Synthetic modification of prostaglandin f(2alpha) indicates different structural determinants for binding to the prostaglandin F receptor versus the prostaglandin transporter

Several principles governing the binding of E series prostaglandins to EP receptors have emerged in recent years. The C-1 carboxyl group binds electrostatically to a conserved arginine residue in the seventh transmembrane segment of the receptor. Prostaglandin E analogs involving bioisosteric replacements of the carboxyl group, such as acylsulfonamide, are also active. In addition, structurally similar esters may also exhibit similar affinity, presumably by virtue of hydrogen bonding. Other regions of the substrate molecule appear to bind to other domains of EP receptors, either via hydrophobic interactions or by hydrogen bonding. Less information is available about the structural requirements for substrate binding to FP receptors. Prostanoids also bind to the prostaglandin transporter PGT. In this case, a conserved C-1 carboxyl group is critically important, since C-1 esters exhibit little affinity. Here we examined the binding of chemically diverse PGF(2alpha) structural analogs to the FP receptor and compared these with binding by the PG transporter. PGT recognized a wide range of anionic substituents. In contrast, the carboxylic acid group was essential for optimal binding to the FP receptor, since replacement by larger moieties with a similar pK(a), such as acylsulfonamide and tetrazole, substantially decreased binding affinity. Interestingly, insertion of cyclic substituents in the omega chain increased binding to the FP receptor but reduced affinity for PGT, and substitution for the 15-hydroxyl group produced only a modest reduction in FP receptor binding, but eliminated binding by PGT. Because extracellular PGF(2alpha) may compete for binding between FP receptors and PGT, these findings have implications for designing PGF(2alpha) analogs for treating disease states.     

Prostaglandin F(2alpha) receptor-dependent regulation of prostaglandin transport

Prostaglandin (PG) F(2alpha) may act on its G protein-coupled receptor (FP) or be imported intracellularly via a transporter, which has high affinity for PGF(2alpha) and PGE(2), but not prostacyclin (PGI(2)). In cells overexpressing the epitope-tagged FP together with the human prostaglandin transporter (hPGT), stimulation of the FP with PGF(2alpha) (1 nM-1 microM), or the less potent FP agonist, the isoprostane 8,12-iso-iPF(2alpha)-III, inhibited prostaglandin uptake via the hPGT. This effect was abolished by pretreatment of the cells with cholera toxin, but not with pertussis toxin. Furthermore, two dominant negative constructs directed against Galpha(s) partially blocked FP-mediated regulation of hPGT function, also suggesting Galpha(s) involvement in this phenomenon. Surprisingly, neither an activator (dibutyryl cyclic AMP) nor an inhibitor (H89) of cyclic AMP-dependent protein kinase had any effect on FP-mediated inhibition of hPGT activity. Furthermore, although PGF(2alpha) increases intracellular cyclic AMP via Galpha(s) activation, it does not induce phosphorylation of the transporter, excluding a role of cyclic AMP-dependent protein kinase in hPGT regulation. Activation of the PGI(2) receptor, which is also coupled to Galpha(s), does not regulate hPGT activity, despite markedly augmenting adenylate cyclase activation. In conclusion, activation of the FP reduces intracellular import of prostaglandins for metabolic inactivation, increasing prostanoid availability for membrane receptor activation. This effect seems to be mediated via Galpha(s), independent of adenylate cyclase and cyclic AMP-dependent protein kinase activation.     

Enhancement of carcinogen-induced malignant cell transformation by prostaglandin F(2 alpha)

The enhancement of carcinogen-induced malignant transformation of C3H/M2 mouse fibroblasts by the tumor promoters 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with the induction of cyclooxygenase expression and the stimulation of prostaglandin (PG) formation. Therefore, the potential of PGs, i.e., PGF(2alpha) and PGE(2), for tumor promotion was studied in the two-step C3H/M2 cell transformation assay, a model of the multi-step process of carcinogenesis. The transformation of fibroblasts was clearly enhanced by the addition of PGF(2alpha) in the promotion phase after pretreatment with a subthreshold dose of a carcinogen (3-methylcholanthrene or N-methyl-N'-nitro-N-nitrosoguanidine). No enhancement of cell transformation was observed in cells without carcinogen-pretreatment, i.e., PGF(2alpha) had no tumor initiating potential. The promotional effect was dose-dependent with a maximum at 16 nM PGF(2alpha). PGE(2) had no significant effect in this assay. Furthermore, PGF(2alpha) (but not PGE(2)) clearly reduced the inhibition of TPA-induced promotion by NS-398, an isozyme-specific inhibitor of cyclooxygenase-2. The inhibition of TPA- or TCDD-induced promotion by the non-specific cyclooxygenase inhibitor indomethacin was not affected by co-treatment with PGF(2alpha) and PGE(2). Our data suggest that PGF(2alpha) acts as an endogenous promoter of cell transformation implying that it may also be critically involved in tumor promoter-induced signalling transfer cascades ultimately triggering the process of carcinogenesis.     

Direct introduction of a formamido group into the 7 alpha (6 alpha)-position of cephalosporins (penicillins)

A novel direct introduction of a formamido group into the 7 alpha (6 alpha)-position of cephalosporins (penicillins) was achieved by treatment of 7 beta (6 beta)-[(3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-1- ylidene)methylimino]cephem (penam) esters with N,N-bis(trimethylsilyl)formamide, followed by deblocking with Girard reagent T to give the corresponding 7 alpha (6 alpha)-formamido-7 beta (6 beta)-amino derivatives. Three 7 alpha-formamidocephalosporins were prepared by the conventional N-acylation of 7 alpha-formamidocephem. All of them were resistant to beta-lactamases, showing similar MIC values against both of a pair of a beta-lactamase-producing strain and the corresponding non or low-producing strain of the same species of bacteria, when tested on Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Proteus vulgaris, Morganella morganii, Enterobacter cloacae and Citrobacter freundii.     

Cyclopentane-1,3-dione: a novel isostere for the carboxylic acid functional group. Application to the design of potent thromboxane (A2) receptor antagonists

Cyclopentane-1,3-diones are known to exhibit pK(a) values typically in the range of carboxylic acids. To explore the potential of the cyclopentane-1,3-dione unit as a carboxylic acid isostere, the physical-chemical properties of representative congeners were examined and compared with similar derivatives bearing carboxylic acid or tetrazole residues. These studies suggest that cyclopentane-1,3-diones may effectively substitute for the carboxylic acid functional group. To demonstrate the use of the cyclopentane-1,3-dione isostere in drug design, derivatives of a known thromboxane A(2) prostanoid (TP) receptor antagonist, 3-(3-(2-(4-chlorophenylsulfonamido)ethyl)phenyl)propanoic acid (12), were synthesized and evaluated in both functional and radioligand-binding assays. A series of mono- and disubstituted cyclopentane-1,3-dione derivatives (41-45) were identified that exhibit nanomolar IC(50) and K(d) values similar to 12. Collectively, these studies demonstrate that the cyclopentane-1,3-dione moiety comprises a novel isostere of the carboxylic acid functional group. Given the combination of the relatively strong acidity, tunable lipophilicity, and versatility of the structure, the cyclopentane-1,3-dione moiety may constitute a valuable addition to the palette of carboxylic acid isosteres.     

Arachidonic acid release and prostaglandin F(2alpha) formation induced by anandamide and capsaicin in PC12 cells

Anandamide, an endogenous agonist of cannabinoid receptors, activates various signal transduction pathways. Anandamide also activates vanilloid VR(1) receptor, which was a nonselective cation channel with high Ca(2+) permeability and had sensitivity to capsaicin, a pungent principle in hot pepper. The effects of anandamide and capsaicin on arachidonic acid metabolism in neuronal cells have not been well established. We examined the effects of anandamide and capsaicin on arachidonic acid release in rat pheochromocytoma PC12 cells. Both agents stimulated [3H]arachidonic acid release in a concentration-dependent manner from the prelabeled PC12 cells even in the absence of extracellular CaCl(2). The effect of anandamide was neither mimicked by an agonist nor inhibited by an antagonist for cannabinoid receptors. The effects of anandamide and capsaicin were inhibited by phospholipase A(2) inhibitors, but not by an antagonist for vanilloid VR(1) receptor. In PC12 cells preincubated with anandamide or capsaicin, [3H]arachidonic acid release was marked and both agents were no more effective. Co-addition of anandamide or capsaicin synergistically enhanced [3H]arachidonic acid release by mastoparan in the absence of CaCl(2). Anandamide stimulated prostaglandin F(2alpha) formation. These findings suggest that anandamide and capsaicin stimulated arachidonic acid metabolism in cannabinoid receptors- and vanilloid VR(1) receptor-independent manner in PC12 cells. The possible mechanisms are also discussed.     

Adrenomedullin inhibits spontaneous and bradykinin-induced but not oxytocin- or prostaglandin F(2alpha)-induced periodic contraction of rat uterus

In isolated rat uterine strips, adrenomedullin (AM) inhibited the spontaneous periodic contraction in a concentration-dependent manner (IC(50)=22.3+/-0.7 nM). The inhibitory effect of AM was prevented by either AM(22-52), a putative antagonist for AM receptors, or calcitonin gene-related peptide (CGRP)(8-37), a putative antagonist for CGRP receptors. AM also attenuated bradykinin (BK)-induced periodic uterine contraction, which was blocked by AM(22-52) or CGRP(8-37), whereas AM had no effect on the periodic contraction caused by oxytocin or prostaglandin F(2alpha) (PGF(2alpha)). RT-PCR analysis showed that mRNAs for calcitonin receptor-like receptor (CRLR), receptor-activity-modifying protein (RAMP)1, RAMP2 and RAMP3 were expressed in the rat uterus. These results demonstrate that AM selectively inhibits spontaneous and BK-induced periodic contraction via activating receptors for AM and CGRP.     

Behavioral responses to intramuscular injections of prostaglandin F(2 alpha) in female pigs

This study investigated the effect of different doses (0-1.25 mg/kg IM) of prostaglandin (PG) F(2 alpha) on the behavior of female pigs (Sus scrofa). Six-month-old cyclic nulliparous sows (gilts) were housed and tested individually in strawed pens (2.8 x 1.7 m). All doses of PGF(2)alpha induced rooting, pawing at the ground, and gathering straw. In the hour following treatment the frequency of pawing increased with increasing dose to reach a maximum level with the highest dose given. The frequency to gather straw was highest in pigs treated with the lowest dose (0.008 mg/kg). The frequency of oronasal contact with the floor and pen walls was unaffected by dose. Scratching, locomotion, and changes in body posture were highest following treatment with the three highest doses of PGF(2 alpha). Many of the behaviors observed following PGF(2 alpha) treatment are characteristic of prepartum nesting behavior in pregnant sows. We conclude that two key components of maternal nest-building behavior, pawing, and gathering straw, are affected differentially by different doses of PGF(2 alpha). The implications of these results on the mechanisms underlying maternal nest building in pigs are discussed.     

Differential regulation of prostaglandin F(2alpha) receptor isoforms by protein kinase C

Prostaglandin F(2alpha) receptors (FP) are G protein-coupled receptors that bind prostaglandin F(2alpha) (PGF(2alpha)), resulting in the activation of an inositol phosphate (IP) second messenger pathway. Alternative mRNA splicing generates two FP receptor isoforms. These isoforms, designated FP(A) and FP(B), are otherwise identical except for their carboxyl termini. FP(B) is essentially a truncated version of FP(A) that lacks the 46 carboxyl-terminal amino acids, including four putative protein kinase C (PKC) phosphorylation sites. Until now, functional differences between these FP receptor isoforms have not been identified. We now report that pretreatment with the PKC inhibitor bisindolylmaleimide I enhanced PGF(2alpha)-stimulated IP accumulation in transfected cells stably expressing the FP(A) isoform but not in cells stably expressing the FP(B) isoform. Whole-cell phosphorylation experiments showed a strong agonist-dependent phosphorylation of the FP(A) isoform but little or no phosphorylation of the FP(B). Pretreatment of cells with bisindolylmaleimide I decreased PGF(2alpha)-stimulated phosphorylation of the FP(A) isoform consistent with a PKC-dependent phosphorylation. In vitro phosphorylation of an FP(A) carboxyl-terminal fusion protein by recombinant PKCalpha showed that the carboxyl terminus of the FP(A) is a substrate for PKC. These results suggest that PKC-dependent phosphorylation is responsible for differential regulation of second messenger signaling by FP prostanoid receptor isoforms.     

Effects of caffeine and paracetamol alone or in combination with acetylsalicylic acid on prostaglandin E(2) synthesis in rat microglial cells

Paracetamol has mild analgesic and antipyretic properties and is, along with acetylsalicylic acid, one of the most popular "over the counter" analgesic agents. However, the mechanism underlying its clinical effects is unknown. Another drug whose mechanism of action is unknown is caffeine, which is often used in combination with other analgesics, augmenting their effect. We investigated the inhibitory effect of paracetamol and caffeine on lipopolysaccharide (LPS)-induced cyclooxygenase (COX)- and prostaglandin (PG)E(2)-synthesis in primary rat microglial cells and compared it with the effect of acetylsalicylic acid, salicylic acid, and dipyrone. Furthermore, combinations of these drugs were used to investigate a possible synergistic inhibitory effect on PGE(2)-synthesis. Both paracetamol (IC(50)=7.45 microM) and caffeine (IC(50)=42.5 microM) dose-dependently inhibited microglial PGE(2) synthesis. In combination with acetylsalicylic acid (IC(50)=3.12 microM), both substances augmented the inhibitory effect of acetylsalicylic acid on LPS-induced PGE(2)-synthesis. Whereas paracetamol inhibited only COX enzyme activity, caffeine also inhibited COX-2 protein synthesis. These results are compatible with the view that the clinical activity of paracetamol and caffeine is due to inhibition of COX. Furthermore, these results may help explain the clinical experience of an adjuvant analgesic effect of caffeine and paracetamol when combined with acetylsalicylic acid.     

Replacement of aromatic or heteroaromatic groups in nonsteroidal antiinflammatory agents with the ferrocene group

Ferrocene analogues of the antiinflammatory agents tolmetin (1), fenbufen (2), flurbiprofen (3), and fenclofenac (4) were synthesized and tested for biological activity. The derivatives exhibited little or no antiarthritic or platelet antiaggregatory activity, indicating that the ferrocene moiety is a poor bioisostere for aromatic or heteroaromatic groups in nonsteroidal antiinflammatory agents.     

Synthesis of 5-[1-hydroxy(or methoxy)-2-bromo(or chloro)ethyl]-2'-deoxyuridines and related halohydrin analogues with antiviral and cytotoxic activity

A series of new 5-(1-hydroxy-2-haloethyl)-2'-deoxyuridines (3, 6, 8) were synthesized in 60-70% yields by addition of HOX (X = Br, Cl, I) to the vinyl substituent of the respective 5-vinyl-2'-deoxyuridines (2, 5, 7). Treatment of 3a,b with methanolic sulfuric acid afforded the corresponding 5-(1-methoxy-2-haloethyl)-2'-(deoxyuridines (4a,b). The 5-(1-hydroxy-2-chloroethyl) (3b), 5-(1-methoxy-2-bromoethyl) (4a), 5-(1-hydroxy-2-bromo-2-(ethoxycarbonyl)ethyl) (6a), and 5-(1-hydroxy-2-iodo-2-(ethoxycarbonyl)ethyl) (6b) derivatives exhibited in vitro antiviral activity (ID50 = 0.1-1 microgram/mL range) against herpes simplex virus type 1 (HSV-1). 5-(1-Hydroxy-2-bromo-2-(ethoxycarbonyl)-ethyl)-2'-deoxyuridine (6a) was the most active cytotoxic agent in the in vitro L1210 screen exhibiting an ED50 of 11 micrograms/mL relative to melphalan (ED50 = 0.15 micrograms/mL).     

Protection of the hydroxyl function of hydroxyproline and serine in peptide synthesis by the 2-methoxyethoxymethyl (Mem) group

Some tri- and hexapeptides containing hydroxyproline and serine were synthesized using the 2-methoxyethoxymethyl group for the protection of the hydroxyl function. For N-protection not only the NpS and the Z group but also the Boc group can be used. As compared to the But group the Mem group is much more stable towards TFA. Application of Mem protection allows a complete deblocking of the Boc group without affecting the Mem group.     

Studies on the in vitro inhibition of prostaglandin synthetase by fenclorac (alpha, m-dichloro-p-cyclohexylphenylacetic acid) and indomethacin.

Analysis of the effect of a new nonsteroidal anti-inflammatory compound, fenclorac (αm-di-chloro-p-cyclohexylphenylacetic acid), on prostaglandin biosynthesis in vitro has shown the drug to be a potent inhibitor of this enzyme system. Fenclorac was ten times as active as indomethacin in inhibiting prostaglandin synthesis in bovine seminal vesicle microsomes. Inhibition by fenclorac was not appreciably dependent upon preincubation of drug with enzyme for maximal activity, and was reversible by dilution. The inhibitory activity of indomethacin was time-dependent and not readily reversible. Kinetic analysis indicated that fenclorac is a competitive inhibitor of prostaglandin synthe- tase, irrespective of preincubation conditions, whereas indomethacin inhibits noncompetitively if pre- incubated with enzyme, and competitively in the absence of any preincubation. Both drugs protected or stabilized prostaglandin synthetase activity under extended preincubation conditions.