Kilian W. Conde‐Frieboes

A novel Ca2+-independent phospholipase A2 (PLA2) has recently been purified from the murine macrophage-like cell line P388D1 (Ackermann, E. J., Kempner, E. S., and Dennis, E. A.(1994) J. Biol. Chem. 269, 9227-9233). This enzyme is now shown to be inhibited by palmitoyl trifluoromethyl ketone (PACOCF3), arachidonyl trifluoromethyl ketone (AACOCF3), and a bromoenol lactone (BEL). Both PACOCF3 and AACOCF3 were found to inhibit the macrophage PLA2 in a concentration-dependent manner. PACOCF3 was found to be ~4-fold more potent than AACOCF3, with IC50 values of 3.8 μM (0.0075 mol fraction) and 15 μM (0.028 mol fraction), respectively. Reaction progress curves in the presence of either inhibitor were found to be linear, and the PACOCF3•PLA2 complex rapidly dissociated upon dilution.BEL was also found to inhibit the macrophage PLA2 in a concentration-dependent manner, with half-maximal inhibition observed at 60 nM after a 5-min preincubation at 40°C. Inhibition was not reversed after extensive dilution of the enzyme into assay buffer. Treatment of the PLA2 with BEL resulted in a linear, time-dependent inactivation of activity, and the rate of this inactivation was diminished in the presence of PACOCF3. In addition, PLA2 treated with [3H]BEL resulted in the covalent labeling of a major band at Mr 80,000. Inactivation of the PLA2 by 5,5′-dithiobis(2-nitrobenzoic acid) prior to treatment with [3H]BEL resulted in the near complete lack of labeling consistent with covalent irreversible suicide inhibition of the enzyme. The labeling of a Mr 80,000 band rather than a Mr 40,000 band upon treatment with [3H]BEL distinguishes the macrophage Ca2+-independent PLA2 from a previously identified myocardial Ca2+-independent PLA2 and provides strong evidence that the Mr 80,000 protein is the catalytic subunit. A novel Ca2+-independent phospholipase A2 (PLA2) has recently been purified from the murine macrophage-like cell line P388D1 (Ackermann, E. J., Kempner, E. S., and Dennis, E. A.(1994) J. Biol. Chem. 269, 9227-9233). This enzyme is now shown to be inhibited by palmitoyl trifluoromethyl ketone (PACOCF3), arachidonyl trifluoromethyl ketone (AACOCF3), and a bromoenol lactone (BEL). Both PACOCF3 and AACOCF3 were found to inhibit the macrophage PLA2 in a concentration-dependent manner. PACOCF3 was found to be ~4-fold more potent than AACOCF3, with IC50 values of 3.8 μM (0.0075 mol fraction) and 15 μM (0.028 mol fraction), respectively. Reaction progress curves in the presence of either inhibitor were found to be linear, and the PACOCF3•PLA2 complex rapidly dissociated upon dilution. BEL was also found to inhibit the macrophage PLA2 in a concentration-dependent manner, with half-maximal inhibition observed at 60 nM after a 5-min preincubation at 40°C. Inhibition was not reversed after extensive dilution of the enzyme into assay buffer. Treatment of the PLA2 with BEL resulted in a linear, time-dependent inactivation of activity, and the rate of this inactivation was diminished in the presence of PACOCF3. In addition, PLA2 treated with [3H]BEL resulted in the covalent labeling of a major band at Mr 80,000. Inactivation of the PLA2 by 5,5′-dithiobis(2-nitrobenzoic acid) prior to treatment with [3H]BEL resulted in the near complete lack of labeling consistent with covalent irreversible suicide inhibition of the enzyme. The labeling of a Mr 80,000 band rather than a Mr 40,000 band upon treatment with [3H]BEL distinguishes the macrophage Ca2+-independent PLA2 from a previously identified myocardial Ca2+-independent PLA2 and provides strong evidence that the Mr 80,000 protein is the catalytic subunit.

Cellular levels of free arachidonic acid (AA) are controlled by a deacylation/reacylation cycle whereby the fatty acid is liberated by phospholipases and reincorporated by acyltransferases. We have found that the esterification of AA into membrane phospholipids is a Ca(2+)-independent process and that it is blocked up to 60-70% by a bromoenollactone (BEL) that is a selective inhibitor of a newly discovered Ca(2+)-independent phospholipase A2 (PLA2) in macrophages. The observed inhibition correlates with a decreased steady-state level of lysophospholipids as well as with the inhibition of the Ca(2+)-independent PLA2 activity in these cells. This inhibition is specific for the Ca(2+)-independent PLA2 in that neither group IV PLA2, group II PLA2, arachidonoyl-CoA synthetase, lysophospholipid:arachidonoyl-CoA acyltransferase, nor CoA-independent transacylase is affected by treatment with BEL. Moreover, two BEL analogs that are not inhibitors of the Ca(2+)-independent PLA2--namely a bromomethyl ketone and methyl-BEL--do not inhibit AA incorporation into phospholipids. Esterification of palmitic acid is only slightly affected by BEL, indicating that de novo synthetic pathways are not inhibited by BEL. Collectively, the data suggest that the Ca(2+)-independent PLA2 in P388D1 macrophages plays a major role in regulating the incorporation of AA into membrane phospholipids by providing the lysophospholipid acceptor employed in the acylation reaction.

Large-scale mass spectrometry-based peptidomics for drug discovery is relatively unexplored because of challenges in peptide degradation and identification following tissue extraction. Here we present a streamlined analytical pipeline for large-scale peptidomics. We developed an optimized sample preparation protocol to achieve fast, reproducible and effective extraction of endogenous peptides from sub-dissected organs such as the brain, while diminishing unspecific protease activity. Each peptidome sample was analysed by high-resolution tandem mass spectrometry and the resulting data set was integrated with publically available databases. We developed and applied an algorithm that reduces the peptide complexity for identification of biologically relevant peptides. The developed pipeline was applied to rat hypothalamus and identifies thousands of neuropeptides and their post-translational modifications, which is combined in a resource format for visualization, qualitative and quantitative analyses.

BACKGROUND: Cyclotides are useful scaffolds to stabilize bioactive peptides. RESULTS: Four melanocortin analogues of kalata B1 were synthesized. One is a selective MC4R agonist. CONCLUSION: The analogues retain the native kalata B1 scaffold and introduce a designed pharmacological activity, validating cyclotides as protein engineering scaffolds. SIGNIFICANCE: A novel type of melanocortin agonist has been developed, with potential as a drug lead for treating obesity. Obesity is an increasingly important global health problem that lacks current treatment options. The melanocortin receptor 4 (MC4R) is a target for obesity therapies because its activation triggers appetite suppression and increases energy expenditure. Cyclotides have been suggested as scaffolds for the insertion and stabilization of pharmaceutically active peptides. In this study, we explored the development of appetite-reducing peptides by synthesizing MC4R agonists based on the insertion of the His-Phe-Arg-Trp sequence into the cyclotide kalata B1. The ability of the analogues to fold similarly to kalata B1 but display MC4R activity were investigated. Four peptides were synthesized using t-butoxycarbonyl peptide chemistry with a C-terminal thioester to facilitate backbone cyclization. The structures of the peptides were found to be similar to kalata B1, evaluated by Hα NMR chemical shifts. KB1(GHFRWG;23-28) had a K(i) of 29 nm at the MC4R and was 107 or 314 times more selective over this receptor than MC1R or MC5R, respectively, and had no detectable binding to MC3R. The peptide had higher affinity for the MC4R than the endogenous agonist, α-melanocyte stimulation hormone, but it was less potent at the MC4R, with an EC(50) of 580 nm for activation of the MC4R. In conclusion, we synthesized melanocortin analogues of kalata B1 that preserve the structural scaffold and display receptor binding and functional activity. KB1(GHFRWG;23-28) is potent and selective for the MC4R. This compound validates the use of cyclotides as scaffolds and has the potential to be a new lead for the treatment of obesity.

Studies are reported on two different types of activated ketones as inhibitors of two important intracellular phospholipase A2s (PLA2): the group IV 85 kDa Ca2+-dependent phospholipase A2 (cPLA2) and the P388D1 Ca2+-independent phospholipase A2 (iPLA2). In a mixed micelle assay, we observed that the reaction progress curve of cPLA2 in the presence of a trifluoromethyl ketone (TFMK) is linear at pH 7.4, while at pH 9.0 it is nonlinear and slows with time. An investigation of this discrepancy demonstrated that the TFMKs are slow, tight-binding inhibitors of the cPLA2 at both pH's, that the rate of dissociation of the enzyme−inhibitor complex is the same at both pH's, but that the rate of association of enzyme and inhibitor is slower at pH 7.4 than at pH 9.0. A novel group of activated ketone inhibitors has been synthesized that contain a fatty acyl tricarbonyl. These compounds also inhibit the cPLA2 in the mixed micelle assay. The inhibition of cPLA2 by the tricarbonyls is readily reversible upon dilution and does not involve slow binding. For both types of inhibitor, no preference for fatty acid chain was observed as the palmityl analogs inhibited as well as the arachidonoyl analogs, despite the fact that the cPLA2 shows a strong preference for arachidonoyl-containing phospholipid substrates over palmitoyl-containing substrates. With the iPLA2, the inhibition by TFMKs is reversible and does not involve slow or tight binding. The tricarbonyls also inhibited the iPLA2, but were less potent than the TFMKs. Unlike the cPLA2, the iPLA2 does exhibit a fatty acid preference as the palmityl analogs of both compounds inhibit better than the arachidonoyl analogs. The palmityl TFMK displayed a 10-fold lower IC50 at pH 9.0 than at pH 7.5, whereas the potency of the tricarbonyl was unchanged in this range. Thus, the TFMKs inhibit both the cPLA2 and the iPLA2, but the mechanism of inhibition of the two enzymes appears to be quite different. The tricarbonyls also inhibited both enzymes, but in both cases in a reversible manner and as such appear to be poorer inhibitors than the TFMKs.