Modulation of cellular apoptosis with apoptotic protease-activating factor 1 (Apaf-1) inhibitors.

The programmed cell death or apoptosis plays both physiological and pathological roles in biology. Anomalous activation of apoptosis has been associated with malignancies. The intrinsic mitochondrial pathway of apoptosis activation occurs through a multiprotein complex named the apoptosome. We have discovered molecules that bind to a central protein component of the apoptosome, Apaf-1, and inhibits its activity. These new first-in-class apoptosome inhibitors have been further improved by modifications directed to enhance their cellular penetration to yield compounds that decrease cell death, both in cellular models of apoptosis and in neonatal rat cardiomyocytes under hypoxic conditions.

Combinatorial chemistry in cancer research.

Among the different strategies to treat cancer, chemotherapy approaches are the subject of intense research efforts. There is still a high demand for new anticancer drugs exhibiting improved efficiency and selectivity for their use in combined therapy strategies. The high development of molecular and cellular biology tools has made possible the set up of simple in vitro assays, susceptible to automation, thus bringing about the possibility of rapid screening of hundreds of compounds. Chemistry has reacted to this challenge by developing a new technology: combinatorial chemistry. By this procedure large collections of compounds, known as chemical libraries, can be prepared in a rapid and efficient manner. In recent years, combinatorial chemistry has had a great impact on drug discovery programmes addressed to tackling cancer pharmaceutical targets. In this review, the contribution of this technology to the discovery of anticancer drugs that are currently in clinical trials or already in the market is discussed.

Combinatorial chemistry in cancer research

Among the different strategies to treat cancer, chemotherapy approaches are the subject of intense research efforts. There is still a high demand for new anticancer drugs exhibiting improved efficiency and selectivity for their use in combined therapy strategies. The high development of molecular and cellular biology tools has made possible the set up of simple in vitro assays, susceptible to automation, thus bringing about the possibility of rapid screening of hundreds of compounds. Chemistry has reacted to this challenge by developing a new technology: combinatorial chemistry. By this procedure large collections of compounds, known as chemical libraries, can be prepared in a rapid and efficient manner. In recent years, combinatorial chemistry has had a great impact on drug discovery programmes addressed to tackling cancer pharmaceutical targets. In this review, the contribution of this technology to the discovery of anticancer drugs that are currently in clinical trials or already in the market is discussed (AU)

Small molecule inhibitors of Apaf-1-related caspase- 3/-9 activation that control mitochondrial-dependent apoptosis.

Apoptosis is a biological process relevant to human disease states that is strongly regulated through protein-protein complex formation. These complexes represent interesting points of chemical intervention for the development of molecules that could modulate cellular apoptosis. The apoptosome is a holoenzyme multiprotein complex formed by cytochrome c-activated Apaf-1 (apoptotic protease-activating factor), dATP and procaspase-9 that link mitochondria disfunction with activation of the effector caspases and in turn is of interest for the development of apoptotic modulators. In the present study we describe the identification of compounds that inhibit the apoptosome-mediated activation of procaspase-9 from the screening of a diversity-oriented chemical library. The active compounds rescued from the library were chemically optimised to obtain molecules that bind to both recombinant and human endogenous Apaf-1 in a cytochrome c-noncompetitive mechanism that inhibits the recruitment of procaspase-9 by the apoptosome. These newly identified Apaf-1 ligands decrease the apoptotic phenotype in mitochondrial-mediated models of cellular apoptosis.

New anti-RNS and -RCS products for cosmetic treatment.

Oxidants and free radicals are known to be a very important factor in skin aging, taking an active part in lipidic peroxidation, breakage of proteins and DNA, etc. The most well-known are reactive oxygen species (ROS), for example, superoxide radical anion, or more commonly called, superoxide (O), hydroxyl radical (OH(*)) or hydrogen peroxide (H(2)O(2)). Both free radicals and other oxidants can be generated by metabolic activity within the cell and by other environmental challenges,. In addition, other dangerous species are known such as reactive nitrogen species (RNS) and reactive carbonyl species (RCS). Some of the most important RNS are peroxynitrite (ONOO(-)), nitrogen dioxide radical ((*)NO(2)) and the nitronium ion (NO). For RCS, some of the most important are 4-hydroxynonenal (HNE), acrolein (ACR), malondialdehyde (MDA) or glyoxal (GXL). Both compounds (RNS and RCS) are thought to play an important role in many diseases and in skin aging, for example, collagen cross-linking, DNA damage, protein tyrosine nitration, etc. This work investigates two new specific chemicals: Lipochroman-6((R))- an anti-RNS which shows good results in inhibiting the nitration of tyrosine by peroxynitrite, and Aldenine((R))- a tripeptide anti-RCS which protects cells from reactive carbonyl compounds such as HNE or ACR; it also shows the ability to prevent glycation of proteins, specifically by superoxide dismutase (SOD).

Monoclonal antibody purification by affinity chromatography with ligands derived from the screening of peptide combinatory libraries.

The peptide, Ala-Pro-Ala-Arg (APAR), was selected from the screening of a tetrapeptide combinatorial synthetic library as the ligand for affinity purification of an anti-Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) monoclonal antibody (Mab) developed in mouse ascitis. The affinity chromatographic matrix obtained by attachment of APAR to agarose, having a peptide density of 0.5 micromol ml(-1), showed a maximum capacity of 9.1 mg Mab ml(-1) and a dynamic capacity of 3.9 mg Mab ml(-1). A 95% yield of electrophoretically pure anti-GM-CSF was obtained in a single step.

The organochlorine pesticides gamma-hexachlorocyclohexane (lindane), alpha-endosulfan and dieldrin differentially interact with GABA(A) and glycine-gated chloride channels in primary cultures of cerebellar granule cells.

The neurotoxic organochlorine pesticides gamma-hexachlorocyclohexane, alpha-endosulfan and dieldrin induce in mammals a hyperexcitability syndrome accompanied by convulsions. They reduce the GABA-induced Cl(-) flux. The strychnine-sensitive glycine receptor also regulates Cl(-)-flux inhibitory responses. We studied the effects of these compounds on Cl(-) channels associated with glycine receptors in cultured cerebellar granule cells in comparison to the GABA(A) receptor. Both GABA (EC(50): 5 microM) and glycine (EC(50): 68 microM) increased (36)Cl(-) influx. This increase was antagonized by bicuculline and strychnine, respectively. Lindane inhibited with similar potency both GABA(A) (IC(50): 6.1 microM) and glycine (5.0 microM) receptors. alpha-Endosulfan and dieldrin inhibited the GABA(A) receptor (IC(50) values: 0.4 microM and 0.2 microM, respectively) more potently than the glycine receptor (IC(50) values: 3.5 microM and 3 microM, respectively). Picrotoxinin also inhibited the glycine receptor, although with low potency (IC(50)>100 microM). A 3D pharmacophore model, consisting of five hydrophobic regions and one hydrogen bond acceptor site in a specific three-dimensional arrangement, was developed for these compounds by computational modelling. We propose that the hydrogen bond acceptor moiety and the hydrophobic region were responsible for the affinity of these compounds at the GABA(A) receptor whereas only the hydrophobic region of the molecules was responsible for their interaction with the glycine receptors. In summary, these compounds could produce neuronal hyperexcitability by blocking glycine receptors besides the GABA(A) receptor. We propose that two zones of the polychlorocycloalkane pesticide molecules (a lipophilic zone and a polar zone) differentially contribute to their binding to GABA(A) and glycine receptors.

Metabolism of (R)- and (S)-3-(phenylamino)propane-1,2-diol in C57BL/6- and A/J-strain mice. Identification of new metabolites with potential toxicological significance to the toxic oil syndrome.

The Toxic Oil Syndrome was a massive food-borne intoxication that occurred in Spain in 1981. Epidemiological studies point to 3-(phenylamino)propane-1,2-diol (PAP) derivatives as the putative toxic agents. We report further identification of metabolites cleared in urine of A/J and C57BL/6 mice in which (R)- and (S)-3-(phenylamino)propane-1,2-diol were administered intraperitoneally. This investigation is an extension of previous studies carried out with the racemic compound [Ladona, M. G., Bujons, J., Messeguer, A., Ampurdanés, C., Morató, A., and Corbella, J. (1999) Chem. Res. Toxicol. 12, 1127-1137]. Both PAP enantiomers were extensively metabolized, and several metabolites were eliminated in urine. The HPLC profiles of the urine samples of both mouse strains treated with each enantiomer were qualitatively similar, but differences were found in a relatively higher proportion of several detected metabolites in mice treated with (R)-PAP compared with those treated with (S)-PAP. The main urine metabolite continues to be 2-hydroxy-3-(phenylamino)propanoic acid (1), which confirms our previous results obtained with rac-PAP. In addition to the detection of other metabolites already reported in our previous paper, interesting evidence is provided on the presence of 4-aminophenol and paracetamol conjugates in the urine samples from both mouse strains. The detection of these metabolites suggests the in vivo formation of quinoneimine PAP derivatives. Indeed, some quinoneimine species (11 and 12), as well as other PAP metabolites (13) that bear modifications in the alkyl chain, have been tentatively identified in mouse urine. These metabolic findings might imply a potential toxicological significance for the Toxic Oil Syndrome.

Metabolism of R,S enantiomers of 3-phenylamino-1,2-propanediol, a compound associated with the toxic oil syndrome, in C57BL/6- and A/J-strain mice.

PAP, a very polar substance, is highly metabolized in mice and excreted principally in urine in the form of the 2-hydroxy-3-phenylaminopropanoic acid of each enantiomer. Thus, the major route of PAP elimination in these strains is alkyl chain oxidation. In particular, S-PAP is eliminated principally in the form of that metabolite, whereas R-PAP enantiomer showed further oxidized species at the aromatic ring and alkyl chain, yielding potential decarboxylated compounds and iminoquinones. All these metabolites may have toxicologic implications. On the other hand, OOPAP intestinal hydrolysis in favour of one PAP enantiomer might be expected since lipases show chiral hydrolysis (unpublished data, manuscript in preparation). In this respect, enantiomeric distribution and metabolic differences should be taken into account in the toxicokinetics of these compounds and their potential association with Toxic Oil Syndrome symptoms.

Identification of selective inhibitors of acetylcholinesterase from a combinatorial library of 2,5-piperazinediones.

The potentiation of central cholinergic activity has been proposed as a therapeutic approach for improving cognitive function in patients with Alzheimer's disease. Increasing the acetylcholine concentration in brain by modulating acetylcholinesterase (AChE) activity is among the most promising strategies. We have used a combinatorial approach to identify different 2,5-piperazinediones (DKP) with AChE inhibitory activity. Our goal was to find inhibitors exhibiting high AChE/BuChE (butyrylcholinesterase) selectivity, in order to reduce the undesirable side effects elicited by most of the inhibitors that have been developed to date. Screening of a DKP library constructed on solid-phase using the multiple parallel synthesis format, resulted in the identification of several compounds with moderate efficacy on AChE. In particular, DKP-80 had an IC50 = 2.2 microM with no significant inhibitory activity on BuChE. Moreover, estimated values of Clog P and log BB for the most active compounds fulfilled the bioavailability requirements for enzyme inhibitors acting on the central nervous system. In order to understand the inhibitory properties of the ligand at the molecular level, molecular dynamics simulations were computed on DKP-80 complexed to AChE, and the most relevant binding interactions of this inhibitor to the active center of the enzyme were characterized. Overall the present results indicate that the DKP-based compounds identified are novel AChE inhibitors which may be considered likely lead compounds for further development of drug candidates against Alzheimer's disease.

Biotransformation and clearance of 3-(phenylamino)propane-1,2-diol, a compound present in samples related to toxic oil syndrome, in C57BL/6 and A/J mice.

In May 1981, a massive food-borne intoxication occurred in Spain. The so-called toxic oil syndrome (TOS) was associated with the consumption of aniline-denatured and refined rapeseed oil that was illegally sold as edible olive oil. Fatty acid anilides and fatty acid derivatives of 3-(phenylamino)propane-1,2-diol were detected in oils and implicated as potential toxic agents and markers of toxic oil batches. Epidemiological evidence points to 3-(phenylamino)propane-1,2-diol derivatives as the putative toxic agents, which were generated during the refining process at the ITH refinery. Here we present the biotransformation and clearance of 3-(phenylamino)propane-1,2-diol (PAP) administered intraperitoneally to A/J and C57BL/6 mice that have been proposed as a murine model for the immunological features of TOS. Mice eliminated 6 microCi of [U-(14)C]PAP during a 24 h period, mostly in urine. Animals exhibited urine elimination rates of 70 and 36% in A/J and C57BL/6 strains, respectively. A/J mice exhibited no increase in the elimination rate when induced with beta-naphthoflavone, whereas C57BL/6 did increase the rate of elimination to 57%. Feces contributed to a lesser extent to the elimination rate (0.6 and 3.3% in A/J and C57BL/6 mice, respectively). Radioactivity remaining in organ tissues was lower than 1% (liver, lung, kidney, spleen, heart, and muscle). Metabolic species in urine were identified by HPLC coupled to UV and radioisotope detectors and further GC/MS analyses. 2-Hydroxy-3-(phenylamino)propanoic acid metabolite was the major chemical species excreted in urine in both strains, in both control and induced animal groups. This compound was the main urinary metabolite of PAP, and unmetabolized PAP excreted in urine constituted less than 1% of the total administered dose. Two additional highly polar metabolites also detected in urine were identified as 3-[(4'-hydroxyphenyl)amino]propane-1,2-diol and 2-hydroxy-3-[(4'-hydroxyphenyl)amino]propanoic acid. These findings are the first reported on PAP metabolism and clearance in mice strains and suggest that PAP can be extensively metabolized in vivo and potential reactive species can be generated.

Prevention of glutamate neurotoxicity in cultured neurons by 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6), a scavenger of nitric oxide.

Glutamate neurotoxicity in cerebellar neurons in culture is mediated by excessive production of nitric oxide (NO). We anticipated that 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6) could act as a scavenger of NO since it contains a position (C-5) highly activated towards nitration reaction. The aim of this work was to assess whether CR-6 acts as an NO scavenger and prevents glutamate neurotoxicity in cultures of cerebellar neurons. It was shown that CR-6 reduced, in a dose-dependent manner, glutamate-induced formation of cGMP (EC50 approximately 15 microM) and prevented glutamate neurotoxicity. The protection was approximately 50% at 3-10 microM and nearly complete at 100 microM. CR-6 did not prevent glutamate-induced activation of NO synthase, but interfered with the glutamate-NO-cGMP pathway at a later step. CR-6 reduced the formation of cGMP induced by S-nitroso-N-acetylpenicillamine (SNAP), an NO-generating agent, indicating that CR-6 acts as a scavenger of NO in cultured neurons. This was further supported by experiments showing that in neurons treated with CR-6 and glutamate, the 5-nitro derivative of CR-6 was formed, as determined by GC-MS analyses. Moreover, in vitro incubation of CR-6 with SNAP also produced the 5-nitroderivative, thus confirming that CR-6 directly reacts with NO. The results reported indicate that CR-6 acts as an NO scavenger in neurons and prevents glutamate neurotoxicity.

Complete elimination of interferences in the organotin determination by oxidation with dimethyldioxirane combined with alumina cleanup.

Most of the analytical procedures used in organotin (OT) speciation from sediment involves the Grignard derivatization reaction followed by a cleanup step and a desulfuration reaction since sulfur and/or sulfur species interfere with OT determination by GC/MS or GC-FPD. However, alkyl sulfides are generated from the coextracted elemental sulfur, and they are not removed by conventional desulfurization procedures. We propose here a method based on the oxidation of all the sulfur species with dimethyldioxirane (DMD) to sulfones or sulfur oxides. While sulfones are easily eliminated by alumina adsorption chromatography because they have higher polarity than OTs, the sulfur oxides are spontaneously evaporated. The DMD chemoselectivity favors the oxidation of sulfur compounds to sulfones in a few minutes, whereas OTs remain unreacted. In addition, the excess DMD is easily removed by evaporation under a nitrogen stream before the Al(2)O(3) cleanup step. The effectiveness of the desulfurization reaction combined with the cleanup step is demonstrated for a variety of sediment samples containing up to 3.1% of elemental sulfur, which is completely removed by adding 0.6 molar equiv of DMD. No statistical differences in the OT distribution pattern throughout the DMD intermediate oxidation steps were observed.

Aflatoxin M1 8,9-epoxide: preparation and mutagenic activity.

Treatment of aflatoxin M1 (AFM1) with dimethyldioxirane in an anhydrous mixture of CH2-Cl2 and acetone afforded the corresponding aflatoxin M1 8,9-epoxide (AFM1-E) in practically quantitative yield. This highly reactive intermediate was identified by 1H NMR and characterized by its neat conversion into the corresponding trans-methoxyhydrin derivative 1. The analysis of the 1H NMR spectrum of the above epoxide revealed that one stereoisomer, which should be that with the exo configuration, was present as major component. The mutagenicities of AFM1-E, the parent mycotoxin (AFM1), aflatoxin B1 (AFB1), and its epoxide (AFB1-E) were assessed by using a sensitive improved Ames test with the Salmonella typhimurium strain TA-100. AFM1 and AFB1 had specific mutagenic activities (SMA) of 13 and 121 revertants/ng, respectively, with S9 metabolic activation. AFM1-E was mutagenic with and without metabolic activation showing SMA of 13 and 12 revertants/ng, respectively. AFB1-E had a SMA of 42 and 29 revertants/ng, with and without S9 metabolic enzymes, respectively. These results suggest that the epoxidation of AFM1 can constitute a major route accounting for the cytotoxic effects elicited by this mycotoxin and that AFM1-E is not as active as AFB1-E in reacting with the constituents of the mutagenicity assay.

Inhibition of rat liver microsomal lipid peroxidation elicited by 2,2-dimethylchromenes and chromans containing fluorinated moieties resistant to cytochrome P-450 metabolism.

2,2-Dimethylchromenes and chromans containing cytochrome P-450 resistant 2,2,2-trifluoroethoxy aryl substituents were synthesized and their activity as lipid peroxidation inhibitors evaluated and compared with that exhibited by the corresponding non-fluorinated derivatives. Lipid peroxidation was stimulated in rat liver microsomes by addition of Fe-ascorbate or NADPH, and determined with the TBARS (thiobarbituric acid reactive substances) test. In assays using Fe-ascorbate stimulation, only those derivatives with a OH group at C6 (i.e. 6 and 12) elicited good inhibitory activities (IC50 = 6.0 and 5.3 microM, respectively). In respect to the NADPH dependent incubations, inhibitory activity of compound 11 (IC50 = 6.0 microM) was the highest found within the 6,7-dialkoxy derivatives tested. Results on metabolism assays with this compound showed the generation of phenol 12 (i.e. the putative active antioxidant species); on the other hand, no metabolite resulting from dealkylation at C7 was detected, thus confirming the resistance conferred by the CF3CH2O group to the cytochrome P-450 promoted cleavage. Finally, in assays where incubations in the presence of NADPH were prolonged up to three hours, inhibitory activity of the non-fluorinated 6,7-dialkoxychroman 8 remained constant, thus suggesting that a continued release of the species responsible for the inhibitory activity was produced. However, inhibition elicited by the fluorinated analog 11 showed a small decrease during the third hour of incubation. This decrease could be attributed to the slight inhibition of the cytochrome P-450 metabolism exerted by substrates bearing the CF3CH2O substituent, which would decelerate the generation of the active phenol species.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro metabolism of juvenile hormone III and juvenile hormone III bisepoxide by Drosophila melanogaster and mammalian cytosolic epoxide hydrolase.

In vitro metabolism of juvenile hormone III (JH III) and juvenile hormone III bisepoxide was investigated using purified mouse liver cytosolic epoxide hydrolase (cEH) and cell fractions from Drosophila melanogaster. JH III was metabolized faster than JH III bisepoxide by epoxide hydrolase activity in D. melanogaster cell fractions and by cEH. After incubation with JH III bisepoxide, all cell fractions and cEH produced epoxy-diol, cis- and trans-tetrahydrofuran-diols, and tetraol as metabolites. An increase in the concentration of cEH resulted in an increase in the proportion of tetraol as a JH III bisepoxide metabolite but this trend was not observed in the D. melanogaster cell fractions. Differences between cell fractions in the metabolism of JH III and JH III bisepoxide suggests the presence of juvenile hormone epoxide hydrolase isozymes.

Application of thermospray/high-performance liquid chromatography/mass spectrometry to the identification of glutathione conjugates derived from bioactive epoxides.

Thermospray high-performance liquid chromatography/mass spectrometry (HPLC/MS) in the positive ion mode has proven to be a useful technique for analysing different synthetic models of potential metabolites of 3,4-epoxyprecocene II (1), the postulated bioactive epoxide responsible for the cytotoxic activity exhibited by precocenes on a variety of insect and mammal tissues. Thus, whereas standards of the glutathione adducts 3a afforded poor responses, the corresponding cysteine (3b) and N-acetylcysteine (3c) derivatives gave defined spectral patterns and good sensitivity levels, particularly when analysed as methyl esters. Likewise, 3,4-dihydrodiols (2) also afforded satisfactory responses. In all cases, the peak at m/z 237, assigned to the stabilized electrophilic oxybenzopyranyl species which results from the loss of the sulphur or oxygen substituent at C(4), was present. Finally, the incubation of racemic epoxide (1) with rat liver cytosolic fraction, followed by enzymatic degradation and further thermospray HPLC/MS analysis of the resulting methylated cysteine derivatives, allowed the identification, for the first time in a biological matrix, of the pairs of cis and trans glutathione conjugates (3a).

Determination by HPLC-RIA of immunoreactive prostaglandin E2 in Blattella germanica and Gryllus bimaculatus.

The application of a combined HPLC-RIA methodology to estimate immunoreactive PGE2 levels in two insect species, namely, males of the cricket Gryllus bimaculatus (Orthoptera, Gryllidae) and males and females of Blattella germanica (Dictyoptera, Blattellidae) is reported. From the results obtained, it can be concluded that, whereas in the crickets the presence of a female does exert a stimulating effect on the PGE2 levels of the male, in cockroaches nonsignificant differences on PGE2 contents were observed among virgin and mated individuals under the conditions assayed, thus suggesting that the biosynthetic pattern of this prostanoid in the cricket studied cannot be extended to the case of the Dictyoptera.