Behavioral rules and tail-up display in extra- and intra-pair interactions of the butterflyfish,Chaetodon lunulatus

The butterflyfish,Chaetodon lunulatus, forms monogamous pair bonds. Each pair defends a feeding territory against conspecifics. The tail-up or lateral display of the butterflyfish is performed not only towards non-partners in territorial disputes (extra-pair interaction), but also towards partners (intra-pair interaction). In order to explain this phenomenon, I investigated the two interaction types, and found that a simple model explains both interactions very well. The model consists of four behavioral rules and two provisos for applying the rules. Rule 1: Perform the tail-up display when approached by another individual. Rule 2: Perform the tail-up display when another individual performs the tail-up display. Rule 3: Attack another individual that neither approaches nor performs the tail-up display. Rule 4: Swim with your partner. Proviso 1: Apply Rules 1 and 2 when you do not intend to run away. Proviso 2: Apply Rules 1, 2, and 3 when you do not recognize another individual as your partner. When Rules 1–4 and Provisos 1–2 are applied, the display can prevent improper attacks between partners caused by imperfect partner recognition.     

Behaviors in agonistic interaction of the butterflyfish (Chaetodon lunulatus)

The butterflyfish (Chaetodon lunulatus) forms heterosexual pair bonds. Each pair defends a feeding territory against conspecifics. In the field, I observed the agonistic interactions related to territoriality, and recognized nine behavioral patterns: staring, parallel swimming, rushing, tail-up display, chasing, fleeing, encircling, TT-fighting (two-piled-tops fighting), and attacking. Almost all interactions were conventional fighting in which attacking seldom occurred. In rare cases, interactions escalated to TT-fighting. In these cases, attacks were frequent, and outcomes were significant (e.g., territorial takeover and serious injuries). Received: October 24, 1998 / Accepted: August 23, 1999     

Signal function of tail-up posture in the monogamous butterflyfish (<Emphasis Type="Italic">Chaetodon lunulatus</Emphasis>): dummy experiments in the field

Dummy field experiments were undertaken to test whether the tail-up posture of the butterflyfish Chaetodon lunulatus is a sufficient stimulus to release the tail-up display of other individuals. Two types of dummies were presented to the fish in their home territories: one mimicking the fish in a tail-up posture and the other in a normal posture. The fish performed the tail-up display to the tail-up dummy. This result suggests that the tail-up posture has the signal function of eliciting the tail-up display in other individuals. This signal function is considered to play an important role in behavioral mechanisms for controlling the aggression of the fish.     

Docking based design of diastereoisomeric MTCA as GPIIb/IIIa receptor inhibitor

In GPIIb/IIIa mediated arterial thrombosis platelet activation plays a central role. To discover platelet activation inhibitor the pharmacophores of GPIIb/IIIa receptor inhibitors and anti-thrombotic agents were analyzed. This led to the design of (1R,3S)- and (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acids as GPIIb/IIIa inhibitors. Comparing to (1S,3S)-isomer (1R,3S)-isomer had lower cdocker interaction energy. AFM image showed that the minimal effective concentration of (1S,3S)-isomer and (1R,3S)-isomer inhibiting platelet activation were 10^?5?M and 10^?6?M, respectively. In vivo 1?μmol/kg of oral (1S,3S)-isomer effectively inhibited the rats to form arterial thrombus and down regulated GPIIb/IIIa expression, but the activities were significantly lower than those of 1?μmol/kg of oral (1R,3S)-isomer. Both (1S,3S)-isomer and (1R,3S)-isomer can be safely used for structural modifications, but (1R,3S)-isomer should be superior to (1S,3S)-isomer.     

Oxidation and keto reduction of 12-hydroxy-5,8,10,14-eicosatetraenoic acids in bovine corneal epithelial microsomes

The R and S enantiomers of 12-hydroxyeicosatetraenoic acid (12-HETE) exhibit different biological activities. Although they appear to be produced by different enzymatic pathways, cytochrome P-450 monooxygenase and lipoxygenase, respectively, they display similar metabolism in both corneal epithelium and neutrophils. In corneal epithelial microsomes, both enantiomers are subject to oxidation and keto reduction reactions to form the dihydro metabolite, 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE), via a keto intermediate. The apparent K_m for the formation of 12-HETrE was 17.9 and 20 μM for 12(R)-HETE and 12(S)-HETE, respectively, and the apparent V_max of the reaction was 17.4 and 8.2 pmol/mg per min, respectively. Chiral analysis of the dihydro metabolite demonstrated a product enantiospecificty. Arachidonic acid, 12(R)-HETE, 12(S)-HETE and the intermediate of this reaction, 12-oxo-ETrE, were metabolized predominantly to 12(R)-HETrE in a ratio [12(R)-HETrE: 12(S)-HETrE] of 7.3:1, 4.3:1, 1.5:1 and 2.3:1, respectively. 12(R)-HETrE is a potent vasodilator, chemotactic and angiogenic factor whose synthesis is induced in inflamed tissues; 12(S)HETrE is devoid of these properties. 12(R)-HETE, derived from NADPH-dependent cytochrome P-450 monooxygenases, and 12(S)-HETE, derived from 12-lipoxygenase, may both play an important role in regulating the inflammatory response by serving as substrates for the local synthesis of 12(R)-HETrE.     

Gene Deletion of 7,8-Linoleate Diol Synthase of the Rice Blast Fungus: STUDIES ON PATHOGENICITY, STEREOCHEMISTRY, AND OXYGENATION MECHANISMS*

Linoleate diol synthases (LDS) are heme enzymes, which oxygenate 18:2n-6 sequentially to (8R)-hydroperoxylinoleic acid ((8R)-HPODE) and to (5S,8R)-dihydroxy-, (7S,8S)-dihydroxy-, or (8R,11S)-dihydroxylinoleic acids (DiHODE). The genome of the rice blast fungus, Magnaporthe oryzae, contains two genes with homology to LDS. M. oryzae oxidized 18:2n-6 to (8R)-HPODE and to (7S,8S)-DiHODE, (6S,8R)-DiHODE, and (8R,11S)-HODE. Small amounts of 10-hydroxy-(8E,12Z)-octadecadienoic acid and traces of 5,8-DiHODE were also detected by liquid chromatography-mass spectrometry. The contribution of the 7,8-LDS gene to M. oryzae pathogenicity was evaluated by replacement of the catalytic domain with hygromycin and green fluorescent protein variant (SGFP) cassettes. This genetically modified strain Δ7,8-LDS infected rice leaves and roots and formed appressoria and conidia as the native fungus. The Δ7,8-LDS mutant had lost the capacity to biosynthesize all the metabolites except small amounts of 8-hydroxylinoleic acid. Studies with stereospecifically deuterated linoleic acids showed that (8R)-HPODE was formed by abstraction of the pro-S hydrogen at C-8 and antarafacial oxygenation, whereas (7S,8S)-DiHODE and (8R,11S)-DiHODE were formed from (8R)-HPODE by suprafacial hydrogen abstraction and oxygenation at C-7 and C-11, respectively. A mac1 suppressor mutant (Δmac1 sum1–99) of M. oryzae, which shows cAMP-independent protein kinase A activity, oxygenated 18:2n-6 to increased amounts of (10R)-HPODE and (5S,8R)-DiHODE. Expression of the 7,8-LDS gene but not of the second homologue was detected in the suppressor mutant. This suggests that PKA-mediated signaling pathway regulates the dioxygenase and hydroperoxide isomerase activities of M. oryzae.     

Chemical synthesis and actions of 11,12-thiirano-hepoxilin A3

A novel analog of hepoxilin A3 has been chemically synthesized in which the 11,12-epoxide group has been altered to a thiirano group. This has been accomplished through allylic rearrangement of unnatural (11R,12R)-hepoxilin B₃ under Mitsunobu conditions, first into unnatural (11R,12R)-hepoxilin A₃, followed by conversion of this compound with inversion of the epoxide centers into the thiirano-hepoxilin A₃ having the natural 11S,12S configuration. We also report herein evidence showing that thiirano-hepoxilin A₃ raises intracellular calcium concentrations in intact human neutrophils.     

Steroid sapogenins from Tristagma uniflorum

From bulbs of Tristagma uniflorum the known sapogenins tigogenin, neotigogenin and (20S,22R,25S)-5α-spirostan-3β,25-diol, as well as the new (20S,22R,25R)-5α-spirostan-3β,25-diol, (20S,22S,25S)-5α-furostan-22,25-epoxy-3β,26-diol and (20S,22S,25R) -5α-furostan-22,25-epoxy-3β,26-diol, were isolated and characterized by spectroscopic (IR, ¹H NMR, ¹³C NMR, MS) methods.     

Production of (R)-3-hydroxybutyric acid by fermentation and bioconversion processes with Azohydromonas lata

Feasibility of producing (R)-3-hydroxybutyric acid ((R)-3-HB) using wild type Azohydromonas lata and its mutants (derived by UV mutation) was investigated. A. lata mutant (M5) produced 780 mg/l in the culture broth when sucrose was used as the carbon source. M5 was further studied in terms of its specificity with various bioconversion substrates for production of (R)-3-HB. (R)-3-HB concentration produced in the culture broth by M5 mutant was 2.7-fold higher than that of the wild type strain when sucrose (3% w/v) and (R,S)-1,3-butanediol (3% v/v) were used as carbon source and bioconversion substrate, respectively. Bioconversion of resting cells (M5) with glucose (1% v/w), ethylacetoacetate (2% v/v), and (R,S)-1,3-butanediol (3% v/v), resulted in (R)-3-HB concentrations of 6.5 g/l, 7.3 g/l and 8.7 g/l, respectively.     

Structure–activity relationship study on α1 adrenergic receptor antagonists from beer

Hordatine A and aperidine have been previously isolated from beer as active ingredients, which bind to muscarinic M₃ receptor. In addition, these compounds have exhibited antagonist activity against the α_1A adrenoceptor. Although the relative structures of these two molecules have previously been determined, the absolute stereochemistry was unclear. Hence, to elucidate the absolute stereochemistry of natural hordatine A, we synthesized each enantiomer of hordatine A and aperidine from optically pure dehydrodi-p-coumaric acid. Several additional related compounds were also synthesized for structure–activity relationship studies. Chiral column HPLC analysis demonstrated that the absolute stereochemistry of natural hordatine A is (2S,3S), while based on the isomerization mechanism, the stereochemistry of aperidine is (2R,3S). The α_1A adrenoceptor binding activity of (2R,3R)-hordatine A is the most potent among the enantiomeric pairs of hordatines and aperidines. Furthermore, the related, synthetic compound, (2R,3R)-methyl benzofurancarboxylate exhibits antagonist activity against the α_1A adrenoceptor at a lower concentration than that of hordatine A.     

The interaction of cytosolic epoxide hydrolase with chiral epoxides

• 1.1. The kinetic parameters of the cytosolic epoxide hydrolase were examined with two sets of spectrophotometric substrates. The (2S,3S)- and (2R,3R)-enantiomers of 4-nitrophenyl trans-2,3-epoxy-3-phenylpropyl carbonate had a K_mof 33 and 68 μm and a V_max of 16 and 27 μmol/min/mg, respectively. With the (2S,3S)- and (2R,3R)- enantiomers of 4-nitrophenyl trans-2,3-epoxy-3-(4-nitrophenyl)propyl carbonate, cytosolic epoxide hydrolase had a K_M of 8.0 and 15 μM and a V_max of 7.8 and 5.0 μmol/min/mg, respectively.
• 2.2. Glycidyl 4-nitrobenzoate had the lowest I₅₀ of the compounds tested in the glycidyl 4-nitrobenzoate series (I₅₀= 140 μM). The I₅₀ of the (2R)-enantiomer was 3.7-fold higher. The inhibitor with the lowest i₅₀ in the glycidol series, and the lowest I₅₀ of any compound tested, was (2S,3S)-3-(4-nitrophenyl)glycidol (I₅₀ = 13.0μM). It also showed the greatest difference in I₅₀ between the enantiomers (330-fold).
• 3.3. All enantiomers of glycidyl 4-nitrobenzoates and _trans-3-phenylglycidols gave differential inhibition of cytosolic epoxide hydrolase. However, neither the (S,S)-/(S)- or (R,R)-/(R)-enantiomer always had the lower I₅₀.
• 4.4. Addition of one or more methyl groups to either enantiomer of glycidyl 4-nitrobenzoate resulted in increased I₅₀. However, addition of a methyl group to C₂ of either enantiomer of 3-phenylglycidol resulted in a decreased I₅₀. Finally, when the hydroxyl group of trans-3-(4-nitrophenyl)glycidol was esterified the I₅₀ of the (2S,3S)- but not the (2R,3R)-enantiomer increased.

     

Stereochemical Control in the Still-Wittig Rearrangement Synthesis of Cyclohexyl (Z)-Alkene Inhibitors of Pin1

Three stereoisomeric inhibitors of Pin1: (2R,5S)-, (2S,5R)- and (2S,5S)-Ac–pSer–Ψ[(Z)CH = C]–pipecolyl(Pip)–2-(2-naphthyl)ethylamine 1, that mimic L-pSer–D-Pro, D-pSer–L-Pro, and D-pSer–D-Pro amides respectively, were synthesized by a 13-step route. The newly formed stereogenic centers in the pipecolyl ring were introduced by Luche reduction, followed by stereospecific [2,3]-Still-Wittig rearrangement. The (Z)- to (E)-alkene ratio in the rearrangements were consistently 5.5 to 1. The stereochemistry at the original Ser α-carbon controlled the stereochemistry of the Luche reduction, but it did not affect the stereochemical outcome of the rearrangement, which consistently gave the (Z)-alkene. The epimerized by-product, (2S,5S)-10, resulting from the work-up after Na/NH₃ debenzylation of (2S,5R)-9, was carried on to the (2S,5S)-1 isomer. Compound (2S,5S)-10 was resynthesized from the Luche reduction by-product, (2R,3R)-3, and the stereochemistry was confirmed by comparison of the optical rotations. The IC₅₀ values for (2R,5S)-1, (2S,5R)-1 and (2S,5S)-1 Pin1 inhibition were: 52, 85, and 140 μM, respectively.     

(22<Emphasis Type="Italic">R</Emphasis>,23<Emphasis Type="Italic">R</Emphasis>)-3β-hydroxy-22,23-epoxy-5α-ergost-8(14)-en-15-one: Improved synthesis and toxicity to MCF-7 cells

The chemical synthesis of (22R,23R)-3β-hydroxy-22,23-epoxy-5α-ergost-8(14)-en-15-one from (22A)-3β-acetoxy-5α-ergosta-7,14,22-triene was improved. The stages of obtaining and isomerization of (22A)-3β-acetoxy-14α15α-epoxy-5α-ergosta-7,22-diene were optimized. The introduction of (22R,23R)-epoxide cycle was carried out by alkaline treatment of intermediate (22S,23R)-3β,23-diacetoxy-22-iodo-5α-ergost-8(14)-en-15-one. In cells of human breast carcinoma MCF-7, (22R,23R)-3β-hydroxy-22,23-epoxy-5α-ergost-8(14)-en-15-one showed a high toxicity (TC₅₀ = 0.4±0.1 μM at 48-h incubation in serum-free medium).     

Structure–cytotoxic activity relationship of sesquilignan,morinol A

The cytotoxic activities of sesquilignans, (7S,8S,7′R,8′R)- and (7R,8R,7′S,8′S)-morinol A and (7S,8S,7′S,8′S)- and (7R,8R,7′R,8′R)-morinol B were compared, showing no significant difference between stereoisomers (IC₅₀ = 24–35 μM). As a next stage, the effect of substituents at 7, 7′, and 7″-aromatic ring on the activity was evaluated to find out the higher activity of (7S,8S,7′R,8′R)-7,7′,7″-phenyl derivative 18 (IC₅₀ = 6–7 μM). In the research on the structure–activity relationship of 7″-position of (7S,8S,7′R,8′R)-7,7′,7″-phenyl derivative 18, the most potent compounds were 7,7′,7″-phenyl derivative 18 (IC₅₀ = 6 μM) against HeLa cells. Against HL-60 cells, 7″-(4-nitrophenyl)-7,7′-phenyl derivative 33 and 7″-hexyl-7,7′-phenyl derivative 37 (IC₅₀ = 5 μM) showed highest activity. We discovered the compounds showed four to sevenfold potent activity than that of natural (7S,8S,7′R,8′R)-morinol A. It was also confirmed that the 7′-benzylic hydroxy group have an important role for exhibiting activity, on the other hand, the resonance system of cinnamyl structure is not crucial for the potent activity.     

Synthesis,NMR characterization and divergent biological actions of 2′-hydroxy-ceramide/dihydroceramide stereoisomers in MCF7 cells

A straightforward method for the simultaneous preparation of (2S,3R,2′R)- and (2S,3R,2′S)-2′-hydroxy-ceramides (2′-OHCer) from (2S,3R)-sphingosine acetonide precursors and racemic mixtures of 2-hydroxy fatty acids (2-OHFAs) is described. The obtained 2′-OH-C4-, -C6-, -C12-, -C16-Cer and 2′-OH-C6-dhCer pairs of diastereoisomers were characterized thoroughly by TLC, MS, NMR, and optical rotation. Dynamic and multidimensional NMR studies provided evidence that polar interfaces of 2′-OHCers are extended and more rigid than observed for the corresponding non-hydroxylated analogs. Stereospecific profile on growth suppression of MCF7 cells was observed for (2′R)- and (2′S)-2′-OH-C6-Cers and their dihydro analogs. The (2′R)-isomers were more active than the (2′S)-isomers (IC₅₀ ～3 μM/8 μM and IC₅₀ ～8 μM/12 μM, respectively), surpassing activity of the ordinary C6-Cer (IC₅₀ ～12 μM) and C6-dhCer (IC₅₀ ～38 μM). Neither isomer of 2′-OH-C6-Cers and 2′-OH-C6-dhCers was metabolized to their cellular long chain 2′-OH-homologs. Surprisingly, the most active (2′R)-isomers did not influence the levels of the cellular Cers nor dhCers. Contrary to this, the (2′S)-isomers generated cellular Cers and dhCers efficiently. In comparison, the ordinary C6-Cer and C6-dhCer also significantly increased the levels of their cellular long chain homologs. These peculiar anabolic responses and SAR data suggest that (2′R)-2′-OHCers/dhCers may interact with some distinct cellular regulatory targets in a specific and more effective manner than their non-hydroxylated analogs. Thus, stereoisomers of 2′-OHCers can be potentially utilized as novel molecular tools to study lipid–protein interactions, cell signaling phenomena and to understand the role of hydroxylated sphingolipids in cancer biology, pathogenesis and therapy.     

The scope of phage display for membrane proteins

Numerous examples of phage display applied to soluble proteins demonstrate the power of the technique for protein engineering, affinity reagent discovery and structure-function studies. Recent reports have expanded phage display to include membrane proteins (MPs). The scope and limitations of MP display remain undefined. Therefore, we report data from the phage display of representative types of membrane-associated proteins including plasma, nuclear, peripheral, single and multipass. The peripheral MP neuromodulin displays robustly with packaging by conventional M13-KO7 helper phage. The monotopic MP Nogo-66 can also display on the phage surface, if packaged by the modified M13-KO7⁺ helper phage. The modified phage coat of KO7⁺ can better mimic the zwitterionic character of the plasma membrane. Four examples of putatively α-helical, integral MPs failed to express as fusions to an anchoring phage coat protein and therefore did not display on the phage surface. However, the β-barrel MPs ShuA (Shigella heme uptake A) and MOMP (major outer membrane protein), which pass through the membrane 22 and 16 times, respectively, can display surprisingly well on the surfaces of both conventional and KO7⁺ phages. The results provide a guide for protein engineering and large-scale mutagenesis enabled by the phage display of MPs.     

Cloning,expression and characterization of glycerol dehydrogenase involved in 2,3-butanediol formation in Serratia marcescens H30

The meso-2,3-butanediol dehydrogenase (meso-BDH) from S. marcescens H30 is responsible for converting acetoin into 2,3-butanediol during sugar fermentation. Inactivation of the meso-BDH encoded by budC gene does not completely abolish 2,3-butanediol production, which suggests that another similar enzyme involved in 2,3-butanediol formation exists in S. marcescens H30. In the present study, a glycerol dehydrogenase (GDH) encoded by gldA gene from S. marcescens H30 was expressed in Escherichia coli BL21(DE3), purified and characterized for its properties. In vitro conversion indicated that the purified GDH could catalyze the interconversion of (3S)-acetoin/meso-2,3-butanediol and (3R)-acetoin/(2R,3R)-2,3-butanediol. (2S,3S)-2,3-Butanediol was not a substrate for the GDH at all. Kinetic parameters of the GDH enzyme showed lower K _m value and higher catalytic efficiency for (3S/3R)-acetoin in comparison to those for (2R,3R)-2,3-butanediol and meso-2,3-butanediol, implying its physiological role in favor of 2,3-butanediol formation. Maximum activity for reduction of (3S/3R)-acetoin and oxidations of meso-2,3-butanediol and glycerol was observed at pH 8.0, while it was pH 7.0 for diacetyl reduction. The enzyme exhibited relative high thermotolerance with optimum temperature of 60 °C in the oxidation–reduction reactions. Over 60 % of maximum activity was retained at 70 °C. Additionally, the GDH activity was significantly enhanced for meso-2,3-BD oxidation in the presence of Fe²⁺ and for (3S/3R)-acetoin reduction in the presence of Mn²⁺, while several cations inhibited its activity, particularly Fe²⁺ and Fe³⁺ for (3S/3R)-acetoin reduction. The properties provided potential application for single configuration production of acetoin and 2,3-butanediol .     

Enantiomeric lignans with anti-β-amyloid aggregation activity from the twigs and leaves of Pithecellobium clypearia Benth

To develop potential agents for slowing the progression of Alzheimer′s disease, two pairs of new enantiomeric lignans, including a couple of rarely 8′,9′-dinor-3′,7-epoxy-8,4′-oxyneolignanes named (7S, 8S)- and (7R, 8R)-pithecellobiumin A (1a/1b) and a pair of 2′,9′-epoxy-arylnaphthalenes named (7R, 8R, 8′R)- and (7S, 8S, 8′S)-pithecellobiumin B (2a/2b) were separated by chiral high performance liquid chromatography (HPLC). Their planar structures were elucidated by spectroscopic data analyses. The absolute configurations were determined by comparing of experimental and calculated electronic circular dichroism (ECD). The inhibitory activity on Aβ aggregation of all optical pure compounds was tested by ThT assay. Interestingly, enantiomeric inhibitors 1a (62.1%) and 1b (81.6%) exhibited different degrees of anti-Aβ aggregation activity. However, 2a (65.4%) and 2b (68.4%) showed similar inhibition rate. The different inhibition profiles were explained by molecular dynamics and docking simulation studies.     

Synthesis and antioxidant evaluation of (S,S)- and (R,R)-secoisolariciresinol diglucosides (SDGs)

Secoisolariciresinol diglucosides (SDGs) (S,S)-SDG-1 (major isomer in flaxseed) and (R,R)-SDG-2 (minor isomer in flaxseed) were synthesized from vanillin via secoisolariciresinol (6) and glucosyl donor 7 through a concise route that involved chromatographic separation of diastereomeric diglucoside derivatives (S,S)-8 and (R,R)-9. Synthetic (S,S)-SDG-1 and (R,R)-SDG-2 exhibited potent antioxidant properties (EC₅₀ = 292.17 ± 27.71 μM and 331.94 ± 21.21 μM, respectively), which compared well with that of natural (S,S)-SDG-1 (EC₅₀ = 275.24 ± 13.15 μM). These values are significantly lower than those of ascorbic acid (EC₅₀ = 1129.32 ± 88.79 μM) and α-tocopherol (EC₅₀ = 944.62 ± 148.00 μM). Compounds (S,S)-SDG-1 and (R,R)-SDG-2 also demonstrated powerful scavenging activities against hydroxyl [natural (S,S)-SDG-1: 3.68 ± 0.27; synthetic (S,S)-SDG-1: 2.09 ± 0.16; synthetic (R,R)-SDG-2: 1.96 ± 0.27], peroxyl [natural (S,S)-SDG-1: 2.55 ± 0.11; synthetic (S,S)-SDG-1: 2.20 ± 0.10; synthetic (R,R)-SDG-2: 3.03 ± 0.04] and DPPH [natural (S,S)-SDG-1: EC₅₀ = 83.94 ± 2.80 μM; synthetic (S,S)-SDG-1: EC₅₀ = 157.54 ± 21.30 μM; synthetic (R,R)-SDG-2: EC₅₀ = 123.63 ± 8.67 μM] radicals. These results confirm previous studies with naturally occurring (S,S)-SDG-1 and establish both (S,S)-SDG-1 and (R,R)-SDG-2 as potent antioxidants and free radical scavengers for potential in vivo use.     

Interaction of the N-(3-Methylpyridin-2-yl)amide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode

Background

Combined fatty acid amide hydrolase (FAAH) and cyclooxygenase (COX) inhibition is a promising approach for pain-relief. The Flu-AM1 and Ibu-AM5 derivatives of flurbiprofen and ibuprofen retain similar COX-inhibitory properties and are more potent inhibitors of FAAH than the parent compounds. However, little is known as to the nature of their interaction with FAAH, or to the importance of their chirality. This has been explored here.

Methodology/Principal Findings

FAAH inhibitory activity was measured in rat brain homogenates and in lysates expressing either wild-type or FAAH^T488A-mutated enzyme. Molecular modelling was undertaken using both docking and molecular dynamics. The (R)- and (S)-enantiomers of Flu-AM1 inhibited rat FAAH with similar potencies (IC₅₀ values of 0.74 and 0.99 μM, respectively), whereas the (S)-enantiomer of Ibu-AM5 (IC₅₀ 0.59 μM) was more potent than the (R)-enantiomer (IC₅₀ 5.7 μM). Multiple inhibition experiments indicated that both (R)-Flu-AM1 and (S)-Ibu-AM5 inhibited FAAH in a manner mutually exclusive to carprofen. Computational studies indicated that the binding site for the Flu-AM1 and Ibu-AM5 enantiomers was located between the acyl chain binding channel and the membrane access channel, in a site overlapping the carprofen binding site, and showed a binding mode in line with that proposed for carprofen and other non-covalent ligands. The potency of (R)-Flu-AM1 was lower towards lysates expressing FAAH mutated at the proposed carprofen binding area than in lysates expressing wild-type FAAH.

Conclusions/Significance

The study provides kinetic and structural evidence that the enantiomers of Flu-AM1 and Ibu-AM5 bind in the substrate channel of FAAH. This information will be useful in aiding the design of novel dual-action FAAH: COX inhibitors.