Imidazo[5,1-f][1,2,4]triazin-2-amines as novel inhibitors of polo-like kinase 1.

The synthesis and biological activities of imidazo[5,1-f][1,2,4]triazin-2-amines (imidazotriazines) as novel polo-like kinase 1 inhibitors are reported.

Structure of the Tie2 RTK domain: self-inhibition by the nucleotide binding loop, activation loop, and C-terminal tail.

BACKGROUND: Angiogenesis, the formation of new vessels from the existing vasculature, is a critical process during early development as well as in a number of disease processes. Tie2 (also known as Tek) is an endothelium-specific receptor tyrosine kinase involved in both angiogenesis and vasculature maintenance. RESULTS: We have determined the crystal structure of the Tie2 kinase domain to 2.2 A resolution. The structure contains the catalytic core, the kinase insert domain (KID), and the C-terminal tail. The overall fold is similar to that observed in other serine/threonine and tyrosine kinase structures; however, several unique features distinguish the Tie2 structure from those of other kinases. The Tie2 nucleotide binding loop is in an inhibitory conformation, which is not seen in other kinase structures, while its activation loop adopts an "activated-like" conformation in the absence of phosphorylation. Tyr-897, located in the N-terminal domain, may negatively regulate the activity of Tie2 by preventing dimerization of the kinase domains or by recruiting phosphatases when it is phosphorylated. CONCLUSION: Regulation of the kinase activity of Tie2 is a complex process. Conformational changes in the nucleotide binding loop, activation loop, C helix, and the C-terminal tail are required for ATP and substrate binding.

Searching for antiviral drugs for human papillomaviruses.

The human papillomaviruses (HPVs) are ubiquitous human pathogens that cause a wide variety of benign and pre-malignant epithelial tumours. Of the almost 100 different types of HPV that have been characterized to date, approximately two dozen specifically infect genital and oral mucosa. Mucosal HPVs are most frequently sexually transmitted and, with an incidence roughly twice that of herpes simplex virus infection, are considered one of the most common sexually transmitted diseases throughout the world. A subset of genital HPVs, termed 'high-risk' HPVs, is highly associated with the development of genital cancers including cervical carcinoma. The absence of a simple monolayer cell culture system for analysis and propagation of the virus has substantially retarded progress in the development of diagnostic and therapeutic strategies for HPV infection. In spite of these difficulties, great progress has been made in the elucidation of the molecular controls of virus gene expression, replication and pathogenesis. With this knowledge and some important new tools, there is great potential for the development of improved diagnostic and prognostic tests, prophylactic and therapeutic vaccines, and traditional antiviral medicines.

Potent dipeptide inhibitors of the pp60c-src SH2 domain.

The design, synthesis, and evaluation of dipeptide analogues as ligands for the pp60c-src SH2 domain are described. The critical binding interactions between Ac-Tyr-Glu-N(n-C5H11)2 (2) and the protein are established and form the basis for our structure-based drug design efforts. The effects of changes in both the C-terminal (11-27) and N-terminal (51-69) portions of the dipeptide are explored. Analogues with reduced overall charge (92-95) are also investigated. We demonstrate the feasibility of pairing structurally diverse subunits in a modest dipeptide framework with the goal of increasing the druglike attributes without sacrificing binding affinity.

The formation of a covalent complex between a dipeptide ligand and the src SH2 domain.

The X-ray crystal structure of the src SH2 domain revealed the presence of a thiol residue (Cys 188) located proximal to the phosphotyrosine portion of a dipeptide ligand. An aldehyde bearing ligand (1) was designed to position an electrophilic carbonyl group in the vicinity of the thiol. X-ray crystallographic and NMR examination of the complex formed between (1) and the src SH2 domain revealed a hemithioacetal formed by addition of the thiol to the aldehyde group with an additional stabilizing hydrogen bond between the acetal hydroxyl and a backbone carbonyl.

Peptide ligands of pp60(c-src) SH2 domains: a thermodynamic and structural study.

Thermodynamic measurements, structural determinations, and molecular computations were applied to a series of peptide ligands of the pp60(c-src) SH2 domain in an attempt to understand the critical binding determinants for this class of molecules. Isothermal titration calorimetry (ITC) measurements were combined with structural data derived from X-ray crystallographic studies on 12 peptide-SH2 domain complexes. The peptide ligands studied fall into two general classes: (1) dipeptides of the general framework N-acetylphosphotyrosine (or phosphotyrosine replacement)-Glu or methionine (or S-methylcysteine)-X, where X represents a hydrophobic amine, and (2) tetra- or pentapeptides of the general framework N-acetylphosphotyrosine-Glu-Glu-Ile-X, where X represents either Glu, Gln, or NH2. Dipeptide analogs which featured X as either hexanolamine or heptanolamine were able to pick up new hydrogen bonds involving their hydroxyl groups within a predominantly lipophilic surface cavity. However, due to internal strain as well as the solvent accessibility of the new hydrogen bonds formed, no net increase in binding affinity was observed. Phosphatase-resistant benzylmalonate and alpha,alpha-difluorobenzyl phosphonate analogs of phosphotyrosine retained some binding affinity for the pp60(c-src) SH2 domain but caused local structural perturbations in the phosphotyrosine-binding site. In the case where a reversible covalent thiohemiacetal was formed between a formylated phosphotyrosine analog and the thiol side chain of Cys-188, deltaS was 25.6 cal/(mol K) lower than for the nonformylated phosphotyrosine parent. Normal mode calculations show that the dramatic decrease in entropy observed for the covalent thiohemiacetal complex is due to the inability of the phosphotyrosine moiety to transform lost rotational and translational degrees of freedom into new vibrational modes.

Construction of a synthetic gene for the metalloregulatory protein MerR and analysis of regionally mutated proteins for transcriptional regulation.

The transcriptional control protein MerR is a metalloregulatory switch, activating transcription of a mercury resistance operon in the presence of mercuric ions and repressing transcription in their absence. We report here the construction and utilization of a synthetic merR gene and a single-copy merT'-lacZ fusion reporter for mutagenic analysis of the MerR protein's function. Site-directed mutagenesis of clustered acidic residues within the central region of the MerR protein indicated that these residues are important to the protein's ability to repress transcription. Quadruple or sextuple mutations involving residues E83 and E84 and other nearby acidic residues result in a repression-deficient (RD) phenotype. One of the mutant proteins was purified and shown by gel shift assay to retain binding to its operator DNA with an affinity similar to wild-type protein, suggesting that transcriptional repression does not correlate with MerR binding affinity. A small region of merR corresponding to residues 81-92 also was mutagenized in a search for other RD mutants and for mutants displaying sufficient transcriptional activation in the absence of mercuric ion to be classified as constitutive activation (CA) mutants. In this case, oligonucleotide-directed randomization of the target region and a screening/selection protocol were employed. Sixteen different mutants with an RD phenotype were identified, as well as eight different mutants with a CA phenotype. A high frequency of S87C mutations is evident in the RD set of mutants. The CA mutants have a high incidence of S86C and A89V mutations. The CA double mutant S86C/A89V was purified and found to bind to its DNA site with an affinity similar to that of the wild-type protein. Chemical nuclease activity assays indicate that the nonmercurated S86C/A89V CA mutant has a DNA distortion activity identical to that of mercurated wild-type MerR. A unique disulfide bond bridging this CA mutant's dimer interface was found and is proposed to constrain protein conformation in a manner analogous to mercuric ion binding in the wild-type protein.

Escherichia coli biotin holoenzyme synthetase/bio repressor crystal structure delineates the biotin- and DNA-binding domains.

The three-dimensional structure of BirA, the repressor of the Escherichia coli biotin biosynthetic operon, has been determined by x-ray crystallography and refined to a crystallographic residual of 19.0% at 2.3-A resolution. BirA is a sequence-specific DNA-binding protein that also catalyzes the formation of biotinyl-5'-adenylate from biotin and ATP and transfers the biotin moiety to other proteins. The level of biotin biosynthetic enzymes in the cell is controlled by the amount of biotinyl-5'-adenylate, which is the BirA corepressor. The structure provides an example of a transcription factor that is also an enzyme. The structure of BirA is highly asymmetric and consists of three domains. The N-terminal domain is mostly alpha-helical, contains a helix-turn-helix DNA-binding motif, and is loosely connected to the remainder of the molecule. The central domain consists of a seven-stranded mixed beta-sheet with alpha-helices covering one face. The other side of the sheet is largely solvent-exposed and contains the active site. The C-terminal domain comprises a six-stranded, antiparallel beta-sheet sandwich. The location of biotin binding is consistent with mutations that affect enzymatic activity. A nearby loop has a sequence that has been associated with phosphate binding in other proteins. It is inferred that ATP binds in this region, adjacent to the biotin. It is proposed that the binding of corepressor to monomeric BirA may promote DNA binding by facilitating the formation of a multimeric BirA-corepressor-DNA complex. The structural details of this complex remain an open question, however.

Mutagenesis of the cysteines in the metalloregulatory protein MerR indicates that a metal-bridged dimer activates transcription.

Bacterial resistance to mercury(II) compounds is controlled by the metalloregulatory MerR protein, a transcriptional repressor and a mercuric ion dependent activator of the mer operon. Site-directed mutagenesis of all four cysteine residues in the Tn501 MerR protein has led to the specific replacement of C82, C115, and C117 with alanine and of C126 with serine. Mutation of C82 and C126 abolishes transcriptional activation in vivo while mutation of C115 and C117 leads to a slight increase and dramatic decrease in transcriptional activation, respectively. All four mutants are competent, to varying degrees, to repress mer transcription. Characterization of the four purified mutant proteins in vitro demonstrates that only the C126S MerR mutant is most notably deficient in stoichiometric Hg(II) binding. All four mutant proteins possess similar DNA binding properties, and the C82 mutant is most affected in the ability to form stable dimers. Given an observed stoichiometry of one Hg(II) per MerR dimer, it is likely that the transcriptionally activating MerR species is a metal-bridged dimer. It is most likely that one C126 per subunit provides high-avidity bidentate ligation to Hg(II), but it remains possible that C82 may be a secondary Hg(II) ligand (e.g., in a tetracoordinate thiol ligation array).

Transcriptional switching by the MerR protein: activation and repression mutants implicate distinct DNA and mercury(II) binding domains.

Bacterial resistance to mercuric compounds is controlled by the MerR metalloregulatory protein. The MerR protein functions as both a transcriptional repressor and a mercuric ion dependent transcriptional activator. Chemical mutagenesis of the cloned merR structural gene has led to the identification of mutant proteins that are specifically deficient in transcriptional repression, activation, or both. Five mutant proteins have been overproduced, purified to homogeneity, and assayed for ability to dimerize, bind mer operator DNA, and bind mercuric ion. A mutation in the recognition helix of a proposed helix-turn-helix DNA binding motif (E22K) yields protein deficient in both activation and repression in vivo (a-r-) and deficient in operator binding in vitro. In contrast, mutations in three of the four MerR cysteine residues are repression competent but activation deficient (a-r+) in vivo. In vitro, the purified cysteine mutant proteins bind to the mer operator site with near wild-type affinity but are variably deficient in binding the in vivo inducer mercury(II) ion. A subset of the isolated proteins also appears compromised in their ability to form dimers at low protein concentrations. These data, taken with the results in the preceding paper (Shewchuk et al., 1989), support a model in which DNA-bound MerR dimer binds one mercuric ion and transmits this occupancy information to a protein region involved in transcriptional activation.

Transcriptional switching by the metalloregulatory MerR protein: initial characterization of DNA and mercury (II) binding activities.

The MerR protein from the Tn501 mercury resistance operon is a metalloregulatory transcriptional switch, converting from repressor to activator on binding of Hg(II). We have determined via binding studies with 203Hg(II) that a single Hg(II) atom binds to the MerR dimer (32 kDa) with a half-saturation concentration of 10(-7) M in the presence of up to 10(-3) M exogenous thiols. This 10(4) selective binding is specific for the binding of Hg(II) and corresponds to concentrations of metal that induce mercury(II) resistance in vivo. Kd values for MerR binding, in the absence and presence of Hg(II), to a 305 bp DNA fragment containing the 18 bp dyad symmetry element, DS1, located at -35 to -10 upstream of the mer structural genes, were determined by a gel shift assay. A Kd of 10(-10) M for free MerR and 10(-11) M for Hg(II)-MerR complexes was revealed. Measurements of koff values, by this assay, show equally long-lived complexes of MerR-DNA (51-min half-life) and Hg(II)-MerR-DNA (49-min half-life), suggesting that Hg(II) accelerates MerR binding to DNA rather than influencing the dissociation rate of the protein-DNA complex. In contrast, 203Hg(II) studies reveal that mercuric ions rapidly dissociate and associate with MerR-DNA complexes. Extensive footprinting studies by DNase I, methylation protection, and hydroxyl radicals indicate MerR stays bound to DS1 even on addition of Hg(II) and shares no interaction in vitro with a second dyad symmetry element, DS2, centered at -79/-80.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular basis of bacterial resistance to organomercurial and inorganic mercuric salts.

Bacteria mediate resistance to organomercurial and inorganic mercuric salts by metabolic conversion to nontoxic elemental mercury, Hg(0). The genes responsible for mercury resistance are organized in the mer operon, and such operons are often found in plasmids that also bear drug resistance determinants. We have subcloned three of these mer genes, merR, merB, and merA, and have studied their protein products via protein overproduction and purification, and structural and functional characterization. MeR is a metalloregulatory DNA-binding protein that acts as a repressor of both its own and structural gene transcription in the absence of Hg(II); in addition it acts as a positive effector of structural gene transcription when Hg(II) is present. MerB, organomercury lyase, catalyzes the protonolytic fragmentation of organomercurials to the parent hydrocarbon and Hg(II) by an apparent SE2 mechanism. MerA, mercuric ion reductase, is an FAD-containing and redox-active disulfide-containing enzyme with homology to glutathione reductase. It has evolved the unique catalytic capacity to reduce Hg(II) to Hg(0) and thereby complete the detoxification scheme.