Alternative nano-structured thin-film materials used as durable thermal nanoimprint lithography templates.

Nanoimprint templates made of diamond-like carbon (DLC) and amorphous silicon carbide (SiC) thin films and fluorine-doped associated materials, i.e. F-DLC and F-SiC were investigated in the context of thermal nanoimprint lithography (NIL) with respect to their release properties. Their performances in terms of durability and stability were evaluated and compared to those of conventional silicon or silica molds coated with antisticking molecules applied as a self-assembled monolayer. Plasma-enhanced chemical vapor deposition parameters were firstly tuned to optimize mechanical and structural properties of the DLC and SiC thin films. The impact of the amount of fluorine dopant on the deposited thin films properties was then analyzed. A comparative analysis of DLC, F-DLC as well as SiC and F-SiC molds was then carried out over multiple imprints, performed into poly (methyl methacrylate) (PMMA) thermo-plastic resist. The release properties of un-patterned films were evaluated by the measurement of demolding energies and surface energies, associated with a systematic analysis of the mold surface contamination. These analyses showed that the developed materials behave as intrinsically easy-demolding and contamination-free molds over series of up to 40 imprints. To our knowledge, it is the first time that such a large number of imprints has been considered within an exhaustive comparative study of materials for NIL. Finally, the developed materials went through standard e-beam lithography and plasma etching processes to obtain nanoscale-patterned templates. The replicas of those patterned molds, imprinted into PMMA, were shown to be of high fidelity and good stability after several imprints.

Safety of PEGylated recombinant human full-length coagulation factor VIII (BAX 855) in the overall context of PEG and PEG conjugates.

INTRODUCTION: BAX 855 is a PEGylated human full-length recombinant factor VIII (rFVIII) based on licensed rFVIII (ADVATE). The applied PEGylation technology has been optimized to retain functionality of the FVIII molecule, improve its pharmacokinetic properties and allow less frequent injections while maintaining efficacy. AIM: The aim of this study was to confirm that the excellent safety profile of ADVATE remains unchanged after PEGylation. METHODS: Non-clinical safety studies with BAX 855 and its respective unbound polyethylene glycol (PEG) were conducted in several species. The distribution of a single dose of radiolabelled BAX 855 was further investigated in rats. Publically available safety data on PEG alone and PEGylated biomolecules were summarized and reviewed for specific safety findings attributable to PEG or PEGylated biopharmaceuticals. RESULTS: Safety pharmacology studies in rabbits and macaques and repeated dose toxicity studies in rats and macaques identified no safety issues. Results of a distribution study in rats administered radiolabelled BAX 855 showed that radioactivity was completely excreted; urine was the major elimination route. A 28-day study in rats dosed with the unbound PEG constituent (PEG2ru20KCOOH) of BAX 855 showed no adverse or non-adverse effects. Safety data for PEG and PEG-protein conjugates indicate no safety concerns associated with PEG at clinically relevant dose levels. Although vacuolation of certain cell types has been reported in mammals, no such vacuolation was observed with BAX 855 or with the unbound PEG constituent. CONCLUSION: Non-clinical safety evaluation of PEG and BAX 855 identified no safety signals; the compound is now in clinical development for the treatment of patients with haemophilia A.

BAX 855, a PEGylated rFVIII product with prolonged half-life. Development, functional and structural characterisation.

A longer acting recombinant FVIII is expected to serve patients' demand for a more convenient prophylactic therapy. We have developed BAX 855, a PEGylated form of Baxter's rFVIII product ADVATE™ based on the ADVATE™ manufacturing process. The conjugation process for preparing BAX 855 uses a novel PEG reagent. The production process was adjusted to yield a rFVIII conjugate with a low PEGylation degree of about 2 moles PEG per FVIII molecule. This optimised modification degree resulted in an improved PK profile for rFVIII without compromising its specific activity. PEGylation sites were identified by employing various HPLC- and MS-based methods. These studies not only indicated that about 60% of the PEG chains are localised to the B-domain, which is cleaved off upon physiological activation during the coagulation process, but also demonstrated an excellent lot to lot consistency with regard to PEGylation site distribution. Detailed biochemical characterization further showed that PEGylated FVIII retained all the physiological functions of the FVIII molecule with the exception of binding to the LRP clearance receptor which was reduced for BAX 855 compared to ADVATE™. This might provide an explanation for the prolonged circulation time of BAX 855 as reduced receptor binding might slow-down clearance. Preclinical studies showed improved pharmacokinetic behaviour and clinically relevant prolonged efficacy compared to ADVATE™ without any signs of toxicity or elevated immunogenicity. The comprehensive preclinical data package formed the basis for approval of the phase 1 clinical study by European authorities which started in 2011.

Forced fluid removal in critically ill patients with acute kidney injury.

PURPOSE: The aim was to test the feasibility of protocol-driven fluid removal with continuous renal replacement therapy (CRRT) in patients in whom standard fluid balance prescription did not result in substantial negative fluid balances. MATERIALS AND METHODS: In 10 mechanically ventilated patients with sepsis or signs of inflammation and acute kidney injury [age 65 (48-78 years; median, range), simplified acute physiology score II 66 (39-116)], fluid removal was guided by mean arterial pressure (MAP), cardiac index (CI), mixed venous oxygen saturation (SvO(2)), lactate/base excess, peripheral circulation, and filling pressures, and adjusted hourly with the goal to maximize volume removal for up to 3 days. RESULTS: Fluid removal rates during the 3 days before and during the study period [66 (36-72) h] were 11 (-30 to +36) ml/kg/day and -59 (-85 to -31) ml/kg/day, respectively (P = 0.002). In 12% of a total of 594 fluid removal rate evaluations, fluid removal had to be decreased or stopped. Most frequent reasons leading to decreasing fluid removal were (n, % of all instances, median lowest value from all patients): SvO(2) (44, 28%, 59%), MAP (36, 23%, 57 mmHg), CI (26, 17%, 2.4 l/min/m(2)), low peripheral temperature (22, 14%, 'cold'). Overall, systemic hemodynamics remained stable throughout the study period. CONCLUSIONS: In these patients, protocolized fluid removal with CRRT was associated with large negative fluid balances.

PEGylation of cyanovirin-N, an entry inhibitor of HIV.

Cyanovirin-N (CV-N) is a potent inhibitor of human immunodeficiency virus and many other viruses. It has a high potential for use as a systemic compound to control viral load or in the development of microbicides to prevent primary viral infection. Due to its cyanobacterial origin it is likely to show the typical drawbacks associated with pharmaceutical use of foreign proteins such as short plasma half-life, proteolysis and immunogenicity. Several strategies were used to covalently bond poly(ethylene glycol) (PEGylate) to CV-N. Random PEGylation at lysine residues resulted in poor retention of antiviral activity. Many site-directed mutants were made to test site-specific PEGylation. One mutant, where glutamine 62 was replaced with cysteine (CV-N(Q62C)) and PEGylated with maleimide activated PEG, retained significant anti-HIV activity in vitro.

Fibrocartilaginous embolism of the spinal cord (FCE) in juvenile Irish Wolfhounds.

This study describes the occurrence of fibrocartilaginous embolism of the spinal cord (FCE) in eight juvenile Irish Wolfhounds that were presented within a period of 16 months (1996-1997). The dogs, seven males and one female between eight and 13 weeks of age, were presented because of an acute onset of abnormal locomotion. Five dogs were euthanized and FCE was diagnosed by the histomorphological presence of focal myelomalacia and Alcian blue-positive-nucleus-pulposus material in the spinal cord vasculature. Three dogs, which were thought to have FCE because of their clinical symptoms, improved with partial or almost complete return to normal locomotion. Although the observed high incidence may be a coincidence, oral information from breeders and lay reports of similar cases in journals for dog breeders from various countries suggest that FCE is a common disorder in young Irish Wolfhounds.

Design and synthesis of diaminopyrrolidinone inhibitors of human osteoclast cathepsin K.

The structure-based design and synthesis of lactam-constrained azapeptide inhibitors of human cathepsin K are described. Enhanced stability to proteolytic cleavage over acyclic analogues is discussed.

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.

Peptide aldehyde inhibitors of cathepsin K inhibit bone resorption both in vitro and in vivo.

We have shown previously that cathepsin K, a recently identified member of the papain superfamily of cysteine proteases, is expressed selectively in osteoclasts and is the predominant cysteine protease in these cells. Based upon its abundant cell type-selective expression, potent endoprotease activity at low pH and cellular localization at the bone interface, cathepsin K has been proposed to play a specialized role in osteoclast-mediated bone resorption. In this study, we evaluated a series of peptide aldehydes and demonstrated that they are potent cathepsin K inhibitors. These compounds inhibited osteoclast-mediated bone resorption in fetal rat long bone (FRLB) organ cultures in vitro in a concentration-dependent manner. Selected compounds were also shown to inhibit bone resorption in a human osteoclast-mediated assay in vitro. Chz-Leu-Leu-Leu-H (in vitro enzyme inhibition Ki,app = 1.4 nM) inhibited parathyroid hormone (PTH)-stimulated resorption in the FRLB assay with an IC-50 of 20 nM and inhibited resorption by isolated human osteoclasts cultured on bovine cortical bone slices with an IC-50 of 100 nM. In the adjuvant-arthritic (AA) rat model, in situ hybridization studies demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss in the distal tibia. Cbz-Leu-Leu-Leu-H (30 mg/kg, intraperitoneally) significantly reduced this bone loss, as well as the associated hind paw edema. In the thyroparathyriodectomized rat model, Cbz-Leu-Leu-Leu-H inhibited the increase in blood ionized calcium induced by a 6 h infusion of PTH. These data indicate that inhibitors of cathepsin K are effective at reducing osteoclast-mediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or osteoporosis.

Design of potent and selective human cathepsin K inhibitors that span the active site.

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups flanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention.

Proteolytic activity of human osteoclast cathepsin K. Expression, purification, activation, and substrate identification.

Human cathepsin K is a recently identified protein with high primary sequence homology to members of the papain cysteine protease superfamily including cathepsins S, L, and B and is selectively expressed in osteoclasts (Drake, F.H., Dodds, R., James I., Connor J., Debouck, C., Richardson, S., Lee, E., Rieman, D., Barthlow, R., Hastings, G., and Gowen, M. (1996) J. Biol., Chem. 271, 12511-12516). To characterize its catalytic properties, cathepsin K has been expressed in baculovirus-infected SF21 cells and the soluble recombinant protein isolated from growth media was purified. Purified protein includes an inhibitory pro-leader sequence common to this family of protease. Conditions for enzyme activation upon removal of the pro-sequence have been identified. Fluorogenic peptides have been identified as substrates for mature cathepsin K. In addition, two protein components of bone matrix, collagen and osteonectin, have been shown to be substrates of the activated protease. Cathepsin K is inhibited by E-64 and leupeptin, but not for by pepstatin, EDTA, phenylmethylsulfonyl fluoride, or phenanthroline, consistent with its classification within the cysteine protease class. Leupeptin has been characterized as a slow binding inhibitor of cathepsin K (kobs/[I] = 273,000 m(-1).s(-1)). Cathepsin K may represent the elusive protease implicated in degradation of protein matrix during bone resorption and represents a novel molecular target in treatment of disease states associated with excessive bone loss such as osteoporosis.

Structure-activity studies of rapamycin analogs: evidence that the C-7 methoxy group is part of the effector domain and positioned at the FKBP12-FRAP interface.

BACKGROUND: Rapamycin is an immunosuppressant natural product, which blocks T-cell mitogenesis and yeast proliferation. In the cytoplasm, rapamycin binds to the immunophilin FKBP12 and the complex of these two molecules binds to a recently discovered protein, FRAP. The rapamycin molecule has two functional domains, defined by their interaction with FKBP12 (binding domain) or with FRAP (effector domain). We previously showed that the allylic methoxy group at C-7 of rapamycin could be replaced by a variety of different substituents. We set out to examine the effects of such substitutions on FKBP12 binding and on biological activity. RESULTS: Rapamycin C-7-modified analogs of both R and S configurations were shown to have high affinities for FKBP12, yet these congeners displayed a wide range of potencies in splenocyte and yeast proliferation assays. The X-ray crystal structures of four rapamycin analogs in complexes with FKBP12 were determined and revealed that protein and ligand backbone conformations were essentially the same as those observed for the parent rapamycin-FKBP12 complex and that the C-7 group remained exposed to solvent. We then prepared a rapamycin analog with a photoreactive functionality as part of the C-7 substituent. This compound specifically labeled, in an FKBP12-dependent manner, a protein of approximately 250 kDa, which comigrates with recombinant FRAP. CONCLUSIONS: We conclude that the C-7 methoxy group of rapamycin is part of the effector domain. In the ternary complex, this group is situated in close proximity to FRAP, at the interface between FRAP and FKBP12.

Catalytic and ligand binding properties of the FK506 binding protein FKBP12: effects of the single amino acid substitution of Tyr82 to Leu.

The binding of FK506 and rapamycin to their cytosolic receptor FKBP12 is an intermediate step in the paths leading to their potent immunosuppressive properties. One of the amino acids defining the hydrophobic binding cleft for the macrocycles is Tyr82, which is thought to form a hydrogen bond with the amide oxygens of the common pipecolyl structural element within the two macrolides. To understand better the influence of this amino acid residue in catalytic activity (cis-trans peptidyl prolyl isomerization) and ligand binding properties, a Tyr82 to Leu site-specific modification of FKBP12 was prepared, purified and characterized. Kinetic experiments have demonstrated that the Tyr82 to Leu modification has a greater effect on catalytic properties than on ligand binding affinities, a result which indicates that these inhibitors may not be binding as true transition-state analogues. In an additional test for cellular function, expression of both wild-type and mutant human FKBP12 in a strain of Saccharomyces cerevisiae rendered resistant to rapamycin by deletion of the gene encoding a cytosolic rapamycin binding protein (RPB1), the yeast homologue of FKBP12, restored wild-type drug sensitivity.

The tyrosine89 residue of yeast FKBP12 is required for rapamycin binding.

Rapamycin (Rm) is a macrolide antifungal agent related to FK506 that exhibits potent immunosuppressive properties which are mediated through interaction with specific cytoplasmic receptors (FKBPs or RBPs, for FK506- and Rm-binding proteins, respectively). These proteins possess peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro which is inhibited by the binding of Rm and FK506. In Saccharomyces cerevisiae, Rm sensitivity (Rms) is mediated by binding of the drug to RBP1, a homolog of the 12-kDa human FK506-binding protein (FKBP12); null mutations in the yeast RBP1 gene result in a recessive drug resistance phenotype. To identify missense mutations that define amino acid (aa) residues in RBP1 involved in drug sensitivity, we selected and genetically characterized over 250 independent RmR rbp1 mutants and screened them for both RBP1-specific mRNA and protein expression. Whereas all rbp1 mutants expressed abundant levels of RBP1 mRNA, stable RBP1 protein production was detected in only one mutant strain. The RBP1 gene was PCR-generated (in triplicate) from several rbp1 mutants and independent clones were sequenced. Most of the immunoblot-negative alleles were found to contain various types of null mutations; however, some alleles contained specific missense mutations that apparently affect protein stability in vivo. The single immunoblot-positive allele was found to contain a mutation altering a specific residue (Tyr89) which is conserved among the known FKBPs, and which, based on the solution and x-ray structures of human FKBP12, has been proposed to be part of a hydrophobic drug-binding pocket for FK506 and Rm.(ABSTRACT TRUNCATED AT 250 WORDS)

The yeast cyclophilin multigene family: purification, cloning and characterization of a new isoform.

Cyclophilins (Cyps) constitute a highly conserved family of proteins present in a wide variety of organisms. Historically, Cyps were first identified by their ability to bind the immunosuppressive agent cyclosporin A (CsA) with high affinity; they later were found to have peptidyl-prolyl cis-trans isomerase (PPIase) activity, which catalyzes the folding of oligopeptides at proline-peptide bonds in vitro and may be important for protein folding in vivo. Cells of Saccharomyces cerevisiae contain at least two distinct Cyp-related PPIases encoded by the genes CYP1 and CYP2. A yeast strain (GL81) containing genomic disruptions of three known yeast PPIase-encoding genes [CYP1, CYP2 and RBP1 (for rapamycin-binding protein); Koltin et al., Mol. Cell. Biol. 11 (1991) 1718-1723] was previously constructed and found to be viable. Soluble fractions of these cells possess residual CsA-sensitive PPIase activity (2-5% of that present in wild-type cells as assayed in vitro). We have purified an approx. 18-kDa protein exhibiting PPIase activity from a soluble fraction of GL81 cells and determined that its N-terminal amino acid (aa) sequence exhibits significant homology (but nonidentity) to the Cyp1 and Cyp2 proteins. We designate the gene for this new protein, CYP3. Using a degenerate oligodeoxyribonucleotide (oligo) based on the N-terminal aa sequence, plus an internal oligo homologous to a conserved region within the portion of CYP1 and CYP2 that had been deleted in the genome, a CYP3-specific DNA fragment was generated by the polymerase chain reaction (PCR) using GL81 genomic DNA as a substrate. This PCR fragment was used as a probe to isolate CYP3 genomic and cDNA clones.(ABSTRACT TRUNCATED AT 250 WORDS)

The CYP2 gene of Saccharomyces cerevisiae encodes a cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase with an N-terminal signal sequence.

Cells of Saccharomyces cerevisiae contain a major cytosolic cyclophilin (Cyp)-related peptidyl-prolyl cis-trans isomerase (PPIase) which is the target for cyclosporin A (CsA) cytotoxicity and which is encoded by the CYP1 gene [Haendler et al., Gene 83 (1989) 39-46]. We recently identified a second Cyp-related gene in yeast, CYP2 [Koser et al., Nucleic Acids Res. 18 (1990) 1643] which predicts a protein with a hydrophobic leader sequence. A sequence lacking 33 codons from the 5'-end of the CYP2 open reading frame was generated by the polymerase chain reaction and engineered for expression in Escherichia coli. The corresponding recombinant truncated protein was purified and found to exhibit PPIase activity which was inhibited by CsA. The CYP2 gene is genetically unlinked to CYP1. As with CYP1, genomic disruption of CYP2 had no effect on haploid cell viability. Disruption of all three of the known yeast PPIase-encoding genes [CYP1, CYP2, and RBP1 for rapamycin-binding protein; Koltin et al., Mol. Cell. Biol. 11 (1991) 1718-1723] in the same haploid cell also resulted in no apparent cellular phenotype, suggesting either that none of these enzymes have an essential function or that additional PPIases can compensate for their specific absence. Whereas cells containing a genomic disruption of CYP1 exhibited a CsA-resistant phenotype, genomic disruption of CYP2 had no effect on CsA sensitivity. This suggests that the CYP1 gene product is the primary cellular target for CsA toxicity in yeast. Since both purified Cyps display CsA sensitivity in vitro, our data suggest that Cyp1 and Cyp2 differ in terms of their cellular function and/or localization.

Effects of a novel lanosterol 14 alpha-demethylase inhibitor on the regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in Hep G2 cells.

A 32-carboxylic acid derivative of lanosterol (SKF 104976) was found to be a potent inhibitor of lanosterol 14 alpha-demethylase (14 alpha DM). 14 alpha DM activity in a Hep G2 cell extract was inhibited 50% by 2 nM SKF 104976. Exposure of intact cells to similar concentrations of the compound resulted in the inhibition of incorporation of [14C]acetate into cholesterol with concomitant accumulation of lanosterol as well as a 40-70% decrease in 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity. SKF 104976 did not effect low density lipoprotein uptake and degradation in Hep G2 cells, suggesting that HMGR and low density lipoprotein receptor activity were not coordinately regulated under these conditions. Reduction of the flux of carbon units in the sterol synthetic pathway by as much as 80% did not alter the suppressing effect of SKF 104976 on HMGR activity. However, under conditions where sterol synthesis was almost completely blocked by lovastatin, HMGR activity was not suppressed by SKF 104976. Mevalonate, at concentrations that did not decrease HMGR activity, was able to restore the inhibiting effect of SKF 104976 on HMGR activity. The rapid inhibition (2-3 h) of HMGR activity by SKF 104976 to 30-60% of the level in controls was not dependent on the initial amount of HMGR enzyme present. These findings suggest that upon inhibition of 14 alpha DM by SKF 104976, a mevalonate-derived precursor regulates HMGR activity, even when the sterol synthetic rate is considerably reduced and when HMGR protein levels are very high. In Hep G2 cells, formation of oxylanostenols from [3H]mevalonate reached a maximum between 1 and 10 nM SKF 104976 and was negligible at higher concentrations. This result suggests that oxylanostenols are not the key mediators of the modulation of HMGR in Hep G2 cells upon 14 alpha DM inhibition.

A single Trp121 to Ala121 mutation in human cyclophilin alters cyclosporin A affinity and peptidyl-prolyl isomerase activity.

Fluorescence and NMR spectral data have suggested an interaction between the single tryptophan in cyclophilin (CyP) and its high affinity ligand cyclosporin A (CsA). To study this interaction, a site mutation of Trp121 to Ala was introduced into human cyclophilin (CyP) and the encoded protein was expressed in E. coli. The Ala121 mutant was shown to catalyze the peptidyl-prolyl cis-trans isomerase (rotomase) reaction with several peptide substrates, albeit at less than ten percent the rate of the purified recombinant human CyP. Values for the apparent inhibition constant (Ki,app) of cyclosporin A with the human CyP and the Ala121 mutant were determined to be 1.6 +/- 0.4 nM and 640 +/- 90 nM, respectively by tight-binding inhibition analysis. The greater loss of affinity for CsA binding (400-fold) than for rotomase catalysis (20 fold) suggests that the catalytic and CsA binding properties associated with CyP can be decoupled as has been observed with an homologous protein found in E. coli (Liu, J. & Walsh, C.T. (1990) Proc. Natl. Acad. Sci. USA 87, 4028-4032).

Use of isotope effects to characterize intermediates in mechanism-based inactivation of dopamine beta-monooxygenase by beta-chlorophenethylamine.

A mechanism for beta-chlorophenethylamine inhibition of dopamine beta-monooxygenase has been postulated in which bound alpha-aminoacetophenone is generated followed by an intramolecular redox reaction to yield a ketone-derived radical cation as the inhibitory species (Mangold, J.B., and Klinman, J.P. (1984) J. Biol. Chem. 259, 7772-7779). Based on the assumption that the ketone radical is the inhibitory intermediate, an analogous system was predicted and verified (Bossard, M.J., and Klinman, J.P. (1986) J. Biol. Chem. 261, 16421-16427). In the present study, the role of alpha-aminoacetophenone as the proposed intermediate in the inactivation by beta-chlorophenethylamine was examined in greater detail. From the interdependence of tyramine and alpha-aminoacetophenone concentrations, ketone inactivation is concluded to occur at the substrate site as opposed to potential binding at the reductant-binding site. Using beta-[2-1H]- and beta-[2-2H]chlorophenethylamine, the magnitude of the deuterium isotope effect on inactivation under second-order conditions has been found to be identical to that observed under catalytic turnover, D(kappa inact/Ki) = D(kappa cat/Km) = 6-7. By contrast, the isotope effect on inactivation under conditions of substrate and oxygen saturation, D kappa inact = 2, is 3-fold smaller than that seen on catalytic turnover, D kappa cat = 6. This reduced isotope effect for inactivation is attributed to a normal isotope effect on substrate hydroxylation followed by an inverse isotope effect on the partitioning of the enol of alpha-aminoacetophenone between oxidation to a radical cation versus protonation to regenerate ketone. These findings are unusual in that two isotopically sensitive steps are present in the inactivation pathway whereas only one is observable in turnover.

Mechanism-based inhibition of dopamine beta-monooxygenase by aldehydes and amides.

A mechanism for beta-chlorophenethylamine inhibition of dopamine beta-monooxygenase has been postulated in which enzyme-bound alpha-aminoacetophenone is generated, followed by an intramolecular redox reaction to yield a ketone-derived radical cation as the enzyme inhibitory species (Mangold, J. B., and Klinman, J. P. (1984) J. Biol. Chem. 259, 7772-7779). If correct, additional compounds capable of producing enzyme-bound (formula; see text) reductant should inhibit dopamine beta-monooxygenase. Phenylacetaldehyde was chosen to test this model, since beta-hydroxyphenylacetaldehyde is expected to function as a reductant in a manner analogous to alpha-aminoacetophenone. Phenylacetaldehyde exhibits the properties of a mechanism-based inhibitor. Kinetic parameters are comparable to beta-chlorophenethylamine under both initial velocity and inactivation conditions. Since phenylacetaldehyde bears little resemblance to beta-chlorophenethylamine, its analogous inhibitory action provides support for an intramolecular redox reaction (via beta-hydroxyphenylacetaldehyde oxidation to a radical cation) in dopamine beta-monooxygenase inactivation. beta-Hydroxyphenylacetaldehyde was identified as the enzymatic product of phenylacetaldehyde turnover. As predicted, this product behaves both as a time-dependent inhibitor of dopamine beta-monooxygenase and as an electron donor in enzyme-catalyzed hydroxylation of tyramine to octopamine. Phenylacetamide and p-hydroxyphenylacetamide are also found to be mechanism-based inhibitors of dopamine beta-monooxygenase. In this case the product of hydroxylation (beta-hydroxyphenylacetamide) is redox inactive and, therefore, is unable to function as either a reductant or an inhibitor. Thus, mechanism-based inhibitors are divided into two types: type I, which undergoes hydroxylation prior to inactivation, and type II, which only requires hydrogen atom abstraction. A general mechanism for dopamine beta-monooxygenase inactivation is described, in which a common mechanistic radical intermediate is formed from both pathways.