Phosphorylation of a constrained azacyclic FTY720 analog enhances anti-leukemic activity without inducing S1P receptor activation.

The frequency of poor outcomes in relapsed leukemia patients underscores the need for novel therapeutic approaches. The Food and Drug Administration-approved immunosuppressant FTY720 limits leukemia progression by activating protein phosphatase 2A and restricting nutrient access. Unfortunately, FTY720 cannot be re-purposed for use in cancer patients due to on-target toxicity associated with S1P receptor activation at the elevated, anti-neoplastic dose. Here we show that the constrained azacyclic FTY720 analog SH-RF-177 lacks S1P receptor activity but maintains anti-leukemic activity in vitro and in vivo. SH-RF-177 was not only more potent than FTY720, but killed via a distinct mechanism. Phosphorylation is dispensable for FTY720's anti-leukemic actions. However, chemical biology and genetic approaches demonstrated that the sphingosine kinase 2 (SPHK2)-mediated phosphorylation of SH-RF-177 led to engagement of a pro-apoptotic target and increased potency. The cytotoxicity of membrane-permeant FTY720 phosphonate esters suggests that the enhanced potency of SH-RF-177 stems from its more efficient phosphorylation. The tight inverse correlation between SH-RF-177 IC50 and SPHK2 mRNA expression suggests a useful biomarker for SH-RF-177 sensitivity. In summary, these studies indicate that FTY720 analogs that are efficiently phosphorylated but fail to activate S1P receptors may be superior anti-leukemic agents compared to compounds that avoid cardiotoxicity by eliminating phosphorylation.

Small constrained SP1-7 analogs bind to a unique site and promote anti-allodynic effects following systemic injection in mice.

Previous results have shown that the substance P (SP) N-terminal fragment SP1-7 may attenuate hyperalgesia and produce anti-allodynia in animals using various experimental models for neuropathic pain. The heptapeptide was found to induce its effects through binding to and activating specific sites apart from any known neurokinin or opioid receptor. Furthermore, we have applied a medicinal chemistry program to develop lead compounds mimicking the effect of SP1-7. The present study was designed to evaluate the pharmacological effect of these compounds using the mouse spared nerve injury (SNI) model of chronic neuropathic pain. Also, as no comprehensive screen with the aim to identify the SP1-7 target has yet been performed we screened our lead compound H-Phe-Phe-NH2 toward a panel of drug targets. The extensive target screen, including 111 targets, did not reveal any hit for the binding site among a number of known receptors or enzymes involved in pain modulation. Our animal studies confirmed that SP1-7, but also synthetic analogs thereof, possesses anti-allodynic effects in the mouse SNI model of neuropathic pain. One of the lead compounds, a constrained H-Phe-Phe-NH2 analog, was shown to exhibit a significant anti-allodynic effect.

Inhibition of hepatocyte gap junctional intercellular communication by endosulfan, chlordane and heptachlor.

The cyclodiene pesticides endosulfan, chlordane and heptachlor have been reported to be non-genotoxic rodent hepatocarcinogens. These three compounds and several metabolites of endosulfan (endosulfan sulfate, endosulfan ether and endosulfan lactone) were examined for their effects on gap junctional intercellular communication (GJIC) in primary cultured male F344 rat hepatocytes and B6C3F1 mouse hepatocytes. GJIC was evaluated by Lucifer Yellow CH dye-coupling. Endosulfan and endosulfan sulfate inhibited rat and mouse hepatocyte GJIC in a dose-responsive manner (50-200 microM) after 4 h treatment. Endosulfan ether inhibited rat hepatocyte GJIC only at 200 microM and had no effect on mouse hepatocytes. Endosulfan lactone did not affect rat or mouse hepatocyte GJIC. Chlordane and heptachlor inhibited both mouse and rat hepatocyte GJIC at concentrations of 50-200 microM. The inhibition of GJIC by the cyclodienes showed similar dose-response relationships and kinetics of onset of inhibition and reversibility for both mouse and rat hepatocytes. Concomitant treatment of the cells with inhibitors of cytochrome P450 monooxygenases (SKF-525A, piperonyl butoxide or carbon monoxide) did not alter the inhibition of GJIC by the cyclodienes, suggesting that cytochrome P450 metabolism was not involved in the inhibitory mechanism. Dibutyryl cyclic AMP (0.5 mM), however, decreased the inhibition of GJIC by the cyclodienes and may indicate that these compounds inhibit intercellular communication through a cAMP-dependent process. The inhibition of mouse and rat hepatocyte GJIC by the cyclodienes correlated with previous reports indicating that these compounds are non-genotoxic rodent liver carcinogens.

Changes in cytosolic CA2+ are not involved in DDT-induced loss of gap junctional communication in WB-F344 cells.

Recent studies have demonstrated that the insecticide DDT is a tumor promoting agent. Similar to many other tumor promoting agents, DDT has been shown to inhibit gap junctional intercellular communication (GJIC) between cells in culture, and it has been suggested that DDT-induced loss of communication between adjacent cells may depend on changes in cytosolic free Ca2+ concentration [( Ca2+]i). In the present study, the role of [Ca2+]i in DDT-induced loss of GJIC was investigated in WB-F344 in rat liver cells using the scrape-loading/dye transfer assay (SLDT) and the Ca2+ fluorescent indicator, fura-2. Our results show that DDT at noncytotoxic concentrations caused a reversible loss of GJIC. Inhibition of GJIC was not associated with detectable increases in [Ca2+]i, and was not prevented by loading cells with the intracellular Ca2+ chelator, BAPTA. In addition, the hydroquinone, tBuBHQ, which caused a 2-3 fold sustained increase in [Ca2+]i, did not inhibit GJIC. Conversely, when untreated cells were loaded with increasing BAPTA concentrations, GJIC were lost. These results indicate that increases in [Ca2+]i are not responsible for DDT-induced loss of communication and that, in general, an increase in [Ca2+]i within physiological levels is not sufficient to abolish GJIC. However, Ca2(+)-dependent processes that are active at normal resting [Ca2+]i appear to be required for the maintenance of GJIC.

Tumour promotion related effects by the cyclodiene insecticide endosulfan studied in vitro and in vivo.

The cyclodiene insecticide endosulfan is structurally related to the tumour promoting pesticides chlordane and heptachlor. Divergent conclusions have been reported regarding the carcinogenic activity of endosulfan. In this study we have investigated if endosulfan and four of its metabolites possess tumour promotion related effects. Two in vitro test systems detecting inhibition of intercellular communication were used; the Chinese hamster lung fibroblast (V79) metabolic cooperation assay and a scrape loading/dye transfer assay using rat liver WB epithelial cells. At non-cytotoxic concentrations, technical grade endosulfan, analytical grade endosulfan (alpha- and beta-isomers and an alpha beta-isomer mixture) and endosulfan-sulfate inhibited gap junctional communication in both assay systems. In addition, the metabolite endosulfan-ether was effective in the rat liver WB epithelial cells. Endosulfan was also studied for enhancement of gamma-glutamyl transpeptidase positive enzyme altered foci incidence in partially hepatectomized, nitrosodiethylamine-initiated male Sprague-Dawley rats. However, endosulfan administered orally (1 or 5 mg/kg/day) five days a week for ten weeks did not enhance enzyme altered foci incidence. These apparently contradictory results with regard to possible tumour promoting activity of endosulfan are discussed in relation to metabolism, systemic toxicity and tissue/species specificity in tumour promotion.

Calmodulin involvement in TPA and DDT induced inhibition of intercellular communication.

The organochlorine pesticide DDT is a liver tumour promoter and a potent inhibitor of intercellular communication. Present knowledge of the mechanism by which DDT inhibits intercellular communication is limited but it has been suggested that increased intracellular free calcium induced by DDT could be of importance. As the effects of calcium are closely associated with the multifunctional protein calmodulin (CaM) in most cells the potential binding of DDT to CaM and subsequent effects on CaM-stimulated Ca2+/Mg2+-ATPase activity were studied. DDT inhibited CaM-stimulated Ca2+/Mg2+-ATPase activity and bound to CaM in a manner similar to established CaM-inhibitors. Subsequently an in vitro assay for measuring inhibition of metabolic cooperation between 6-thioguanine (TG)-sensitive and TG-resistant Chinese hamster (V79) cells was used to investigate the possible involvement of CaM in the regulation of intercellular communication. Calmidazolium (CzM), a potent CaM inhibitor, was tested alone or in combination with the tumour promoters 12-O-tetradecanoyl phorbol-13-acetate (TPA) or DDT known inhibitors of intercellular communication. The results showed that CzM alone was without effect with regard to inhibition of metabolic cooperation but potentiated the response induced by TPA, an effect not noticed with DDT. These results suggest different mechanisms of action of TPA and DDT on metabolic cooperation and support the hypothesis that with calcium CaM may be of importance for drug-induced inhibition of intercellular communication and tumour promotion.