DNA methylation epi-signature is associated with two molecularly and phenotypically distinct clinical subtypes of Phelan-McDermid syndrome.

BACKGROUND: Phelan-McDermid syndrome is characterized by a range of neurodevelopmental phenotypes with incomplete penetrance and variable expressivity. It is caused by a variable size and breakpoint microdeletions in the distal long arm of chromosome 22, referred to as 22q13.3 deletion syndrome, including the SHANK3 gene. Genetic defects in a growing number of neurodevelopmental genes have been shown to cause genome-wide disruptions in epigenomic profiles referred to as epi-signatures in affected individuals. RESULTS: In this study we assessed genome-wide DNA methylation profiles in a cohort of 22 individuals with Phelan-McDermid syndrome, including 11 individuals with large (2 to 5.8 Mb) 22q13.3 deletions, 10 with small deletions (< 1 Mb) or intragenic variants in SHANK3 and one mosaic case. We describe a novel genome-wide DNA methylation epi-signature in a subset of individuals with Phelan-McDermid syndrome. CONCLUSION: We identified the critical region including the BRD1 gene as responsible for the Phelan-McDermid syndrome epi-signature. Metabolomic profiles of individuals with the DNA methylation epi-signature showed significantly different metabolomic profiles indicating evidence of two molecularly and phenotypically distinct clinical subtypes of Phelan-McDermid syndrome.

Predictors of vitamin D status in subjects that consume a vitamin D supplement.

BACKGROUND/OBJECTIVE: Although dietary supplement use has increased significantly among the general population, the interplay between vitamin D supplementation and other factors that influence vitamin D status remains unclear. The objective of this study was to identify predictor variables of vitamin D status in free-living subjects to determine the extent to which vitamin D supplements and other factors influence vitamin D status. SUBJECTS/METHODS: This was a retrospective, cross-sectional study involving 743 volunteers. Serum 25-hydroxy-vitamin D (25(OH)D) level and the variables diet, supplement usage, latitude of residence, ethnicity, age and body mass index (BMI) were used to predict vitamin D status in a summer and winter cohort. RESULTS: Supplemental vitamin D3 consumption was the most significant positive predictor, whereas BMI was the most significant negative predictor, of vitamin D status in each cohort. Other positive predictors were fortified beverage and dairy consumption in the summer and winter cohort, respectively. Negative predictors were: African American, Asian and Hispanic race in the summer; latitude of residence >36°N, Asian and Hispanic ethnicity in the winter. Mean(± s.d.) 25(OH)D levels were 101.1 (± 42.1) and 92.6 (± 39.0) nmol/l in summer and winter, respectively. Comparing non-supplement vs supplement users, approximately 38 vs 2.5% in the winter and 18 vs 1.4% in the summer had vitamin D levels <50 nmol/l. CONCLUSIONS: Vitamin D supplementation was the most significant positive predictor of vitamin D status. Collectively, these data point to the practicality of utilizing vitamin D supplements to reduce hypovitaminosis D in adults throughout the United States.

SB-242235, a selective inhibitor of p38 mitogen-activated protein kinase. II: in vitro and in vivo metabolism studies and pharmacokinetic extrapolation to man.

1. Inhibition of p38 MAP kinase has been investigated extensively as a potential therapy for cytokine-mediated diseases such as autoimmune and inflammatory diseases. SB-242235 (1-(4-piperidinyl)-4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl) imidazole) is a potent and selective p38 MAP kinase inhibitor; the preclinical pharmacokinetics of SB-242235 have been described previously. The present studies were conducted to describe the in vitro metabolic rates and routes of SB-242235 metabolism, to characterize its in vivo preclinical metabolism, and to use these data to aid in the prediction of the pharmacokinetic behaviour of SB-242235 in man. 2. SB-242235 was metabolically stable in rat, dog, monkey and human hepatic microsomes, isolated hepatocytes and liver slices in vitro. The in vivo preclinical metabolism studies were consistent with the in vitro findings; SB-242235 was minimally metabolized, and was primarily excreted unchanged in the urine (45 and 67% of the administered dose in the rat and monkey, respectively). 3. Allometric scaling using various correction factors predicted that SB-242235 would have low clearance in man with a predicted half-life ranging from 11.5 to 18.7h. This prediction was consistent with the observed mean half-life of 16.4h in the first-in-man study for SB-242235. An allometric scaling method with a correction for interspecies differences in glomerular filtration rate provided the most accurate prediction of the pharmacokinetic behaviour of SB-242235 in humans, although the clinical data also highlight potential difficulties in conducting prospective allometry.

SB-242235, a selective inhibitor of p38 mitogen-activated protein kinase. I: preclinical pharmacokinetics.

1. SB-242235 (1-(4-piperidinyl)-4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl) imidazole) is a potent and selective p38 MAP kinase inhibitor that may be an effective therapy for cytokine-mediated diseases such as autoimmune or inflammatory diseases. The present studies were conducted to evaluate the pharmacokinetics of SB-242235 in several preclinical species, including rat, dog and monkey. 2. SB-242235 demonstrates generally favourable pharmacokinetic properties in all species examined. Systemic plasma clearance was high in rat, but in the non-rodent species SB-242235 demonstrated low to moderate clearance with plasma half-lives > 4h. Oral bioavailability in each preclinical species was high. In rat and monkey, SB-242235 demonstrated non-linear elimination kinetics that manifested as a decrease in clearance with increasing dose and apparent oral bioavailability > 100% at high oral doses. Furthermore, SB-242235 displayed concentration-dependent plasma protein binding over a concentration range of 1000-10,000 ng ml(-1). 3. In conclusion, SB-242235 demonstrates high oral bioavailability across the major preclinical species, and may thus be a useful tool compound for investigation of the role of p38 inhibition in various disease states. However, the observations of non-linear protein binding and disposition also suggest the need for caution in the design of and data interpretation from such studies.

SB-239063, a potent and selective inhibitor of p38 map kinase: preclinical pharmacokinetics and species-specific reversible isomerization.

PURPOSE: A series of studies was conducted to evaluate the preclinical pharmacokinetics of SB-239063 (trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-[(2-methoxy)pyrimidin-4-yl] imidazole), a potent and selective p38 MAP kinase inhibitor. METHODS: SB-239063 was administered both i.v. and p.o. in the rat, dog, cynomolgus monkey, and rhesus monkey, with standard pharmacokinetic parameters generated from the concentration vs. time data. RESULTS: Initial rat studies suggested possible nonlinear disposition, however, assay refinement revealed an in vivo trans-cis isomerization of SB-239063 to a metabolite with nearly identical chromatographic and mass spectral properties. SB-239063 exhibited low to moderate clearance and good bioavailability in the rat and dog, but poor bioavailability in the cynomolgus monkey. Substantial in vivo trans-cis isomerization occurred in the rat and cynomolgus monkey, but occurred to a far lesser extent in the dog. The isomerization reaction was reversible, with a recycled fraction of 0.20 and 0.0003 in the rat and cynomolgus monkey, respectively. In the rhesus monkey, bioavailability was also poor. but no in vivo isomerization was observed. Conclusions. These studies demonstrate the necessity of exercising vigilance in conducting high-throughput analytical method development, and the importance of using a variety of preclinical species when evaluating the disposition of new drug candidates.

Cellular response of antioxidant metalloproteins in Cu/Zn SOD transgenic mice exposed to hyperoxia.

Ceruloplasmin, metallothionein, and ferritin are metal-binding proteins with potential antioxidant activity. Despite evidence that they are upregulated in pulmonary tissue after oxidative stress, little is known regarding their influence on trace metal homeostasis. In this study, we have used copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) transgenic-overexpressing and gene knockout mice and hyperoxia to investigate the effects of chronic and acute oxidative stress on the expression of these metalloproteins and to identify their influence on copper, zinc, and iron homeostasis. We found that the oxidative stress-mediated induction of ceruloplasmin and metallothionein in the lung had no effect on tissue levels of copper, iron, or zinc. However, Cu/Zn SOD expression had a marked influence on hepatic copper and iron as well as circulating copper homeostasis. These results suggest that ceruloplasmin and metallothionein may function as antioxidants independent of their role in trace metal homeostasis and that Cu/Zn SOD functions in copper homeostasis via mechanisms distinct from its superoxide scavenging properties.

Potent and selective nonpeptide inhibitors of caspases 3 and 7.

5-Dialkylaminosulfonylisatins have been identified as potent, nonpeptide inhibitors of caspases 3 and 7. The most active compound within this series (34) inhibited caspases 3 and 7 in the 2-6 nM range and exhibited approximately 1000-fold selectivity for caspases 3 and 7 versus a panel of five other caspases (1, 2, 4, 6, and 8) and was at least 20-fold more selective versus caspase 9. Sequence alignments of the active site residues of the caspases strongly suggest that the basis of this selectivity is due to binding in the S2 subsite comprised of residues Tyr204, Trp206, and Phe256 which are unique to caspases 3 and 7. These compounds inhibit apoptosis in three cell-based models: human Jurkat T cells, human chondrocytes, and mouse bone marrow neutrophils.

Development of an in vivo preclinical screen model to estimate absorption and bioavailability of xenobiotics.

The purpose of this study was to develop an in vivo screening method for rapid preclinical characterization of absorption and bioavailability of large numbers of compounds. This effort involved several steps. First, a pharmacokinetic characterization of a reference compound was conducted in the monkey. These data were used to verify theoretical calculations of a maximal portal dose-normalized area under the concentration-time curve. Next, a monkey screen was implemented using mixtures of up to five compounds each (i.e., cassettes) to estimate the bioavailability of approximately 200 compounds. Cassettes were administered as a single intraduodenal dose to a single monkey followed by simultaneous portal and systemic blood sampling. Definitive studies were then conducted to determine absolute bioavailability of 14 of these compounds. The studies with the reference compound demonstrated that the theoretical methodology based on a single intraduodenal dose with portal and systemic sampling provided consistent estimates of bioavailability. In the screen studies, approximately 75% of the test compounds were excluded from further evaluation due to poor absorption. Of the 14 compounds selected for follow-up evaluation from both well and poorly absorbed compounds, the absolute bioavailability of 10 of them were correctly classified from the screening data. The remaining 4 compounds were false positives, which showed low bioavailability; no false negatives were encountered. This approach allows for a rapid and reliable screen to evaluate absorption and bioavailability using a single dose in a preclinical model.

Preclinical pharmacokinetics of SB-203580, a potent inhibitor of p38 mitogen-activated protein kinase.

1. SB-203580 (4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)imidazole) is a potent, selective inhibitor of p38 MAP kinase used extensively as a tool inhibitor in various pharmacological and toxicological models. This study was designed to evaluate the pharmacokinetics of SB-203580 in several preclinical species, both to assist with the interpretation of existing studies and to aid in the design of future studies with this inhibitor. 2. In vitro, SB-203580 was stable in mouse, rat, dog, monkey and human plasma over 24 h. However, species differences in plasma protein binding were observed; SB-203580 was 96-97% bound in human plasma and 78-92% bound in other species. These data suggest that protein binding may influence the results of in vitro studies using SB-203580, particularly when comparing results from different in vitro systems that incorporate plasma components. In vivo, SB-203580) demonstrated moderate to high clearance in all species tested, with non-linear elimination observed in the rat at plasma concentrations > 1,000 ngml(-1). Although good solution bioavailability was observed in non-rodents (78% in dog, 32% in monkey), lower and more variable bioavailability was observed in the rat and mouse (3 -48%). 3. These interspecies differences in bioavailability, and the non-linear pharmacokinetics observed in rat, highlight the importance of monitoring SB-203580 systemic exposure in parallel witb the pharmacological endpoint during in vivo pharmacology

Inhibition of caspase-3-like activity prevents apoptosis while retaining functionality of human chondrocytes in vitro.

Apoptosis was induced in a human chondrocyte cell line, T/C 28a4, by treatment with various stimuli, including camptothecin, tumor necrosis factor-alpha, staurosporine, okadaic acid, and reduced serum conditions. All stimuli induced a cytosolic DEVDase activity, coincident with apoptosis. Caspase activities in the lysates were characterized and quantitated with peptide cleavage profiles. To confirm that the results were not related to the immortalized nature of the cell line, primary human chondrocytes also were shown to undergo apoptosis under similar conditions, which resulted in increased cytosolic DEVDase activity. There was little or no caspase-1 (interleukin-1beta-converting enzyme) or caspase-8-like activity in the apoptotic cells. In all cases, the irreversible nonselective caspase inhibitor, Z-VAD-FMK, and the caspase-3-selective inhibitor, Ac-DMQD-CHO, inhibited DEVDase activity and apoptosis, whereas the caspase-1-selective inhibitor, Ac-YVAD-CHO, had no effect. Human chondrocytes were stably and transiently transfected with a type-II collagen gene (COL2A1) regulatory sequence driving a luciferase reporter as a specific marker of chondrocyte gene expression. Treatment of the cells with camptothecin or tumor necrosis factor-alpha plus cycloheximide significantly inhibited COL2A1 transcriptional activity. Significantly, cotreatment with Z-VAD-FMK or Ac-DMQD-CHO maintained COL2A1-reporter gene activity, indicating that the prevention of apoptosis by caspase-3 inhibition was sufficient to maintain cell functionality as assessed by the retention of type-II collagen promoter activity.

Assembly and preferential localization of Nup116p on the cytoplasmic face of the nuclear pore complex by interaction with Nup82p.

The yeast Saccharomyces cerevisiae nucleoporin Nup116p serves as a docking site for both nuclear import and export factors. However, the mechanism for assembling Nup116p into the nuclear pore complex (NPC) has not been resolved. By conducting a two-hybrid screen with the carboxy (C)-terminal Nup116p region as bait, we identified Nup82p. The predicted coiled-coil region of Nup82p was not required for Nup116p interaction, making the binding requirements distinct from those for the Nsp1p-Nup82p-Nup159p subcomplex (N. Belgareh, C. Snay-Hodge, F. Pasteau, S. Dagher, C. N. Cole, and V. Doye, Mol. Biol. Cell 9:3475-3492, 1998). Immunoprecipitation experiments using yeast cell lysates resulted in the coisolation of a Nup116p-Nup82p subcomplex. Although the absence of Nup116p had no effect on the NPC localization of Nup82p, overexpression of C-terminal Nup116p in a nup116 null mutant resulted in Nup82p mislocalization. Moreover, NPC localization of Nup116p was specifically diminished in a nup82-Delta108 mutant after growth at 37 degrees C. Immunoelectron microscopy analysis showed Nup116p was localized on both the cytoplasmic and nuclear NPC faces. Its distribution was asymmetric with the majority at the cytoplasmic face. Taken together, these results suggest that Nup82p and Nup116p interact at the cytoplasmic NPC face, with nucleoplasmic Nup116p localization utilizing novel binding partners.

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality.

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

Evaluation of the use of accelerated infusions for the determination of pharmacokinetic linearity.

Accelerated infusions are potentially useful in the investigation of pharmacokinetic linearity. However, little information exists to validate this technique or to demonstrate its limitations. This investigation was performed to determine whether accelerated infusion regimens reliably estimate the range of pharmacokinetic linearity for molecules of varying pharmacokinetic properties, to evaluate the ability of accelerated infusions to identify pharmacokinetic nonlinearity, and to validate the accelerated infusion technique using compounds with known pharmacokinetic parameters. Simulations incorporating accelerated infusion as the input function resulted in the anticipated concentration-time profiles that contained an initial lag phase before reaching a linear slope. This lag phase increased with increasing distributional volume and in some instances was sufficiently great to obscure or prevent the linear portion of the profile. These simulations also revealed that clearance estimated from the apparently linear portion of the concentration-time profile can be erroneous under some conditions, as for large-volume compounds. Simulations of structured nonlinearity produced the predicted profiles for compounds with low to moderate volumes of distribution while demonstrating that modeling of data derived from compounds with large volumes of distribution may be inaccurate. Finally, experiments using accelerated infusions with various test compounds further demonstrated the usefulness of this technique while presenting limits imposed on the interpretation of the data. The results of this investigation indicate that the accelerated infusion may be used to determine pharmacokinetic linearity for compounds within certain pharmacokinetic boundaries, but that appropriate caution should be exercised in the extent of interpretation that should be extracted from such studies.

A substrate combinatorial array for caspases.

An efficient strategy for the synthesis of a tetrapeptidyl substrate combinatorial array directed toward the caspases is described. Testing of this array with caspases 1 and 4 gave substrate hydrolytic profiles characteristic of each caspase, and permitted the identification of efficiently processed substrates. A comparison of this approach to that using a positional scanning library is presented.

Potent dipeptidylketone inhibitors of the cysteine protease cathepsin K.

Cathepsin K (EC 3.4.22.38) is a cysteine protease of the papain superfamily which is selectively expressed within the osteoclast. Several lines of evidence have pointed to the fact that this protease may play an important role in the degradation of the bone matrix. Potent and selective inhibitors of cathepsin K could be important therapeutic agents for the control of excessive bone resorption. Recently a series of peptide aldehydes have been shown to be potent inhibitors of cathepsin K. In an effort to design more selective and metabolically stable inhibitors of cathepsin K, a series of electronically attenuated alkoxymethylketones and thiomethylketones inhibitors have been synthesized. The X-ray co-crystal structure of one of these analogues in complex with cathepsin K shows the inhibitor binding in the primed side of the enzyme active site with a covalent interaction between the active site cysteine 25 and the carbonyl carbon of the inhibitor.

Structure-based design of non-peptide, carbohydrazide-based cathepsin K inhibitors.

Using binding models which were based on the X-ray crystal structure of an amino acid-based active site-spanning inhibitor complexed with cathepsin K, Cbz-leucine mimics have been developed, leading ultimately to the design of a potent cathepsin K inhibitor free of amino acid components. These mimics, which consist of alpha-substituted biphenylacetyl groups in place of Cbz-leucine moieties, effectively mimic all aspects of the Cbz-leucine moieties which are important for inhibitor binding. The predicted directions of binding for the inhibitors were confirmed by mass spectral analysis of their complexes with cathepsin K, which gave results consistent with acylation of the enzyme and loss of the acylhydrazine portion of the inhibitor which binds on the S' side of the active site. The binding models were found to be very predictive of relative inhibitor potency as well as direction of inhibitor binding. These results strengthen the validity of a strategy involving iterative cycles of structure-based design and inhibitor synthesis and evaluation for the discovery of non-peptide inhibitors.

Synergistic effects of concurrent challenge with bovine respiratory syncytial virus and 3-methylindole in calves.

OBJECTIVE: To evaluate the potential synergy between bovine respiratory syncytial virus (BRSV) and 3-methylindole (3MI) in inducing respiratory disease in cattle. ANIMALS: 20 mixed-breed beef calves. PROCEDURE: A 2 X 2 factorial design was used, with random assignment to the following 4 treatment groups: unchallenged control, BRSV challenge exposure (5 X 10(4) TCID50 by aerosolization and 5.5 X 10(5) TCID50 by intratracheal inoculation), 3MI challenge exposure (0.1 g/kg of body weight, PO), and combined BRSV-3MI challenge exposure. Clinical examinations were performed daily. Serum 3MI concentrations, WBC counts, PCV, total plasma protein, and fibrinogen concentrations were determined throughout the experiment. Surviving cattle were euthanatized 7 days after challenge exposure. Pulmonary lesions were evaluated at postmortem examination. RESULTS: Clinical respiratory disease was more acute and severe in cattle in the BRSV-3MI challenge-exposure group than in cattle in the other groups. All 5 cattle in this group and 3 of 5 cattle treated with 3MI alone died or were euthanatized prior to termination of the experiment. Mean lung displacement volume was greatest in the BRSV-3MI challenge-exposure group. Gross and histologic examination revealed that pulmonary lesions were also most severe for cattle in this group. CONCLUSIONS AND CLINICAL RELEVANCE: Feedlot cattle are commonly infected with BRSV, and 3MI is produced by microflora in the rumen of all cattle. Our results suggest that there is a synergy between BRSV and 3MI. Thus, controlling combined exposure may be important in preventing respiratory disease in feedlot cattle.

Mechanism of inhibition of cathepsin K by potent, selective 1, 5-diacylcarbohydrazides: a new class of mechanism-based inhibitors of thiol proteases.

The nature of the inhibition of thiol proteases by a new class of mechanism-based inhibitors, 1,5-diacylcarbohydrazides, is described. These potent, time-dependent, active-site spanning inhibitors include compounds that are selective for cathepsin K, a cysteine protease unique to osteoclasts. The 1,5-diacylcarbohydrazides are slow substrates for members of the papain superfamily with inhibition resulting from slow enzyme decarbamylation. Enzyme-catalyzed hydrolysis of 2,2'-N, N'-bis(benzyloxycarbonyl)-L- leucinylcarbohydrazide is accompanied by formation of a hydrazide-containing product and a carbamyl-enzyme intermediate that is sufficiently stable to be observed by mass spectrometry and NMR. Stopped-flow studies yield a saturation limited value of 43 s(-)(1) for the rate of cathepsin K acylation by 2,2'N, N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide. Inhibition potency varies among proteases tested as reflected by 2-3 orders of magnitude differences in K(i) and K(obs)/I, but all eventually form the same stable covalent intermediate. Reactivation rates are equivalent for all enzymes tested (1 x 10(-)(4) s(-)(1)), indicating hydrolysis of a common carbamyl-enzyme form. NMR spectroscopic studies with cathepsin K and 2,2'-N,N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide provide evidence of inhibitor cleavage to generate a covalent carbamyl-enzyme intermediate rather than a tetrahedral complex. The product Cbz-leu-hydrazide does not appear enzyme-bound after cleavage in the NMR spectra, suggesting that the stable inhibited form of the enzyme is the thioester complex. 1, 5-diacylcarbohydrazides represent a new class of unreactive cysteine protease inhibitors that share a common mechanism of action across members of the papain superfamily. Both S and S' subsite interactions are exploited in achieving high selectivity and potency.

Structure-based design of cathepsin K inhibitors containing a benzyloxy-substituted benzoyl peptidomimetic.

Peptidomimetic cathepsin K inhibitors have been designed using binding models which were based on the X-ray crystal structure of an amino acid-based, active site-spanning inhibitor complexed with cathepsin K. These inhibitors, which contain a benzyloxybenzoyl group in place of a Cbz-leucine moiety, maintained good inhibitory potency relative to the amino acid-based inhibitor, and the binding models were found to be very predictive of relative inhibitor potency. The binding mode of one of the inhibitors was confirmed by X-ray crystallography, and the crystallographically determined structure is in close qualitative agreement with the initial binding model. These results strengthen the validity of a strategy involving iterative cycles of structure-based design, inhibitor synthesis and evaluation, and crystallographic structure determination for the discovery of peptidomimetic inhibitors.