Kinetic and structural characterization of caspase-3 and caspase-8 inhibition by a novel class of irreversible inhibitors.

Because of their central role in programmed cell death, the caspases are attractive targets for developing new therapeutics against cancer and autoimmunity, myocardial infarction and ischemic damage, and neurodegenerative diseases. We chose to target caspase-3, an executioner caspase, and caspase-8, an initiator caspase, based on the vast amount of information linking their functions to diseases. Through a structure-based drug design approach, a number of novel beta-strand peptidomimetic compounds were synthesized. Kinetic studies of caspase-3 and caspase-8 inhibition were carried out with these urazole ring-containing irreversible peptidomimetics and a known irreversible caspase inhibitor, Z-VAD-fmk. Using a stopped-flow fluorescence assay, we were able to determine individual kinetic parameters of caspase-3 and caspase-8 inhibition by these inhibitors. Z-VAD-fmk and the peptidomimetic inhibitors inhibit caspase-3 and caspase-8 via a three-step kinetic mechanism. Inhibition of both caspase-3 and caspase-8 by Z-VAD-fmk and of caspase-3 by the peptidomimetic inhibitors proceeds via two rapid equilibrium steps followed by a relatively fast inactivation step. However, caspase-8 inhibition by the peptidomimetics goes through a rapid equilibrium step, a slow-binding reversible step, and an extremely slow inactivation step. The crystal structures of inhibitor complexes of caspases-3 and -8 validate the design of the inhibitors by illustrating in detail how they mimic peptide substrates. One of the caspase-8 structures also shows binding at a secondary, allosteric site, providing a possible route to the development of noncovalent small molecule modulators of caspase activity.

Outlet density as a predictor of alcohol use in early adolescence.

INTRODUCTION: Recent national youth surveys suggest that alcohol availability plays a role in determining use. One measure of availability receiving recent attention is outlet density; however, few studies have examined the effects of outlet density in younger populations. METHODS: Data were collected from a national sample of the United States (N = 5,903) followed between 6th and 8th grades, as part of a study funded by the Office of Juvenile Justice and Delinquency Prevention (OJJDP). Measures of outlet density were also acquired. RESULTS: Students in high off-site density communities increased their alcohol use; however, students attending schools in low outlet density communities had higher initial levels of alcohol use that remained relatively stable. DISCUSSION: The implications and limitations of these findings are discussed.

Escherichia coli expression, purification and characterization of functional full-length recombinant alpha2beta2gamma3 heterotrimeric complex of human AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is an energy-sensing serine/threonine protein kinase that plays a central role in whole-body energy homeostasis. AMPK is a heterotrimeric enzyme with a catalytic (alpha) subunit and two regulatory (beta and gamma) subunits. The muscle-specific AMPK heterotrimeric complex (alpha2beta2gamma3) is involved in glucose and fat metabolism in skeletal muscle and therefore has emerged as an attractive target for drug development for diabetes and metabolic syndrome. To date, expression of recombinant full-length human AMPK alpha2beta2gamma3 has not been reported. Here we describe the expression, purification and biochemical characterization of functional full-length AMPK alpha2beta2gamma3 heterotrimeric complex using an Escherichia coli expression system. All three subunits of AMPK alpha2beta2gamma3 were transcribed as a single tricistronic transcript driven by the T7 RNA polymerase promoter, allowing spontaneous formation of the heterotrimeric complex in the bacterial cytosol. The self-assembled trimeric complex was purified from the cell lysate by nickel-ion chromatography using the hexahistidine tag fused exclusively at the N-terminus of the alpha 2 domain. The un-assembled beta 2 and gamma 3 domains were removed by extensive washing of the column. Further purification of the heterotrimer was performed using size exclusion chromatography. The final yield of the recombinant AMPK alpha2beta2gamma3 complex was 1.1mg/L culture in shaker flasks. The E. coli expressed enzyme was catalytically inactive after purification, but was activated in vitro by upstream kinases such as CaMKKbeta and LKB1. The kinase activity of activated AMPK alpha2beta2gamma3 complex was significantly enhanced by AMP (an allosteric activator) but not by thienopyridone A-769662, a known small molecule activator of AMPK. Mass spectrometric characterization of recombinant AMPK alpha2beta2gamma3 showed significant heterogeneity before and after activation that could potentially hamper crystallographic studies of this complex.

Expression, purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain.

Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono- (Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non- and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69mg per 20L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3mg per 10L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliper-based microfluidics assay was used to determine the kinetic parameters (K(m) and k(cat)) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4A. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.

Substituted pyrazoles as hepatoselective HMG-CoA reductase inhibitors: discovery of (3R,5R)-7-[2-(4-fluoro-phenyl)-4-isopropyl-5-(4-methyl-benzylcarbamoyl)-2H-pyrazol-3-yl]-3,5-dihydroxyheptanoic acid (PF-3052334) as a candidate for the treatment of hypercholesterolemia.

In light of accumulating evidence that aggressive LDL-lowering therapy may offer increased protection against coronary heart disease, we undertook the design and synthesis of a novel series of HMG-CoA reductase inhibitors based upon a substituted pyrazole template. Optimizing this series using both structure-based design and molecular property considerations afforded a class of highly efficacious and hepatoselective inhibitors resulting in the identification of (3 R,5 R)-7-[2-(4-fluoro-phenyl)-4-isopropyl-5-(4-methyl-benzylcarbamoyl)-2 H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic (PF-3052334) as a candidate for the treatment of hypercholesterolemia.

Does tailoring matter? Meta-analytic review of tailored print health behavior change interventions.

Although there is a large and growing literature on tailored print health behavior change interventions, it is currently not known if or to what extent tailoring works. The current study provides a meta-analytic review of this literature, with a primary focus on the effects of tailoring. A comprehensive search strategy yielded 57 studies that met inclusion criteria. Those studies-which contained a cumulative N = 58,454-were subsequently meta-analyzed. The sample size-weighted mean effect size of the effects of tailoring on health behavior change was found to be r = .074. Variables that were found to significantly moderate the effect included (a) type of comparison condition, (b) health behavior, (c) type of participant population (both type of recruitment and country of sample), (d) type of print material, (e) number of intervention contacts, (f) length of follow-up, (g) number and type of theoretical concepts tailored on, and (h) whether demographics and/or behavior were tailored on. Implications of these results are discussed and future directions for research on tailored health messages and interventions are offered.

Structural basis for AMPK activation: natural and synthetic ligands regulate kinase activity from opposite poles by different molecular mechanisms.

AMP-activated protein kinase (AMPK) is a principal metabolic regulator affecting growth and response to cellular stress. Comprised of catalytic and regulatory subunits, each present in multiple forms, AMPK is best described as a family of related enzymes. In recent years, AMPK has emerged as a desirable target for modulation of numerous diseases, yet clinical therapies remain elusive. Challenges result, in part, from an incomplete understanding of the structure and function of full-length heterotrimeric complexes. In this work, we provide the full-length structure of the widely expressed α1ß1γ1 isoform of mammalian AMPK, along with detailed kinetic and biophysical characterization. We characterize binding of the broadly studied synthetic activator A769662 and its analogs. Our studies follow on the heels of the recent disclosure of the α2ß1γ1 structure and provide insight into the distinct molecular mechanisms of AMPK regulation by AMP and A769662.

Activation of AMP-activated protein kinase revealed by hydrogen/deuterium exchange mass spectrometry.

AMP-activated protein kinase (AMPK) monitors cellular energy, regulates genes involved in ATP synthesis and consumption, and is allosterically activated by nucleotides and synthetic ligands. Analysis of the intact enzyme with hydrogen/deuterium exchange mass spectrometry reveals conformational perturbations of AMPK in response to binding of nucleotides, cyclodextrin, and a synthetic small molecule activator, A769662. Results from this analysis clearly show that binding of AMP leads to conformational changes primarily in the γ subunit of AMPK and subtle changes in the α and ß subunits. In contrast, A769662 causes profound conformational changes in the glycogen binding module of the ß subunit and in the kinase domain of the α subunit, suggesting that the molecular binding site of the latter resides between the α and ß subunits. The distinct short- and long-range perturbations induced upon binding of AMP and A769662 suggest fundamentally different molecular mechanisms for activation of AMPK by these two ligands.

A short-term, quasi-experimental evaluation of D.A.R.E.'s revised elementary school curriculum.

We present the short-term results of a quasi-experimental evaluation of the revised D.A.R.E. (Drug Abuse Resistance Education) curriculum. Study outcomes examined were D.A.R.E.'s effects on three substances, namely students' lifetime and 30-day use of tobacco, alcohol, and marijuana, as well as their school attendance and academic performance. The study comprised students in 17 urban schools, each of which served as its own control; 5th graders in the 2006-2007 school year constituted the comparison group (n = 1490), and those enrolled as 5th graders in the 2007-2008 school year constituted the intervention group (n= 1450). We found no intervention effect on students' substance use for any of the substance use outcomes assessed. We did find that students were more likely to attend school on days they received D.A.R.E. lessons and that students in the intervention group were more likely to have been suspended. Study findings provide little support for the implementation and dissemination of the revised D.A.R.E. curriculum.

Discovering expectant mothers' beliefs about oral health: an application of the Centering Pregnancy Smiles program.

Research shows expectant mothers with oral infections may have an increased risk for delivering preterm, low birth weight babies. Difficulty accessing dental services, limited resources, and beliefs about dental care put expectant mothers from rural communities at a greater risk for oral health problems, which can have adverse health consequences for themselves and their unborn children. There is a need to better educate these women on proper oral health practices to decrease oral infections and increase the likelihood of delivering healthy babies. Using the Extended Parallel Process Model (EPPM) [1], this study examines the impact of a prenatal program, Centering Pregnancy Smiles (CPS), on changing rural expectant mothers' attitudes and beliefs about maintaining good oral health during pregnancy. Results showed the CPS program had a primarily positive impact on changing expectant mothers' attitudes and beliefs regarding oral health. Implications for educational prenatal programs on oral health in rural areas are discussed.

Thermodynamic and structure guided design of statin based inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Clinical studies have demonstrated that statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) inhibitors, are effective at lowering mortality levels associated with cardiovascular disease; however, 2-7% of patients may experience statin-induced myalgia that limits compliance with a treatment regimen. High resolution crystal structures, thermodynamic binding parameters, and biochemical data were used to design statin inhibitors with improved HMGR affinity and therapeutic index relative to statin-induced myalgia. These studies facilitated the identification of imidazole 1 as a potent (IC 50 = 7.9 nM) inhibitor with excellent hepatoselectivity (>1000-fold) and good in vivo efficacy. The binding of 1 to HMGR was found to be enthalpically driven with a Delta H of -17.7 kcal/M. Additionally, a second novel series of bicyclic pyrrole-based inhibitors was identified that induced order in a protein flap of HMGR. Similar ordering was detected in a substrate complex, but has not been reported in previous statin inhibitor complexes with HMGR.

Design and synthesis of novel, conformationally restricted HMG-CoA reductase inhibitors.

Using structure-based design, a novel series of conformationally restricted, pyrrole-based inhibitors of HMG-CoA reductase were discovered. Leading analogs demonstrated potent inhibition of cholesterol synthesis in both in vitro and in vivo models and may be useful for the treatment of hypercholesterolemia and related lipid disorders.

Design and synthesis of hepatoselective, pyrrole-based HMG-CoA reductase inhibitors.

This manuscript describes the design and synthesis of a series of pyrrole-based inhibitors of HMG-CoA reductase for the treatment of hypercholesterolemia. Analogs were optimized using structure-based design and physical property considerations resulting in the identification of 44, a hepatoselective HMG-CoA reductase inhibitor with excellent acute and chronic efficacy in a pre-clinical animal models.

Co-crystal structure and inhibition of factor Xa by PD0313052 identifies structurally stabilized active site residues of factor Xa and prothrombinase.

The enzyme complex prothrombinase plays a pivotal role in fibrin clot development through the production of thrombin, making this enzyme complex an attractive target for therapeutic regulation. This study both functionally and structurally characterizes a potent, highly selective, active site directed inhibitor of human factor Xa and prothrombinase, PD0313052, and identifies structurally conserved residues in factor Xa and prothrombinase. Analyses of the association and dissociation of PD0313052 with human factor Xa identified a reversible, slow-onset mechanism of inhibition and a simple, single-step bimolecular association between factor Xa and PD0313052. This interaction was governed by association (k(on)) and dissociation (k(off)) rate constants of (1.0 +/- 0.1) x 10(7) M(-1) s(-1) and (1.9 +/- 0.5) x 10(-3) s(-1), respectively. The inhibition of human factor Xa by PD0313052 displayed significant tight-binding character described by a Ki* = 0.29 +/- 0.08 nM. Similar analyses of the inhibition of human prothrombinase by PD0313052 also identified a slow-onset mechanism with a Ki* = 0.17 +/- 0.03 nM and a k(on) and k(off) of (0.7 +/- 0.1) x 10(7) M(-1) s(-1) and (1.7 +/- 0.8) x 10(-3) s(-1), respectively. Crystals of factor Xa and PD0313052 demonstrated hydrogen bonding contacts within the S1-S4 pocket at residues Ser195, Asp189, Gly219, and Gly216, as well as interactions with aromatic residues within the S4 pocket. Overall, these data demonstrate that the inhibition of human factor Xa by PD0313052 occurs via a slow, tight-binding mechanism and indicate that active site residues of human factor Xa, including the catalytic Ser195, are effectively unaltered following assembly into prothrombinase.

Inhibitors of HCV NS5B polymerase. Part 1: Evaluation of the southern region of (2Z)-2-(benzoylamino)-3-(5-phenyl-2-furyl)acrylic acid.

A novel series of nonnucleoside HCV NS5B polymerase inhibitors were prepared from (2Z)-2-(benzoylamino)-3-(5-phenyl-2-furyl)acrylic acid, a high throughput screening lead. SAR studies combined with structure based drug design focusing on the southern heterobiaryl region of the template led to the synthesis of several potent and orally bioavailable lead compounds. X-ray crystallography studies were also performed to understand the interaction of these inhibitors with HCV NS5B polymerase.

Inhibitors of HCV NS5B polymerase. Part 2: Evaluation of the northern region of (2Z)-2-benzoylamino-3-(4-phenoxy-phenyl)-acrylic acid.

A novel series of non-nucleoside HCV NS5B polymerase inhibitors was prepared from a (2Z)-2-benzoylamino-3-(4-phenoxy-phenyl)-acrylic acid template. Solution and solid phase analog synthesis focused on the northern region of the template combined with structure based design led to the discovery of several potent and orally bioavailable lead compounds.

Physical methods to determine the binding mode of putative ligands for hepatitis C virus NS3 helicase.

Several small molecules identified by high-throughput screening (HTS) were evaluated for their ability to bind to a nonstructural protein 3 (NS3) helicase from hepatitis C virus (HCV). Equilibrium dissociation constants (K(d)'s) of the compounds for this helicase were determined using several techniques including an assay measuring the kinetics of isothermal enzyme denaturation at several concentrations of the test molecule. Effects of two nonhydrolyzable ATP analogs on helicase denaturation were measured as controls using the isothermal denaturation (ITD) assay. Two compounds, 4-(2,4-dimethylphenyl)-2,7,8-trimethyl-4,5-quinolinediamine and 2-phenyl-N-(5-piperazin-1-ylpentyl)quinazolin-4-amine, were identified from screening that inhibited the enzyme and had low micromolar dissociation constants for NS3 helicase in the ITD assay. Low micromolar affinity of the quinolinediamine to helicase was also confirmed by nuclear magnetic resonance experiments. Unfortunately, isothermal titration calorimetry (ITC) experiments indicated that a more water-soluble analog bound to the 47/23-mer oligonucleotide helicase substrate with low micromolar affinity as did the substituted quinazolinamine. There was no further interest in these templates as helicase inhibitors due to the nonspecific binding to enzyme and substrate. A combination of physical methods was required to discern the mode of action of compounds identified by HTS and remove undesirable lead templates from further consideration.

Crystal structure of type II peptide deformylase from Staphylococcus aureus.

The first crystal structure of Class II peptide deformylase has been determined. The enzyme from Staphylococcus aureus has been overexpressed and purified in Escherichia coli and the structure determined by x-ray crystallography to 1.9 A resolution. The purified iron-enriched form of S. aureus peptide deformylase enzyme retained high activity over many months. In contrast, the iron-enriched form of the E. coli enzyme is very labile. Comparison of the two structures details many differences; however, there is no structural explanation for the dramatic activity differences we observed. The protein structure of the S. aureus enzyme reveals a fold similar, but not identical to, the well characterized E. coli enzyme. The most striking deviation of the S. aureus from the E. coli structure is the unique conformation of the C-terminal amino acids. The distinctive C-terminal helix of the latter is replaced by a strand in S. aureus which wraps around the enzyme, terminating near the active site. Although there are no differences at the amino acid level near the active site metal ion, significant changes are noted in the peptide binding cleft which may play a role in the design of general peptide deformylase inhibitors.

Co-crystallization of Staphylococcus aureus peptide deformylase (PDF) with potent inhibitors.

In bacteria the biosynthesis of all nascent polypeptides begins with N-formylmethionine. The post-translational removal of the N-formyl group is carried out by peptide deformylase (PDF). Processing of the N-formyl group from critical bacterial proteins is required for cell survival. This formylation/deformylation cycle is unique to eubacteria and is not utilized in eucaryotic cytosolic protein biosynthesis. Thus, inhibition of PDF would halt bacterial growth, spare host cell-function, and would be a novel mechanism for a new class of antibiotic. Diffraction-quality Se-met crystals of S. aureus PDF were prepared that belong to space group C222(1) with unit cell parameters of a = 94.1 b = 121.9 c = 47.6 A. Multiple anomalous dispersion data were collected at the Advanced Photon Source 17-ID beamline and used to solve the PDF structure to 1.9 A resolution. Crystals were also prepared with three PDF inhibitors: thiorphan, actinonin and PNU-172550. The thiorphan and actinonin co-crystals belong to space group C222(1) with similar unit-cell dimensions. Repeated attempts to generate a complex structure of PDF with PNU-172550 from the orthorhombic space group were unsuccessful. Crystallization screening identified an alternate C2 crystal form with unit-cell dimensions of a = 93.4 b = 42.5 c = 104.1 A, beta = 93 degrees.