A novel class of azetidinone acid-derived dual PPARalpha/gamma agonists has been synthesized for the treatment of diabetes and dyslipidemia. The preferred stereochemistry in this series for binding ...and functional agonist activity against both PPARalpha and PPARgamma receptors was shown to be 3S,4S. Synthesis, in vitro and in vivo activities of compounds in this series are described. A high-yielding method for N-arylation of azetidinone esters is also described.
Structure and specificity of the anti-digoxinantibody 40–50 Jeffrey, Philip D.; Schildbach, Joel F.; Chang, ChiehYing Y. ...
Journal of Molecular Biology/Journal of molecular biology,
1995, 1995-00-00, Letnik:
248, Številka:
2
Journal Article
Recenzirano
Odprti dostop
We determined the sequence, specificity for structurally related cardenolides,and three-dimensional structure of the anti-digoxin antibody 40–50 Fab in complex with ouabain. The 40–50 antibody does ...not share close sequence homology with other high-affinity anti-digoxin antibodies. Measurement of the binding constants of structurally distinct digoxin analogs indicated a well-defined specificity pattern also distinct from other anti-digoxin antibodies. The 40–50—ouabain Fab complex crystallizes in space group C2 with cell dimensions of
a = 93.7 Å,
b = 84.8 Å,
c = 70.1 Å,
β = 128.0°. The structure of the complex was determined by X-ray crystallography and refined at a resolution of 2.7 Å. The hapten is bound in a pocket extending as a groove from the center of the combining site across the light chain variable domain, with five of the six complementarity-determining regions involved in interactions with the hapten. Approximately three-quarters of the hapten surface area is buried in the complex; two hydrogen bonds are formed between the antibody and hapten. The surface area of the antibody combining site buried by ouabain is contributed equally by the light and heavy chain variable domains. Over half of the surface area buried on the Fab consists of the aromatic side-chains. The surface complementarity between hapten and antibody is sufficient to make the complex specific for only one lactone ring conformation in the hapten. The crystal structure of the 40–50—ouabain complex allows qualitative explanation of the observed fine specificities of 40–50, including that for the binding of haptens substituted at the 16 and 12 positions. Comparison of the crystal structures of 40–50 complexed with ouabain and the previously determined 26-10 anti-digoxin Fab complexed with digoxin, demonstrates that the antibodies bind these structurally related haptens in different orientations, consistent with their different fine specificities. These results demonstrate that the immune system can generate antibodies that provide diverse structural solutions to the binding of even small molecules.
Compound 3 (BMS-536924), a novel small-molecule inhibitor of the insulin-like growth factor receptor kinase with equal potency against the insulin receptor is described. The in vitro and in vivo ...biological activity of this interesting compound is also reported.
CTLA‐4 (CD152) is involved in T‐lymphocyte co‐stimulatory pathways modulating both humoral and cellular immune response. The membrane‐external domain has been prepared and crystallized. The unit‐cell ...parameters are a = b = 43, c = 143 Å with the symmetry of space group P3121 or its enantiomer and the crystals diffract to 2.7 Å resolution at synchrotron beamlines.
The synthesis and follow-up SAR studies of our development candidate 1 by incorporating 2-aryl-4-oxazolylmethoxy and 2-aryl-4-thiazolylmethoxy moieties into the oxybenzylglycine framework of the ...PPARalpha/gamma dual agonist muraglitazar is described. SAR studies indicate that different substituents on the aryloxazole/thiazole moieties as well as the choice of carbamate substituent on the glycine moiety can significantly modulate the selectivity of PPARalpha versus PPARgamma. Potent, highly selective PPARalpha activators 2a and 2l, as well as PPARalpha activators with significant PPARgamma activity, such as 2s, were identified. The in vivo pharmacology of these compounds in preclinical animal models as well as their ADME profiles are discussed.
The structure of the complex formed between bovine β‐trypsin and the highly potent synthetic inhibitor ...2‐{3′‐5′‐methoxy‐2′‐(N‐p‐diaminomethylphenyl)amido‐1′‐pyrido}‐5‐(N‐2″‐t‐butylethanol)amidobenzoic acid (C28H32N5O6) has been determined at 0.97 Å resolution. X‐ray intensity data were collected to 0.97 Å under cryocooled conditions. The structure was refined anisotropically using REFMAC5 and SHELX‐97 to Rcryst factors of 13.4 and 12.6% and Rfree factors of 15.7 and 16.3%, respectively. Several regions of the main chain and side chains that have not been previously observed were clearly defined in the present structure. H atoms are indicated as significant peaks in an |Fo−Fc| difference map, which accounts for an estimated 35% of all H atoms at the 2.5σ level. The C, N and O atoms are definitively differentiated in the electron‐density maps. The amido part of the inhibitor occupies the specificity pocket and the remainder fills the remaining part of the ligand‐binding cleft and interacts with the enzyme through an extensive network of hydrogen bonds. The inhibitor distorts the stereochemistry of the catalytic triad, Ser195–His57–Asp102, thereby blocking the proton‐relay process of the active site by preventing the formation of the crucial hydrogen bond between His57 Nδ1 and Asp102 Oδ1.
Uridine diphosphate‐N‐acetylmuramate:L‐alanine ligase (EC 6.3.2.8, UNAM:L‐Ala ligase or MurC gene product) catalyzes the ATP‐dependent ligation of the first amino acid to the sugar moiety of the ...peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram‐positive and gram‐negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady‐state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studied included: cation specificity, ionic strength, buffer composition and pH. At 37 °C and pH 8.0, kcat was equal to 980 40 min−1, while Km values for ATP, UNAM, and L‐alanine were, 130 10, 44 3, and 48 6 μM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1. pH profiles of Vmax/Km for the three substrates and of Vmax were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X‐ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 Å, β = 105°, and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L‐alanine. This L‐alanine independent activity is dependent upon the concentrations of both ATP and UNAM; kcat for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L‐alanine. Numerous L‐alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L‐alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L‐alanine analogs tested as inhibitors were competitive versus L‐alanine.
The three-dimensional structure of the antibody N10 Fab fragment complexed with staphylococcal nuclease (SNase) has been determined to 2.9 Å resolution.Eighteen residues from ...six-complementarity-determining regions (CDR) recognize an epitope of five distinct SNase segments with a total of 17 residues. The overall shape of the antibody- antigen interface is U-shaped rather than the more or less rectangular seen in other antibody-protein antigen interfaces. Despite the U-shaped interface, the amount of surface buried in the complex, 828 Å
2for SNase and 793 Å
2for N10, is typical of antibody-protein antigen complexes. Contributing to the shape of the interface is the shortest antibody heavy chain-CDR3 loop reported to date, which probably allows access of bulk solvent in the center of the “U” interface. Another nusual feature of the N10 antibody is the 15 residue antibody light chain-CDR1, a length seen in only three other reported antibodies. Antibody light chain-CDR1 displays a previously nobserved conformation in its distal portion. Finally, although some of the movement observed in the antibody-bound SNase may be due to crystal contacts, it is clear that some side-chain rearrangements are the result of antigen-antibody interaction.
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