Neurotensin analog selective for hypothermia over antinociception and exhibiting atypical neuroleptic-like properties.

Neurotensin (NT) is a tridecapeptide neurotransmitter in the central nervous system. It has been implicated in the therapeutic effects of neuroleptics. Central activity of NT can only be demonstrated by direct injection into the brain, since it is readily degraded by peptidases in the periphery. We have developed many NT(8-13) analogs that are resistant to peptidase degradation and can cross the blood-brain barrier (BBB). In this study, we report on one of these analogs, NT77L. NT77L induced hypothermia (ED(50)=6.5 mg/kg, i.p.) but induced analgesia only at the highest dose examined (20 mg/kg, i.p.). Like the atypical neuroleptic clozapine, NT77L blocked the climbing behavior in rats induced by the dopamine agonist apomorphine (600 microg/kg) with an ED(50) of 5.6 mg/kg (i.p.), without affecting the licking and the sniffing behaviors. By itself NT77L did not cause catalepsy, but it moderately reversed haloperidol-induced catalepsy with an ED(50) of 6.0 mg/kg (i.p.). Haloperidol alone did not lower body temperature, but it potentiated the body temperature lowering effect of NT77L. In studies using in vivo microdialysis NT77L showed similar effects on dopamine turnover to those of clozapine, and significantly different from those of haloperidol in the striatum. In the prefrontal cortex, NT77L significantly increased serotonergic transmission as evidenced by increased 5-hydroxyindole acetic acid:5-hydroxytryptamine (5-HIAA:5-HT) ratio. Thus, NT77L selectively caused hypothermia, over antinociception, while exhibiting atypical neuroleptic-like effects.

Antiparkinson-like effects of a novel neurotensin analog in unilaterally 6-hydroxydopamine lesioned rats.

Parkinson's disease is a neuropathological disorder involving the degeneration of dopamine neurons in the substantia nigra, with the resultant loss of their terminals in the striatum. This dopamine loss causes most of the motor disturbances associated with the disease. One animal model of Parkinson's disease involves destruction of the nigrostriatal pathway with a neurotoxin (6-hydroxydopamine) injected into this pathway. In unilaterally lesioned animals, injection of D-amphetamine causes rotation towards the lesioned side, while injection of apomorphine acting upon supersensitive postsynaptic dopamine receptors causes rotation away from the lesioned side. In this study, we tested the effects of acute and subchronic injection of a neurotensin analog (NT69L) on the rotational behavior induced by D-amphetamine (5 mg/kg) or apomorphine (600 microg/kg) in unilaterally 6-hydroxydopamine lesioned rats. Pretreatment of animals with intraperitoneal injections of NT69L (1 mg/kg) resulted in a significant reduction of apomorphine-induced contralateral rotation and D-amphetamine-induced ipsilateral rotation in these lesioned rats with an ED(50) of 40 and 80 microg/kg, respectively. After three daily injections of NT69L, its effects on this rotational behavior were unchanged, suggesting that no tolerance develops to this effect of NT69L.

Altering behavioral responses and dopamine transporter protein with antisense peptide nucleic acids.

The dopamine transporter (DAT) plays a role in locomotion and is an obligatory target for amphetamines. We designed and synthesized an antisense peptide nucleic acid (PNA) to rat DAT to examine the effect of this antisense molecule on locomotion and on responsiveness to amphetamines. Rats were injected intraperitoneally daily for 9 days with either saline, an antisense DAT PNA, a scrambled DAT PNA, or a mismatch DAT PNA. On days 7 and 9 after initial motility measurements were taken, the animals were challenged with 10 mg/kg of amphetamine and scored for motility. On day 7, there was no significant difference between the baseline levels of activity of any of the groups or their responses to amphetamine. On day 9, the antisense PNA-treated rats showed a statistically significant increase in their resting motility (P < 0.01). When these rats were challenged with amphetamine, motility of the saline-, scrambled PNA-, and mismatch PNA-treated animals showed increases of 31-, 36-, and 20-fold, respectively, while the antisense PNA-treated animals showed increases of only 3.4-fold (P < 0.01). ELISA results revealed a 32% decrease in striatal DAT in antisense PNA-treated rats compared with the saline, scrambled PNA, and mismatch PNA controls (P < 0.001). These results extend our previous findings that brain proteins can be knocked down in a specific manner by antisense molecules administered extracranially. Additionally, these results suggest some novel approaches for the treatment of diseases dependent upon the function of the dopamine transporter.

Quantitative measurement of porphobilinogen in urine by stable-isotope dilution liquid chromatography-tandem mass spectrometry.

BACKGROUND: Measurement of porphobilinogen (PBG) is useful in the diagnosis of the acute neurologic porphyrias. Currently used colorimetric assays lack analytical and clinical sensitivity and specificity. METHODS: We developed a liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the measurement of PBG in 1 mL of urine, using 5-(aminoethyl)-4-(carboxymethyl) 1H-2,4-[(13)C]pyrolle-3-propanoic acid ([2,4-(13)C]PBG; 2.75 microg) as internal standard. After solid-phase extraction, LC-MS/MS analysis was performed in the selected-reaction monitoring (SRM) mode. PBG and [2,4-(13)C]PBG were monitored through their own precursor and product ion settings (m/z 227 to 210 and m/z 229 to 212, respectively). The retention time of PBG and [2,4-(13)C]PBG was 1.0 min in a 2.3-min analysis. RESULTS: Daily calibrations (n = 6) between 0.1 and 2.0 mg/L were linear and reproducible. Inter- and intraassay CVs were 3.2-3.5% and 2.6-3.1%, respectively, at mean concentrations of 0.24, 1.18, and 2.15 mg/L. The regression equation for the comparison between an anion-exchange column method (y) and the LC-MS/MS method (x) was: y = 0.84x + 0.74 (S(y/x) = 5.8 mg/24 h; r = 0.85; n = 100). In 47 volunteers, PBG excretion was 0.02-0.42 mg/24 h, lower than reported reference intervals (up to 2.0 mg/24 h) based on colorimetric methods. In 85 samples with PBG < or =0.5 by LC-MS/MS, 8 (9.4%) had values > or =2.0 mg/24 h by the anion-exchange method (mean +/- SD, 4.3 +/- 1.8 mg/24 h). In 11 patients with confirmed diagnoses of acute porphyria and increased PBG by LC-MS/MS, 2 had values within the reported reference intervals by a quantitative anion-exchange method. CONCLUSIONS: The quantitative LC-MS/MS method for PBG measurement exhibits greater analytical specificity and improved clinical sensitivity and specificity than currently available methods.

A novel neurotensin analog blocks cocaine- and D-amphetamine-induced hyperactivity.

Neurotensin is a tridecapeptide that exhibits selective anatomic and neurochemical interactions with dopaminergic systems. Since dopaminergic neurotransmission underlies many of the behavioral properties of psychostimulants, and since neurotensin has been implicated in modulating dopaminergic neurotransmitter systems, we tested the effect of our novel neurotensin analog, NT69L (N-methyl-Arg(8),L-Lys(9),L-neo-Trp(11),tert-Leu(12)]neurotensin-(8-13)), on hyperactivity caused by cocaine and D-amphetamine. Previously, we showed that NT69L reduces body temperature, blocks apomorphine-induced climbing, and haloperidol-induced catalepsy. In this study, NT69L blocked the hyperactivity induced by both cocaine and D-amphetamine administered at three different doses each, when this peptide was injected intraperitoneally. These results provide further evidence for the involvement of the neurotensin system in some of the behavioral properties of psychostimulants and suggest that NT69L may find clinical application in patients who abuse this class of compounds.

Intraperitoneal injection of antisense peptide nucleic acids targeted to the mu receptor decreases response to morphine and receptor protein levels in rat brain.

To determine the effectiveness of peptide nucleic acids (PNAs) in vivo, we designed and synthesized PNAs antisense to the mu receptor, the molecular target of morphine for inducing antinociception. Responsiveness of rats to morphine and the levels of mu receptor expression after treatment was measured. We delivered intraperitoneal injections of antisense PNAs targeted to the mu receptor (AS-MOR), mismatch PNAs (AS-MOR MM), antisense PNAs targeted to the neurotensin receptor subtype 1 (AS-NTR1), or saline and then challenged the rats with 5 mg/kg morphine (intraperitonally) or neurotensin directly into the periaqueductal gray region of the brain. To avoid tolerance, separate groups of animals were tested at 24, 48, and 72 h post-PNA treatment. Only animals treated with the AS-MOR showed a reduction in their antinociceptive response to morphine. The lack of effect of morphine on the AS-MOR rats was profound at 24 and 48 h, but animals tested at 72 h were similar to control groups. At 24 h the AS-MOR rats had a significant 55% decrease in the levels of mu receptor in their periaqueductal gray region, while AS-MOR MM rats showed no significant change. Lastly, the AS-MOR rats continued to show a normal antinociceptive response to neurotensin. This study, therefore, provides additional support for the use of PNAs to target proteins within brain by systemically administered PNAs.

Cell-free assays for gamma-secretase activity.

The amyloid b-protein (Ab) deposited in Alzheimer's disease (AD) is a normally secreted proteolytic product of the amyloid b-protein precursor (APP). Generation of Ab from the APP requires two sequential proteolytic events: an initial b-secretase cleavage at the amino terminus of the Ab sequence followed by g-secretase cleavage at the carboxyl terminus of Ab. We describe the development of a robust in vitro assay for g-secretase cleavage by showing de novo Ab production in vitro and establish that this assay monitors authentic gamma-secretase activity by documenting the production of a cognate g-CTF, confirming the size of the Ab produced by mass spectrometry, and inhibiting cleavage in this system with multiple inhibitors that alter g-secretase activity in living cells. Using this assay, we demonstrate that the g-secretase activity 1) is tightly associated with the membrane, 2) can be solubilized, 3) has a pH optimum of 6.8 but is active from pH 6.0 to pH >8.4, and 4) ascertain that activities of the g-40 and g-42 are indeed pharmacologically distinct. These studies should facilitate the purification of the protease or proteases that are responsible for this unusual activity, which is a major therapeutic target for the treatment of AD.

Presenilin 1 regulates pharmacologically distinct gamma -secretase activities. Implications for the role of presenilin in gamma -secretase cleavage.

Presenilins (PSs) are polytopic membrane proteins that have been implicated as potential therapeutic targets in Alzheimer's disease because of their role in regulating the gamma-secretase cleavage that generates the amyloid beta protein (Abeta). It is not clear how PSs regulate gamma-secretase cleavage, but there is evidence that PSs could be either essential cofactors in the gamma-secretase cleavage, gamma-secretase themselves, or regulators of intracellular trafficking that indirectly influence gamma-secretase cleavage. Using presenilin 1 (PS1) mutants that inhibit Abeta production in conjunction with transmembrane domain mutants of the amyloid protein precursor that are cleaved by pharmacologically distinct gamma-secretases, we show that PS1 regulates multiple pharmacologically distinct gamma-secretase activities as well as inducible alpha-secretase activity. It is likely that PS1 acts indirectly to regulate these activities (as in a trafficking or chaperone role), because these data indicate that for PS1 to be gamma-secretase it must either have multiple active sites or exist in a variety of catalytically active forms that are altered to an equivalent extent by the mutations we have studied.

A novel neurotensin peptide analog given extracranially decreases food intake and weight in rodents.

Neurotensin decreases food intake in the rat when injected into the cerebral ventricles. We tested the effect of a novel neurotensin analog (NT69L), injected intra-peritoneally (i.p.), on weight gain and food intake in rats. Sprague-Dawley rats (270 g) were injected i. p. with either saline or NT69L at 0.001 or 0.010 mg/kg. In further experiments, larger rats at a more steady state on the growth curve (400 g) were injected with either saline or 0.010 or 1 mg/kg NT69L. Food intake, water consumption and body weight were recorded daily. Weight gain was significantly reduced in the smaller rats injected with 0.001 or 0.010 mg/kg, showing only a 8.5 and 9.0% increase in original weight, respectively, as compared to a 29% increase for the controls. The larger rats injected with 1 mg/kg, had a significant reduction in body weight with a 3.0% decrease in original body weight as compared to a 2.4% increase for the controls. Food intake was significantly reduced suggesting that the weight loss observed after injection of NT69L was attributable in part to a reduction in food intake. The genetically obese Zucker rats injected with NT69L (1 mg/kg) had a significant reduction in weight gain and food intake. NT69L significantly increased blood glucose and corticosterone levels and decreased TSH and T4 in Sprague-Dawley and Zucker rats, an effect that was only transitory. NT69L also caused a decrease in norepinephrine in both the hypothalamus and nucleus accumbens, and an increase in dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and serotonin. In this study, NT69L exhibited a consistent and dramatic effect on body weight and food intake in Sprague-Dawley and obese Zucker rats, and enabled us to study the role that NT plays in weight control and the functional interactions of NT with brain amines, and metabolic and endocrinological parameters.

Effects of a novel neurotensin peptide analog given extracranially on CNS behaviors mediated by apomorphine and haloperidol.

Neurotensin (NT) is a neuropeptide neurotransmitter in the central nervous system. It has been implicated in the therapeutic and in the adverse effects of neuroleptics. Activity of NT in brain can only be shown by direct injection of the peptide into that organ. However, we have developed a novel analog of NT(8-13), NT69L, which is active upon intraperitoneal (i.p.) injection. Like atypical neuroleptics, NT69L blocked the climbing behavior in rats, but not the licking and sniffing behaviors of a high dose (600 microgram/kg) of the non-selective dopamine agonist apomorphine. Its blockade of climbing was very potent with an ED(50) (effective dose at 50% of maximum) of 16 microgram/kg. Both apomorphine and NT69L caused a long-lasting hypothermia, which was greater with the peptide but not synergistic in combination with apomorphine. The ED(50) of NT69L for hypothermia was 390 microgram/kg. NT69L (up to 5 mg/kg i.p.) did not produce catalepsy. However, when given before haloperidol, NT69L, but not clozapine, completely prevented catalepsy. When given after haloperidol, NT69L, but not clozapine, reversed haloperidol's cataleptic effects with an ED(50) of 260 microg/kg. There was no significant difference between the ED(50)s for hypothermia and anticataleptic effects of NT69L. However, the ED(50) for blocking the effects of apomorphine was significantly lower than the other two. These data suggest that NT69L may have neuroleptic properties in humans and may be useful in the treatment of extrapyramidal side effects caused by typical neuroleptics such as haloperidol.

An empirical model of gamma-secretase activity.

gamma-Secretase catalyzes the cleavage at the carboxyl terminus of A beta to release it from the APP. While gamma-secretase is a major therapeutic drug target for the treatment of Alzheimer's disease (AD), it appears to be an unusual proteolytic activity, and, to date, no protease responsible for this activity has been identified. Based on studies of APP transmembrane domain (TMD) mutants, it is apparent that there are multiple pharmacologically distinct gamma-secretase activities that are spatially restricted and that presenilins (PS) regulate cleavage by gamma-secretases in a protease independent fashion. Based on these studies, we propose a multiprotease model for gamma-secretase activity and predict that the gamma-secretases are likely to be closely related proteases.

The intraflavin hydrogen bond in human electron transfer flavoprotein modulates redox potentials and may participate in electron transfer.

Electron-transfer flavoprotein (ETF) serves as an intermediate electron carrier between primary flavoprotein dehydrogenases and terminal respiratory chains in mitochondria and prokaryotic cells. The three-dimensional structures of human and Paracoccus denitrificans ETFs determined by X-ray crystallography indicate that the 4'-hydroxyl of the ribityl side chain of FAD is hydrogen bonded to N(1) of the flavin ring. We have substituted 4'-deoxy-FAD for the native FAD and investigated the analog-containing ETF to determine the role of this rare intra-cofactor hydrogen bond. The binding constants for 4'-deoxy-FAD and FAD with the apoprotein are very similar, and the energy of binding differs by only 2 kJ/mol. The overall two-electron oxidation-reduction potential of 4'-deoxy-FAD in solution is identical to that of FAD. However, the potential of the oxidized/semiquinone couple of the ETF containing 4'-deoxy-FAD is 0.116 V less than the oxidized/semiquinone couple of the native protein. These data suggest that the 4'-hydoxyl-N(1) hydrogen bond stabilizes the anionic semiquinone in which negative charge is delocalized over the N(1)-C(2)O region. Transfer of the second electron to 4'-deoxy-FAD reconstituted ETF is extremely slow, and it was very difficult to achieve complete reduction of the flavin semiquinone to the hydroquinone. The turnover of medium chain acyl-CoA dehydrogenase with native ETF and ETF containing the 4'-deoxy analogue was essentially identical when the reduced ETF was recycled by reduction of 2,6-dichlorophenolindophenol. However, the steady-state turnover of the dehydrogenase with 4'-deoxy-FAD was only 23% of the turnover with native ETF when ETF semiquinone formation was assayed directly under anaerobic conditions. This is consistent with the decreased potential of the oxidized semiquinone couple of the analog-containing ETF. ETF containing 4'-deoxy-FAD neither donates to nor accepts electrons from electron-transfer flavoprotein ubiquinone oxidoreductase (ETF-QO) at significant rates (

In vitro binding and CNS effects of novel neurotensin agonists that cross the blood-brain barrier.

Animal studies with neurotensin (NT) directly injected into brain suggest that it has pharmacological properties similar to those of antipsychotic drugs. Here, we present radioligand binding data for some novel hexapeptide analogs of NT(8-13) at the molecularly cloned rat and human neurotensin receptors (NTR-1), along with behavioral and physiological effects of several of these peptides after intraperitoneal (i.p.) administration in rats. One unique analog, NT66L, which had high affinity (0.85 nM) for the molecularly cloned rat neurotensin receptor (NTR-1), caused a drop in body temperature and antinociception at doses as low as 0.1 mg/kg after i.p. injection. At 30 min post-injection, the ED50 for NT66L-induced hypothermia (rectal temperature) and antinociception (hot plate test) was 0.5 and 0.07 mg/kg, respectively. At a dose of 1 mg/kg i.p., NT66L caused 100% of the maximum possible effect for antinociception for up to 2 h after administration. At this dose body temperature lowering was greater than -2.5 degrees C from 20 to 120 min after i.p. administration. These results in animals suggest that NT66L has agonist properties at NTR-1 in vivo after extracranial administration and provide support for its further study in behavioral tests predictive of neuroleptic activity.

Peptide nucleic acids targeted to the neurotensin receptor and administered i.p. cross the blood-brain barrier and specifically reduce gene expression.

Intraperitoneal injection of an unmodified antisense peptide nucleic acid (PNA) complementary to mRNA of the rat neurotensin (NT) receptor (NTR1) was demonstrated by a gel shift assay to be present in brain, thus indicating that the PNA had in fact crossed the blood-brain barrier. An i.p. injection of this antisense PNA specifically inhibited the hypothermic and antinociceptive activities of NT microinjected into brain. These results were associated with a reduction in binding sites for NT both in brain and the small intestine. Additionally, the sense-NTR1 PNA, targeted to DNA, microinjected directly into the brain specifically reduced mRNA levels by 50% and caused a loss of response to NT. To demonstrate the specificity of changes in behavioral, binding, and mRNA studies, animals treated with NTR1 PNA were tested for behavioral responses to morphine and their mu receptor levels were determined. Both were found to be unaffected in these NTR1 PNA-treated animals. The effects of both the antisense and sense PNAs were completely reversible. This work provides evidence that any antisense strategy targeted to brain proteins can work through i. p. delivery by crossing the normal blood-brain barrier. Equally important was that an antigene strategy, the sense PNA, was shown in vivo to be a potentially effective therapeutic treatment.

Systemic and intestinal pharmacokinetics of methotrexate in patients with inflammatory bowel disease.

BACKGROUND: The pharmacokinetics of low-dose subcutaneous methotrexate have not been determined throughout the standard weekly dosing interval. It is not known whether methotrexate concentrations in the gastrointestinal tract are sufficient for pharmacologic activity in inflammatory bowel disease. METHODS: Ten patients with inflammatory bowel disease participated in the study. After the patients started taking 15 or 25 mg subcutaneous methotrexate once a week, erythrocyte methotrexate concentration was measured every 2 weeks. The absorption, rectal distribution, metabolism, and elimination of methotrexate were measured. The effect of methotrexate on proliferation of an intestinal epithelial cell line was determined. RESULTS: After weekly subcutaneous administration of methotrexate was begun, trough erythrocyte concentration rose to reach a plateau after 6 to 8 weeks, ranging from 150 to 300 nmol/L. More than 90% of subcutaneously administered methotrexate was rapidly excreted in the urine. The methotrexate plasma time course after subcutaneous administration fit a 2-compartment first-order model with biphasic elimination and trough concentration of about 1 nmol/L. Trough and peak methotrexate concentrations (mean value +/- SD) were 64 +/- 33 and 206 +/- 64 fmol/mg in the rectal mucosa and 4 +/- 3 and 51 +/- 26 nmol/L in the rectal lumen. These methotrexate concentrations were in the range found to be pharmacologically active against Caco-2 cell growth, that is, a 50% inhibitory concentration from 10 to 46 nmol/L. CONCLUSION: Subcutaneous methotrexate was well absorbed and distributed to the site of the lesions in patients with inflammatory bowel disease. Methotrexate was concentrated intracellularly in blood and in the rectum. The methotrexate concentration in the rectal mucosa remained within a pharmacologically active range throughout the dosing interval. The findings represent a pharmacologic explanation for the sustained efficacy of weekly methotrexate therapy.

Inhibition of recombinant human mitochondrial aldehyde dehydrogenase by two intermediate metabolites of disulfiram.

Disulfiram is used in aversion therapy for alcoholism. S-Methyl-N,N-diethylthiocarbamate (MeDTC) sulfoxide, a potent inhibitor of the target enzyme mitochondrial aldehyde dehydrogenase (ALDH2), is thought to be the principal active metabolite of disulfiram in vivo. We examined the effects on recombinant human ALDH2 of two intermediate metabolites of disulfiram, S-methyl-N,N-diethyldithiocarbamate (MeDDC) sulfoxide and MeDDC sulfine. MeDDC sulfoxide was a potent inhibitor of ALDH2 with an IC50 of 2.2 +/- 0.5 microM (mean +/- SD, N = 4) after preincubation with enzyme for 30 min. MeDDC sulfine was a relatively weak inhibitor of ALDH2 under the same conditions with an IC50 value of 62 +/- 14 microM. The inhibition of ALDH2 by both compounds was irreversible and did not require the cofactor NAD. The latter finding demonstrates that inactivation of ALDH2 is independent of the dehydrogenase activity of the enzyme. GSH blocked almost completely the inhibition by 20 microM of MeDDC sulfoxide and greatly diminished the inhibition by 200 microM of MeDDC sulfine. Inactivation by MeDDC sulfoxide was time dependent. MeDTC sulfoxide was a more potent inhibitor of recombinant human ALDH2 (IC50 = 1.4 +/- 0.3 microM after preincubation for 15 min) than either of the intermediate metabolites, and its inhibition was unaffected by GSH. Our results suggest that these newer intermediate metabolites of disulfiram, especially the more potent MeDTC sulfoxide, have the potential to inhibit the target enzyme ALDH2 in patients receiving disulfiram. However, until the significance of the interactions of the inhibitors with GSH is more fully understood, the contribution of MeDDC sulfine and MeDDC sulfoxide to the pharmacological effects of disulfiram in vivo is uncertain.

Defining the minimal structural requirements for partial agonism at the type I myo-inositol 1,4,5-trisphosphate receptor.

The novel synthetic analogues D-3-fluoro-myo-inositol 1,5-bisphosphate-4-phosphorothioate, [3F-Ins(1,5)P2-4PS], D-3-fluoro-myo-inositol 1,4-bisphosphate-5-phosphorothioate [3F-Ins(1,4)P2-5PS], and D-3-fluoro-myo-inositol 1-phosphate-4,5-bisphosphorothioate [3F-Ins(1)P-(4,5)PS2] were utilised to define the structure-activity relationships which could produce partial agonism at the Ca2+ mobilising myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor. Based on prior structure-activity data we hypothesised that the minimal structural requirements for lns(1,4,5)P3 receptor partial agonism, were phosphorothioate substitution of the crucial vicinal 4,5-bisphosphate pair accompanied by another structural perturbation, such fluorination of 3-position of the myo-inositol ring. All the analogues fully displaced [3H]Ins(1,4,5)P3 from a single Ins(1,4,5)P3 binding site in pig cerebellar membranes [3F-Ins(1,5)P2-4PS (1C50 = 26 nM), 3F-Ins(1,4)P2-5PS (IC50 = 80 nM) and 3F-Ins(1)P-(4,5)PS2 (IC50 = 109 nM) cf. Ins(1,4,5)P3 (IC50 = 11 nM)]. In contrast, 3F-Ins(1,5)P2-4PS (IC50 = 424 nM) and 3F-Ins(1,4)P2-5PS (IC50 = 3579 nM) were weak full agonists at the Ca2+ mobilising Ins(1,4,5)P3 receptor of permeabilised SH-SY5Y neuroblastoma cells, being respectively 4- and 36-fold less potent than Ins(1,4,5)P3 (EC50 = 99 nM). While 3F-Ins(1)P-(4,5)PS2 (EC50 = 11345 nM) was a partial agonist releasing only 64.3 +/- 1.9% of the Ins(1,4,5)P3-sensitive intracellular Ca2+ pools. 3F-Ins(1)P-(4,5)PS2 was unique among the Ins(1,4,5)P3 receptor partial agonists so far identified in having a relatively high affinity for the Ins(1,4,5)P3 binding site, accompanied by a significant loss of intrinsic activity for Ca2+ mobilisation. This improved affinity was probably due to the retention of the 1-position phosphate, which enhances interaction with the Ins-(1,4,5)P3 receptor. 3F-Ins(1)P-(4,5)PS2 may be an important lead compound for the development of efficient Ins(1,4,5)P3 receptor antagonists.

Cloning of a cDNA for short/branched chain acyl-Coenzyme A dehydrogenase from rat and characterization of its tissue expression and substrate specificity.

The acyl-CoA dehydrogenases are a family of related enzymes which catalyze the alpha,beta-dehydrogenation of acyl-CoA esters, transferring electrons to electron-transferring flavoprotein. A cDNA for human short/branched chain acyl-CoA dehydrogenase has recently been cloned, and it has been suggested that this enzyme represents the human homolog for the previously reported 2-methyl branched chain acyl-CoA dehydrogenase purified from rat liver. We now report the cloning and expression of rat short/branched chain acyl-CoA dehydrogenase and characterization of its substrate specificity. The rat enzyme is more active toward longer carbon side chains than its human counterpart, while the human enzyme can utilize substrates with longer primary carbon chains. In addition, short/branched chain acyl-CoA dehydrogenase can utilize valproyl-CoA as a substrate. Northern blotting of mRNA shows ubiquitous tissue expression of both the rat and human enzyme. Further study of these enzymes will be helpful in understanding structure/function relationships in this gene family.

S-methyl-N,N-diethylthiocarbamate sulfoxide and S-methyl-N,N-diethylthiocarbamate sulfone, two candidates for the active metabolite of disulfiram.

The mechanism of action of disulfiram involves inhibition of hepatic aldehyde dehydrogenase (ALDH). Although disulfiram inhibits ALDH in vitro, it is believed that the drug is too short-lived in vivo to inhibit the enzyme directly. The ultimate inhibitor is thought to be a metabolite of disulfiram. In this study, we examined the effects of S-methyl-N,N-diethylthiocarbamate (MeDTC) sulfoxide and S-methyl-N,N-diethylthiocarbamate sulfone (confirmed and proposed metabolites of disulfiram, respectively) on rat liver mitochondrial low K(m) ALDH. MeDTC sulfoxide and MeDTC sulfone, in 10-min incubations with detergent-solubilized mitochondria, inhibited ALDH activity with an IC50 (mean +/- SD) of 0.93 +/- 0.04 and 0.53 +/- 0.11 microM, respectively, compared with 7.4 +/- 1.0 microM for the parent drug disulfiram. Inhibition by MeDTC sulfone and MeDTC sulfoxide, both at 0.6 microM, was time-dependent, following apparent pseudo-first-order kinetics with a t1/2 of inactivation of 3.5 and 8.8 min, respectively. Dilution of ALDH inhibited by either sulfoxide or sulfone did not restore activity, an indication of irreversible inhibition. Addition of glutathione (50 to 1000 microM) to ALDH before the inhibitors did not alter the inhibition by MeDTC sulfoxide. In contrast, the inhibition by MeDTC sulfone was decreased > 10-fold (IC50 = 6.3 microM) by 50 microM of glutathione and almost completely abolished by 500 microM of glutathione. The cofactor NAD, in a concentration-dependent manner, protected ALDH from inhibition by MeDTC sulfoxide and MeDTC sulfone. In incubations with intact mitochondria, the potency of the two compounds was reversed (IC50 of 9.2 +/- 3.6 and 0.95 +/- 0.30 microM for the MeDTC sulfone and sulfoxide, respectively). Our results suggest that MeDTC sulfone is highly reactive with normal cellular constituents (e.g., glutathione), which may protect ALDH from inhibition, unless this inhibitor is formed very near the target enzyme. In contrast, MeDTC sulfoxide is a better candidate for the ultimate active metabolite of disulfiram, because it is more likely to be sufficiently stable to diffuse from a distant site of formation, such as the endoplasmic reticulum, penetrate the mitochondria, and react with ALDH located in the mitochondrial matrix.