Some 1,2-diphenylethane derivatives as inhibitors of retinoic acid--metabolising enzymes.

In a search for novel inhibitors of RA-metabolising enzyme inhibitors as potential anti-cancer agents some 1,2-ethandiones, 2-hydroxyethanones and 1-ethylenedioxyethanones based on aryl-substituted 1,2-diphenylethane have been examined. Several of the compounds were weak inhibitors of the non-specific rat liver microsomal P450 enzymes and moderate inhibitors of the RA-induced enzymes in cultured human genital fibroblasts, where the RA-specific enzyme CYP26 is probably expressed. The 2-hydroxyethanone (13) with a 1-(4-dimethylaminophenyl) substituent was overall the most potent compound for rat liver microsomal enzyme (IC50 = 52.1 microM; ketoconazole, 2.8 microM) and the RA-induced enzyme (100 microM, 65.9% inhibition; ketoconazole, 20 microM, 75.0%). Modification of the dimethylamino group in (13) with more hydrophobic dialkylamino functions or separate modification of the 2-(2,4-dichlorophenyl) function did not improve potency.

Estimating pKa values for pentaoxyphosphoranes.

pKa values are estimated independently, by two entirely different methods, for the ionizations of the apical and equatorial OH groups of two representative hydroxyphosphoranes. A bond length-pKa correlation based on crystal structures of cyclohexanol derivatives gives values of 13.5 +/- 1.5 and 8.62 +/- 1.87, respectively, for the apical and equatorial OH groups of tetracyclohexyloxyhydroxyphosphorane, and an ab initio molecular dynamics calculation gives values of 14.2 and 9.8 for the corresponding first ionizations of pentahydroxyphosphorane.

On the magnitude and specificity of medium effects in enzyme-like catalysts for proton transfer.

Medium effects are normally studied by comparing the rates of reactions in different solvents. However, medium effects at the active site of enzymes differ dramatically from bulk solvents, both in their diversity (the presence of more than one type of "solvent") and in their spatial arrangement. We describe medium effects in a simple catalytic system, obtained by systematic alkylation of a polymeric scaffold bearing amine groups to give synzymes that catalyze the Kemp elimination of benzisoxazoles with remarkable efficiency. Our analysis indicates that catalysis by these synzymes is driven primarily by specific, localized enzyme-like medium effects, and these effects seem to differ dramatically from the nonspecific medium effects (i.e., desolvation activation) exhibited by solvents. Ligand-binding studies indicate that the synzyme active sites provide localized microenvironments affording a combination of hydrophobic and apolar regions on one hand and dipolar, protic, and positively charged on the other. Such localized microenvironments are not available in bulk solvents. A Brønsted (leaving group) analysis indicates that, in comparison to solvent catalysis, the efficiency of synzyme catalysis shows little sensitivity to leaving group pK(a). We show that enzyme-like medium effects alone, in the absence of efficient positioning of the catalytic amine base relative to the substrate, can give rise to rate accelerations as high as 10(5), for both activated and nonactivated substrates. Supported by the accidental identification of active sites on the surfaces of noncatalytic proteins and the promiscuous activities found in many enzymes, our findings suggest that the interfaces of protein surfaces and their hydrophobic cores provide a microenvironment that is intrinsically active and may serve as a basis for further evolutionary improvements to give proficient and selective enzymes.

Rational approaches to the design of cationic gemini surfactants for gene delivery.

We report a new class of amphiphilic gemini surfactants as vehicles for gene delivery into cells, and the beginnings of a systematic structure-activity study. Preliminary results suggest that combining gemini surfactants with dioleoylphosphatidylethanolamine (DOPE) should allow the preparation of liposomes of various sizes and lipid compositions. Control of such colloidal changes could be as significant as the changes in the molecular composition of the gemini surfactants in delivering optimum gene expression in animal models.

Novel spermine-based cationic gemini surfactants for gene delivery.

Two types of spermine-based gemini surfactants have been synthesised; structure-activity studies have shown one type to be far superior in gene transection than the other.

Efficient intramolecular general acid catalysis: a preview from a crystal structure.

The crystal structure of acetal acid 5 reveals changes in geometry representing progress along the reaction coordinate for acetal cleavage.

Evaluation of 7-hydroxy-flavones as inhibitors of oestrone and oestradiol biosynthesis.

A series of 4-aryl substituted 7-hydroxy-flavones were prepared using the three-step Baker-Venkataraman synthesis in good overall yields. The flavones were all evaluated in vitro for inhibitory activity against aromatase (P450AROM, CYP19), using human placental microsomes, and for inhibitory activity against 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD-1) using human placental cytosol. The phenyl, 4-fluoro-phenyl and 4-bromo-phenyl derivatives displayed moderate inhibitory activity against P450AROM (IC50 17.2, 13.5 and 10.1 microM, respectively), none of the flavones, including the standard genistein, displayed any inhibitory activity against 17beta-HSD type 1 at 100 microM concentration.

1-[(Benzofuran-2-yl)phenylmethyl]triazoles as steroidogenic inhibitors: synthesis and in vitro inhibition of human placental CYP19 aromatase.

Hormone-dependent breast cancer is stimulated by the female hormones oestrone and oestradiol, therefore compounds which inhibit the specific enzymes involved in the formation of the nitogenic hormones, namely CYP19 aromatase (P450arom) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 1, are targets of therapeutic interest for the treatment of breast cancer. A series of novel 1-[(benzofuran-2-yl)phenylmethyl]1,2,4-triazoles were prepared using a three-step synthesis and evaluated for their inhibitory activity against human placental aromatase in vitro, using [1,2,6,7-3H]androstenedione as the substrate for the aromatase enzyme. Inhibitory activity was dependent on both substituent and position of substitution, with introduction of small electron-withdrawing groups in the phenyl ring showing optimum activity (IC50 ranging from 0.065 to 2.02 microm). Substitution in the benzofuran ring resulted in a loss of activity when substituted at C-5 (IC50 > 20 microm). The compounds were all shown to exhibit weak inhibitory activity against rat testes P450 17 (17,20-lyase), indicating good selectivity towards P450arom.

Vesicles accelerate proton transfer from carbon up to 850-fold.

[reaction: see text] We have analyzed the different catalytic effects of surfactant aggregates upon the rate-determining hydroxide ion induced deprotonation reaction of 1. Vesicles are more effective catalysts than micelles, most likely providing a more apolar microenvironment at the substrate binding sites. We suggest that this leads to a catalytic reaction involving less strongly hydrated hydroxide ions. In the case of DODAB and DODAC vesicles, binding of cholesterol to the bilayer further increases the catalytic efficiency.

Nonspecific catalysis by protein surfaces.

Catalytic antibodies are the best available all-around enzyme mimics. They provide a unique experimental approach and some special insights into general questions about catalysis by enzymes. They offer enantiospecific reactions and levels of substrate binding that compare well with typical enzyme reactions, but not--so far--comparable catalytic efficiency. We and others have used the Kemp elimination as a probe of catalytic efficiency in antibodies. We compare these reactions with nonspecific catalysis by other proteins, and with catalysis by enzymes. Several simple reactions are catalyzed by the serum albumins with Michaelis-Menten kinetics, and can be shown to involve substrate binding and catalysis by local functional groups. Here we report the details of one investigation, which implicate known binding sites on the protein surface, and discuss implications for catalyst design and efficiency.

The antioxidant activity of Chinese herbs for eczema and of placebo herbs--I.

A standardized mixture of Chinese herbs has recently been demonstrated to be an effective treatment for chronic atopic eczema in placebo controlled trials in the UK. Aqueous decoctions of this formulated mixture (PSE 222), the placebo mixture, and their component herbs were examined for antioxidant activity to determine whether antioxidant activity could account for the anti-eczema activity. Two measures of antioxidant activity were employed: the DPPH assay for non-specific hydrogen atom (or electron) donating activity and a superoxide scavenging assay. Antioxidant activity was detected in some components of both the active and placebo mixtures, but the formulated active mixture (PSE 222) was significantly more effective than the formulated placebo mixture. Further studies are needed to elucidate the in vivo significance of this result.

[Preparation and characteristics of antibodies to a sulfur-containing hapten, mimicking the transition state in lactamization reactions]. / Poluchenie i kharakteristika antitel k serosoderzhashchemu gaptenu, imitiruiushchemu perekhodnoe sostoianie v reaktsiiakh laktamizatsii.

A sulfur-containing hapten was proposed to be an analogue of the tetrahedral transition state of lactamization reactions. The dynamics of the immune response to this hapten upon using different immunization schemes was studied. Sixty-one clones of mouse anti-hapten antibodies were obtained. Three clones efficiently bound not only the transition state analogue (the hapten) but also potential substrates of lactamization reactions. Antibody-induced changes in the optical absorption spectra of substrate solutions were studied for a substrate with stabilized active conformation (2-aminomethylbenzoate) and an unmodified substrate (gamma-aminobutyric acid), which demonstrated that the catalytic antibodies obtained significantly accelerated the lactam ring formation.

Virologic and serologic markers of rapid progression to AIDS after HIV-1 seroconversion.

The association between early virologic and immunologic events after human immunodeficiency virus type 1 (HIV-1) infection and progression of HIV-1 infection to acquired immunodeficiency syndrome (AIDS) was studied among 59 homosexual men with documented time of seroconversion. Epidemiologic factors, such as number of lifetime sexual partners, history of sexually transmitted diseases, and other factors, also were studied. All 17 seroconverters in the cohort who developed AIDS within 3 years (rapid progressors = RPs) were compared with 42 men without AIDS for at least 6 years seroconversion (nonrapid progressors = non-RPs). Plasma levels of HIV-1 RNA, p24 antigen, antibodies to HIV-1 structural genes, beta-2 microglobulin, neopterin, and interferon-alpha were measured at four time points: (a) the last seronegative visit, (b) the first seropositive visit, (c) the visit closest to AIDS (or the corresponding visit for the non-RPs) and (d) 6 years after seroconversion (for non-RPs). Up to seroconversion, the RPs had a significantly higher number of lifetime sexual partners than non-RPs (503 versus 171, respectively). At the first seropositive visit, RPs had significantly higher concentrations of plasma HIV-1 RNA (p < 0.01) and prevalence of p24 antigenemia (p < 0.001) and significantly lower levels of antibodies to the HIV-1 gag proteins p17 and p24 (p < 0.01-0.001) compared with non-RPs. These differences increased during follow-up visits. Antibodies to p66 and gp120 were significantly different only at the visit closet to AIDS (p < 0.001), as were beta-2 microglobulin and interferon alpha. These findings suggest that early virologic-immunologic events after HIV-1 infection may determine the rate of progression to AIDS. Anti-gag immune response may prevent rapid progression of HIV-1 disease and should be considered for future vaccine studies.

Off-the-shelf proteins that rival tailor-made antibodies as catalysts.

Mimicking the efficiency of enzyme catalysis is a daunting challenge. An enzyme selectively binds and stabilizes the transition state (s) for a particular reaction. Artificial host systems can bind ground states just as efficiently, and rate enhancements comparable to those in enzymatic reactions can be achieved by bringing catalytic and substrate groups together in intramolecular reactions. But the combination of selective binding and efficient catalysis remains elusive. The best enzyme mimics currently known are catalytic antibodies. They bind transition-state analogues with high affinity, but their catalytic efficiency generally falls far short of that of enzymes. Thorn et al. recently described an antibody that catalyses the eliminative ring-opening of a benziosoxazole "exceptionally efficiently" using carboxylate as the general base, raising the intriguing possibility that this high efficiency derives from precise positioning of catalytic and substrate groups. Here we show that familiar 'off-the-shelf' proteins--serum albumins--catalyse the same reaction at similar rates, using a lysine side-chain amino group as the catalytic general base. Comparisons suggest that formal general base catalysis is of only modest efficiency in both systems, and that the antibody catalysis is boosted by a non-specific medium effect.

The potential of catalytic antibodies.

The idea that antibodies raised against transition state analogues should show specific catalytic activity is beautiful and seductive. In the tenth year since the idea became an experimental reality it is possible to make at least a preliminary assessment of their potential. It is concluded that their high stereoselectivity makes abzymes excellent prospects for asymmetric synthesis, though their practical usefulness is currently limited by their catalytic efficiency.

Kinetics and mechanism of the cleavage of the peptide bond next to asparagine.

The spontaneous cleavage reaction of the tetra-peptide Piv-Gly Asn-Sar-Gly-NHtBu to the C-terminal dipeptide and N-terminal succinimide dipeptide proceeds through pre-equilibrium deprotonation of the amide group of the asparagine side chain, followed by intramolecular nucleophilic attack of nitrogen on the peptide carbonyl carbonyl carbon atom. General acid-catalyzed breakdown of the intermediate then gives the products. According to this mechanism, the reaction rate strongly increases with pH and buffer concentration.

Long-term survivors with HIV-1 infection: incubation period and longitudinal patterns of CD4+ lymphocytes.

To characterize long-term survival with HIV-1, we need to estimate the proportion of seroconverters remaining free from clinical AIDS for long periods. We predict that approximately 13% of homosexual/bisexual men infected at a young age may remain so for > 20 years. Since studies have not followed individuals for such periods, long-term survivors must be characterized using stability of immunologic markers. In a cohort of 1,809 seropositive men followed since 1984-85, 15% (187/1,214) of those with at least two consecutive visits early in the study showed no decline in CD4+ cell count. From these, 67 men with long follow-up and no use of zidovudine were identified as cases to investigate correlates of protection against HIV-1-induced immunodepletion. Two matched control subgroups, one with moderate and one with rapid CD4+ lymphocyte decline, produced 56 triplets of individuals to be contrasted. Analysis of data from early in the study on demographics, sexual behavior, and sexually transmitted diseases revealed no significant differences among the three groups. Men showing no decline in CD4+ lymphocytes persistently showed a healthier profile with respect to onset of clinical AIDS, survival, and concomitant hematologic variables. Moderate decliners had rates of clinical AIDS and death significantly higher than those in the stable group but lower than the fast decliners.