Novel selective butyrylcholinesterase inhibitors incorporating antioxidant functionalities as potential bimodal therapeutics for Alzheimer’s Disease

Isosorbide-2-carbamates-5-aryl esters are highly potent and very selective butyrylcholinesterase inhibitors. The objective of the present work was to address the hypothesis that the isosorbide-aryl-5-ester group could be replaced with an antioxidant functionality while maintaining inhibitor effects and selectivity. We successfully incorporated ferulic acid or lipoic acid groups producing potent selective inhibitors of butyrylcholinesterase (BuChE). The hybrid compounds were non-toxic to the murine hippocampal cell line HT-22 and lipoate esters were neuroprotective at 10 and 25 mu M when the cells were challenged with glutamate (5 mM) in a similar manner to the positive control quercetin. The benzyl carbamate 7a was a potent inhibitor of BuChE (IC50 150 nM) and it was effective in reducing glutamate toxicity to neuronal cells at >5 mu M. Representative compounds exhibited an antioxidant effect in the oxygen radical absorbance capacity assay as the lipoate 7d was not active, whereas the ferulate 8a showed a weak, but significant, activity with 0.635 +/- 0.020 Trolox Equivalent

Platelet factor 4 impairs the anticoagulant activity of activated protein C.

Platelet factor 4 (PF4) is an abundant platelet -granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the -carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anticoagulant activity. PF4 inhibited both protein S-dependentAPC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q/ R679Q and FVa-R306Q/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg306 by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activatio

Dissociation of activated protein C functions by elimination of protein S cofactor enhancement.

Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain

Ca2+-Regulatory Muscle Proteins in the Alcohol-Fed Rat

Alcoholic myopathy is characterized by muscle weakness and difficulties in gait and locomotion. It is one of the most prevalent skeletal muscle disorders in the Western hemisphere, affecting between 40% and 60% of all chronic alcohol misusers. However, the pathogenic mechanisms are unknown, although recent studies have suggested that membrane defects occur as a consequence of chronic alcohol exposure. It was our hypothesis that alcohol ingestion perturbs membrane-located proteins associated with intracellular signalling and contractility, in particular those relating to calcium homeostasis. To test this, we fed male Wistar rats nutritionally complete liquid diets containing ethanol as 35% of total dietary energy. Controls were pair-fed identical amounts of the same diet in which ethanol was replaced by isocaloric glucose. At the end of 6 weeks, rats were killed and skeletal muscles dissected. These were used to determine important ion-regulatory skeletal muscle proteins including sarcalumenin (SAR), sarcoplasmic-endoplasmic reticulum Ca(2+)-adenosine triphosphatase (ATPase) (SERCA1), the junctional face protein of 90 kd (90-JFP), alpha(1)- and alpha(2)-dihydropyridine receptor (alpha(1)-DHPR and alpha(2)-DHPR), and calsequestrin (CSQ) by immunoblotting. The relative abundance of microsomal proteins was determined by immunoblotting using the enhanced chemiluminescence (ECL) technique. The data showed that alcohol-feeding significantly reduced gastrocnemius and hind limb muscle weights (P .2 in all instances). Reductions in CSQ were of marginal significance (P =.0950). We conclude that upregulation of SERCA1 protein and Ca(2+)-ATPase activity may be an adaptive mechanism and/or a contributory process in the pathology of alcohol-induced muscle disease

Design of Barbiturate–Nitrate Hybrids that Inhibit MMP-9 Activity and Secretion

We describe a new type of barbiturate-based matrix metalloproteinase (MMP) inhibitor incorporating a nitric oxide (NO) donor/mimetic group (series 1). The compounds were designed to inhibit MMP at enzyme level and to attenuate MMP-9 secretion arising from inflammatory signaling. To detect effects related to the nitrate, we prepared and studied an analogous series of barbiturate C5-alkyl alcohols that were unable to release NO (series 2). Both series inhibited recombinant human MMP-2/9 activity with nanomolar potency. Series 1 consistently inhibited the secretion of MMP-9 from TNFα/IL1β stimulated Caco-2 cells at 10 μM, which could be attributed to NO related effects because the non-nitrate panel did not affect enzyme levels. Several compounds from series 1 (10 μM) inhibited tumor cell invasion but none from the non-nitrate panel did. The work shows that MMP-inhibitory barbiturates are suitable scaffolds for hybrid design, targeting additional facets of MMP pathophysiology, with potential to improve risk-benefit ratios

Platelet Factor 4 Impairs the Anticoagulant Activity of Activated Protein C

Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anticoagulant activity. PF4 inhibited both protein S-dependentAPC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q/R679Q and FVa-R306Q/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg306 by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation