Challenges and Hurdles to Business as Usual in Drug Development for Treatment of Rare Diseases.

Only 10-15 first-in-class new medicines are approved each year by the global pharmaceutical industry for all diseases, of which less than a third is for rare (orphan) diseases. The drug discovery processes to identify rare and common diseases are similar, suggesting it will be impossible to discover new drugs for even a small fraction of the rare diseases using the current paradigm. Different approaches are required to address this large unmet medical need.

Quantitative expression analysis of the cellular specificity of HECT-domain ubiquitin E3 ligases.

We evaluated the expression of 28 gene sequences with homology to the carboxy terminal of HECT E3 ubiquitin ligases in nine human cell lines using RT-PCR, to determine whether gene expression could be associated with cell-specific functions (HECT is "homologous to E6AP C-terminus"). In general, HECT-domain E3 ligases are constitutively expressed at low levels with a broad range between cell types. hecth3, 21, and 23 had higher levels in three leukocytic lines (Jurkat, MM6, THP1); hecth11 was more abundant in HepG2 and A495; and hecth15 and hecth12 were differentially expressed in lung fibroblasts derived from normal and severe emphysema patients (CCD16 and CCD29, respectively). Absolute quantitation showed that most HECT E3s have about 20-100 copies of mRNA per Jurkat cell. By comparison, UBCH7 (an ubiquitin-conjugating E2) is 10-fold more abundant in Jurkat cells and 30-fold more abundant than E2 UBCH5A. We interpret the broad range of transcript levels to be consistent with the hypothesis that the concentrations of E3 are important for ubiquitination selectivity, leading us to conclude that substrate activation is necessary but not sufficient for selectivity.

Cloning and functional expression of the cDNA encoding rat lanosterol 14-alpha demethylase.

Lanosterol 14 alpha-demethylase (LDM) is a cytochrome P-450 enzyme in the biosynthetic pathway of cholesterol. As such, it represents a target for cholesterol-lowering drugs. Rat LDM (rLDM) has been purified from the livers of rats treated with cholestyramine. The purified protein was used to generate tryptic fragments which were then sequenced. The amino acid (aa) sequences were used to design oligodeoxyribonucleotide primers and a DNA fragment was generated by RT-PCR to probe a phagemid library. A clone encoding rLDM was isolated from the livers of cholestyramine-treated rats. The clone contains an open reading frame encoding a polypeptide of 486 aa and a predicted molecular mass of 55 045 Da. The deduced aa sequence shows a high degree of identity to the yeast LDM sequences, as well as sequences which match typical P-450 sequence motifs. When produced in a baculovirus/insect cell culture system, LDM activity was detected and inhibited by the specific inhibitor azalanstat with an IC50 value of less than 2 nM. The isolation of this full-length coding sequence should facilitate research into understanding the direct and indirect effects of LDM in the regulation of cholesterol biosynthesis and the search for cholesterol-lowering drugs.

Stereoisomers of ketoconazole: preparation and biological activity.

The four stereoisomers of the antifungal agent ketoconazole (1) were prepared and evaluated for their selectivity in inhibiting a number of cytochrome P-450 enzymes. Large differences in selectivity among the isomers were observed for inhibition of the cytochromes P-450 involved in steroid biosynthesis, whereas little differences was observed for inhibition of those associated with hepatic drug metabolism. The cis-(2S,4R) isomer 2 was the most effective against rat lanosterol 14 alpha-demethylase, (2S,4R)-2 greater than (2R,4S)-4 much greater than (2R,4R)-3 = (2S,4S)-5, and progesterone 17 alpha,20-lyase, (2S,4R)-2 much greater than (2S,4S)-5 greater than (2R,4R)-3 = (2R,4S)-4, whereas the cis-(2R,4S) isomer 4 was more effective against cholesterol 7 alpha-hydroxylase, (2R,4S)-4 greater than (2S,4S)-5 greater than (2R,4R)-3, greater than (2S,4R)-2, and the trans-(2S,4S) isomer 5 was the most effective against aromatase, (2S,4R)-5 much greater than (2R,4R)-3 = (2R,4S)-4 greater than (2S,4R)-2. The cis-(2S,4R) and trans-(2R,4R) isomers 2 and 3 are equipotent in inhibiting corticoid 11 beta-hydroxylase and much more effective than their antipodes. Little selectivity was observed for inhibition of cholesterol side chain cleavage or xenobiotic hydroxylase. These data indicate that the affinity of azoles for cytochrome P-450 enzymes involved in steroid synthesis is highly dependent on the stereochemistry of the entire molecule, whereas binding to drug metabolizing enzymes is a less selective process.

A novel rat hepatic clofibrate-inducible cytochrome P450 that is not a lauric acid hydroxylase.

2-Methoxy-6-[1-methylethyl]naphthalene (MMEN) was hydroxylated in an NADPH-dependent manner to the (omega-1)-alcohol and the (R)-omega- and (S)-omega-alcohols by rat hepatic microsomes. (S)-omega-Hydroxylation was selectively induced 7-fold by clofibrate treatment. Phenobarbital, 3-methyl-cholanthrene, dexamethasone, cholestyramine, and MMEN did not induce this activity to the same extent. Incubation of the racemic omega-alcohols with microsomes isolated from rats resulted in a greater rate of degradation of the (S)- than the (R)-omega-alcohol confirming (S)-omega-hydroxylation to be an initial catalytic event. MMEN and lauric acid were not competitive inhibitors of each other in microsomes from clofibrate-treated rats, indicating the (S)-omega-MMEN hydroxylase to be a different enzyme from the characterized clofibrate-inducible lauric acid hydroxylases, CYP4A1 and CYP4A3. This was confirmed by the observations that (1) lauric acid hydroxylation was inhibited by 0.02% Tween 20 or Tween 80 and 25 microM capric or myristic acids, whereas omega-MMEN hydroxylation was not, (2) omega-MMEN hydroxylation was inhibited by ketoconazole, cholesterol and acetone, whereas lauric acid hydroxylation was not, and (3) CYP4A1 and CYP4A3 expressed in Hep G2 cells did not catalyze MMEN hydroxylation. Microsomes from the lungs of rabbits treated with progesterone and kidney of untreated rats did not support selective (S)-omega-MMEN hydroxylation, indicating that this activity is not associated with CYP4A4 or CYP4A2, respectively. Leukotriene B4 (LTB4) hepatic microsomal hydroxylation was not inhibited by MMEN and microsomes from human neutrophils did not support the reaction. These data identify a hitherto uncharacterized cytochrome P450 which is selectively induced by clofibrate and does not catalyze the omega-hydroxylation of the fatty acids or prostaglandins investigated. It is proposed that the enzyme catalyzing the selective (S)-omega-hydroxylation of MMEN is a novel rat P450 and that it is either a new member of the CYP4 family or a clofibrate-inducible P450 from another gene family.

Azalanstat (RS-21607), a lanosterol 14 alpha-demethylase inhibitor with cholesterol-lowering activity.

Agents that inhibit hepatic cholesterol biosynthesis reduce circulating cholesterol levels in experimental animals and humans, and may be of pharmacological importance in the prevention of atherosclerosis. Azalanstat (RS-21607), a synthetic imidazole, has been shown to inhibit cholesterol synthesis in HepG2 cells, human fibroblasts, hamster hepatocytes and hamster liver, by inhibiting the cytochrome P450 enzyme lanosterol 14 alpha-demethylase. When administered orally to hamsters fed regular chow, RS-21607 (50 mg/kg/day) lowered serum cholesterol in a dose-dependent manner (ED50 = 62 mg/kg) in a period of 1 week. It preferentially lowered low density lipoprotein (LDL) cholesterol and apo B relative to high density lipoprotein (HDL) cholesterol and apo A-1. It also lowered plasma cholesterol levels in hamsters fed a high saturated fat and cholesterol diet. RS-21607 inhibited hepatic microsomal hydroxymethylglutaryl-CoA (HMG-CoA) reductase activity in hamsters in a dose-dependent manner (ED50 = 31 mg/kg), and this was highly correlated with serum cholesterol lowering (r = 0.97). Cholesterol lowering by azalanstat and cholestyramine was additive, and the increase in HMG-CoA reductase brought about by cholestyramine was attenuated significantly by azalanstat. In vitro studies with HepG2 cells indicated that this modulation of reductase activity was indirect, occurring at a post-transcriptional step, and it is proposed that a regulatory oxysterol derived from dihydrolanosterol (or lanosterol) may be responsible for this regulation. Azalanstat does not appear to lower circulating cholesterol in the hamster by up-regulation of the hepatic LDL receptor, suggesting that other mechanisms are involved. Orally administered azalanstat (50-75 mg/kg) stimulated hepatic microsomal cholesterol 7 alpha-hydroxylase activity by 50-400% in hamsters, and it is postulated that this may result from modified cholesterol absorption and bile acid synthesis.

Phenotypic vs. target-based drug discovery for first-in-class medicines.

Current drug discovery strategies include both molecular and empirical approaches. The molecular approaches are predominantly hypothesis-driven and are referred to as target-based. The empirical approaches are referred to as phenotypic because they rely on phenotypic measures of response. A recent analysis revealed the phenotypic approaches to be the more successful strategy for small-molecule, first-in-class medicines. The rationalization for this success was the unbiased identification of the molecular mechanism of action (MMOA).

Progesterone metabolism in hepatic microsomes. Effect of the cytochrome P-450 inhibitor, ketoconazole, and the NADPH 5 alpha-reductase inhibitor, 4-MA, upon the metabolic profile in human, monkey, dog, and rat.

Progesterone was incubated in the presence of NADPH with hepatic microsomes isolated from male and female human, monkey, dog, and rat and the effect of 17 beta-NN-diethylcarbamoyl-4-methyl-4-aza-5 alpha- androstan-3-one (4-MA), an NADPH 5 alpha-reductase inhibitor, and ketoconazole, a cytochrome P-450 inhibitor, upon oxidative metabolism was evaluated. 4-MA caused an increase in detectable oxidative products only with microsomes isolated from rat. An increase in 2 alpha- and 16 alpha-hydroxylation was observed in male rat, and an increase in the formation rate of nine products was observed in female rat. delta 6-Progesterone, 6 beta-, 15 alpha-, 16 alpha-, and 21-hydroxyprogesterone (6 beta-, 15 alpha-, 16 alpha-, and 21-OHP) were common products in both sexes of all species studied. Differences were observed in the formation rate of 2 alpha-, 2 beta-, 6 alpha-, 7 alpha-, and 17 alpha-OHPs. At the 2-carbon, microsomes isolated from both sexes of primates hydroxylated progesterone exclusively at the 2 beta-position. Microsomes from both dog sexes and female rat formed 2 alpha- and 2 beta-OHP, while microsomes isolated from male rat formed exclusively 2 alpha-OHP. 7 alpha-Hydroxylation was detected exclusively in rat, and 6 alpha-hydroxylation was detected in both dog and rat. 17 alpha-Hydroxylase activity in primates was detected only in microsomes from male human. IC50 values associated with ketoconazole inhibition of progesterone metabolism differed among species.(ABSTRACT TRUNCATED AT 250 WORDS)

Targeting protein ubiquitination for drug discovery. What is in the drug discovery toolbox?

Protein ubiquitination regulates the half-lives of many proteins by targeting them for degradation. Ubiquitination is a specific process associated with several highly regulated biological outcomes including cell cycle progression, differentiation, antigen presentation, retrovirus assembly, apoptosis, signal transduction, transcriptional activation, biological clocks, receptor downregulation and endocytosis. Newly discovered families of ubiquitination and deubiquitination enzymes participate in these processes. These enzymes could provide new families of drug targets and new ways of intervention in many human diseases; however, much work is required to validate this approach. This review will discuss what is in the drug discovery toolbox to assist in the validation of ubiquitination enzymes as therapeutic targets.

Androgen formation by cytochrome P450 CYP17. Solvent isotope effect and pL studies suggest a role for protons in the regulation of oxene versus peroxide chemistry.

CYP17 catalyzes the cleavage of the C-17 side chain of progesterone to form androstenedione. The two-step reaction involves an initial 17 alpha-hydroxylation catalyzed by oxene chemistry followed by cleavage of the C-17 side chain. We have recently shown that C-17 side-chain cleavage may involve the rearrangement of a peroxy intermediate via a Baeyer-Villiger rearrangement [Mak, A. Y., & Swinney, D. C. (1992) J. Am. Chem. Soc. 114, 8309]. Accordingly, CYP17 is proposed to catalyze oxidations via both oxene and peroxide chemistry. This study was initiated to investigate the possibility that protons may play a determining role in differentiating between the oxene and peroxide chemistries associated with product formation. The pL dependence of the deuterium solvent isotope effects associated with progesterone oxidation to 17 alpha-hydroxyprogesterone and 17-O-acetyltestosterone and 17 alpha-hydroxyprogesterone oxidation to androstenedione was determined in microsomes from pig testes. The formation of 17 alpha-hydroxyprogesterone is assumed to occur via oxene chemistry and the formation of 17-O-acetyltestosterone and androstenedione by peroxide chemistry. The initial rate of progesterone oxidation to 17 alpha-hydroxyprogesterone was associated with a pL-independent inverse solvent isotope effect (Hk/Dk = 0.75-0.95, in 30% DOD), whereas the rate of oxidation to 17-O-acetyltestosterone was associated with a pL-independent positive solvent isotope effect in the presence of 30% DOD (Hk/Dk approximately 2). In contrast, DOD inhibited the formation of androstenedione from 17 alpha-hydroxyprogesterone in a noncompetitive, pL-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Intramolecular isotope effects associated with meta-hydroxylation of biphenyl catalyzed by cytochrome P-450.

Intramolecular isotope effects and the degree of deuterium retention were determined for the meta-hydroxylation of biphenyl as catalyzed by microsomal cytochrome P-450 obtained from rats pretreated with phenobarbital. The percent deuterium retention after meta-hydroxylation of 3,5,3',5'-[2H4]-biphenyl was found to be 77.3% +/- 1.9. The intramolecular isotope effects associated with 3,3'-[2H2]-biphenyl and 3,5-[2H2]-biphenyl were found to be 0.90 +/- .05 and 1.05 +/- .06, respectively. These data demonstrate conclusively that a direct insertion or abstraction mechanism is not operable in the meta-hydroxylation of biphenyl and suggest the possibility of an addition-rearrangement mechanism as opposed to initial and direct arene oxide formation.

Isotopically labeled chlorobenzenes as probes for the mechanism of cytochrome P-450 catalyzed aromatic hydroxylation.

Noncompetitive and competitive intermolecular deuterium isotope effects were measured for the cytochrome P-450 catalyzed hydroxylation of a series of selectively deuterated chlorobenzenes. An isotope effect of 1.27 accompanied the meta hydroxylation of chlorobenzene-2H5 as determined by two totally independent methods (EC-LC and GC-MS assays). All isotope effects associated with the meta hydroxylation of chlorobenzenes-3,5-2H2 and -2,4,6-2H3 were approximately 1.1. In contrast, competitive isotope studies on the ortho and para hydroxylation of chlorobenzenes-4-2H1, -3,5-2H2, and -2,4,6-2H3 resulted in significant inverse isotope effects (approximately 0.95) when deuterium was substituted at the site of oxidation whereas no isotope effect was observed for the oxidation of protio sites. These results eliminate initial epoxide formation and initial electron abstraction (charge transfer) as viable mechanisms for the cytochrome P-450 catalyzed hydroxylation of chlorobenzene. The results, however, can be explained by a mechanism in which an active triplet-like oxygen atom adds to the pi system in a manner analogous to that for olefin oxidation. The resulting tetrahedral intermediate can then rearrange to phenol directly or via epoxide or ketone intermediates.

Accumulation of intermediates and isotopically sensitive enolization distinguish between aromatase (cytochrome P450 CYP19) from rat ovary and human placenta.

Aromatase activity in microsomes from human placenta (HPM) and rat ovary (ROM) was compared by measuring the accumulation of 19-oxygenated intermediates, the effect of tritium substitution upon rates, and the distribution of tritium in products. A considerable accumulation of intermediates (19-hydroxyandrogen and 19-al-androgen) and a lag in product formation (estrogen and water) was observed with ROM but not HPM. Addition of purified NADPH-cytochrome P450 reductase to ROM decreased the accumulation of 19-hydroxyandrostenedione and increased the rate of estrone formation. This difference could not be attributed to the microsomal reductase concentration since its concentration was similar in both tissues. Estrogen formation by aromatase from these tissues was not associated with a significant kinetic isotope effect when androstenedione was labeled with tritium at C-1 and C-2. Isotopically sensitive switching (branching) from the 19-al-androstenedione enzyme complex to form free 19-al-androstenedione rather than estrogen was not observed. These data suggest that for aromatase in both tissues, an enzymatic step exists between the 19-al-androstenedione intermediate and hydrogen abstraction or enolization that has a large commitment to catalysis. The distribution of tritium into the products, water and estrogen, was dependent upon substrate, enzyme source, and position of the label. Incubation of 1 beta, 2 beta-[3H]androstenedione and testosterone with ROM and 1 beta,2 beta-[3H]testosterone with HPM resulted in approximately 50% of the label being retained in the estrogen and 50% being lost in water. The majority of the label was lost in water upon incubation of 1 beta-labeled substrates with microsomes from both sources. Unexpectedly, no label was lost to water upon incubation of the specifically 1 alpha,2 alpha-labeled substrates with either enzyme source. Only incubation of 1 beta,2 beta-[3H]androstenedione with HPM resulted in loss of tritium from the 2-position. These data were interpreted to indicate that, for androstenedione metabolism by ROM and testosterone metabolism by both ROM and HPM, enolization occurs nonspecifically in an isotopically sensitive manner following deformylation, but for HPM metabolism of androstenedione enolization occurs in an enzyme-assisted manner. The studies show that aromatase located in ROM differs from that in HPM by its accumulation of intermediates and inability to selectively remove the 2 beta-tritium from androstenedione.

The kinetic factors that determine the affinity and selectivity for slow binding inhibition of human prostaglandin H synthase 1 and 2 by indomethacin and flurbiprofen.

We present here for the first time a method for determining the rate constants associated with slow binding inhibition of prostaglandin H synthase (PGHS). The rate constants were determined by a method using initial steady-state conditions, which minimize the impact of catalytic autoinactivation of the enzyme. The currently available methods for determining the kinetic constants associated with slow binding enzyme inhibition do not distinguish between rate decreases due to enzyme inhibition or due to autoinactivation of the enzyme. A mathematical model was derived assuming a rapid reversible formation of an initial enzyme-inhibitor complex (EI) followed by a slow reversible formation of a second enzyme-inhibitor complex (EI*). The two enzyme inhibitor complexes are assumed to be in slow equilibrium. This method was used to evaluate the kinetic parameters associated with the binding and selectivity of the nonsteroidal antinflammatory drugs (NSAIDs), flurbiprofen and indomethacin. The KI values associated with the formation of the first reversible complex (EI) for flurbiprofen with PGHS1 and PGHS2 were 0.53 +/- 0. 06 and 0.61 +/- 0.08 microM, respectively; the rate constants for the forward isomerization, k2, into the second reversible complex (EI*) were 0.97 +/- 0.99 and 0.11 +/- 0.01 s-1, respectively, and rates of the reverse isomerization from EI*, k-2, were 0.031 +/- 0.004 and 0.0082 +/- 0.0008 s-1, respectively. Indomethacin was estimated to form the EI complex with the same affinity for both PGHS1 and PGHS2, 10.0 +/- 2.8 microM and 11.2 +/- 2.0 microM, respectively, and dissociate from EI* at approximately the same rate 0.0011 +/- 0.0002 s-1 and 0.0031 +/- 0.0003 s-1, respectively. However, the rate of isomerization into EI* from EI was much greater for PGHS1 than PGHS2, 0.33 +/- 0.08 s-1 as compared with 0.034 +/- 0.004 s-1. These results show that the overall affinity for the inhibition of PGHS1 versus PGHS2 was 30-fold greater for indomethacin (KI* = 0.032 +/- 0.005 and 1.02 +/- 0.08 microM, respectively) and 3-fold greater for flurbiprofen (KI* = 0.017 +/- 0.002 and 0.045 +/- 0.004 microM, respectively). The results also show that for both PGHS1 and PGHS2, flurbiprofen was bound tighter to the initial EI complex than indomethacin; however, the rate of dissociation from EI* was slower for indomethacin than flurbiprofen. The rate of the forward isomerization to EI* is primarily responsible for the selectivity of both NSAIDs for PGHS1. This analysis shows the quantitative importance of the different kinetic parameters upon the overall binding affinity of these NSAIDs and should greatly assist in our understanding of the structural interactions that promote enzyme-inhibitor binding.

Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g.

Purified cytochrome P-450g, a male-specific rat hepatic isozyme, was observed to metabolize progesterone to two primary metabolites (6 beta-hydroxyprogesterone and 16 alpha-hydroxyprogesterone), two secondary metabolites (6 beta,16 alpha-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16 alpha-hydroxyprogesterone). The Km,app for the formation of these products from progesterone was determined to be approximately 0.5 microM, while the Km,app for metabolism of 6 beta- and 16 alpha-hydroxyprogesterone was found to be 5-10 microM. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 microM, and a lag in formation of secondary metabolites was not observed in 1-min incubations. Concerted oxidation of progesterone to secondary products without the intermediate products leaving the active site was suggested by these results and confirmed by isotopic dilution experiments in which little or no dilution of metabolically formed 6 beta,16 alpha-dihydroxyprogesterone and 6-keto-16 alpha-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6 beta-hydroxyprogesterone or 16 alpha-hydroxyprogesterone. Incubation of 6 beta-hydroxyprogesterone with a reconstituted system in an atmosphere of 18O2 resulted in greater than 90% incorporation of 18O in the 16 alpha-position of 6 beta,16 alpha-dihydroxyprogesterone but no incorporation of 18O into 6-ketoprogesterone, even though the reaction was dependent upon enzyme and O2, and not inhibited by mannitol, catalase, or superoxide dismutase. Factors which characterize the metabolism of progesterone by cytochrome P-450g in terms of active-site constraints and the catalytic competence of the enzyme in microsomes were also explored.

Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes.

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.

Pharmacology of a Ca(2+)-influx pathway activated by emptying the intracellular Ca2+ stores in HL-60 cells: evidence that a cytochrome P-450 is not involved.

In HL-60 cells, inhibition of the endoplasmic-reticular Ca2+ pump by thapsigargin leads to the emptying of this intracellular Ca2+ store and a subsequent activation of plasma-membrane Ca2+ influx through a non-voltage-dependent pathway. The elevated intracellular free Ca2+ concentration ([Ca2+]i) produced and maintained by this Ca2+ inflow was used to examine the potency of various compounds to inhibit this influx mechanism. As expected, specific blockers of known Ca2+ channels, such as nifedipine, omega-conotoxin GVIA and ryanodine were without effect. The less selective inhibitors La3+, SKF-96365 and L-651,582, which are thought to inhibit both voltage-dependent and voltage-independent Ca2+ channels, decreased [Ca2+]i back to resting levels, with pIC50 values of 5.2, 5.9 and 6.2 respectively. It has been proposed that a cytochrome P-450 is involved in activating Ca(2+)-influx pathways in thymocytes, neutrophils and platelets. Consistent with this idea, the imidazole cytochrome P-450 inhibitors miconazole, econazole, clotrimazole and ketoconazole inhibited the thapsigargin-elevated [Ca2+]i with pIC50 values of 7.1, 7.1, 7.1 and 5.8 respectively. The high affinity of imidazoles for cytochromes P-450 is due to co-ordinate binding to the haem. This interaction is greatly decreased in 2-substituted imidazoles. We examined whether the inhibition of Ca2+ influx was due to an interaction of the inhibitor imidazole nitrogen with the haem iron of the putative cytochrome P-450 by comparing the activity of two compounds, identical except that one was methylated at the imidazole 2-position. They were found to block thapsigargin-activated Ca2+ influx with equal potency. These results strongly suggest that a cytochrome P-450 is not involved in the activation of the Ca2+ influx produced by emptying the intracellular Ca2+ stores.

Structural characterization of arachidonyl radicals formed by prostaglandin H synthase-2 and prostaglandin H synthase-1 reconstituted with mangano protoporphyrin IX.

A tyrosyl radical generated in the peroxidase cycle of prostaglandin H synthase-1 (PGHS-1) can serve as the initial oxidant for arachidonic acid (AA) in the cyclooxygenase reaction. Peroxides also induce radical formation in prostaglandin H synthase-2 (PGHS-2) and in PGHS-1 reconstituted with mangano protoporphyrin IX (MnPGHS-1), but the EPR spectra of these radicals are distinct from the initial tyrosyl radical in PGHS-1. We have examined the ability of the radicals in PGHS-2 and MnPGHS-1 to oxidize AA, using single-turnover EPR studies. One wide singlet tyrosyl radical with an overall EPR line width of 29-31 gauss (G) was generated by reaction of PGHS-2 with ethyl hydroperoxide. Anaerobic addition of AA to PGHS-2 immediately after formation of this radical led to its disappearance and emergence of an AA radical (AA.) with a 7-line EPR, substantiated by experiments using octadeuterated AA. Subsequent addition of oxygen resulted in regeneration of the tyrosyl radical. In contrast, the peroxide-generated radical (a 21G narrow singlet) in a Y371F PGHS-2 mutant lacking cyclooxygenase activity failed to react with AA. The peroxide-generated radical in MnPGHS-1 exhibited a line width of 36-38G, but was also able to convert AA to an AA. with an EPR spectrum similar to that found with PGHS-2. These results indicate that the peroxide-generated radicals in PGHS-2 and MnPGHS-1 can each serve as immediate oxidants of AA to form the same carbon-centered fatty acid radical that subsequently reacts with oxygen to form a hydroperoxide. The EPR data for the AA-derived radical formed by PGHS-2 and MnPGHS-1 could be accounted for by a planar pentadienyl radical with two strongly interacting beta-protons at C10 of AA. These results support a functional role for peroxide-generated radicals in cyclooxygenase catalysis by both PGHS isoforms and provide important structural characterization of the carbon-centered AA..

Differential allosteric regulation of prostaglandin H synthase 1 and 2 by arachidonic acid.

Prostaglandins are synthesized by prostaglandin H synthase (PGHS) 1 and 2. PGHS2 is regulated through inducible expression. We report here the regulation of PGHS1 activity by substrate-dependent cooperative activation. The cooperativity is characterized by a Hill coefficient of 1.29 +/- 0.06, a curved Eadie-Scatchard plot, and activation by low concentrations of competitive inhibitors. The activation also appears to induce a conformational change in the cyclooxygenase site. The cooperativity produces a 2-4-fold greater rate of PGHS2-dependent prostaglandin formation compared with PGHS1-dependent prostaglandin formation at arachidonic acid concentrations below 0.5 microM. A consequence of the PGHS1 cooperativity is that the affinity of many cyclooxygenase inhibitors for PGHS1 decreases in parallel to the activation by arachidonic acid. In contrast, the affinity of these inhibitors for PGHS2 is unaffected by the changes in arachidonic acid concentration. This results in a dramatic difference in PGHS2/PGHS1 selectivity at different arachidonic acid concentrations.