Prevention of polymerization of M and Z alpha1-Antitrypsin (alpha1-AT) with trimethylamine N-oxide. Implications for the treatment of alpha1-at deficiency.

alpha1-Antitrypsin (alpha1-AT) is the most abundant circulating proteinase inhibitor. The Z variant results in profound plasma deficiency as the mutant polymerizes within hepatocytes. The retained polymers are associated with cirrhosis, and the lack of circulating protein predisposes to early onset emphysema. We have investigated the role of the naturally occurring solute trimethylamine N-oxide (TMAO) in modulating the polymerization of normal M and disease-associated Z alpha1-AT. TMAO stabilized both M and Z alpha1-AT in an active conformation against heat-induced polymerization. Spectroscopic analysis demonstrated that this was due to inhibition of the conversion of the native state to a polymerogenic intermediate. However, TMAO did not aid the refolding of denatured alpha1-AT to a native conformation; instead, it enhanced polymerization. These data show that TMAO can be used to control the conformational transitions of folded alpha1-AT but that it is ineffective in promoting folding of the polypeptide chain within the secretory pathway.

Azepanone-based inhibitors of human and rat cathepsin K.

The synthesis, in vitro activities, and pharmacokinetics of a series of azepanone-based inhibitors of the cysteine protease cathepsin K (EC 3.4.22.38) are described. These compounds show improved configurational stability of the C-4 diastereomeric center relative to the previously published five- and six-membered ring ketone-based inhibitor series. Studies in this series have led to the identification of 20, a potent, selective inhibitor of human cathepsin K (K(i) = 0.16 nM) as well as 24, a potent inhibitor of both human (K(i) = 0.0048 nM) and rat (K(i,app) = 4.8 nM) cathepsin K. Small-molecule X-ray crystallographic analysis of 20 established the C-4 S stereochemistry as being critical for potent inhibition and that unbound 20 adopted the expected equatorial conformation for the C-4 substituent. Molecular modeling studies predicted the higher energy axial orientation at C-4 of 20 when bound within the active site of cathepsin K, a feature subsequently confirmed by X-ray crystallography. Pharmacokinetic studies in the rat show 20 to be 42% orally bioavailable. Comparison of the transport of the cyclic and acyclic analogues through CaCo-2 cells suggests that oral bioavailability of the acyclic derivatives is limited by a P-glycoprotein-mediated efflux mechanism. It is concluded that the introduction of a conformational constraint has served the dual purpose of increasing inhibitor potency by locking in a bioactive conformation as well as locking out available conformations which may serve as substrates for enzyme systems that limit oral bioavailability.

Cyclic ketone inhibitors of the cysteine protease cathepsin K.

Cathepsin K (EC 3.4.22.38), a cysteine protease of the papain superfamily, is predominantly expressed in osteoclasts and has been postulated as a target for the treatment of osteoporosis. Crystallographic and structure--activity studies on a series of acyclic ketone-based inhibitors of cathepsin K have led to the design and identification of two series of cyclic ketone inhibitors. The mode of binding for four of these cyclic and acyclic inhibitors to cathepsin K is discussed and compared. All of the structures are consistent with addition of the active site thiol to the ketone of the inhibitors with the formation of a hemithioketal. Cocrystallization of the C-3 diastereomeric 3-amidotetrahydrofuran-4-one analogue 16 with cathepsin K showed the inhibitor to occupy the unprimed side of the active site with the 3S diastereomer preferred. This C-3 stereochemical preference is in contrast to the X-ray cocrystal structures of the 3-amidopyrrolidin-4-one inhibitors 29 and 33 which show these inhibitors to prefer binding of the 3R diastereomer. The 3-amidopyrrolidin-4-one inhibitors were bound in the active site of the enzyme in two alternate directions. Epimerization issues associated with the labile alpha-amino ketone diastereomeric center contained within these inhibitor classes has proven to limit their utility despite promising pharmacokinetics displayed in both series of compounds.

Cloning and characterization of a novel human dual flavin reductase.

Flavoprotein reductases play a key role in electron transfer in many physiological processes. We have isolated a cDNA with strong sequence similarities to cytochrome P-450 reductase and nitric-oxide synthase. The cDNA encodes a protein of 597 amino acid residues with a predicted molecular mass of 67 kDa. Northern blot analysis identified a predicted transcript of 3.0 kilobase pairs as well as a larger transcript at 6.0 kilobase pairs, and the gene was mapped to chromosome 9q34.3 by fluorescence in situ hybridization analysis. The amino acid sequence of the protein contained distinct FMN-, FAD-, and NADPH-binding domains, and in order to establish whether the protein contained these cofactors, the coding sequence was expressed in insect cells and purified. Recombinant protein bound FMN, FAD, and NADPH cofactors and exhibited a UV-visible spectrum with absorbance maxima at 380, 460, and 626 nm. The purified enzyme reduced cytochrome c, with apparent K(m) and k(cat) values of 21 microM and 1.3 s(-1), respectively, and metabolized the one-electron acceptors doxorubicin, menadione, and potassium ferricyanide. Immunoblot analysis of fractionated MCF7 cells with antibodies to recombinant NR1 showed that the enzyme is cytoplasmic and highly expressed in a panel of human cancer cell lines, thus indicating that this novel reductase may play a role in the metabolic activation of bioreductive anticancer drugs and other chemicals activated by one-electron reduction.

Lipoprotein-associated phospholipase A(2), platelet-activating factor acetylhydrolase, is expressed by macrophages in human and rabbit atherosclerotic lesions.

We studied the expression of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), an enzyme capable of hydrolyzing platelet-activating factor (PAF), PAF-like phospholipids, and polar-modified phosphatidylcholines, in human and rabbit atherosclerotic lesions. Oxidative modification of low-density lipoprotein, which plays an important role in atherogenesis, generates biologically active PAF-like modified phospholipid derivatives with polar fatty acid chains. PAF is known to have a potent proinflammatory activity and is inactivated by its hydrolysis. On the other hand, lysophosphatidylcholine and oxidized fatty acids released from oxidized low-density lipoprotein as a result of Lp-PLA(2) activity are thought to be involved in the progression of atherosclerosis. Using combined in situ hybridization and immunocytochemistry, we detected Lp-PLA(2) mRNA and protein in macrophages in both human and rabbit atherosclerotic lesions. Reverse transcriptase-polymerase chain reaction analysis indicated an increased expression of Lp-PLA(2) mRNA in human atherosclerotic lesions. In addition, approximately 6-fold higher Lp-PLA(2) activity was detected in atherosclerotic aortas of Watanabe heritable hyperlipidemic rabbits compared with normal aortas from control rabbits. It is concluded that (1) macrophages in both human and rabbit atherosclerotic lesions express Lp-PLA(2), which could cleave any oxidatively modified phosphatidylcholine present in the lesion area, and (2) modulation of Lp-PLA(2) activity could lead to antiatherogenic effects in the vessel wall.

Nitric oxide synthases catalyze the activation of redox cycling and bioreductive anticancer agents.

Nitric oxide synthases (NOSs) play a crucial role in the control of blood flow, memory formation, and the immune response. These proteins can be structurally divided into oxygenase and reductase domains. The reductase domain shares a high degree of sequence homology with P450 reductase, which is thought to be the major enzyme responsible for the one-electron reduction of foreign compounds, including bioreductive antitumor agents currently undergoing clinical trials. In view of the structural similarities between NOS and P450 reductase, we investigated the capacity of NOS to reduce the hypoxic cytotoxin tirapazamine, the antitumor agent doxorubicin, and also the redox cycling compound menadione. All three isoforms exhibited high levels of activity toward these compounds. In the case of doxorubicin and menadione, the activity of NOS II was 5-10-fold higher than the other enzymes, whereas with tirapazamine, the activities were broadly similar. NOS-mediated metabolism of tirapazamine resulted in a large increase in plasmid DNA strand breaks, demonstrating that the reduction was a bioactivation process. In addition, tirapazamine inhibited NOS activity. Because nitric oxide is implicated in maintaining tumor vascular homeostasis, it is conceivable that tirapazamine could potentiate its own toxicity by increasing the degree of hypoxia. This study suggests that the NOSs could play a key role in the therapeutic effects of tirapazamine, particularly because NOS activity is markedly increased in several human tumors. In addition, the presence of NOS in the heart indicates that these enzymes may contribute to the cardiotoxicity of redox cycling drugs, such as doxorubicin.

Inhibition of human cytomegalovirus protease by enedione derivatives of thieno[2,3-d]oxazinones through a novel dual acylation/alkylation mechanism.

Enedione derivatives of thieno[2,3-d]oxazinones are nanomolar inhibitors of CMV protease which act through a novel dual acylation of the catalytic serine and alkylation of the protease cysteine 161 via a Michael addition to the enedione moiety of the inhibitor.

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.

Solid-phase synthesis of a combinatorial array of 1,3-bis(acylamino)-2-butanones, inhibitors of the cysteine proteases cathepsins K and L.

To more rapidly prepare members of the 1,3-bis(acylamino)-2-butanone class of cysteine protease inhibitors, a solid-phase synthesis was developed. 1-Azido-3-amino-2,2-dimethoxybutane (4), which has the two amino groups differentiated and the ketone protected as a a ketal, served as a surrogate for the 1,3-diamino-2-butanone core. Amine (4) was coupled to the BAL-resin-linked carboxylic acids derived from alpha-amino acid esters. Evaluation of a small combinatorial array by measuring inhibition constants (Ki,appS) against cathepsins K, L, and B provided some structure-activity relationship trends with respect to selectivity and potency. Novel, potent inhibitors of cathepsins K and L were identified.

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.

The role of ATP citrate-lyase in the metabolic regulation of plasma lipids. Hypolipidaemic effects of SB-204990, a lactone prodrug of the potent ATP citrate-lyase inhibitor SB-201076.

ATP citrate (pro-S)-lyase (EC 4.1.3.8), a cytosolic enzyme that generates acetyl-CoA for cholesterol and fatty acid synthesis de novo, is a potential target for hypolipidaemic intervention. Here we describe the biological effects of the inhibition of ATP citrate-lyase on lipid metabolism in Hep G2 cells, and plasma lipids in rats and dogs, by using SB-204990, the cell-penetrant gamma-lactone prodrug of the potent ATP citrate-lyase inhibitor SB-201076 (Ki=1 microM). Consistent with an important role of ATP citrate-lyase in the supply of acetyl-CoA units for lipid synthesis de novo, SB-204990 inhibited cholesterol synthesis and fatty acid synthesis in Hep G2 cells (dose-related inhibition of up to 91% and 82% respectively) and rats (76% and 39% respectively). SB-204990, when administered orally to rats, was absorbed into the systemic circulation; pharmacologically relevant concentrations of SB-201076 were recovered in the liver. When administered in the diet (0.05-0. 25%, w/w) for 1 week, SB-204990 caused a dose-related decrease in plasma cholesterol (by up to 46%) and triglyceride levels (by up to 80%) in rats. This hypolipidaemic effect could be explained, at least in part, by a decrease (up to 48%) in hepatic very-low-density lipoprotein (VLDL) production as measured by the accumulation of VLDL in plasma after injection of Triton WR-1339. SB-204990 (25 mg/kg per day) also decreased plasma cholesterol levels (by up to 23%) and triglyceride levels (by up to 38%) in the dog, preferentially decreasing low-density lipoprotein compared with high-density lipoprotein cholesterol levels. Overall these results are consistent with the concept that ATP citrate-lyase is an important enzyme in controlling substrate supply for lipid synthesis de novo and a potential enzyme target for hypolipidaemic intervention.

Napsins: new human aspartic proteinases. Distinction between two closely related genes.

cDNA sequences were elucidated for two closely related human genes which encode the precursors of two hitherto unknown aspartic proteinases. The (pro)napsin A gene is expressed predominantly in lung and kidney and its translation product is predicted to be a fully functional, glycosylated aspartic proteinase (precursor) containing an RGD motif and an additional 18 residues at its C-terminus. The (pro)napsin B gene is transcribed exclusively in cells related to the immune system but lacks an in-frame stop codon and contains a number of polymorphisms, one of which replaces a catalytically crucial Gly residue with an Arg. Consideration is given to whether (pro)napsin B may be a transcribed pseudogene or whether its putative protein product undergoes rapid intracellular degradation.

ATP-citrate lyase as a target for hypolipidemic intervention. Design and synthesis of 2-substituted butanedioic acids as novel, potent inhibitors of the enzyme.

ATP-citrate lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. Inhibitors of the enzyme represent a potentially novel class of hypolipidemic agent, which are anticipated to have combined hypocholesterolemic and hypotriglyceridemic properties. A series of 2-substituted butanedioic acids have been designed and synthesized as inhibitors of the enzyme. The best compounds, 58, 68, 71, 74 have reversible Ki's in the 1-3 microM range against the isolated rat enzyme. As representative of this compound class, 58, has been shown to exert its inhibitory action through a mainly competitive mechanism with respect to citrate and a noncompetitive one with respect to CoA. None of the inhibitors were able to inhibit cholesterol and/or fatty acid synthesis in HepG2 cells. This has been attributed to the adverse physicochemical properties of the molecules leading to a lack of cell penetration. Despite this, a lead structural class of compound has been identified with the potential for modification into potent, cell-penetrant, and efficacious inhibitors of ATP-citrate lyase.