[W206R]-procaspase 3: an inactivatable substrate for caspase 8.

We report here the cloning and high-level expression of a soluble proform of human caspase 3 (Ser(24)-H(277)) engineered to contain a short stretch of N-terminal sequence (MTISDSPREQD) from the prosegment of procaspase 8 and a C-terminal heptahistidine tag. The precursor protein isolated from extracts of recombinant Escherichia coli by immobilized metal-ion affinity chromatography was predominantly unprocessed and migrated as a 32-kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gels. Incubation of this protein with recombinant human caspase 8 produced fragments characteristic of the properly processed caspase 3, but the product was inactive. Amino-terminal sequence analysis of the caspase 3 polypeptides proved that caspase 8 had specifically cleaved the Asp(175)-Ser(176) bond to yield the expected p18 and p12 subunits, with partial cleavage at the Asp(28)-Ser(29) bond to release the prosegment. The lack of caspase 3 activity was found to be the result of a fortuitous mutation in which Trp(206) in the S4 subsite was replaced by arginine (W206R). This mutant procaspase 3, which we call m-pro3, serves as a useful reagent with which to test the efficacy of caspase 8 inhibitors in blocking processing of the natural polypeptide substrate of this enzyme and may be valuable as a source of "proenzyme" for crystallographic analysis.

Caspase 8: an efficient method for large-scale autoactivation of recombinant procaspase 8 by matrix adsorption and characterization of the active enzyme.

A gene coding for a truncated form of human procaspase 8 has been cloned and expressed in Escherichia coli. This construct contains M(206) through D(479) of human procaspase 8, preceded by an N-terminal polyhistidine tag. The recombinant protein, containing 286 amino acids, was expressed in high yield in the form of inclusion bodies (IB). The IB were solubilized in guanidinium chloride and dialyzed against 50% acetic acid. The solution was mixed with 9 volumes of H(2)O and then rapidly diluted from the acidic medium to one containing 1.0 M Tris, pH 8.0, and 5 mM DTT. SDS-PAGE analysis of the soluble, dilute protein solution (20-30 microgram of protein/ml) showed a single 33-kDa band corresponding to the nonprocessed, inactive procaspase 8. Concentration of the dilute protein to levels as high as 2 mg/ml resulted in only modest (1-10%) autocatalytic conversion to the 19- and 11-kDa polypeptide subunits which are characteristic of the activated enzyme. Further concentration of these protein solutions to a near-dry state on the ultrafiltration membrane, followed by washing of the membrane with buffer, led to extracts containing high yields of enzyme showing a specific activity of 8.43 micromol/min/mg against the chromogenic substrate Ac-IETD-pNA. SDS-PAGE, protein sequencing, and mass spectrometric analysis of these extracts showed complete conversion of the 33-kDa procaspase 8 to the 19- and 11-kDa subunits of activated caspase 8. This method allows for preparation of 100-mg quantities of highly pure and active recombinant human caspase 8. Enzyme activity was shown to be associated with a heterotetrameric complex that is converted to an inactive dimer upon storage.

Stereoselective hydroxylation of nonpeptidic HIV protease inhibitors by CYP2D6.

PNU-106893, N-{3-[1-(4-hydroxy-2-oxo-6-phenyl-6-propyl-5, 6-dihydro-2H-pyran-3-yl)-2, 2-dimethylpropyl]phenyl}-1-methyl-1H-imidazole-4-sulfonamide, is a selective HIV aspartyl protease inhibitor under evaluation as a potential oral treatment of acquired immunodeficiency disease. PNU-106893 is a mixture of four stereoisomers, designated PNU-109165 (3alphaR, 6S), PNU-109166 (3alphaR, 6R), PNU-109167 (3alphaS, 6S), and PNU-109168 (3alphaS, 6R). The major P450 isoforms involved in the metabolism of PNU-106893 and its pure stereoisomers are identified as CYP2D6 and CYP3A4. The major oxidative biotransformation pathway of PNU-106893 which occurs in microsomal incubations appears to be hydroxylation of the phenylethyl side chain attached to the C-6 carbon of the dihydropyrone ring. This hydroxylation is mediated by CYP2D6 only and the process is stereoselective for the 6R absolute stereochemistry. The configuration at position 3 appears to play a minor role in the CYP2D6 mediated hydroxylation. These insights have impacted drug candidate selection for this class of compounds.

The atomic-resolution structure of human caspase-8, a key activator of apoptosis.

BACKGROUND: Caspases are a family of cysteine proteases that have important intracellular roles in inflammation and apoptosis. Caspase-8 activates downstream caspases which are unable to carry out autocatalytic processing and activation. Caspase-8 is designated as an initiator caspase and is believed to sit at the apex of the Fas- or TNF-mediated apoptotic cascade. In view of this role, the enzyme is an attractive target for the design of inhibitors aimed at blocking the undesirable cell death associated with a range of degenerative disorders. RESULTS: The structure of recombinant human caspase-8, covalently modified with the inhibitor acetyl-Ile-Glu-Thr-Asp-aldehyde, has been determined by X-ray crystallography to 1.2 A resolution. The asymmetric unit contains the p18-p11 heterodimer; the biologically important molecule contains two dimers. The overall fold is very similar to that of caspase-1 and caspase-3, but significant differences exist in the substrate-binding region. The structure answers questions about the enzyme-inhibitor complex that could not be explained from earlier caspase structures solved at lower resolution. CONCLUSIONS: The catalytic triad in caspase-8 comprises Cys360, His317 and the backbone carbonyl oxygen atom of Arg258, which points towards the Nepsilon atom of His317. The oxygen atom attached to the tetrahedral carbon in the thiohemiacetal group of the inhibitor is hydrogen bonded to Ndelta of His317, and is not in a region characteristic of a classical 'oxyanion hole'. The N-acetyl group of the inhibitor is in the trans configuration. The caspase-8-inhibitor structure provides the basis for understanding structure/function relationships in this important initiator of the proteolytic cascade that leads to programmed cell death.

Activated sulfonamides are cleaved by glutathione-S-transferases.

In preclinical pharmacokinetic studies and in in vitro rat, dog, and human primary hepatocyte incubations, the sulfonamide (-NH-SO(2)-) bond of a potent inhibitor of the HIV-1 protease containing the p-cyanopyridinyl moiety (PNU-109112), undergoes metabolic cleavage to form the corresponding amine metabolite (PNU-143070). Strikingly, a compound, PNU-140690, obtained by substituting the cyanopyridinyl group of PNU-109112 with a trifluoropyridinyl moiety, was stable under the same in vivo and in vitro conditions used for PNU-109112. The apparent "sulfonamidase activity" present in liver was localized to the cytosolic fraction and shown to be an enzyme-mediated reaction requiring reduced glutathione (GSH). The enzyme responsible was purified in a single step on a GSH immobilized gel and was identified as glutathione-S-transferase (GST) by sequence analysis of peptides obtained by tryptic digestion of the purified protein. Moreover, a mixture of GST isoenzymes purified from rat liver, and three recombinant human GST isoforms, A1-1, M1-1, and P1-1, were active toward PNU-109112 sulfonamide cleavage; the three isoforms exhibited differential rates of PNU-109112 cleavage, demonstrating isoenzyme selectivity.

Mechanism, structure-activity studies, and potential applications of glutathione S-transferase-catalyzed cleavage of sulfonamides.

The mechanism of sulfonamide cleavage of PNU-109112, a potent HIV-1 protease inhibitor, by glutathione-S-transferase (GST) was investigated in the presence of reduced GSH. GST-catalyzed sulfonamide cleavage takes place via the nucleophilic attack of GSH on the pyridine moiety of the substrate with formation of the GS-para-CN-pyridinyl conjugate, the corresponding amine, and sulfur dioxide. Structure activity studies with a variety of sulfonamides indicate that an electrophilic center alpha to the sulfonyl group is required for cleavage. Substituents that withdraw electron density from the carbon atom alpha- to the sulfonyl group facilitate nucleophilic attack by the GS(-) thiolate bound to GST. The rate of sulfonamide cleavage is markedly affected by the nature of the electrophilic group; replacement of para-CN by para-CF(3) on the pyridine ring of PNU-109112 confers stability against sulfonamide cleavage. On the other hand, stability of sulfonamides is less dependent on the nature of the amine moiety. These principles can be applied to the synthesis of sulfonamides, labile toward cellular GST, that may serve as prodrugs for release of bioactive amines. Tumors are particularly attractive targets for these sulfonamide prodrugs as GST expression is significantly up-regulated in many cancer cells. Another potential application could be in organic synthesis, where protection of amines as the corresponding activated sulfonamides can be reversed by GST/GSH under mild conditions.

Equilibrium distribution of HIV antiviral drugs into human peripheral blood mononuclear cells (PBMC) is controlled by free drug concentration in the extracellular medium.

Effect of protein binding on the equilibrium distribution of selected HIV antiviral drugs into isolated human peripheral blood mononuclear cells (PBMC, mainly lymphocytes) was investigated. Human PBMC from a single healthy human donor were isolated, purified, and cryopreserved. Uptake of non-peptide HIV-1 protease inhibitors PNU-96988 and PNU-103017 by these cells in vitro was evaluated as a function of increasing concentration of human serum in the cell incubation media. Both PNU-96988 and PNU-103017 were extensively bound to serum proteins. Uptake/efflux kinetics were very rapid such that accumulation by the cells was thermodynamically, not kinetically, controlled. Accumulation by human PBMCs in vitro was directly proportional to the free and not the total drug concentration in the media. For comparative purposes, the serum protein binding effect on the distribution of two HIV reverse transcriptase (RT) inhibitors, delavirdine (RESCRIPTOR) and zidovudine (AZT), was also evaluated. Like the HIV-1 protease inhibitors, delavirdine was found to be extensively associated with serum proteins and its accumulation by human PBMCs in vitro to be proportional to the free and not total drug concentration. In contrast, AZT was not bound to serum proteins to any significant extent. The uptake of this drug by human PBMCs in vitro was independent of serum concentration. However, the intrinsic cellular accumulation of PNU-96988, PNU-103017 and delavirdine were all greater than AZT. Thus, the extent to which drugs uptake by cells is affected by serum appears proportional to the binding affinity of the serum proteins for the drug.

Microsomal metabolism of delavirdine: evidence for mechanism-based inactivation of human cytochrome P450 3A.

Administration of delavirdine, an HIV-1 reverse transcriptase inhibitor, to rats or monkeys resulted in apparent loss of hepatic microsomal CYP3A and delavirdine desalkylation activity. Human CYP3A catalyzes the formation of desalkyl delavirdine and 6'-hydroxy delavirdine, an unstable metabolite, while CYP2D6 catalyzes only desalkyl delavirdine. CYP2D6 catalyzed desalkyl delavirdine formation was linear with time (up to 30 min) but when catalyzed by cDNA expressed CYP3A4 or human liver microsomes the reaction rate declined progressively with time. Coincubation with triazolam showed that delavirdine caused a time- and NADPH-dependent loss of CYP3A4 activity in human liver microsomes as measured by triazolam 1'-hydroxylation. The catalytic activity loss was saturable and was characterized by a Ki of 21.6 +/- 8.9 microM and a kinact of 0.59 +/- 0.08 min-1. An apparent partition ratio of 41 was determined with cDNA expressed CYP3A4, based on the substrate depletion method. Incubation of [14C]delavirdine with microsomes from several species resulted in irreversible association with an approximately 50 kDa protein, as demonstrated by SDS-PAGE/autoradiography. Binding to the protein was NADPH dependent, glutathione insensitive, proportional to the level of CYP3A expression and was inhibited by ketoconazole, a specific CYP3A inhibitor. NADPH-dependent irreversible binding to human and rat total microsomal protein was demonstrated following exhaustive extraction of microsomal protein. Binding was decreased in the presence of glutathione and appeared to be related to expression level of CYP3A. These results suggest that delavirdine can inactivate CYP3A and has the potential to slow the metabolism of coadministered CYP3A substrates.

Contribution of serum protein association to discrepancy between the in vivo and in vitro UDS results for 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843).

U-89843 has been shown to undergo biotransformation, both in vitro and in vivo, to form U-97924 as a major primary metabolite. U-89843 was found to be positive in an in vitro UDS mutagenesis screen conducted with primary rat hepatocytes in serum-free media. In contrast to in vitro results, no evidence of genetic toxicity of U-89843 was observed in rats in the in vivo/in vitro version of the UDS test with single oral doses up to 1400 mg/kg. The negative results may be related to more robust in vivo detoxification mechanisms or relatively lower exposure to reactive metabolites formed by bioactivation of U-89843 as compared to that observed in the serum-free in vitro hepatocyte test system. Further studies showed rat serum suppressed the in vitro metabolism of U-89843 as well as the formation of the corresponding hydroxylated metabolite, U-97924, the putative precursor of proposed reactive electrophilic metabolite. The measured in vivo systemic clearance of U-89843 (0.53 l/h/kg) in rats was about 1000-fold slower than the in vitro intrinsic clearance (606 l/h/kg) estimated by measuring the formation of U-97924 in rat liver microsomal incubations. Since U-89843 is extensively associated with serum proteins a poor extraction ratio into the liver may account for the slower biotransformation of U-89843 in vivo as compared to that exhibited in in vitro serum-free hepatocyte incubations. Addition of bovine serum albumin (1-40 mg/ml) to the in vitro UDS assay medium decreased the UDS mean net grains per nucleus response of U-89843. These results suggest that the effect of serum protein should be considered when comparing serum-free in vitro UDS and in vivo UDS results for highly serum protein bound compounds.

Bioactivation of 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843) to reactive intermediates that bind covalently to macromolecules and produce genotoxicity.

U-89843 is a novel pyrrolo[2,3-d]pyrimidine antioxidant with prophylactic activity in animal models of lung inflammation. During preclinical safety evaluation, U-89843 was found to give a positive response in the in vitro unscheduled DNA synthesis (UDS) assay, an assay which measures DNA repair following chemically-induced DNA damage in metabolically competent rat hepatocytes. Incubation of [14C]U-89843 with liver microsomes resulted in covalent binding of radioactive material to macromolecules by a process that was NADPH-dependent. U-89843 has been shown to undergo C-6 methylhydroxylation to give U-97924, in rat both in vivo and in vitro, in a reaction catalyzed by cytochrome P450 2C11. Synthetical U-97924 is chemically reactive and undergoes dimerization in aqueous solution. The dimerization of U-97924 was significantly inhibited by addition of nucleophiles such as methanol, glutathione, and N-acetylcysteine. Characterization of the corresponding methanol, glutathione, and N-acetylcysteine adducts of U-97924 supported the hypothesis of a reaction pathway involving reactive iminium species formed via dehydration of U-97924. The metabolism-dependent irreversible covalent binding of radioactive material to liver microsomal protein and DNA also is dramatically reduced in the presence of reduced glutathione (GSH). A trifluoromethyl analog of U-89843 was prepared in an effort to block the corresponding metabolic hydroxylation pathway. This new compound (U-107634) was found to be negative in the in vitro UDS assay, and its metabolic susceptibility toward hydroxylation at the C-6 methyl group was eliminated. These observations suggest that the positive in vitro UDS results of U-89843 are mediated by the bioactivation of U-89843, leading to reactive electrophilic intermediates derived from the (hydroxymethyl)pyrrole metabolite U-97924.

Chromatographic measurement of drug-protein interaction: determination of HIV protease inhibitor-serum albumin association.

A chromatographic method for evaluation of the serum protein binding of a large number of non-peptide human immunodeficiency virus (HIV) protease inhibitors in short analysis time and automated fashion was developed. The method utilizes a size exclusion HPLC column. Bovine or human serum albumin is added to the mobile-phase running buffer. Qualitatively, a shift to shorter drug retention time in the presence of protein in the mobile phase is indicative of binding interaction of the drug and protein. The extent of binding of the drug to the protein is quantitated by comparison of the shift in retention time in the presence of protein to the retention time of the drug in the same buffer in the absence of protein (i.e., the drug's "intrinsic" retention time on the column). Binding measurements were carried out a 37 degrees C with a temperature-controlled autosampler and column oven. Results were compared with those obtained by ultrafiltration. The method yields thermodynamically valid binding measurements and is capable of directly detecting differences in the protein binding of individual stereoisomers present in mixtures (either enantiomers or diastereomers) without prior purification of the individual stereoisomers. The method measures overall binding to albumin and is not binding-site specific. Because of this, quantitative comparison of the extent of albumin binding of drugs which bind to the same site, different sites, or nonspecifically (i.e., not at discrete sites) is possible.

In vitro and in vivo biotransformation of 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-D]pyrimidine (U-89843) in the rat.

The biotransformation of 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843) has been studied in rat both in vitro and in vivo. Major metabolites observed by HPLC analysis of rat plasma, liver cytosol, and microsomal incubations were characterized by UV, LC/MS, and comparison with synthetic standards. The structures of the metabolites were shown to be the C-6 hydroxymethyl (U-97924), C-6 formyl (U-97865), and C-6 carboxyl analogs of U-89843. In the male rat, formation of U-97924 is mediated by cytochrome P4502C11. Kinetic analysis of U-97924 formation indicated that it was a high-affinity/high-capacity process (KM = 4.2 +/- 0.5 microM; Vmax = 21.2 +/- 0.8 nmol/mg/min). Formation of U-97865 via enzymatic oxidation from the primary metabolite U-97924 was catalyzed by both the microsomal subcellular fraction in a NADPH-dependent process (presumably via cytochrome P450) and in cytosol by NAD(+)-dependent alcohol dehydrogenase. Upon incubation with cytosolic fractions, U-97865 was found to undergo NAD(+)-dependent oxidation, mediated by aldehyde dehydrogenase, to the corresponding carboxylic acid. Although significant levels of U-89843, U-97924, and U-97865 were observed in vivo in rat plasma, only a minor amount of the carboxylic acid together with larger amounts of unidentified polar metabolites were excreted in urine and feces.

Determination of warfarin-human serum albumin protein binding parameters by an improved Hummel-Dreyer high-performance liquid chromatographic method using internal surface reversed-phase columns.

The Hummel-Dreyer size-exclusion high-performance liquid chromatographic method for the determination of protein binding parameters has been improved and automated by use of an internal surface reversed-phase (ISRP) column (5 cm X 4.6 mm) and a computer-controlled mobile-phase delivery system with low volume syringe mixing. The high-efficiency ISRP columns, which are nonadsorptive and exclusionary to serum proteins but allow partitioning of small molecules with an internal peptide bonded phase, maintain high performance after many injections of human serum albumin (HSA), enable the use of short columns, and provide for the resolution of primary ligand from protein binding displacers. The modified Hummel-Dreyer high-performance liquid chromatographic method was demonstrated by the determination of binding parameters for warfarin-HSA in phosphate buffer, which were found to be n1 = 1.0, n2 = 2.1, K1 = 3.30 X 10(5) M-1, and K2 = 2.03 X 10(4) M-1. The necessary sequence of chromatographic experiments was repeated 4 times at 18 separate warfarin mobile phase concentrations. Each automated sequence required 8 h to complete. The parameters were measured with a precision of less than 10% relative standard deviation.

High-performance liquid chromatographic separation of peptides on a diol-Gly-Phe-Phe tripeptide-bonded phase.

The retention characteristics of some selected peptides (mol. wt. less than 2000 a.m.u.) have been investigated on a diol-Gly-Phe-Phe partitioning phase, bound to 5-microns porous silica. The hydrophobic, positively charged peptides can be separated with mild mobile phases, containing only acetonitrile and phosphate buffer. The peptide selectivity of the diol-Gly-Phe-Phe-bonded phase is uniquely different from that of a C8 column. The dependence of capacity factors on mobile phase pH, ionic strength, and organic solvent concentration demonstrated that the partitioning mechanisms of the diol-Gly-Phe-Phe phase involve multifunctional reversed-phase and cation-exchange processes.