse of cofactor 2,3-bisphosphoglycerate. Although the enzyme has been isolated in only small quantities (c. 100 micrograms), gel filtration and sodium dodecylsulphate polyacrylamide gel electrophoresis indicated that it is monomeric with Mr approximately 23,000, an extremely low value for this enzyme. Preliminary investigations of the kinetic characteristics and the nature of important amino acid side chains have been undertaken.