Metabolism of polycyclic compounds. The metabolism of 9,10-epoxy-9,10-dihydrophenanthrene in rats

1. The main products of the metabolism of 7,12-dimethylbenz[a]anthracene by rat-liver homogenates are the isomeric monohydroxymethyl derivatives. The syntheses of these compounds are described. 2. Two phenolic products and two dihydrodihydroxy compounds were formed, but none of these appeared to have been formed by hydroxylation at the `K region'. There was little evidence for the formation of a glutathione conjugate of the hydrocarbon. 3. The monohydroxymethyl derivatives are products of the hydroxylation of the hydrocarbon in the ascorbic acid–Fe²⁺–oxygen model hydroxylating system.     

The metabolism of methylcyclohexane

1. When [U-¹⁴C]methylcyclohexane is fed to rabbits (dose 2–2·5m-moles/kg. body wt.), 65% of the radioactivity is excreted in the urine as metabolites, 0·5% appears in the faeces and about 15% in the expired air, some 4–5% remaining in the body in about 60hr. after dosing. The 15% of the dose appearing in the expired air consists of unchanged methylcyclohexane (10%) and ¹⁴CO₂ (5%). The low output of ¹⁴CO₂ shows that reactions leading to complete oxidation of methylcyclohexane are of minor importance. 2. The main metabolite found in the urine was the glucuronide of trans-4-methylcyclohexanol which was isolated. Seven methylcyclohexanols were found in the urine as conjugated glucuronides. The amounts of these were determined by isotope dilution to be as follows: cis-2-, 0·6%; trans-2-, 1·2%; cis-3-, 11·5%; trans-3-, 10·5%; cis-4-, 2·4%; trans-4-methylcyclohexanol, 14·7%, cyclohexylmethanol, 0·3%. No 1-methylcyclohexanol was found. There was evidence also that a small amount (approx. 1%) of the hydrocarbon aromatized to benzoic acid, probably via cyclohexylmethanol and cyclohexane-carboxylic acid. 3. The pattern of hydroxylation and the various amounts of the isomers found suggest that the hydroxylation in vivo of methylcyclohexane is dependent on steric factors in the molecule, hydroxylation occurring to the greatest extent at the carbon atom furthest away from the methyl group.     

Design,synthesis and biological evaluation of novel N-phosphorylated and O-phosphorylated tacrine derivatives as potential drugs against Alzheimer’s disease

In this work, we designed, synthesised and biologically investigated a novel series of 14 N- and O-phosphorylated tacrine derivatives as potential anti-Alzheimer’s disease agents. In the reaction of 9-chlorotacrine and corresponding diamines/aminoalkylalcohol we obtained diamino and aminoalkylhydroxy tacrine derivatives. Next, the compounds were acid to give final products 6–13 and 16–21 that were characterised by ¹H, ¹³C, ³¹P NMR and MS. The results of the docking studies revealed that the designed phosphorus hybrids, in theory can bind to AChE and BChE. All compounds exhibited significantly lower AutoDock Vina scores compared to tacrine. The inhibitory potency evaluation was performed using the Ellman’s method. The most inhibitory activity against AChE exhibited compound 8 with an IC₅₀ value of 6.11 nM and against BChE 13 with an IC₅₀ value of 1.97 nM and they were 6- and 12-fold potent than tacrine. Compound 19 showed the lack of hepatocytotoxicity in MTT assay.     

Novel oxindole/benzofuran hybrids as potential dual CDK2/GSK-3β inhibitors targeting breast cancer: design,synthesis, biological evaluation,and in silico studies

The serine/threonine protein kinases CDK2 and GSK-3β are key oncotargets in breast cancer cell lines, therefore, in the present study three series of oxindole-benzofuran hybrids were designed and synthesised as dual CDK2/GSK-3β inhibitors targeting breast cancer (5a–g, 7a–h, and 13a–b). The N¹-unsubstituted oxindole derivatives, series 5, showed moderate to potent activity on both MCF-7 and T-47D breast cancer cell lines. Compounds 5d–f showed the most potent cytotoxic activity with IC₅₀ of 3.41, 3.45 and 2.27 μM, respectively, on MCF-7 and of 3.82, 4.53 and 7.80 μM, respectively, on T-47D cell lines, in comparison to the used reference standard (staurosporine) IC₅₀ of 4.81 and 4.34 μM, respectively. On the other hand, the N¹-substituted oxindole derivatives, series 7 and 13, showed moderate to weak cytotoxic activity on both breast cancer cell lines. CDK2 and GSK-3β enzyme inhibition assay of series 5 revealed that compounds 5d and 5f are showing potent dual CDK2/GSK-3β inhibitory activity with IC₅₀ of 37.77 and 52.75 nM, respectively, on CDK2 and 32.09 and 40.13 nM, respectively, on GSK-3β. The most potent compounds 5d–f caused cell cycle arrest in the G2/M phase in MCF-7 cells inducing cell apoptosis because of the CDK2/GSK-3β inhibition. Molecular docking studies showed that the newly synthesised N¹-unsubstituted oxindole hybrids have comparable binding patterns in both CDK2 and GSK-3β. The oxindole ring is accommodated in the hinge region interacting through hydrogen bonding with the backbone CO and NH of the key amino acids Glu81 and Leu83, respectively, in CDK2 and Asp133 and Val135, respectively, in GSK-3β. Whereas, in series 7 and 13, the N¹-substitutions on the oxindole nucleus hinder the compounds from achieving these key interactions with hinge region amino acids what rationalises their moderate to low anti-proliferative activity.     

Hydroxylation of prostaglandin E1 by kidney cortex microsomal monooxygenase in the guinea pig

The incubation of [5,6-³H]prostaglandin E₁ ([³H]PGE₁) with guinea pig kidney cortex microsomes in the presence of NADPH in an atmosphere of air, resulted in chromatographically polar metabolites. The incubation products were treated with base which converted PGE₁ derivatives into PGB₁ derivatives, with a λ_max = 278 nm and the products were analyzed by TLC and high pressure-liquid chromatography (HPLC). Based on UV absorption, mobility on TLC and retention time in HPLC, as compared with authentic compounds, it was concluded that the two polar UV-absorbing peaks in HPLC represented 19-hydroxy-PGB₁ (19-OH-PGB₁) and 20-hydroxy-PGB₁ (20-OH-PGB₁). Further identification of the metabolites was obtained by derivatizing the incubation products as methyl esters and t-butyldimethylsilyl ethers, followed by co-injection with similarly derivatized authentic compounds in HPLC and gas chromatography. Finally, the derivatized metabolites were identified by comparing their mass fragmentation with that of similarly derivatized authentic compounds. There was an absolute requirement for NADPH, and NADH did not significantly support the hydroxylation of PGE₁. Inhibitors of microsomal monooxygenase (SKF 525A, metyrapone, and cytochrome c) inhibited the hydroxylation of PGE₁ by kidney cortex microsomes. By contrast, carbon monoxide at a CO:O₂ ratio of 5:1 did not inhibit the hydroxylation of PGE₁, pointing to a low or lack of CO sensitivity of the hydroxylation of PGE₁. The addition of PGE₁ or laurate to guinea pig kidney cortex microsomes elicited Type I spectral changes. The spectral dissociation constant (K_s) for PGE₁ was 2.4 × 10^?4m. The kinetic constants for 19- and 20-hydroxylations of PGE₁ were determined. The K_M values for the 19- and 20-hydroxylation pathways were found to be identical, being 3.3 × 10^?4m, suggesting that the same enzyme is involved in both hydroxylations; however, the V_max values for 19-hydroxylation and 20-hydroxylation of PGE₁ were 50 nmol/hr and 20.8 nmol/hr respectively. These results demonstrate that PGE₁ is a substrate for the kidney cortex microsomal monooxygenase. The similarities and differences of the kidney monooxygenase in the guinea pig with that in the rat are discussed.     

Some changes in the reactivity of enzymes resulting from their chemical attachment to water-insoluble derivatives of cellulose

1. Purified ficin was chemically attached to CM-cellulose, and partially purified ATP–creatine phosphotransferase was chemically attached to both CM-cellulose and p-aminobenzylcellulose. 2. The apparent K_m with respect to ATP and Mg²⁺ of ATP–creatine phosphotransferase was observed to increase about tenfold on attachment of the enzyme to CM-cellulose, and to increase by only 23% on its attachment to p-aminobenzylcellulose. 3. The reactivity of both ficin and ATP–creatine phosphotransferase with 5,5′-dithiobis-(2-nitrobenzoic acid) was observed to decrease on chemical attachment of these enzymes to water-insoluble derivatives of cellulose. With derivatives prepared from CM-cellulose, the extent of the reaction with 5,5′-dithiobis-(2-nitrobenzoic acid) was dependent on ionic strength, but with similar derivatives prepared from p-aminobenzylcellulose the extent of this reaction was independent of ionic strength. 4. The effect of diffusion and electrostatic interaction of charged enzyme substrates and charged enzyme supports on the apparent K_m of a water-insoluble derivative of an enzyme is discussed. An equation is derived that satisfactorily describes the observed effects of these factors on the apparent K_m.     

Isolation and Characterization of pso Mutants Sensitive to Photo-Addition of Psoralen Derivatives in SACCHAROMYCES CEREVISIAE

We have isolated mutants sensitive to photo-addition of bi-functional and mono-functional derivatives of psoralen in Saccharomyces cerevisiae. Three of these pso mutants were analyzed in detail. They segregate in meiosis like Mendelian genes and complement each other, as well as existing radiation-sensitive (rad and rev) mutants. The study of heterozygous diploid strains (PSO⁺/pso) indicates that the three pso genes are recessive. The mutant pso1–1 demonstrates a cross-sensitivity to UV and γ-rays, whereas mutants pso2–1 and pso3–1 are specifically sensitive to photo-addition of psoralen derivatives. The comparison of exponentially growing cells to stationary-phase cells demonstrates that for the three mutants the defect in repair capacity of DNA cross-links and monoadducts concerns G1 and early S-phase cells. The pso2–1 mutant is, however, also defective in G2 repair and loses diploid resistance when it is in the homozygous state.—The block in repair capacity in these novel mutants is discussed in relation to the three other repair pathways known to be involved in the repair of furocoumarins photo-induced lesions in yeast DNA.     

Methylglyoxal, an endogenous aldehyde, crosslinks DNA polymerase and the substrate DNA

Methylglyoxal, a known endogenous and environmental mutagen, is a reactive α-ketoaldehyde that can modify both DNA and proteins. To investigate the possibility that methylglyoxal induces a crosslink between DNA and DNA polymerase, we treated a ‘primed template’ DNA and the exonuclease-deficient Klenow fragment (KF^exo–) of DNA polymerase I with methylglyoxal in vitro. When the reaction mixtures were analyzed by SDS–PAGE, we found that methylglyoxal induced a DNA–KF^exo– crosslink. The specific binding complex of KF^exo– and ‘primed template’ DNA was necessary for formation of the DNA–KF^exo– crosslink. Methylglyoxal reacted with guanine residues in the single-stranded portion of the template DNA. When 2′-deoxyguanosine was incubated with Nα-acetyllysine or N-acetylcysteine in the presence of methylglyoxal, a crosslinked product was formed. No other amino acid derivatives tested could generate a crosslinked product. These results suggest that methylglyoxal crosslinks a guanine residue of the substrate DNA and lysine and cysteine residues near the binding site of the DNA polymerase during DNA synthesis and that DNA replication is severely inhibited by the methylglyoxal-induced DNA–DNA polymerase crosslink.     

Papain-catalysed hydrolysis of some hippuric esters. A new mechanism for papain-catalysed hydrolyses

1. The Michaelis–Menten parameters for the papain-catalysed hydrolysis of a number of alkyl, aryl and alkyl-thiol esters of hippuric acid have been determined. 2. For all the aryl esters and most of the alkyl esters studied, the catalytic constant, k₀, is 2–3sec.⁻¹ and most probably represents deacylation of the common intermediate, hippuryl-papain. 3. Two alkyl esters and hippurylamide, however, have catalytic rate constants, k₀, less than 2–3sec.⁻¹. It is possible to interpret all the available kinetic data in terms of a three-step mechanism in which an enzyme–substrate complex is first formed, followed by acylation of the enzyme through an essential thiol group, followed by deacylation of the acyl-enzyme. 4. The logarithm of the ratio of the Michaelis–Menten parameters, which reflect the acylation rate constant, for four aryl esters of hippuric acid studied give a linear Hammett plot against the substituent constant, σ. Arguments are presented that indicate acid as well as nucleophilic catalysis in the acylation process and that the most likely proton donor is an imidazolium ion. 5. It is suggested that this imidazolium ion is part of the same histidine residue that has been tentatively implicated in the deacylation process (Lowe & Williams, 1965b). 6. A new mechanism is proposed for the papain-catalysed hydrolysis of N-acyl-α-amino acid derivatives.     

Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

Cytochrome P450 monooxygenases (P450s), which constitute a superfamily of heme-containing proteins, catalyze the direct oxidation of a variety of compounds in a regio- and stereospecific manner; therefore, they are promising catalysts for use in the oxyfunctionalization of chemicals. In the course of our comprehensive substrate screening for all 27 putative P450s encoded by the Streptomyces griseus genome, we found that Escherichia coli cells producing an S. griseus P450 (CYP154C3), which was fused C terminally with the P450 reductase domain (RED) of a self-sufficient P450 from Rhodococcus sp., could transform various steroids (testosterone, progesterone, Δ⁴-androstene-3,17-dione, adrenosterone, 1,4-androstadiene-3,17-dione, dehydroepiandrosterone, 4-pregnane-3,11,20-trione, and deoxycorticosterone) into their 16α-hydroxy derivatives as determined by nuclear magnetic resonance and high-resolution mass spectrometry analyses. The purified CYP154C3, which was not fused with RED, also catalyzed the regio- and stereospecific hydroxylation of these steroids at the same position with the aid of ferredoxin and ferredoxin reductase from spinach. The apparent equilibrium dissociation constant (K_d) values of the binding between CYP154C3 and these steroids were less than 8 μM as determined by the heme spectral change, indicating that CYP154C3 strongly binds to these steroids. Furthermore, kinetic parameters of the CYP154C3-catalyzed hydroxylation of Δ⁴-androstene-3,17-dione were determined (K_m, 31.9 ± 9.1 μM; k_cat, 181 ± 4.5 s⁻¹). We concluded that CYP154C3 is a steroid D-ring 16α-specific hydroxylase which has considerable potential for industrial applications. This is the first detailed enzymatic characterization of a P450 enzyme that has a steroid D-ring 16α-specific hydroxylation activity.     

Development of naringenin-O-alkylamine derivatives as multifunctional agents for the treatment of Alzheimer’s disease

In this study, a series of naringenin-O-alkylamine derivatives were designed and obtained by introducing an alkylamine fragment into the naringenin skeleton. The in vitro biological activity results revealed that compounds 5f and 7k showed good antioxidant activity with ORAC values of 2.3eq and 1.2eq, respectively. Compounds 5f and 7k were reversible and excellent huAChE inhibitors with IC₅₀ values of 0.91 μM and 0.57 μM, respectively. Moreover, compounds 5f and 7k could inhibit self-induced Aβ_1–42 aggregation with 62.1% and 43.8% inhibition rate, respectively, and significantly inhibited huAChE-Aβ_1–40 aggregation with 51.7% and 43.4% inhibition rate, respectively. In addition, compounds 5f and 7k were selective metal chelators and remarkably inhibited Cu²⁺-induced Aβ_1–42 aggregation with 73.5% and 68.7% inhibition rates, respectively. Furthermore, compounds 5f and 7k could cross the blood-brain barrier in vitro and displayed good neuroprotective effects and anti-inflammatory properties. Further investigation showed that compound 5f did not show obvious hepatotoxicity and displayed a good hepatoprotective effect by its antioxidant activity. The in vivo study displayed that compound 5f significantly improved scopolamine-induced mice memory impairment. Therefore, compound 5f was a potential multifunctional candidate for the treatment of AD.     

Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily

The copper membrane monooxygenases (CuMMOs) are an important group of enzymes in environmental science and biotechnology. Areas of relevance include the development of green chemistry for sustainable exploitation of methane (CH₄) reserves, remediation of chlorinated hydrocarbon contamination and monitoring human impact in the biogeochemical cycles of CH₄ and nitrogen. Challenges for all these applications are that many aspects of the ecology, physiology and structure–function relationships in the CuMMOs are inadequately understood. Here, we describe genetic and physiological characterization of a novel member of the CuMMO family that has an unusual physiological substrate range (C₂–C₄ alkanes) and a distinctive bacterial host (Mycobacterium). The Mycobacterial CuMMO genes (designated hmoCAB) were amenable to heterologous expression in M. smegmatis—this is the first example of recombinant expression of a complete and highly active CuMMO enzyme. The apparent specific activity of recombinant cells containing hmoCAB ranged from 2 to 3 nmol min^–1 per mg protein on ethane, propane and butane as substrates, and the recombinants could also attack ethene, cis-dichloroethene and 1,2-dichloroethane. No detectable activity of recombinants or wild-type strains was seen with methane. The specific inhibitor allylthiourea strongly inhibited growth of wild-type cells on C₂–C₄ alkanes, and omission of copper from the medium had a similar effect, confirming the physiological role of the CuMMO for growth on alkanes. The hydrocarbon monooxygenase provides a new model for studying this important enzyme family, and the recombinant expression system will enable biochemical and molecular biological experiments (for example, site-directed mutagenesis) that were previously not possible.     

Sunscreen Products as Emerging Pollutants to Coastal Waters

A growing awareness of the risks associated with skin exposure to ultraviolet (UV) radiation over the past decades has led to increased use of sunscreen cosmetic products leading the introduction of new chemical compounds in the marine environment. Although coastal tourism and recreation are the largest and most rapidly growing activities in the world, the evaluation of sunscreen as source of chemicals to the coastal marine system has not been addressed. Concentrations of chemical UV filters included in the formulation of sunscreens, such as benzophehone 3 (BZ-3), 4-methylbenzylidene camphor (4-MBC), TiO₂ and ZnO, are detected in nearshore waters with variable concentrations along the day and mainly concentrated in the surface microlayer (i.e. 53.6–577.5 ng L^-1 BZ-3; 51.4–113.4 ng L^-1 4-MBC; 6.9–37.6 µg L^-1 Ti; 1.0–3.3 µg L^-1 Zn). The presence of these compounds in seawater suggests relevant effects on phytoplankton. Indeed, we provide evidences of the negative effect of sunblocks on the growth of the commonly found marine diatom Chaetoceros gracilis (mean EC₅₀ = 125±71 mg L^-1). Dissolution of sunscreens in seawater also releases inorganic nutrients (N, P and Si forms) that can fuel algal growth. In particular, PO₄ ³⁻ is released by these products in notable amounts (up to 17 µmol PO₄ ³⁻ g⁻¹). We conservatively estimate an increase of up to 100% background PO₄ ³⁻ concentrations (0.12 µmol L^-1 over a background level of 0.06 µmol L^-1) in nearshore waters during low water renewal conditions in a populated beach in Majorca island. Our results show that sunscreen products are a significant source of organic and inorganic chemicals that reach the sea with potential ecological consequences on the coastal marine ecosystem.     

2,4,6-Trinitrotoluene Reduction by Carbon Monoxide Dehydrogenase from Clostridium thermoaceticum

Purified CO dehydrogenase (CODH) from Clostridium thermoaceticum catalyzed the transformation of 2,4,6-trinitrotoluene (TNT). The intermediates and reduced products of TNT transformation were separated and appear to be identical to the compounds formed by C. acetobutylicum, namely, 2-hydroxylamino-4,6-dinitrotoluene (2HA46DNT), 4-hydroxylamino-2,6-dinitrotoluene (4HA26DNT), 2,4-dihydroxylamino-6-nitrotoluene (24DHANT), and the Bamberger rearrangement product of 2,4-dihydroxylamino-6-nitrotoluene. In the presence of saturating CO, CODH catalyzed the conversion of TNT to two monohydroxylamino derivatives (2HA46DNT and 4HA26DNT), with 4HA26DNT as the dominant isomer. These derivatives were then converted to 24DHANT, which slowly converted to the Bamberger rearrangement product. Apparent K_m and k_cat values of TNT reduction were 165 ± 43 μM for TNT and 400 ± 94 s⁻¹, respectively. Cyanide, an inhibitor for the CO/CO₂ oxidation/reduction activity of CODH, inhibited the TNT degradation activity of CODH.     

Nitrogen loss by anaerobic oxidation of ammonium in rice rhizosphere

Anaerobic oxidation of ammonium (anammox) is recognized as an important process for nitrogen (N) cycling, yet its role in agricultural ecosystems, which are intensively fertilized, remains unclear. In this study, we investigated the presence, activity, functional gene abundance and role of anammox bacteria in rhizosphere and non-rhizosphere paddy soils using catalyzed reporter deposition–fluorescence in situ hybridization, isotope-tracing technique, quantitative PCR assay and 16S rRNA gene clone libraries. Results showed that rhizosphere anammox contributed to 31–41% N₂ production with activities of 0.33–0.64 nmol N₂ g⁻¹ soil h⁻¹, whereas the non-rhizosphere anammox bacteria contributed to only 2–3% N₂ production with lower activities of 0.08–0.26 nmol N₂ g⁻¹ soil h⁻¹. Higher anammox bacterial cells were observed (0.75–1.4 × 10⁷ copies g⁻¹ soil) in the rhizosphere, which were twofold higher compared with the non-rhizosphere soil (3.7–5.9 × 10⁶ copies g⁻¹ soil). Phylogenetic analysis of the anammox bacterial 16S rRNA genes indicated that two genera of ‘Candidatus Kuenenia'' and ‘Candidatus Brocadia'' and the family of Planctomycetaceae were identified. We suggest the rhizosphere provides a favorable niche for anammox bacteria, which are important to N cycling, but were previously largely overlooked.     

Microbial Hydroxylation of Indole Alkaloids

The hydroxylation of the indole-type alkaloids, yohimbine, α-yohimbine, β-yohimbine, and corynanthine, was achieved with several genera of higher fungi and species of Streptomyces. Microorganisms were found which monohydroxylated these compounds in three different positions. The site of hydroxylation was strain-specific for two strains of Cunninghamella echinulata and C. bainieri.     

Identification of Novel Genetic Loci Associated with Thyroid Peroxidase Antibodies and Clinical Thyroid Disease

Autoimmune thyroid diseases (AITD) are common, affecting 2-5% of the general population. Individuals with positive thyroid peroxidase antibodies (TPOAbs) have an increased risk of autoimmune hypothyroidism (Hashimoto''s thyroiditis), as well as autoimmune hyperthyroidism (Graves'' disease). As the possible causative genes of TPOAbs and AITD remain largely unknown, we performed GWAS meta-analyses in 18,297 individuals for TPOAb-positivity (1769 TPOAb-positives and 16,528 TPOAb-negatives) and in 12,353 individuals for TPOAb serum levels, with replication in 8,990 individuals. Significant associations (P<5×10⁻⁸) were detected at TPO-rs11675434, ATXN2-rs653178, and BACH2-rs10944479 for TPOAb-positivity, and at TPO-rs11675434, MAGI3-rs1230666, and KALRN-rs2010099 for TPOAb levels. Individual and combined effects (genetic risk scores) of these variants on (subclinical) hypo- and hyperthyroidism, goiter and thyroid cancer were studied. Individuals with a high genetic risk score had, besides an increased risk of TPOAb-positivity (OR: 2.18, 95% CI 1.68–2.81, P = 8.1×10⁻⁸), a higher risk of increased thyroid-stimulating hormone levels (OR: 1.51, 95% CI 1.26–1.82, P = 2.9×10⁻⁶), as well as a decreased risk of goiter (OR: 0.77, 95% CI 0.66–0.89, P = 6.5×10⁻⁴). The MAGI3 and BACH2 variants were associated with an increased risk of hyperthyroidism, which was replicated in an independent cohort of patients with Graves'' disease (OR: 1.37, 95% CI 1.22–1.54, P = 1.2×10⁻⁷ and OR: 1.25, 95% CI 1.12–1.39, P = 6.2×10⁻⁵). The MAGI3 variant was also associated with an increased risk of hypothyroidism (OR: 1.57, 95% CI 1.18–2.10, P = 1.9×10⁻³). This first GWAS meta-analysis for TPOAbs identified five newly associated loci, three of which were also associated with clinical thyroid disease. With these markers we identified a large subgroup in the general population with a substantially increased risk of TPOAbs. The results provide insight into why individuals with thyroid autoimmunity do or do not eventually develop thyroid disease, and these markers may therefore predict which TPOAb-positives are particularly at risk of developing clinical thyroid dysfunction.     

Photoaffinity-labeled Cytokinins: Synthesis and Biological Activity

Two new azidopurine derivatives, 2-azido-N⁶-(Δ²-isopentenyl)adenine and 2-azido-N⁶-benzyladenine, have been synthesized as potential photoaffinity labels for probing cytokinin-binding sites. The preparation and the biological activity of these compounds are described.     

A Mechanistic Paradigm for Broad-Spectrum Antivirals that Target Virus-Cell Fusion

LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC₅₀≤0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001''s specific inhibition of virus-cell fusion.The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen (¹O₂) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated ¹O₂. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. ¹O₂-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced ¹O₂-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides.Based on our understanding of LJ001''s mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001''s limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC₅₀<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of ¹O₂ generation), and (4) 10–100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion.     

The fate of the hydrolysis products of thalidomide in the pregnant rabbit

1. The fate in the pregnant New Zealand White rabbit of oral doses of four ¹⁴C-labelled hydrolysis products of thalidomide, namely α-(o-carboxybenzamido)-glutarimide, 2-phthalimidoglutaramic acid, 2-phthalimidoglutaric acid and 2-(o-carboxybenzamido)glutaramic acid, administered on the 192nd hour of pregnancy has been studied. 2. About 60–95% of the administered ¹⁴C of each compound appears in the urine in 58hr. and the remainder is found in the faeces and in the gut and its contents. 3. Radioactivity is present in the plasma, liver, kidney, brain, muscle, fat and embryo. 4. The ¹⁴C-labelled substances in the plasma and embryo consist of the unchanged compounds and their further hydrolysis products. 5. Since the above four thalidomide hydrolysis products are found in the rabbit conceptus together with their further hydrolysis products after their oral administration to the pregnant rabbit, it appears that the teratogenic activity of thalidomide is due to the compound itself rather than to one or more of its hydrolysis products.