Pharmacological inhibition of sphingosine-1-phosphate lyase partially reverses spatial memory impairment in streptozotocin-diabetic rats.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid with strong neuroprotective properties that is important for normal excitability and synaptic transmission in the hippocampal neurons. Considering the above, the aim of the present study was to determine whether increasing brain S1P level is able to reverse spatial memory impairment in streptozotocin-diabetic rats. The experiment was carried out on diabetic (n = 22) and nondiabetic (n = 10) male Wistar rats. Diabetes was induced by a single injection of streptozotocin. Eleven weeks later, 11 diabetic animals received injections of THI (S1P lyase inhibitor) for seven days. During the last five days of the experiment spatial reference memory acquisition and retention were tested in the Morris water maze task. The animals were then anaesthetized and samples of the hippocampus, prefrontal cortex, striatum, and cerebellum were excised. The content of S1P and related sphingolipids was measured using a HPLC method. Diabetes induced a depletion of ceramide in the hippocampus and cerebellum that was associated with impaired spatial memory and learning. Administration of THI to the diabetic animals prevented ceramide depletion in the hippocampus and cerebellum, and induced an increase in S1P content in all examined brain structures. These effects were associated with an improvement in spatial memory. We conclude that pharmacological inhibition of S1P lyase partially reverses the impairment in spatial memory induced by chronic hyperglycemia, and that this effect may be related to the prevention of ceramide depletion in the hippocampus and cerebellum, the increase in brain S1P level, or both.

Nicotinamide phosphoribosyltransferase is a molecular target of potent anticancer agents identified from phenotype-based drug screening.

Phenotypic screening in drug discovery has been revived with the expectation of providing promising lead compounds and drug targets and improving the success rate of drug approval. However, target identification remains a major bottleneck in phenotype-based drug discovery. We identified the lead compounds K542 and K405 with a selective inhibition of cell viability against sphingosine-1-phosphate lyase 1 (SGPL1)-transduced ES-2 cells by phenotypic screening. We therefore performed an in vivo pharmacological examination and observed the antitumor activity of K542 in an HT-1080 tumor-bearing mouse xenograft model. SGPL1 was expected to be a therapeutic target in some cancers, suggesting that these lead molecules might be promising candidates; however, their mechanisms of action still remain unexplained. We therefore synthesized the affinity probe Ind-tag derived from K542 and identified the proteins binding to Ind-tag via a pull-down experiment. Proteomics and biochemical analyses revealed that the target molecule of these lead compounds was Nicotinamide phosphoribosyltransferase (NAMPT). We established K542-resistant DLD-1 and HT-1080 cells, and genetic analyses of these cells identified a missense mutation in the NAMPT-encoding gene. This enzymatic experiment clearly showed that K393 exerts enzymatic inhibition against NAMPT. These proteomics, genetics and biochemical analyses clarified that compounds K542 and K405 were NAMPT inhibitors.

Acquired modification of sphingosine-1-phosphate lyase activity is not related to adrenal insufficiency.

BACKGROUND: Congenital sphingosine-1-phosphate (S1P) lyase deficiency due to biallelic mutations in SGPL1 gene has recently been described in association with primary adrenal insufficiency and steroid-resistant nephrotic syndrome. S1P lyase, on the other hand, is therapeutically inhibited by fingolimod which is an oral drug for relapsing multiple sclerosis (MS). Effects of this treatment on adrenal function has not yet been evaluated. We aimed to test adrenal function of MS patients receiving long-term fingolimod treatment. METHODS: Nineteen patients (14 women) with MS receiving oral fingolimod (Gilenya®, Novartis) therapy were included. Median age was 34.2 years (range; 21.3-44.6 years). Median duration of fingolimod treatment was 32 months (range; 6-52 months) at a dose of 0.5 mg/day. Basal and ACTH-stimulated adrenal steroid measurements were evaluated simultaneously employing LC-MS/MS based steroid panel. Basal steroid concentrations were also compared to that of sex- and age-matched healthy subjects. Cortisol and 11-deoxycortisol, 11-deoxycorticosterone and dehydroepiandrosterone were used to assess glucocorticoid, mineralocorticoid and sex steroid producing pathways, respectively. RESULTS: Basal ACTH concentrations of the patients were 20.8 pg/mL (6.8-37.8 pg/mL) (normal range; 5-65 pg/mL). There was no significant difference in the basal concentrations of cortisol, 11-deoxycortisol, 11-deoxycorticosterone and dehydroepiandrosterone between patients and controls (p = 0.11, 0.058, 0.74, 0.15; respectively). All patients showed adequate cortisol response to 250 mcg IV ACTH stimulation (243 ng/mL, range; 197-362 ng/mL). There was no significant correlation between duration of fingolimod treatment and basal or ACTH-stimulated cortisol or change in cortisol concentrations during ACTH stimulation test (p = 0.57, 0.66 and 0.21, respectively). CONCLUSION: Modification and inhibition of S1P lyase activity by the long-term therapeutic use of fingolimod is not associated with adrenal insufficiency in adult patients with MS. This suggests that S1P lyase has potentially a critical role on adrenal development rather than the function of a fully mature adrenal gland.

Deficiency of sphingosine-1-phosphate lyase impairs lysosomal metabolism of the amyloid precursor protein.

Progressive accumulation of the amyloid ß protein in extracellular plaques is a neuropathological hallmark of Alzheimer disease. Amyloid ß is generated during sequential cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. In addition to the proteolytic processing by secretases, APP is also metabolized by lysosomal proteases. Here, we show that accumulation of intracellular sphingosine-1-phosphate (S1P) impairs the metabolism of APP. Cells lacking functional S1P-lyase, which degrades intracellular S1P, strongly accumulate full-length APP and its potentially amyloidogenic C-terminal fragments (CTFs) as compared with cells expressing the functional enzyme. By cell biological and biochemical methods, we demonstrate that intracellular inhibition of S1P-lyase impairs the degradation of APP and CTFs in lysosomal compartments and also decreases the activity of γ-secretase. Interestingly, the strong accumulation of APP and CTFs in S1P-lyase-deficient cells was reversed by selective mobilization of Ca(2+) from the endoplasmic reticulum or lysosomes. Intracellular accumulation of S1P also impairs maturation of cathepsin D and degradation of Lamp-2, indicating a general impairment of lysosomal activity. Together, these data demonstrate that S1P-lyase plays a critical role in the regulation of lysosomal activity and the metabolism of APP.

Essential cysteines in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase from Escherichia coli: analysis by chemical modification and site-directed mutagenesis.

The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase (EC 4.1. 2.16) (KDO 8-P synthase) that catalyzes the condensation of D-arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP) to give 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) and inorganic phosphate (Pi) was inactivated by the thiol-modifying reagents 5,5-dithiobis (2-nitrobenzoate) (DTNB) and methyl methanethiosulfonate (MMTS). Reaction of cloned native KDO 8-P synthase with DTNB correlated with modification of two of the four cysteine sulfhydryls per monomer of enzyme and total loss of enzymatic activity which could be partially restored by treatment with dithiothreitol (DTT). Cyanolysis of the DTNB-inactivated enzyme with KCN led to the elimination of 2 equiv of 5-thio-2-nitrobenzoate and partial recovery of activity. The presence of either substrate(s) or product(s) provided no protection against inactivation nor affected the number of cysteines modified, indicating that the cysteines modified are most likely not at the active site of KDO 8-P synthase. Titration of denatured enzyme with DTNB resulted in the modification of all four cysteines. After treatment of native enzyme with MMTS, no cysteines could be titrated with DTNB and no enzymatic activity could be detected. Treatment of the MMTS-inactivated KDO 8-P synthase with DTT resulted in restoration of enzymatic activity and the presence of two DTNB-titratable cysteine residues. Based on these observations and a report that KDO 8-P synthase is inactivated in a time-dependent manner with 3-bromopyruvate and that the substrate PEP protects against this inactivation, all four cysteines (38, 166, 206, and 249) were individually mutated to alanines via a modified PCR methodology. The C206A and C249A mutants were both enzymatically active with K(m) and Vmax values approximately identical to those of wild-type KDO 8-P synthase, and both native mutants reacted with DTNB to modify only one of the three remaining cysteine sulfhydryls per monomer of enzyme. Titration of denatured C206A and C249A mutants resulted in the modification of three cysteines. The C38A and C166A mutants were both for the most part enzymatically inactive. Titration of native C38A and C166A with DTNB resulted in modification of two cysteines while titration of the denatured mutant protein resulted in modification of the three remaining cysteines. Circular dichroism measurements of wild-type KDO 8-P synthase and the four C --> A mutants indicate modest but significant changes in the structure of the mutants. These results indicate that C206 and C249 in native KDO 8-P synthase are readily accessible to the modification reagent DTNB and therefore inactivation may result from structural changes in the DTNB-modified KDO 8-P synthase or blockage of access of substrates to the active site. The C38 and C166 in native KDO 8-P synthase are inaccessible to the modification reagent DTNB, indicating that they are located in the interior of KDO 8-P synthase, and loss of activity in the C38A and C166A mutants suggests their essentiality in the KDO 8-P synthase reaction.

Characterization of fructose 6 phosphate phosphoketolases purified from Bifidobacterium species.

Fructose 6 phosphate phosphoketolases (F6PPKs) were purified from Bifidobacterium longum BB536, B. dentium ATCC 27534, B. globosum ATCC 25864, and Bifidobacterium animalis ATCC 25527. Concerning ions (Cu++, Zn++, Ca++, Mg++, Fe++, Co++, Mn++) and common enzyme inhibitors (fructose, ammonium sulfate, iodoacetate, and parachloromercuribenzoic acid), no difference appeared between the enzymes. Cu++, parachloromercuribenzoic acid (pCMB), and mercuric acetate induced high enzymatic inhibition. The study of pCMB demonstrated a noncompetitive inhibition. Additional results showed that the sulfhydryl group was not involved in catalytic reaction. Photooxidation experiments and determination of ionizable group pKas (5.16-7.17) suggested the presence of one or more histidines necessary for the catalytic reaction and explained the inhibition observed with pCMB. In light of the noncompetitive inhibition, this group was not directly involved in substrate binding. Determination of Km demonstrated that the affinities for fructose 6 phosphate in the case of animal and human origin strains were close. In addition, the same enzymatic efficiency (Kcat/Km) was obtained for each strain. The F6PPK activity was regulated by sodium pyrophosphate, ATP, and especially by ADP.

LY207320 (6-methylene-4-pregnene-3,20-dione) inhibits testosterone biosynthesis, androgen uptake, 5 alpha-reductase, and produces prostatic regression in male rats.

LY207320 is an in vitro inhibitor (estimated IC50 = 0.06 microM) of steroid 5 alpha-reductase that catalyzes the conversion of testosterone (T) to dihydrotestosterone (DHT). In contrast, LY207320 was only moderately active against rat prostatic 5 alpha-reductase in vivo (32% inhibition at 50.0 mg/kg single dose). LY207320 did, however, inhibit the in vivo uptake of [3H]-T by the prostate. The antiprostatic and endocrine effects of this agent were evaluated following daily (21 days) administration to castrated, androgen-supplemented castrate, and intact rats. LY207320, which has modest progestational competitive binding activity, does not bind to rat prostatic androgen or uterine estrogen cytosolic receptors. In the castrated male rat, subcutaneously (s.c.) administered LY207320 had no androgen agonist activity, as evidenced by a lack of accessory sex organ weight gains. Administration of s.c. LY207320 to intact rats for 21 days at doses greater than 5.0 mg/kg-day produced significant (P < 0.05) reductions of seminal vesicle and ventral prostatic weights (maximal regression = -65% and -40% from control values, respectively at 50.0 mg/kg-day). The compound had no regressive activity on male accessory sex organs when administered orally. LY207320 did not alter circulating prolactin, LH, or corticosterone levels, but at high doses (> or = 50.0 mg/kg-day), lowered circulating T[-67% from intact control levels (P < 0.05)]. Histological analysis of the rat ventral prostates (RVPs) in LY207320-treated rats was consistent with an androgen-deprived state. Decreased circulating androgens and prostatic regression are associated with inhibition of testicular 17 alpha-hydroxy/C17,20-lyase enzyme activity (IC50 = 0.06 microM). These findings support the contention that LY207320 is a physiological antagonist of androgen action in male rats, and that its effects are mediated primarily through inhibition of testicular androgen production rather than accessory sex organ 5 alpha-reductase.