Bax activation by engagement with, then release from, the BH3 binding site of Bcl-xL.

Bcl-2 homologues (such as Bcl-x(L)) promote survival in part through sequestration of "activator" BH3-only proteins (such as Puma), preventing them from directly activating Bax. It is thus assumed that inhibition of interactions between activators and Bcl-x(L) is a prerequisite for small molecules to antagonize Bcl-x(L) and induce cell death. The biological properties, described here of a terphenyl-based alpha-helical peptidomimetic inhibitor of Bcl-x(L) attest that displacement of Bax from Bcl-x(L) is also critical. Terphenyl 14 triggers Bax-dependent but Puma-independent cell death, disrupting Bax/Bcl-x(L) interactions without affecting Puma/Bcl-x(L) interactions. In cell-free assays, binding of inactive Bax to Bcl-x(L), followed by its displacement from Bcl-x(L) by terphenyl 14, produces mitochondrially permeabilizing Bax molecules. Moreover, the peptidomimetic kills yeast cells that express Bax and Bcl-x(L), and it uses Bax-binding Bcl-x(L) to induce mammalian cell death. Likewise, ectopic expression of Bax in yeast and mammalian cells enhances sensitivity to another Bcl-x(L) inhibitor, ABT-737, when Bcl-x(L) is present. Thus, the interaction of Bcl-x(L) with Bax paradoxically primes Bax at the same time it keeps Bax activity in check, and displacement of Bax from Bcl-x(L) triggers an apoptotic signal by itself. This mechanism might contribute to the clinical efficiency of Bcl-x(L) inhibitors.

Anxiolytic properties of the selective, non-peptidergic CRF(1) antagonists, CP154,526 and DMP695: a comparison to other classes of anxiolytic agent.

The selective, non-peptidergic corticotropin-releasing factor (CRF)(1) receptor antagonists, CP154,526 and DMP695, dose-dependently increased punished responses of rats in a Vogel conflict test and enhanced social interaction (SI) of rats in an unfamiliar environment. They were, however, inactive in a plus-maze procedure and failed to reduce ultrasonic vocalizations (USV) associated with an aversive environment. In contrast, the benzodiazepine, chlordiazepoxide, was effective in all these procedures. Further, the serotonin (5-HT)(1A) agonist, flesinoxan, was active in each paradigm (except the plus-maze) while the 5-HT(2C) antagonist, SB242,084, was effective in the SI and Vogel but not the plus-maze and USV procedures. In contrast to chlordiazepoxide, flesinoxan and SB242,084, CP154,526 did not modify dialysate levels of 5-HT, norepinephrine (NE) and dopamine (DA) in the frontal cortex (FCX) of freely moving rats. In conclusion, CP154,526 and DMP695 possess a common and distinctive profile of anxiolytic action expressed in the absence of an intrinsic influence upon monoamine release.

Protective effect of the antioxidant 6-ethoxy-2,2-pentamethylen-1,2-dihydroquinoline (S 33113) in models of cerebral neurodegeneration.

In a previous study Dorey et al. [Bio. Org. Chem. Lett., 10 (2000) 935] a series of novel dihydroquinoline compounds were developed, based on the potent antioxidant 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline (ethoxyquin), and permitted the selection of the analogue 6-ethoxy-2,2-pentamethylen-1,2-dihydroquinoline (S 33113) lacking the hypothermic effects associated with ethoxyquin at equivalent doses. Herein, an extensive investigation of the neuroprotective capacity of S 33113 in different in vitro and in vivo paradigms of oxidative stress-mediated cellular degeneration was undertaken. In vitro S 33113 was a potent inhibitor (IC(50) = 0.29 microM) of Fenton-reaction-induced lipid peroxidation in mouse cortical membranes. Administration of S 33113 either intraperitoneally (< or =150 mg/kg i.p.) or orally (< or =600 mg/kg p.o.) did not significantly modify body temperature in NMRI mice. Furthermore, S 33113 (150 mg/kg i.p. or 600 mg/kg p.o.) markedly reduced the lethality induced by an intracerebroventricular injection of t-butylhydroperoxide in NMRI (naval medical research institute) mice for up to 5 h. Oral administration of S 33113, significantly attenuated alloxan-mediated hyperglycaemia in NMRI mice at 400 and 600 mg/kg (60%; P < 0.001). Administration of S 33113 (150 mg/kg i.p.) 30 min before transient global ischaemia significantly prevented delayed neuronal cell death in the CA1 region of the rat hippocampal formation, 7 days post-ischaemia (33% cell loss vs. 88% in ischaemia controls; P < 0.001). Similarly, a single pre-administration of S 33113 (150 mg/kg i.p.) prevented kainic acid-induced cell death in the CA3 hippocampal region at 7 days post-exposure (17% cell loss vs. 52% in kainate-treated controls; P < 0.01). Furthermore, D-methamphetamine-mediated dopamine depletion in the striatum of C57BL/6 mice (39-46%) was significantly prevented with S 33113 administered at either (2 x 150mg/kg i.p.) (11%; P < 0.01) or (2x150 mg/kg p.o.) (17%; P < 0.001). In conclusion, S 33113 represents a novel dihydroquinoline compound with potential for the treatment of cerebral pathologies implicating chronic neurodegeneration.

General synthesis of alpha-substituted 3-bisaryloxy propionic acid derivatives as specific MMP inhibitors.

Modulations of alpha and aryl substitutions on 3-aryloxy propionic acid hydroxamates led to novel and potent inhibitors of MMP-2,3,9 and 13, and selectivity versus MMP-1.

New quinolinic derivatives as centrally active antioxidants.

A series of new 1,2-dihydro and 1,2,3,4-tetrahydroquinolines, synthesized from the corresponding propargylaniline intermediates, have been developed as antioxidants for the potential treatment of pathologies implicating central oxidative stress.

General synthesis of 3-substituted alkenyl GABA as potential anticonvulsants.

Stereospecific synthesis of cis and trans 3-substituted vinyl-gamma-aminobutyric acid analogs were obtained by either a Claisen rearrangement or a Wittig reaction from common diene precursors.

Antiretroviral activities of acyclic nucleoside phosphonates [9-(2-phosphonylmethoxyethyl)adenine, 9-(2-phosphonylmethoxyethyl)guanine, (R)-9-(2-phosphonylmethoxypropyl)adenine, and MDL 74,968] in cell cultures and murine sarcoma virus-infected newborn NMRI mice.

From a side-by-side comparative study, the acyclic nucleoside phosphonates (R)-9-(2-phosphonylmethoxypropyl)adenine [(R)-PMPA] and 9-(2-methylidene-3-phosphonomethoxypropyl)guanine (MDL 74,968) proved more selective in their inhibitory effect on human immunodeficiency virus types 1 and 2, feline immunodeficiency virus, and Moloney murine sarcoma virus (MSV) in cell cultures than the 9-(2-phosphonylmethoxyethyl) derivatives of adenine (PMEA) and guanine (PMEG). In particular, PMEG proved quite toxic. PMEA, (R)-PMPA, and MDL 74,968 afforded a marked delay in MSV-induced tumor initiation in MSV-infected newborn NMRI mice and substantially delayed associated animal death at doses as low as 4 to 10 mg/kg of body weight. Treatment of the NMRI mice with PMEA, (R)-PMPA, and MDL 74,968 at 25 or 50 mg/kg resulted in a high percentage of long-term survivors.

Early treatment with 9-(2-phosphonylmethoxyethyl)adenine reduces virus burdens for a prolonged period in SIV-infected rhesus macaques.

We evaluated the effects of a reverse transcriptase inhibitor, 9-(2-phosphonylmethoxyethyl)adenine (PMEA), on simian immunodeficiency virus (SIV) infection in rhesus macaques (Macaca mulatta). Four macaques were given PMEA (20 mg/kg) subcutaneously on days 1 and 2 and inoculated with virus on day 2. Drug treatment was continued for 30 consecutive days, after which the virus burdens and course of infection were monitored for a further 6 months. Four control animals that did not receive PMEA all developed high virus burdens and two of the four developed clinical disease. In contrast, virus burdens remained low in three of the four macaques treated with PMEA and all four remained healthy. Our results show that suppression of virus replication early in infection can result in reduced virus burdens for a much longer period.

MDL 74,968, a new acyclonucleotide analog: activity against human immunodeficiency virus in vitro and in the hu-PBL-SCID.beige mouse model of infection.

The novel acyclonucleotide derivative of guanine, 9-[2-methylidene-3-(phosphonomethoxy)propyl] guanine (MDL 74,968), had antiviral activity comparable to those of 9-(2-phosphonomethoxyethyl) adenine (PMEA) and 2',3'-dideoxyinosine against laboratory strains of both human immunodeficiency virus (HIV) types 1 and 2 cultured in MT-4 cells and several clinical HIV isolates cultured in human peripheral blood mononuclear cells (PBMCs). MDL 74,968 was at least fourfold less toxic than PMEA to MT-4 cells or PBMCs, thereby producing a more favorable in vitro selectivity index for the former compound. Studies of acute toxicity in CD-1 mice showed that MDL 74,968 was not toxic at doses of 1,600 mg/kg of body weight via the intraperitoneal route or at doses of 500 mg/kg via the intravenous route. Furthermore, no adverse effects of MDL 74,968 were apparent when mice were treated at doses of 200 mg/kg twice daily for 5 days. Treatment by continuous subcutaneous infusion of MDL 74,968 or PMEA at the daily dose of 20 mg/kg in the hu-PBL-SCID.beige murine model of HIV infection significantly reduced the severity of infection compared with that in placebo-treated controls. Quantitation of virus recovery by endpoint titration of spleen cells in coculture with mitogen-activated PBMCs demonstrated that MDL 74,968 as well as PMEA significantly reduced the amount of virus (P < 0.02). Moreover, by using DNA extracted from spleens, the mean HIV:HLA PCR product ratio, which takes into account individual variation in immune system reconstitution, were 0.50 and 0.40 for MDL 74,968 and PMEA treatments, respectively, whereas animals receiving the placebo control had significantly higher levels of HIV proviral DNA (mean 0.78; P < 0.02). Taken together, these promising findings suggest that an orally bioavailable prodrug of MDL 74,968 should be developed for the treatment of HIV infection.

Uptake of the antitrypanosomal drug 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (MDL 73811) by the purine transport system of Trypanosoma brucei brucei.

An irreversible inhibitor of S-adenosyl-L-methionine decarboxylase (AdoMetDC), 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (MDL 73811), was found to cure Trypanosoma brucei brucei and multidrug-resistant T. b. rhodesiense infections in mice [Bitonti, Byers, Bush, Casara, Bacchi, Clarkson, McCann & Sjoerdsma (1990) Antimicrob. Agents Chemother. 34, 1485-1490]. Doses of this drug which resulted in a rapid clearance of parasites from T. b. brucei-infected rats resulted in plasma levels of 50-60 microM-MDL 73811 and an intratrypanosomal MDL 73811 concentration of 1.9 mM within 10 min of administration [Byers, Bush, McCann & Bitonti (1991) Biochem. J. 274, 527-533[. Based on this finding we speculated that MDL 73811, which is an adenosine analogue, is a substrate for the trypanosome active purine transport system. We now report evidence that supports this hypothesis. MDL 73811 uptake by T. b. brucei in vitro was time- and temperature-dependent and was saturable over a time course in which MDL 73811 metabolism was undetectable, suggesting that MDL 73811 uptake is a transport-mediated phenomenon. Inhibition of MDL 73811 uptake by purine nucleosides is consistent with the drug being a substrate for the trypanosome purine transport system. The accumulation of MDL 73811 by cultured L1210 mouse leukaemia cells was significantly less than by trypanosomes exposed to the same pharmacologically relevant concentrations of MDL 73811. Given that the half-life of MDL 73811 in the plasma of rats and mice is approx. 10 min, it seems likely that the existence of a highly active parasite transport system for MDL 73811 is crucial for the sensitivity of trypanosomes towards MDL 73811 in vivo, and that the absence of active transport of MDL 73811 by the host's cells may play a role in the selectivity of this drug.

(4S)-4-amino-5,6-heptadienoic acid (MDL 72483): a potent anticonvulsant GABA-T inhibitor.

(4S)-4-Amino-5,6-heptadienoic acid [S)-gamma-allenyl-GABA; MDL 72483) is a potent inactivator of brain GABA-T in mice; (ED50 (i.p.) = 60 mg.kg-1; ED50 (oral) = 70 mg.kg-1). Its anticonvulsant effects against 3-mercaptopropionic acid (MPA)-induced seizures in mice is related to the elevation of whole brain GABA concentrations: The mentioned doses of MDL 72483 which cause a decrease of GABA-T activity by 50%, produce within 5 h after dosing an increase of GABA concentration by about 3 mumol.g-1, and protect 50% of the mice against seizures in this model of presynaptic GABA deficit. When given orally MDL 72483 is about five times more potent than vigabatrin [4R/S)-4-amino-5-hexenoic acid) a known antiepileptic GABA-T inhibitor. Complete protection was achieved with a dose of 150 mg.kg-1. Similar to vigabatrin, MDL 72483 does not protect significantly against metrazol-induced convulsions. However, at a dose of 300 mg.kg-1, the time elapsing between metrazol administration and onset of convulsions was prolonged by a factor of 3.4. Oral administration of MDL 72483 for up to 19 days at a daily dose of 91-96 mg.kg-1 did not produce any obvious behavioral changes in mice, nor was the ED50 of the drug in MPA-seizure tests significantly altered by the pretreatment. These observations indicate that MDL 72483 is a promising drug for the treatment of certain epilepsies.

Irreversible inhibition of rat S-adenosylmethionine decarboxylase by 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine.

5'-([(Z)-4-Amino-2-butenyl]methylamino)-5'-deoxyadenosine [Z)-AbeAdo) was tested in vitro and in vivo as a potential inhibitor of S-adenosyl-L-methionine decarboxylase (AdoMetDC), a pyruvoyl-containing enzyme, purified from rat liver. In vitro (Z)-AbeAdo produces a time- and dose-dependent irreversible inhibition of the enzyme. Saturation kinetics are observed when the enzyme is preincubated with (Z)-AbeAdo in the presence of 50 microM putrescine, a known activator of AdoMetDC. Under these conditions kinetic constants were measured (Ki = 0.56 +/- 0.04 microM; tau 1/2 = 0.51 +/- 0.03 min). The inhibition is not relieved by prolonged dialysis of the inactivated enzyme. The turnover number for (Z)-AbeAdo, i.e. the number of inactivator molecules required to inactivate one enzyme molecule, is approximately 1.5. The selectivity of (Z)-AbeAdo was explored: the compound is not a substrate of adenosine deaminase, mitochondrial monoamine oxidase and diamine oxidase, but is slowly oxidized by benzylamine oxidase from rat aorta. The (E)-isomer of AbeAdo, is at least 100-fold less active than (Z)-AbeAdo as a time-dependent inhibitor of rat liver AdoMetDC. In rats, intraperitoneal administration of (Z)-AbeAdo produces a rapid, long-lasting and dose-dependent decrease of AdoMetDC activity in ventral prostate, testis and brain.

Cure of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense infections in mice with an irreversible inhibitor of S-adenosylmethionine decarboxylase.

A structural analog, 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxy adenosine (MDL 73811), of decarboxy S-adenosyl-L-methionine, the product of the reaction catalyzed by S-adenosyl-L-methionine (AdoMet) decarboxylase (DC), was found to inhibit Trypanosoma brucei brucei AdoMet DC. The inhibition was time dependent (tau 50, 0.3 min), exhibited pseudo-first-order kinetics (Ki, 1.5 microM), and was apparently irreversible. The natural substrate of the reaction, AdoMet, protected the enzyme from inactivation, suggesting that MDL 73811 was directed at the enzyme active site and was probably catalytically activated. Administration of MDL 73811 to T. b. brucei-infected rats resulted in rapid inhibition of AdoMet DC activity, a decrease in spermidine, and an increase in putrescine in the trypanosomes isolated from treated rats. Treatment of T. b. brucei-infected mice with MDL 73811 (20 mg/kg of body weight intraperitoneally twice daily for 4 days) resulted in cures of the trypanosome infections. Additionally, drug-resistant T. brucei rhodesiense infections in mice were cured by either a combination of MDL 73811 (50 mg/kg intraperitoneally three times per day for 5 days) and relatively low oral doses of alpha-difluoromethylornithine or MDL 73811 (50 mg/kg per day for 7 days) administered alone in implanted miniosmotic pumps. These data suggest that MDL 73811 and, perhaps, other inhibitors of AdoMet DC have potential for therapeutic use in various forms of African trypanosomiasis.

Catalytic irreversible inhibition of bacterial and plant arginine decarboxylase activities by novel substrate and product analogues.

Arginine decarboxylase (ADC) activity from Escherichia coli and two plant species (oats and barley) was inhibited by five new substrate (arginine) and product (agmatine) analogues. The five compounds, (E)-alpha-monofluoromethyldehydroarginine (delta-MFMA), alpha-monofluoromethylarginine (MFMA), alpha-monofluoromethylagatine (FMA), alpha-ethynylagmatine (EA) and alpha-allenylagmatine (AA), were all more potent inhibitors of ADC activity than was alpha-difluoromethylarginine (DFMA), the only irreversible inhibitor of this enzyme described previously. The inhibition caused by the five compounds was apparently enzyme-activated and irreversible, since the loss of enzyme activity followed pseudo-first-order kinetics, was time-dependent, the natural substrate of ADC (arginine) blocked the effects of the inhibitors, and the inhibition remained after chromatography of inhibited ADC on Sephadex G-25 or on overnight dialysis of the enzyme. DFMA, FMA, delta-MFMA and MFMA were effective at very low concentrations (10 nM-10 microM) at inhibiting ADC activity in growing E. coli. FMA was also shown to deplete putrescine effectively in E. coli, particularly when combined with an inhibitor of ornithine decarboxylase, alpha-monofluoromethyl-putrescine. The potential uses of the compounds for the study of the role of polyamine biosynthesis in bacteria and plants is discussed.

Antitumor properties of (2R,5R)-6-heptyne-2,5-diamine, a new potent enzyme-activated irreversible inhibitor of ornithine decarboxylase, in rodents.

(2R,5R)-6-Heptyne-2,5-diamine hydrochloride (MDL 72175) is a new, potent, and selective inhibitor of mammalian ornithine decarboxylase. MDL 72175 given p.o. in drinking fluid reduced by 80% the growth of EMT6 sarcoma in mice and of HTC hepatoma in rats. It prolonged the survival of mice bearing L1210 or P388 leukemias and inhibited the development of Lewis lung carcinoma in mice at doses 10- to 20-fold lower than those of alpha-difluoromethylornithine, the most widely used irreversible inhibitor of ornithine decarboxylase. MDL 72175 depleted putrescine and spermidine levels in the tumors to the same extent as did alpha-difluoromethylornithine. In the EMT6 sarcoma, MDL 72175 achieved at low doses a greater maximal antitumor effect than did alpha-difluoromethylornithine. In combination therapy, MDL 72175 plus Adriamycin gave at least additive antitumor effects on solid tumors and experimental leukemias in animals. The combination MDL 72175 plus methylglyoxal bis(guanylhydrazone) also gave additive antitumor effects on P388 leukemia, associated with an increased uptake of methylglyoxal bis(guanylhydrazone); in contrast, antagonistic effects were observed with this combination on EMT6 tumors in mice. Since MDL 72175 did not present toxicity at effective antitumor doses, this new ornithine decarboxylase inhibitor can be considered as a promising antitumor drug.

alpha-Allenyl putrescine, an enzyme-activated irreversible inhibitor of bacterial and mammalian ornithine decarboxylases.

alpha-Allenyl putrescine (5,6-heptadiene-1,4-diamine) was designed as a new potential enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC). This compound, and more specifically its (R)-enantiomer, produced time-dependent inhibitions of E. coli and rat liver ODC. The inhibitions exhibit saturation kinetics and were not relieved by prolonged dialysis of the inactivated enzyme. Selective inactivation of the two types of ODC by the (R)-enantiomer is in agreement with the stereochemistry reported for ornithine decarboxylation by the enzyme. Kinetic constants of E. coli ODC inactivation by alpha-(R)-allenyl putrescine compare favorably with other irreversible inhibitors of this enzyme.

Effects of (2R, 5R)-6-heptyne-2,5-diamine, a potent inhibitor of L-ornithine decarboxylase, on rat hepatoma cells cultured in vitro.

DL-alpha-Difluoromethylornithine (F2MeOrn), the most widely-used inhibitor of L-ornithine decarboxylase, has been a useful tool to demonstrate that polyamine biosynthesis is required to maintain maximum rates of cell proliferation. However, in most eukaryotic cell systems, F2MeOrn exerts cytostatic rather than cytotoxic effects. This may be due to the fact that this inhibitor creates only incomplete polyamine deficiency. In particular, F2MeOrn scarcely depletes intracellular spermine levels. We now demonstrate in rat hepatoma tissue culture (HTC) cells that (2R, 5R)-6-heptyne-2,5-diamine, a more potent irreversible inhibitor of L-ornithine decarboxylase than F2MeOrn, decreases the concentrations of all polyamines including spermine. In parallel with the depletion of these amines, there is a progressive decrease in the rate of cell proliferation and in cell viability. Restoration of the intracellular polyamine content, by addition to the medium of polyamines or a high concentration of L-ornithine, the substrate of L-ornithine decarboxylase, further demonstrates that the antiproliferative effects of (2R, 5R)-6-heptyne-2,5-diamine do result from polyamine deficiency. These findings support the concept that polyamines play an essential function in the cell division processes and emphasize the vital function of spermine in mammalian cells.