David S. Menaldino

Acylation of Naturally Occurring and Synthetic 1-Deoxysphinganines by Ceramide Synthase FORMATION OF N-PALMITOYL-AMINOPENTOL PRODUCES A TOXIC METABOLITE OF HYDROLYZED FUMONISIN, AP1, AND A NEW CATEGORY OF CERAMIDE SYNTHASE INHIBITOR

Fumonisin B1 (FB1) is the predominant member of a family of mycotoxins produced byFusarium moniliforme (Sheldon) and related fungi. Certain foods also contain the aminopentol backbone (AP1) that is formed upon base hydrolysis of the ester-linked tricarballylic acids of FB1. Both FB1 and, to a lesser extent, AP1 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 AP1 (but not FB1, under the conditions tested) to N-palmitoyl-AP1 (PAP1) with a V max/K m of approximately 1. The toxicity of PAP1 was evaluated using HT29 cells, a human colonic cell line. PAP1 was at least 10 times more toxic than FB1 or AP1 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- andthreo-isomers are acylated with the highest apparentV max/K m for theerythro-analogs; and AP1 is acylated to PAP1, a new category of ceramide synthase inhibitor as well as a toxic metabolite that may play a role in the diseases caused by fumonisins.

A stereoselective synthesis of sphingosine, a protein kinase c inhibitor

Abstract A stereoselective synthesis of each of the four enantiomers of sphingosine from either L- or D-serine is reported here. The key steps in the sequence involve: (a) the diastereoselective addition of 1-lithiopentadecyne to aldehyde 4 and (b) Mitsunobu inversion of propargyl alcohol 5 .

FUMONISIN TOXICITY AND SPHINGOLIPID BIOSYNTHESIS

Fumonisins are inhibitors of sphinganine (sphingosine) N-acyltransferase (ceramide synthase) in vitro, and exhibit competitive-type inhibition with respect to both substrates of this enzyme (sphinganine and fatty acyl-CoA). Removal of the tricarballylic acids from fumonisin B1 reduces the potency by at least 10 fold; and fumonisin A1 (which is acetylated on the amino group) is essentially inactive. Studies with diverse types of cells (hepatocytes, neurons, kidney cells, fibroblasts, macrophages, and plant cells) have established that fumonisin B1 not only blocks the biosynthesis of complex sphingolipids; but also, causes sphinganine to accumulate. Some of the sphinganine is metabolized to the 1-phosphate and degraded to hexadecanal and ethanolamine phosphate, which is incorporated into phosphatidylethanolamine. Sphinganine is also released from cells and, because it appears in blood and urine, can be used as a biomarker for exposure. The accumulation of these bioactive compounds, as well as the depletion of complex sphingolipids, may account for the toxicity, and perhaps the carcinogenicity, of fumonisins.

Sphingoid bases and de novo ceramide synthesis: enzymes involved, pharmacology and mechanisms of action

The sphingoid base backbones of sphingolipids (sphingosines, sphinganines, 4-hydroxysphinganines and others) are highly bioactive species directly and-in most cases-as their metabolites, the N-acyl-sphingoid bases (ceramides) and sphingoid base 1-phosphates. The complexity of these compounds affords many opportunities to prepare synthetic analogs for studies of sphingolipid metabolism and the functions of the sphingoid bases and metabolites. Described in this review are methods for the preparation of libraries of sphingoid bases, including a series of 1-deoxy-analogs, as well as information about their metabolism and biological activities. Findings with these compounds have uncovered some of the complications of working with compounds that mimic a naturally occurring biomodulator-such as that they are sometimes metabolized by enzymes that handle the endogenous compounds and the products may have potent (and unexpected) biological activities. Through studying such compounds, there is now a greater understanding of the metabolism and mechanism(s) of action of naturally occurring sphingoid bases as well as of these analogs.

Main group metal halide complexes with sterically hindered thioureas XI. Complexes of antimony(III) and bismuth(III) chlorides with a new bidentate thiourea — 1,1′-methylenebis(3-methyl-2H-imidazole-2-thione)

Abstract A new bidentate ligand, mbit, [mbit=1,1′-methylenebis (3-methyl-2H-imidazole-2-thione)] was synthesized and characterized along with two new pnictogen complexes - SbCl3mbit and BiCl3mbit. A single crystal X-ray structure of SbCl3mbit gave the following cell parameters: space group P21/c, a = 11.812(9), b = 7.699(4), c = 18.100(9) A, β = 101.29(5)°, Z = 4, V = 1614.2 A3, Do = 1.92 g/cm3, Dc = 1.927 g/cm3 (λ = 0.71069 A). The structure refined to a conventional R=0.042. The local geometry around the antimony atom was distorted octahedral with bridging sulfur atoms creating long zigzag chains of octahedra sharing vertices cis to each other. Two chlorine atoms are trans to each other, and the third chlorine is trans to the bridging sulfur. The Sb—S bridges are 3.41 A in length. There is no strong evidence for a stereoactive lone pair in the coordination sphere of antimony. The ligand forms an eight-membered ring with the antimony atom included. Solid state IR data showed little change in ligand vibrational modes normally sensitive to coordination, and solution state proton NMR gave evidence for a high degree of association.

Context-Dependent GluN2B-Selective Inhibitors of NMDA Receptor Function Are Neuroprotective with Minimal Side Effects

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 noninjured 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.

Inhibition of skin carcinomas but not papillomas by sphingosine, N‐methylsphingosine, and N‐acetylsphingosine

The sphingoid base backbones of sphingolipids are highly bioactive compounds that affect cell growth, differentiation, diverse cell behaviors, and programmed cell death. Therefore, the efficacy of sphingosine (SPH) and the analogs N-acetylsphingosine (NAS), N-methylsphingosine (NMS), octylamine (OCT), and sterylamine (STR) in the prevention of skin cancer was assessed in female Sencar mice by measuring effects on the induction of epidermal ornithine decarboxylase (ODC) activity and hyperplasia by 12-O-tetradecanoylphorbol-13-acetate (TPA) and effects on the induction of skin tumors by 7, 12-dimethylbenz[a]anthracene (DMBA) and TPA. ODC was measured in the shaved dorsal skin of mice treated topically with 0.05-20 mumol of these compounds 30 minutes before application of 8.5 nmol of TPA in 0.2 ml of acetone. ODC activity was inhibited by > or = 5 mumol of SPH and STR, > or = 10 mumol of NAS and NMS, and 20 mumol of OCT. In contrast, the induction of hyperplasia was not inhibited by application of these compounds 30 minutes before TPA. Two carcinogenesis studies were conducted with 10 nmol of DMBA as the initiator and 3.2 nmol of TPA (2x/wk for 15 wk) as the promoter. In the first study, NAS, NMS, OCT, and STR (0.05 and 0.5 mumol) were applied before each TPA application. Papilloma incidence and multiplicity were not inhibited, but NAS (0.05 mumol) and NMS (0.05 and 0.50 mumol) increased cancer-free survival. In the second experiment, SPH, NAS, and NMS (0.05 and 0.5 mumol) were applied 30 minutes before each TPA treatment and twice weekly for 10 weeks after the final TPA treatment. Papilloma incidence and multiplicity were not inhibited; however, the proportion of mice without carcinoma was increased by both doses of SPH and by 0.5 mumol of NAS. Thus low doses of sphingolipids that were not effective in inhibiting ODC activity, reducing hyperplasia, or preventing epidermal papilloma development were, nonetheless, effective in inhibiting carcinoma development.

Novel Synthesis and Biological Evaluation of Enigmols as Therapeutic Agents for Treating Prostate Cancer

Enigmol is a synthetic, orally active 1-deoxysphingoid base analogue that has demonstrated promising activity against prostate cancer. In these studies, the pharmacologic roles of stereochemistry and N-methylation in the structure of enigmols were examined. A novel enantioselective synthesis of all four possible 2S-diastereoisomers of enigmol (2-aminooctadecane-3,5-diols) from l-alanine is reported, which features a Liebeskind-Srogl cross-coupling reaction between l-alanine thiol ester and (E)-pentadec-1-enylboronic acid as the key step. In vitro biological evaluation of the four enigmol diastereoisomers and 2S,3S,5S-N-methylenigmol against two prostate cancer cell lines (PC-3 and LNCaP) indicates that all but one diastereomer demonstrate potent oncolytic activity. In nude mouse xenograft models of human prostate cancer, enigmol was equally effective as standard prostate cancer therapies (androgen deprivation or docetaxel), and two of the enigmol diastereomers, 2S,3S,5R-enigmol and 2S,3R,5S-enigmol, also caused statistically significant inhibition of tumor growth. A pharmacokinetic profile of enigmol and N-methylenigmol is also presented.

Influences of sphingosine on two-stage skin tumorigenesis in Sencar mice

Sphingosine (SPH) was studied as an inhibitor of skin tumor promotion in skin cancer initiated by 7,12-dimethylbenz[a]-anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). Two tumorigenesis studies were conducted using female Sencar mice treated with 10 nmol DMBA in 0.2 ml acetone at 8 weeks of age and promoted beginning 1 week later with 3.2 nmol TPA applied twice per week. In the high-dose study, 10 mumol SPH was applied 30 min before each TPA treatment. The low-dose study used 0.5, 0.05, or 0.01 mumol treatments with SPH 30 min before TPA. In the high-dose study SPH treatment alone following initiation by DMBA, and SPH treatment preceding TPA in DMBA-initiated mice accelerated the development of papillomas in comparison with the DMBA/TPA-treated group. The low-dose experiment showed no consistent alteration of tumorigenesis by SPH in the DMBA/TPA-treated groups, and low doses of SPH following DMBA did not promote skin tumorigenesis. SPH treatment did not alter body weight in either experiment.

Ceramide Synthase and Ceramidases in the Regulation of Sphingoid Base Metabolism

Ceramides and sphingoid bases (such as sphingosine and sphinganine) are not only intermediates of complex sphingolipid biosynthesis and turnover, but also highly bioactive compounds that have been proposed to mediate cellular responses to growth factors, cytokines, and other agents (including γ-irradiation and some anti-cancer drugs). In studying the involvement of these compounds in growth, differentiation, diverse cell functions, and cell death, sphingoid bases and/or ceramides are often added to cells exogenously, or the amounts of the endogenous compounds are analyzed. A complicating factor in such analyses is that sphingoid bases and ceramides can be interconverted by acylation and deacylation reactions, respectively, catalyzed by ceramide synthase and ceramidase. This review describes the characteristics of ceramide synthesis and turnover, properties of the enzymes responsible for these reactions, and how inhibitors are useful in studying the function of these compounds. Furthermore, one class of ceramide synthase inhibitor (the fumonisins) are responsible for severe diseases of plants and animals; therefore information is presented about how fumonisins disrupt sphingolipid metabolism, alter cell behavior, and cause disease.