Françoise Heymans

Biosynthesis of platelet-activating factor: I. Evidence for an acetyl-transferase activity in murine macrophages

Platelet-activating factor (PAF-acether; 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) is released from murine peritoneal adherent cells by inflammatory and non-inflammatory stimuli. We have found, in extracts from these cells, an enzyme activity that synthesizes. PAF-acether from synthetic lyso-PAF-acether by transferring the acetyl moiety of acetyl-coenzyme A onto the lyso-PAF-acether molecule. The enzyme is stabilized by 1 mM dithiothreitol, is calcium-dependent, has an apparent Km of 172 microM for acetyl-CoA and is active in a 6-8 pH range. When the acetyl-CoA substrate is replaced by propionyl-CoA, an ether lipid is produced which turns out to be as potent an aggregating agent as PAF-acether. In all cases, the products of the reaction were characterized by their behaviour in platelet-aggregation tests and their high-pressure liquid chromatography (HPLC) elution profiles. The precise definition of this acetyl-transferase is of primary importance for the development of new pharmacological agents capable of moduling a potent platelet aggregating factor.

Platelet activating factor (PAF-acether): total synthesis of 1-O-octadecyl 2-O-acetyl sn-glycero-3-phosphoryl choline

Platelet-activating factor (PAF), a mediator of anaphylaxis and inflammation, is released from IgE-sensitized antigen-stimulated basophils [l-3]. PAF is a low molecular weight phospholipid which is sensitive to hydrolysis by phospholipases AZ, C, and D, but not by lipase from Rhizopus arrhizus or sphingomyelinase C [4]. PAF appeared to be a glycerophospholipid with a choline polar head group devoid of an ester function at position 1 [4,5]. The presence of PAF has been demonstrated in cells from several species including man [3,6,7], rabbit [l-3], rat [8] and pig [4]. PAF has been obtained from rat and mouse peritoneal macrophages upon stimulation by the ionophore A23187 or exposure to phagocytable particles [9]. PAF has been shown to induce aggregation of blood platelets from rabbit [1,3],man [lO],rat [ll] andguineapig [12].The structure of the molecule responsible for PAF activity has been elucidated [13,14]. This was obtained by acetylation of 2-lyso plasmalogens possessing a 30phosphoryl choline group [ 131 or a 3-O-phosphoryl ethanolamine group followed by reaction with diazomethane and quaternization with methyl iodide [ 141. This result lead us to propose a 1 -O-alkyl 2-0-acetyl sn-glycero3-phosphoryl choline structure and the terminology ‘PAF-acether’ for this substance. However, total synthesis was necessary to confirm the structure of PAF-acether. Here, we report the total synthesis of l-O-octadecyl20acetyl sn-glycero3-phosphoryl choline, which has the same biological activity as the natural PAF-acether.

Alkyl analogs of diacylglycerol as activators of protein kinase C

Diacylglycerols which activate protein kinase C have the 1,2‐sn configuration. Short‐chain saturated fatty acids or long‐chain unsaturated fatty acids are required for supporting the potency of these lipids. Using alkyl analogs such as 1‐O‐decyl‐2‐O‐decanoylglycerol, 1‐O‐decanoyl‐2‐O‐decylglycerol, 1,2‐O‐didecyl‐glycerol, 1‐O‐hexadecyl‐2‐O‐acetylglycerol and 1‐O‐decyl‐2‐O‐acetylglycerol, we showed that the ether bond was consistently associated with a loss of activity to varying extents. The results suggest that the ester bond in the 1‐position is a major determinant of diacylglycerol‐mediated protein kinase activation.

Pharmacological profile of PMS777, a new AChE inhibitor with PAF antagonistic activity

The key pathophysiological mechanisms in Alzheimers disease involve the selective loss of cholinergic neurons and pro-inflammatory mediator-related chronic inflammatory responses in the brain, therefore interventions of these processes are crucial to the treatment of this disease. In the present study, the pharmacological profile of PMS777, a new acetylcholinesterase (AChE) inhibitor with platelet-activating factor (PAF) antagonistic activity, has been evaluated in vitro and in vivo. PMS777 (1-100 microM) dose-dependently inhibited PAF-induced rabbit platelet aggregation by competing with [3H]PAF for its receptor on platelets, and protected a human neuroblastoma cell line SH-SY5Y against PAF-induced neurotoxicity. Moreover, it markedly inhibited brain AChE activity in mice and showed a modest selectivity for AChE (AChE: IC50=2.48+/-0.12 microM; butyrylcholinesterase: IC50=4.47+/-0.15 microM). Ex vivo, PMS777 (5, 10, 20 or 40 mg/kg i.p.) reduced brain AChE activity in a dose-dependent manner. In-vivo studies revealed that PMS777 (0.25, 0.5, 1, 2.5 or 5 mg/kg i.p.) could reverse scopolamine-induced memory retrieval deficits in mice, and displayed a typical bell-shaped dose-response relationship. Taken together, these results demonstrate that PMS777 possesses dual activities for PAF receptor antagonism and AChE inhibition, suggesting that this compound may be a promising lead compound for further investigation related to the treatment for Alzheimers disease.

Structure–activity relationships in platelet-activating factor (PAF). 11-From PAF-antagonism to phospholipase A2 inhibition: syntheses and structure–activity relationships in 1-arylsulfamido-2-alkylpiperazines

1-Benzoyl-2-alkyl piperazines are strong inhibitors of Group I and II secreted PLA(2)s. An improvement of their activity was obtained by replacing the amide function by a sulfamide and by introduction of electrodonor substituents on the para position of the benzenesulfonyl moiety. Neither the position on one of the carbon of the piperazine ring nor the absolute configuration of this carbon have an effect on the affinity for one or the other group of PLA(2), but the lipophilicity remains for these series an essential parameter. In addition structure-activity relationships allow new hypothesis on interaction of these piperazine derivatives with the catalytic site of PLA(2)s.

4-Alkoxybenzamidines as new potent phospholipase A2 inhibitors.

A series of 4-alkoxybenzamidines was synthesized, varying the number of carbons of the alkyl chain, and their potency as phospholipase A2 (PLA2) inhibitors was evaluated. The relationship between their capacity to inhibit PLA2 activity and their lipophilicity was examined. The optimum of the inhibitory effect against two extracellular PLA2S from rabbit platelets and bovine pancreas was observed with compounds bearing an alkyl chain of 12 and 14 carbons. These 4-dodecyl and tetradecyloxbenzamidines inhibited bovine pancreatic and rabbit platelet lysate PLA2S with IC50 values of 3 microM and 5-5.8 microM, respectively. The mechanism of inhibition was of the competitive type. In addition, 4-tetradecyloxbenzamidine was shown to exert an antiinflammatory effect in vivo on the carrageenan-induced rat paw oedema. These results show that 4-tetradecyloxybenzamidine will serve as an interesting tool to investigate the physiological role of mammalian-secreted PLA2, both in vitro and in vivo.

PAF receptor structure: a hypothesis.

Different hypotheses of the structure of platelet-activating factor (PAF) receptor based on structure-activity relationships of agonists and antagonists are reviewed. For an agonistic effect, strong hydrophobic interactions and an ether function are required in position-1 of the glycerol backbone; chain length limitations and steric hindrance demand a small group in position-2. The unusual structural properties of non-PAF-like antagonists required 3-D electrostatic potential calculations. This method applied to seven potent antagonists suggests a strong “Cache-oreilles” (ear-muff) effect,i.e., two strong electronegative wells (isocontour at −10 Kcal/mole) are located at 180° to each other and at a relatively constant distance. Initial consideration of the “Cache-oreilles” effect implied the structure of a bipolarized cylinder of 10–12 Å diameter for the receptor. However, very recent results on studies with agonists and antagonists structurally similar to PAF suggest that the receptor may in fact be a multi-polarized cylinder.

Differential effect of PMS777, a new type of acetylcholinesterase inhibitor, and galanthamine on oxidative injury induced in human neuroblastoma SK-N-SH cells.

In the search for highly selective and potent cholinesterase inhibitors (AChEI) being able to improve oxidative injury, PMS777, a tetrahydrofuran derivative, was designed as a novel dual PAF and acetylcholinesterase inhibitor. The aim of this study was to investigate the modulatory effects of PMS777 and galanthamine, another AChEI, on the oxidative injury induced in neuronal cells. The SK-N-SH cells stimulated with LPS+IL-(1beta) were selected to investigate the direct inhibitory effect of PMS777 and galanthamine. LPS+IL-(1beta) induced oxidative injury as assessed by ROS production (29%), GSH depletion (11%) and loss of mitochondrial activity (22%). GSH depletion was never decreased by either drug. In contrast, ROS production and mitochondrial activity were totally prevented by addition of PMS777 but not galanthamine. PMS777 also inhibits butylcholinesterase and it shows selectivity for acetylcholinesterase. Thus, this PAF antagonist inaugurates a new type of AChEI, able to fight oxidative injury. Therefore, PMS777 could be of interest on patients with cognitive impairments and inflammatory damage, as in AD.

Novel piperazine derivative PMS1339 exhibits tri-functional properties and cognitive improvement in mice

Amyloid-beta-induced neuroinflammation plays a central role in the extensive loss of cholinergic neurons and cognitive decline in Alzheimers disease. The acetylcholinesterase (AChE) inhibitors are the first class of drugs used to enhance surviving cholinergic activities. However, their limited effectiveness following long-term treatment raises a need for new multi-target therapies. We report herein a novel piperazine derivative compound PMS1339 possesses multifunctional properties including anti-platelet-activating factor, AChE inhibition, Abeta aggregation inhibition and cognitive improvement. PMS1339 could significantly inhibit both mice brain AChE (IC50=4.41+/-0.63 microM) and sera butyrylcholinesterase (BuChE, IC50=1.09+/-0.20 microM). PMS1339 was also found to inhibit neuronal AChE secreted by SH-SY5Y cell line (IC50=17.95+/-2.31 microM). Enzyme kinetics experiments performed on electric eel AChE indicated that PMS1339 acts as a mixed type competitive AChE inhibitor. Molecular docking studies using the X-ray crystal structure of AChE from Torpedo californica elucidated the interactions between PMS1339 and AChE: PMS1339 is well buried inside the active-site gorge of AChE interacting with Trp84 at the bottom, Tyr121 halfway down and Trp279 at the peripheral anionic site (PAS). Thioflavin T-based fluorimetric assay revealed the ability of PMS1339 to inhibit AChE-induced Abeta aggregation. In-vivo study indicated PMS1339 (1 mg/kg i.p.) reversed scopolamine-induced memory impairment in mice. Overall, these findings indicated that PMS1339 exhibits tri-functional properties in vitro and cognitive improvement in vivo, and revealed the emergence of a multi-target-directed ligand to tackle the determinants of Alzheimers disease.

PAF receptor and “cache-oreilles” effect. Simple PAF antagonists

Nine simple and structurally flexible PAF antagonists were synthesized and their inhibitory effects on PAF induced platelet aggregation were measured. Compounds with PAF antagonistic activity exhibited a negative electrostatic potential generated by two trimethoxyphenyl groups (isocontour at −10 Kcal/mole) at various distances between the negative clouds. The optimal distance between the atoms generating the “cache-oreilles” system for exhibiting potent PAF antagonistic activity is estimated to be 11–13 Å. In the flexible molecules studied, the dispersion of the electronic distribution is not necessarily favorable for anti-PAF activity. The data support the simple bipolarized model for the PAF receptor that has been proposed by the authors.