Robert S. Jacobs

Molecular pharmacology of manoalide: Inactivation of bee venom phospholipase A2☆

The marine natural product manoalide (MLD) was shown to directly inactivate bee venom phospholipase A2 (PLA2). Inactivation was pH dependent (maximum inactivation occurred at pH 8.0), time dependent and concentration dependent. The IC50 was estimated at 0.05 microM and virtually complete inactivation of the enzyme occurred at 4.0 microM. The time-dependent loss of PLA2 activity suggested that inactivation does not follow typical Michaelis-Menten kinetics. Reversibility was studied directly by dilution and dialysis; both methods were ineffective in dissociating the MLD-PLA2 complex. A kinetic plot of initial velocity (v) versus [PLA2] supported our hypothesis that MLD apparently inactivates bee venom PLA2 by an irreversible mechanism.

Scytonemin-a marine natural product inhibitor of kinases key in hyperproliferative inflammatory diseases

Chronic inflammatory diseases are associated with the persistent production of proinflammatory mediators and tissue hyperplasia. Protein kinases play an important role in regulating the signaling events controlling mediator release and cell proliferation. For instance, in in vivo models, the tumor promoter, phorbol myristate acetate (PMA), is used as an initiator of inflammation, both acute and chronic. PMA produces these effects by activating protein kinase C (PKC), an enzyme involved in a number o f cellular activities, including growth, differentiation, and mediator formation [1]. In addition, cell cycle progression is highly regulated predominantly through kinase activity. One example is polo-like kinase 1 (PLK1), an enzyme important for the G2-M transition and mitotic spindle formation. Therefore, by targeting certain kinases involved in both processes, it may be possible to reduce the adverse nature o f chronic inflammatory disorders. Here we report the novel pharmacological properties o f scy-

Pharmacological characterization of the pseudopterosins: Novel anti-inflammatory natural products isolated from the Caribbean soft coral, Pseudopterogorgia elisabethae

Pseudopterosin E (PSE), a C-10 linked fucose glycoside and pseudopterosin A (PSA), a C-9 xylose glycoside isolated from the marine gorgonian Pseudopterogorgia elisabethae were both effective in reducing PMA-induced mouse ear edema when administered topically (ED50 (microg/ear) PSE(38), PSA(8)) or systemically (ED50 (mg/kg, i.p.) PSE (14), PSA (32)). Both compounds exhibited in vivo analgesic activity in phenyl-p-benzoquinone-induced writhing (ED50 (mg/kg, i.p.) PSE(14), PSA(4). PSE inhibited zymosan-induced writhing (ED50 = 6 mg/kg, i.p.), with a concomitant dose-dependent inhibition of peritoneal exudate 6-keto-prostaglandin F1alpha (ED50 = 24 mg/kg) and leukotriene C4 (ED50 = 24 mg/kg). In vitro, the pseudopterosins were inactive as inhibitors of phospholipase A2, cyclooxygenase, cytokine release, or as regulators of adhesion molecule expression. PSA inhibited prostaglandin E2 and leukotriene C4 production in zymosan-stimulated murine peritoneal macrophages (IC50 = 4 microM and 1 microM, respectively); however, PSE was much less effective. These data suggest that the pseudopterosins may mediate their anti-inflammatory effects by inhibiting eicosanoid release from inflammatory cells in a concentration and dose-dependent manner.

A marine natural product, patellamide D, reverses multidrug resistance in a human leukemic cell line

A cyclic octapeptide (patellamide D) isolated from the marine tunicate, Lissaclinum patella, acts as a resistance-modifying agent in the multidrug resistant CEM/VLB100 human leukemic cell line. A three-day microculture tetrazolium proliferation assay was used to determine the 50% inhibitory concentration (IC50) for vinblastine, colchicine and adriamycin and calculate the degree of resistance modulation. Patellamide D at 3.3 microM was compared with 5.1 microM verapamil in modulating drug resistance in vitro. The IC50 for vinblastine was reduced from 100 ng/ml to 1.5 ng/ml in the presence of patellamide D or to 2.1 ng/ml when exposed to verapamil. Colchicine cytotoxicity was enhanced only 1.4-fold by verapamil, as compared with 2.8-fold using patellamide D (IC50 was reduced from 140 ng/ml to 100 ng/ml or 50 ng/ml). Adriamycin toxicity was reduced from IC50 > 1000 ng/ml to 110 ng/ml and 160 ng/ml when coexposed to patellamide D and verapamil, respectively. Our results indicate that patellamide D acts as a selective antagonist in multidrug resistance and stresses the importance of investigating marine-derived compounds as a potential new source for modulators of the drug-resistance phenotype.

Inactivation of human synovial fluid phospholipase a2 by the marine natural product, manoalide

The marine natural product, manoalide (MLD), was investigated to determine if this drug inhibited purified human synovial fluid phospholipase A2 (HSF-PLA2). Utilizing classical Michaelis-Menten kinetics, apparent Km and Vmax values for HSF-PLA2 of 1.34 mM and 0.47 mumol [3H]palmitic acid released/min/mg protein were obtained using dipalmitoylphosphatidylcholine (DPPC) as the substrate, and 38.0 microM and 18.8 mumol [3H]arachidonic acid released/min/mg protein with Escherichia coli as a natural substrate. These kinetic parameters were utilized subsequently to evaluate the inhibitory effects of manoalide on HSF-PLA2. Inhibition of HSF-PLA2 by MLD was concentration and time dependent with IC50 values of 0.2 and 0.02 microM for DPPC and E. coli respectively. Dialysis studies and examination of DPPC or E. coli hydrolysis versus enzyme concentration indicate that MLD is an irreversible inhibitor of HSF-PLA2. Substrate specificity was also examined in the absence and presence of MLD using dipalmitoylphosphatidylethanolamine (DPPE) as a substrate. MLD inhibited the hydrolysis of DPPE (greater than 90% inhibition at 2 microM), and preliminary results indicate that DPPC was more readily hydrolyzed than DPPE under the substrate conditions of the assay. While the cellular source of secreted HSF-PLA2 is unknown, these studies indicate that MLD can inactivate secreted phospholipase A2 isolated from patients with inflammatory joint disease.

Identification of anti-inflammatory diterpenes from the marine gorgonian Pseudopterogorgia elisabethae

Analysis of the terpene metabolites of Pseudopterogorgia elisabethae collected from the Florida Keys has resulted in the identification of a novel hydroxyquinone, elisabethadione (1), as well as new pseudopterosins and seco-pseudopterosins. Anti-inflammatory assays indicate that elisabethadione is more potent than the well characterized pseudopterosin A and E. This report also describes the co-occurrence of pseudopterosins and seco-pseudopterosins, diterpenes with amphilectane and serrulatane skeletons, respectively. This together with our previously described isolation of elisabethatriene as the sole diterpene cyclase product in P. elisabethae suggests that the amphilectane and serrulatane families of diterpenes are derived from the same geranylgeranyl diphosphate cyclase product.

Luffariellolide, an anti-inflammatory sesterterpene from the marine spongeLuffariella sp.

Luffariellolide (2) is a sesterterpene from the Palauan spongeLuffariella sp. that has useful anti-inflammatory properties. In contrast with the irreversible action of manoalide (1) on phospholipase A2, luffariellolide (2) is a slightly less potent but partially reversible PLA2 inhibitor.

Two-step inactivation of bee venom phospholipase A2 by scalaradial.

Scalaradial (SLD), a marine natural product isolated from the sponge (Cacospongia sp., possesses anti-inflammatory properties in vivo and in vitro (Pharmacologist 32: 168, 1990). In this study we characterize its effects against bee venom phospholipase A2 (PLA2; EC 3.1.1.4). SLD is a potent inactivator of bee venom PLA2 with an IC50 value of 0.07 microM. Inactivation of bee venom PLA2 occurred in a time-dependent, irreversible manner. The rate of inactivation followed first-order reaction kinetics and was dependent on the concentration of SLD. Kinetic analysis suggested a two-step mechanism of inactivation: an initial apparent noncovalent binding (Ki = 4.5 x 10(-5) M) followed by covalent modification. The rate of inactivation was reduced markedly in the presence of excess phosphatidylcholine, suggesting that modification of the enzyme occurs at or near the substrate binding site.

In vitro inactivation of the neurotoxic action of β-bungarotoxin by the marine natural product, manoalide

The irreversible neurotoxic action of β-bungarotoxin (β-BuTx) can be prevented by preincubation of the toxin with manoalide, a non-steroidal anti-inflammatory agent. Manoalide was also found to inactivate purified phospholipase A2 and thus prevent hydrolysis of phosphatidylcholine. PLA2 is a component found in several neurotoxic venoms and is also a rate limiting enzyme important in phospholipid metabolism and prostaglandin synthesis in man.

Structure and biosynthesis of novel conjugated polyene fatty acids from the marine green algaAnadyomene stellata

Novel polyunsaturated fatty acids with four conjugated double bonds were found in extracts of the green macroalga,Anadyomene stellata. The isolation of five of these with different chain lengths and varying degrees of unsaturation −16∶5, 18∶4, 20∶5, 20∶6, and 22∶7—was accomplished by organic extraction followed by a combination of vaccum and high-performarce liquid chromatography. One of these that was a novel substance (22∶7) was characterized as 4ZZ,7Z,9E,13Z,16Z,19Z-docosaheptaenoic acid and assigned the trivial name stellaheptaenoic acid. The structure of this new compound, isolated as its methyl ester derivative, was deduced from detailed nuclear magnetic resonance, gas chromatography/mass spectrometry (GC/MS), and other spectroscopic methods. Incubation of a chloroplast preparation, isolated from a crude algal homogenate by differential centrifugation, with six unsaturated fatty acids (palmitoleic, 6Z,9Z,12Z,15Z-octadecatetraenoic acid, arachidonic acid, eicosapentaenoic acid, 7Z,10Z,13Z,16Z-docosatetraenoic acid, and 4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid) resulted in substantially increased synthesis of unique tetraene compouds as detected by ultraviolet spectrophotometry and tentatively identified by GC/MS.