Sphingosine, sphinganine, and other long-chain (sphingoid) bases inhibit protein kinase C in vitro and block cellular responses to agonists that are thought to act via this enzyme. To gain further insight into the mechanism of this inhibition, a series of long-chain analogues differing in alkyl chain length (11-20 carbon atoms), stereochemistry, and headgroup were examined for (a) inhibition of protein kinase C activity in vitro, (b) the neutrophil respiratory burst in response to phorbol myristate acetate (PMA), (c) the PMA-induced differentiation of HL-60 cells, and (d) the growth of Chinese hamster ovary cells. In every instance, the effects were maximal with the 18-carbon homologues, which are the same length as the predominant naturally occurring long-chain base (sphingosine). The lower potency of the shorter chain homologues was partially due to decreased uptake by cells. Small differences were obtained with the four stereoisomers of sphingosine (i.e., D and L forms of erythro- and threo-sphingosine), with N-methyl derivatives of the different sphingosine homologues, and with simpler alkylamines (e.g., stearylamine). The potency of the different headgroup analogues may be affected by the degree of protonation at the assay pH. The pKa of sphingosine was measured to be 6.7; the pKa varied among the analogues. These findings establish that the major structural features required for inhibition of protein kinase C and cellular processes dependent on this enzyme are the presence of a free amino group and an aliphatic side chain and that other groups have more subtle effects.(ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARY Stroke remains a significant problem despite decades of work on neuroprotective strategies. NMDA receptor (NMDAR) antagonists are neuroprotective in preclinical models, but have been clinically unsuccessful, in part due to side effects. Here we describe a prototypical GluN2B-selective antagonist with an IC50 value that is 10-fold more potent at acidic pH 6.9 associated with ischemic tissue compared to pH 7.6, a value close to the pH in healthy brain tissue. This should maximize neuroprotection in ischemic tissue while minimizing on-target side-effects associated with NMDAR blockade in non-injured brain regions. We have determined the mechanism underlying pH-dependent inhibition and demonstrate the utility of this approach in vivo. We also identify dicarboxylate dimers as a novel proton sensor in proteins. These results provide insight into the molecular basis of pH-dependent neuroprotective NMDAR block, which could be beneficial in a wide range of neurological insults associated with tissue acidification.

N-Methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate excitatory synaptic transmission and have been implicated in numerous neurological disorders. NMDARs typically comprise two GluN1 and two GluN2 subunits. The four GluN2 subtypes (GluN2A-GluN2D) have distinct functional properties and gene expression patterns, which contribute to diverse functional roles for NMDARs in the brain. Here, we present a series of GluN2C/2D-selective negative allosteric modulators built around a N-aryl benzamide (NAB) core. The prototypical compound, NAB-14, is >800-fold selective for recombinant GluN2C/GluN2D over GluN2A/GluN2B in Xenopus oocytes and has an IC value of 580 nM at recombinant GluN2D-containing receptors expressed in mammalian cells. NAB-14 inhibits triheteromeric (GluN1/GluN2A/GluN2C) NMDARs with modestly reduced potency and efficacy compared to diheteromeric (GluN1/GluN2C/GluN2C) receptors. Site-directed mutagenesis suggests that structural determinants for NAB-14 inhibition reside in the GluN2D M1 transmembrane helix. NAB-14 inhibits GluN2D-mediated synaptic currents in rat subthalamic neurons and mouse hippocampal interneurons, but has no effect on synaptic transmission in hippocampal pyramidal neurons, which do not express GluN2C or GluN2D. This series possesses some druglike physical properties and modest brain permeability in rat and mouse. Altogether, this work identifies a new series of negative allosteric modulators that are valuable tools for studying GluN2C- and GluN2D-containing NMDAR function in brain circuits, and suggests that the series has the potential to be developed into therapies for selectively modulating brain circuits involving the GluN2C and GluN2D subunits.

Fumonisin B 1 (FB 1 ) is the predominant member of a family of mycotoxins produced by Fusarium moniliforme (Sheldon) and related fungi. Certain foods also contain the aminopentol backbone (AP 1 ) that is formed upon base hydrolysis of the ester-linked tricarballylic acids of FB 1 . Both FB 1 and, to a lesser extent, AP 1 inhibit ceramide synthase due to structural similarities between fumonisins (as 1-deoxy-analogs of sphinganine) and sphingoid bases. To explore these structure-function relationships further, erythro-and threo-2-amino, 3-hydroxy-(and 3, 5-dihydroxy-) octadecanes were prepared by highly stereoselective syntheses. All of these analogs inhibit the acylation of sphingoid bases by ceramide synthase, and are themselves acylated with V max /K m of 40 -125 for the erythro-isomers (compared with approximately 250 for D-erythro-sphinganine) and 4 -6 for the threo-isomers. Ceramide synthase also acylates AP 1 (but not FB 1 , under the conditions tested) to N-palmitoyl-AP 1 (PAP 1 ) with a V max /K m of approximately 1. The toxicity of PAP 1 was evaluated using HT29 cells, a human colonic cell line. PAP 1 was at least 10 times more toxic than FB 1 or AP 1 and caused sphinganine accumulation as an inhibitor of ceramide synthase. These studies demonstrate that: the 1-hydroxyl group is not required for sphingoid bases to be acylated; both erythro-and threo-isomers are acylated with the highest apparent V max /K m for the erythro-analogs; and AP 1 is acylated to PAP 1 , a new category of ceramide synthase inhibitor as well as a toxic metabolite that may play a role in the diseases caused by fumonisins.Fumonisins are mycotoxins produced by Fusarium moniliforme (Sheldon) and related fungi that are common contaminants of maize and certain other foods (1). Fumonisin B 1 (FB 1 ) 1 is comprised of a long chain aminopentol (AP 1 ) with two of the side chain hydroxyls esterified to tricarballylic acids ( Fig. 1) (2, 3). Although numerous fumonisins have been characterized (1-4), FB 1 is usually the most abundant in contaminated food, except when corn has been treated with base to produce masa flour for tortillas, which hydrolyzes FB 1 to AP 1 (1).Fumonisins are responsible for at least two diseases, equine leukoencephalomalacia and porcine pulmonary edema (5, 6). Studies with these and other animals have uncovered a wide spectrum of toxicologic effects, which include hepatotoxicity, nephrotoxicity, neurotoxicity, developmental toxicity, and immunosuppression (and immunostimulation under some conditions) (for reviews, see Refs. 1, 7, and 8). Fumonisins are also hepatocarcinogenic in rats (1) and have been implicated in esophageal cancer in humans in South Africa and China (9 -11).The diverse effects of these mycotoxins appear to be due to inhibition of ceramide synthase, the enzyme that catalyzes the acylation of sphinganine, sphingosine, and other sphingoid bases (12, 13). Inhibition has been shown in vitro with intact cells and for animals exposed to fumonisins (7) and is manifested by up to several hundredfold incre...

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