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.

Cyclooxygenase-2 blockade does not impair endothelial vasodilator function in healthy volunteers: randomized evaluation of rofecoxib versus naproxen on endothelium-dependent vasodilatation.

BACKGROUND: From a cardiovascular standpoint, the safety of cyclooxygenase-2 (COX-2) blockers has been a topic of increasing concern. This concern stemmed from observations indicating that the COX-2 isoform is the major source of endothelium-derived prostacyclin and, hence, that selective blockade of this enzyme may impair endothelial health. To investigate this matter, we examined the effects of 7 days of treatment with rofecoxib versus naproxen on endothelial function in healthy volunteers. METHODS AND RESULTS: Thirty-five healthy volunteers were randomized to receive 7-day treatment with either rofecoxib (25 mg/d, n=18) or naproxen (750 mg/d, n=17). Vascular response measurements were conducted using forearm strain-gauge plethysmography. Changes in forearm blood flow in response to the endothelium-dependent vasodilator acetylcholine (3, 10, and 30 microg/min) and the endothelium-independent vasodilator sodium nitroprusside (1 and 10 microg/min) were assessed before and after treatment. Acetylcholine evoked a dose-dependent increase in forearm blood flow in all groups. Importantly, treatment resulted in no change in acetylcholine-mediated increases in forearm blood flow in either group (naproxen, P=0.27; rofecoxib, P=0.58). Similarly, there was no change in forearm blood flow in response to sodium nitroprusside (naproxen, P=0.55; rofecoxib, P=0.63). CONCLUSIONS: We herein describe, for the first time, the effects of COX-2-selective inhibition on endothelium-dependent vasodilatation in healthy adults. COX-2 blockade, when used at the doses employed therapeutically (which are known to inhibit vascular prostacyclin production) did not result in significant changes in endothelial vasodilator responses in healthy volunteers. The effects of COX-2 inhibitors on vasodilator responses in patients with coronary artery disease remain to be determined.

Oxindole-based inhibitors of cyclin-dependent kinase 2 (CDK2): design, synthesis, enzymatic activities, and X-ray crystallographic analysis.

Two closely related classes of oxindole-based compounds, 1H-indole-2,3-dione 3-phenylhydrazones and 3-(anilinomethylene)-1,3-dihydro-2H-indol-2-ones, were shown to potently inhibit cyclin-dependent kinase 2 (CDK2). The initial lead compound was prepared as a homologue of the 3-benzylidene-1,3-dihydro-2H-indol-2-one class of kinase inhibitor. Crystallographic analysis of the lead compound bound to CDK2 provided the basis for analogue design. A semiautomated method of ligand docking was used to select compounds for synthesis, and a number of compounds with low nanomolar inhibitory activity versus CDK2 were identified. Enzyme binding determinants for several analogues were evaluated by X-ray crystallography. Compounds in this series inhibited CDK2 with a potency approximately 10-fold greater than that for CDK1. Members of this class of inhibitor cause an arrest of the cell cycle and have shown potential utility in the prevention of chemotherapy-induced alopecia.

Prevention of chemotherapy-induced alopecia in rats by CDK inhibitors.

Most traditional cytotoxic anticancer agents ablate the rapidly dividing epithelium of the hair follicle and induce alopecia (hair loss). Inhibition of cyclin-dependent kinase 2 (CDK2), a positive regulator of eukaryotic cell cycle progression, may represent a therapeutic strategy for prevention of chemotherapy-induced alopecia (CIA) by arresting the cell cycle and reducing the sensitivity of the epithelium to many cell cycle-active antitumor agents. Potent small-molecule inhibitors of CDK2 were developed using structure-based methods. Topical application of these compounds in a neonatal rat model of CIA reduced hair loss at the site of application in 33 to 50% of the animals. Thus, inhibition of CDK2 represents a potentially useful approach for the prevention of CIA in cancer patients.

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.

Binding mode of the 4-anilinoquinazoline class of protein kinase inhibitor: X-ray crystallographic studies of 4-anilinoquinazolines bound to cyclin-dependent kinase 2 and p38 kinase.

4-Anilinoquinazolines represent an important class of protein kinase inhibitor. Modes of binding for two members of this inhibitor class were determined by X-ray crystallographic analysis of one inhibitor (4-[3-hydroxyanilino]-6,7-dimethoxyquinazoline) in complex with cyclin-dependent kinase 2 (CDK2) and the other (4-[3-methylsulfanylanilino]-6,7-dimethoxyquinazoline) in complex with p38 kinase. In both inhibitor/kinase structures, the 4-anilinoquinazoline was bound in the ATP site with the quinazoline ring system oriented along the peptide strand that links the two domains of the protein and with the anilino substituent projecting into a hydrophobic pocket within the protein interior. In each case, the nitrogen at position-1 of the quinazoline accepted a hydrogen bond from a backbone NH (CDK2, Leu-83; p38, Met-109) of the domain connector strand, and aromatic hydrogen atoms at C2 and C8 interacted with backbone carbonyl oxygen atoms of the peptide strand. The anilino group of the CDK2-bound compound was essentially coplanar with the quinazoline ring system and occupied a pocket between Lys-33 and Phe-80. For the p38-bound inhibitor, the anilino group was angled out of plane and was positioned between Lys-53 and Thr-106 in a manner similar to that observed for the aryl substituent of the pyridinylimidazole class of inhibitor.

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.

Exercise-induced muscle injury: a calpain hypothesis.

It is well established that periods of increased contractile activity result in significant changes in muscle structure and function. Such morphological changes as sarcomeric Z-line disruption and sarcoplasmic reticulum vacuolization are characteristic of exercise-induced muscle injury. While the precise mechanism(s) underlying the perturbations to muscle following exercise remains to be elucidated, it is clear that disturbances in Ca2+ homeostasis and changes in the rate of protein degradation occur. The resulting elevation in intracellular [Ca2+] activates the non-lysosomal cysteine protease, calpain. Because calpain cleaves a variety of protein substrates including cytoskeletal and myofibrillar proteins, calpain-mediated degradation is thought to contribute to the changes in muscle structure and function that occur immediately following exercise. In addition, calpain activation may trigger the adaptation response to muscle injury. The purpose of this paper is to: (i) review the chemistry of the calpain-calpastatin system; (ii) provide evidence for the involvement of the non-lysosomal, calcium-activated neutral protease (calpain) in the response of skeletal muscle protein breakdown to exercise (calpain hypothesis); and (iii) describe the possible involvement of calpain in the inflammatory and regeneration response to exercise.

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.

Dietary L-glutamine does not improve lymphocyte metabolism or function in exercise-trained rats.

Decreased glutamine availability is proposed as a mechanism for changes in immune function with intense exhaustive exercise. Less is known about the immunomodulatory effects of regular nonexhaustive exercise. To determine the effects of low intensity regular exercise and dietary glutamine supplementation on plasma glutamine concentrations, lymphocyte metabolism, and immune function, male (278 +/- 5 g) and female (182 +/- 1 g) Sprague-Dawley Buffalo rats were fed nutritionally complete casein-based semi-purified diets +/- 2% w/w glutamine. Rats were trained (21 d), as confirmed by higher (P < 0.05) succinate dehydrogenase activity in soleus muscle, to swim 2 or 4 h.d-1 or remained sedentary. Exercise lowered plasma concentrations of tryptophan, glutamate, methionine, alanine, threonine, aspartate, asparagine, and ornithine and increased the lysine concentration (P < 0.05). Neither diet nor exercise altered plasma glutamine concentrations, lymphocyte phenotypes in spleen, or the in vitro rates of splenocyte energy metabolism (production of glucose and glutamine metabolites or ATP concentrations in the incubation media). Compared with nonsupplemented rats, splenic cytolytic activity (lysis of 51Cr labeled YAC-1 cells) was reduced (P < 0.05) in the glutamine-supplemented exercising group. Under these conditions, glutamine supplementation does not appear to provide any added benefit to the exercise-trained animal.

Dietary L-glutamine supplementation reduces the growth of the Morris Hepatoma 7777 in exercise-trained and sedentary rats.

Dietary glutamine supplementation and exercise have been reported independently to enhance immune function and reduce tumor growth. We study the effect of both of these interventions on the growth of the Morris Hepatoma 7777, implanted in 59 female Sprague-Dawley Buffalo rats. Rats were fed a nutritionally complete, purified diet with or without L-glutamine 20 g/kg diet and randomized to swim 3 h/d or to remain sedentary. After 14 d, the mean tumor weight of glutamine-supplemented rats was lower (P < 0.0001) than that of unsupplemented rats (5.8 +/- 0.4 vs. 8.7 +/- 0.5 g, respectively). Exercise did not alter tumor growth. Glutamine supplementation increased [3H] thymidine incorporation by splenocytes incubated with Concanavalin A and the proportion of natural killer cells in spleen, but not cytotoxic activity against YAC-1 cells. Glutamine supplementation did not alter glutamine concentrations in plasma (691 +/- 12 mumol/L) or soleus muscle (5328 +/- 102 pmol/mg) but resulted in higher (P < 0.004) plasma concentrations of leucine, isoleucine and valine, precursors of glutamine. Splenocytes from exercised rats had a higher (P < 0.001) mitogen response than those from sedentary rats. Isolated tumor cells demonstrated high rates of non-oxidative glucose and glutamine metabolism and consumption of glutamine, tryptophan and methionine. However, neither diet nor exercise significantly affected glucose or glutamine metabolism by tumor cells. The precise mechanism of tumor growth suppression by oral glutamine supplementation is not clear but may be related to changes in substrate availability, improved tumor-directed natural killer cytotoxic activity or a faster response to an immune challenge.

Reduced splenocyte metabolism and immune function in rats implanted with the Morris Hepatoma 7777.

Although the immune system is important in antitumor defense, little is know about the immune response during progressive tumor growth. Sprague-Dawley rats (171 +/- 3g) of the Buffalo strain were implanted with the Morris Hepatoma 7777 ([MH 7777] a poorly differentiated, rapidly growing tumor) and killed either 2 (T2) or 3 (T3) weeks postimplantation when the tumor weighed 3.0 +/- 0.4 and 14 +/- 1 g, respectively. Splenocytes were isolated and their phenotypes, metabolism (metabolite production from glucose and glutamine), proliferative response ([3H]thymidine incorporation in response to polyclonal mitogens), and natural killer (NK) cytotoxicity (lysis of YAC-1 cells) were determined. Five rats were killed with the T2 group to serve as non-tumor-bearing controls (T0). Food intake and nontumor body weight decreased (P < .01) 14 days after tumor implantation. There was a progressive decrease (T3 < T2 < T0) in splenic mitogen responses (P < .05) and plasma essential and nonessential amino acid concentrations (P < .05). Compared with T0, NK cytotoxic activity was significantly (P < .05) lower at T2 and higher at T3. The presence of the tumor at both T2 and T3 resulted in lower production of metabolites from glucose and glutamine by splenocytes. The proportion of CD8+ cells was lower (P < .05) and the proportion of B cells and macrophages higher (P < .05) in spleens from tumor-bearing rats. In conclusion, the presence of even a small tumor burden (1.4% of body weight) significantly altered the host's immune function and metabolism. A larger tumor burden (6% of body weight) increased NK cytotoxic activity and further reduced cell-mediated immune function.

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.

Structure of a hinge-bending bacteriophage T4 lysozyme mutant, Ile3-->Pro.

The mutant T4 phage lysozyme in which isoleucine 3 is replaced by proline (I3P) crystallizes in an orthorhombic form with two independent molecules in the asymmetric unit. Relative to wild-type lysozyme, which crystallizes in a trigonal form, the two I3P molecules undergo large hinge-bending displacements with the alignments of the amino-terminal and carboxy-terminal domains changed by 28.9 degrees and 32.9 degrees, respectively. The introduction of the mutation, together with the hinge-bending displacement, is associated with repacking of the side-chains of Phe4, Phe67 and Phe104. These aromatic residues are clustered close to the site of the mutation and are at the junction between the amino and carboxyl-terminal domains. As a result of this structural rearrangement the side-chain of Phe4 moves from a relatively solvent-exposed conformation to one that is largely buried. Mutant I3P also crystallizes in the same trigonal form as wild-type and, in this case, the observed structural changes are restricted to the immediate vicinity of the replacement. The main change is a shift of 0.3 to 0.5 A in the backbone of residues 1 to 5. The ability to crystallize I3P under similar conditions but in substantially different conformations suggests that the molecule undergoes large-scale hinge-bending displacements in solution. It is also likely that these conformational excursions are associated with repacking at the junction of the N-terminal and C-terminal domains. On the other hand, the analysis is complicated by possible effects of crystal packing. The different I3P crystal structures show substantial differences in the binding of solvent, both at the site of the Ile3-->Pro replacement and at other internal sites.

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.