Mechanistic information and structure-based design methods have been used to design a series of nonpeptide cyclic ureas that are potent inhibitors of human immunodeficiency virus (HIV) protease and HIV replication. A fundamental feature of these inhibitors is the cyclic urea carbonyl oxygen that mimics the hydrogen-bonding features of a key structural water molecule. The success of the design in both displacing and mimicking the structural water molecule was confirmed by x-ray crystallographic studies. Highly selective, preorganized inhibitors with relatively low molecular weight and high oral bioavailability were synthesized.

Factor VIII (fVIII) is a serum protein in the coagulation cascade that nucleates the assembly of a membrane-bound protease complex on the surface of activated platelets at the site of a vascular injury. Hemophilia A is caused by a variety of mutations in the factor VIII gene and typically requires replacement therapy with purified protein. We have determined the structure of a fully active, recombinant form of factor VIII (r-fVIII), which consists of a heterodimer of peptides, respectively containing the A1-A2 and A3-C1-C2 do IntroductionThe principal mechanism used to stop the loss of blood in mammals following vascular injury consists of a pair of overlapping proteolytic cascades called the extrinsic and intrinsic pathways. [1][2][3][4] The process of blood coagulation requires extraordinary spatial and temporal regulation, which is accomplished by assembling and tethering the central proteolytic activities of these cascades at the location of transiently exposed biomolecules and cellular surfaces ( Figure 1A). This includes an integral membrane protein called "tissue factor" that initiates the rapid up-regulation of the short-lived extrinsic pathway, 5 and the surfaces of activated platelets, which modulate the activation of the longer-lived intrinsic pathway. 6 A total of 2 homologous procoagulants, factors V and VIII (fV and fVIII), are each localized on the surface of these platelets, where they nucleate the assembly of multiprotein proteolytic complexes.When fVIII is bound to activated platelets at the site of vascular injury, it recruits the serine protease fIXa into a complex that then catalyzes the proteolytic activation of fX. 1,4,7 The proteolytic activity of fIXa is enhanced by approximately 200 000-fold through its interaction with fVIII, calcium, and the phospholipid bilayer, 8 corresponding to an increase of approximately 10 9 in k cat /K M .The full-length, unprocessed fVIII protein consists of 2332 amino acid residues and has the domain structure A1-A2-B-A3-C1-C2 9-12 ( Figure 1B). The 3 A domains are each approximately 330 residues, and approximately 40% identical to each other and to the copper-binding protein ceruloplasmin. 13 The C domains are smaller (approximately 160 residues) and are more distantly related to various members of the discoidin protein fold family, such as galactose oxidase. [14][15][16][17] The B domain has no known structural homologs, is heavily glycosylated, and is relatively dispensible for procoagulant activity. fVIII is initially processed by proteolytic cleavage events that remove a large portion of the B domain, generating a heterodimer that circulates in a tight complex with von Willebrand factor (VWF). 18 This interaction is essential for maintaining stable levels of fVIII in circulation. 19 Upon vascular injury, further proteolytic processing generates activated factor VIIIa (fVIIIa), a heterotrimer (A1/A2/A3-C1-C2) that is released from VWF and binds to activated platelets. 18 The carboxy-terminal 159 amino acids of fVIII comprise its C2 domain, which is invo...

The molecular replacement method has been succesfully used to provide a structure for the photosynthetic reaction center of Rhodopseudomonas sphaeroides at 3.7 A resolution. Atomic coordinates derived from the R. viridu reaction center were used in the search structure. The crystallographic R-factor is 0.39 for reflections between 8 and 3.7 A. Validity of the resulting model is further suggested by the visualization of amino acid side chains not included in the R. viridis search structure, and by the arrangements of the reaction centers in the unit cell. In the initial calculations quinones or pigments were not included; nevertheless, in the resulting electron density map, electron density for both quinones QA and Qa appears along with the bacteriochlorophylls and bacteriopheophytins. Kinetic analysis of the charge recombination shows that the secondary quinone is fully functional in the R. sphaeroides crystal. Photosynthesis(Rhodopseudomonas sphaeroides) Reaction center X-ray crystallograph? Molecular replacement

High-resolution X-ray structures of the complexes of HIV-1 protease (HIV-1PR) with peptidomimetic inhibitors reveal the presence of a structural water molecule which is hydrogen bonded to both the mobile flaps of the enzyme and the two carbonyls flanking the transition-state mimic of the inhibitors. Using the structure−activity relationships of C 2-symmetric diol inhibitors, computed-aided drug design tools, and first principles, we designed and synthesized a novel class of cyclic ureas that incorporates this structural water and preorganizes the side chain residues into optimum binding conformations. Conformational analysis suggested a preference for pseudodiaxial benzylic and pseudodiequatorial hydroxyl substituents and an enantiomeric preference for the RSSR stereochemistry. The X-ray and solution NMR structure of the complex of HIV-1PR and one such cyclic urea, DMP323, confirmed the displacement of the structural water. Additionally, the bound and “unbound” (small-molecule X-ray) ligands have similar conformations. The high degree of preorganization, the complementarity, and the entropic gain of water displacement are proposed to explain the high affinity of these small molecules for the enzyme. The small size probably contributes to the observed good oral bioavailability in animals. Extensive structure-based optimization of the side chains that fill the S2 and S2‘ pockets of the enzyme resulted in DMP323, which was studied in phase I clinical trials but found to suffer from variable pharmacokinetics in man. This report details the synthesis, conformational analysis, structure−activity relationships, and molecular recognition of this series of C 2-symmetry HIV-1PR inhibitors. An initial series of cyclic ureas containing nonsymmetric P2/P2‘ is also discussed.