Discovery of a selective, state-independent inhibitor of NaV1.7 by modification of guanidinium toxins.

The voltage-gated sodium channel isoform NaV1.7 is highly expressed in dorsal root ganglion neurons and is obligatory for nociceptive signal transmission. Genetic gain-of-function and loss-of-function NaV1.7 mutations have been identified in select individuals, and are associated with episodic extreme pain disorders and insensitivity to pain, respectively. These findings implicate NaV1.7 as a key pharmacotherapeutic target for the treatment of pain. While several small molecules targeting NaV1.7 have been advanced to clinical development, no NaV1.7-selective compound has shown convincing efficacy in clinical pain applications. Here we describe the discovery and characterization of ST-2262, a NaV1.7 inhibitor that blocks the extracellular vestibule of the channel with an IC50 of 72 nM and greater than 200-fold selectivity over off-target sodium channel isoforms, NaV1.1-1.6 and NaV1.8. In contrast to other NaV1.7 inhibitors that preferentially inhibit the inactivated state of the channel, ST-2262 is equipotent in a protocol that favors the resting state of the channel, a protocol that favors the inactivated state, and a high frequency protocol. In a non-human primate study, animals treated with ST-2262 exhibited reduced sensitivity to noxious heat. These findings establish the extracellular vestibule of the sodium channel as a viable receptor site for the design of selective ligands targeting NaV1.7.

Pharmacological profile of the isomers of the GluR-specific agonist ATPA.

We have synthesized the (R)- and (S)-isomers of 2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoate (ATPA) by stereochemically certain routes. Our studies in the rat cortical wedge preparation indicate that (R)-ATPA has no observable excitatory effect, while (S)-ATPA has an apparent K(D) of 16 microM. This excitatory response is unaffected by the specific N-methyl-D-aspartate (NMDA) antagonist, D-2-amino-5-phosphonopentanoic acid (DAP5) but is partially blocked by 6-nitro-sulfamoyl[f]quinoxalinedione (NBQX) at concentrations that attenuate the effects of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA), the effects are however reduced by the nonspecific antagonist kynurenate (KYN), indicating an interaction with a class of kainate receptor.

Phase transition properties of aqueous dispersions of homologues of all-trans 2,3-dipalmitoylcyclopentano-1-phosphocholine.

In a previous publication, (Singer, M.A., Jain, M.K., Sable, H.Z., Pownall, H.H., Mantulin, W.W., Lister, M.D. and Hancock, A.J. (1983) Biochim. Biophys. Acta, 731, 373-377), we reported the properties of aqueous dispersions of the six diastereo-isomers of cyclopentanoid analogues of dipalmitoylphosphatidylcholine. Two of these isomers displayed unusually high enthalpies of transition, about double that of dipalmitoylphosphatidylcholine. One of the high enthalpy isomers whose configuration is all-trans has now been modified by the insertion of extra methylene residues (n = 3 through 9) between the nitrogen and phosphorus atoms of the headgroup. Vesicles were formed from these lipids and studied by 22Na permeability measurements, differential scanning calorimetry, fluorescence polarization, 31P-NMR, and freeze-fracture electron microscopy. Vesicles composed of lipids with n = 2 or 3 exhibit a sharp transition at 46 degrees C or 49 degrees C, respectively, and a high enthalpy with no detectable sub- or pretransitions. Lipids with n greater than 3 exhibit a main transition between 38 and 43 degrees C with enthalpies less than 10 kcal/mol and after prolonged coding (more than 3 days at 4 degrees C) a broad endotherm at about 20 +/- 3 degrees C with enthalpies greater than 4 kcal/mol. These same dispersions display a permeability peak at 20-25 degrees C and a second increase in 22Na efflux in the temperature range 30-40 degrees C. The results of 31P-NMR measurements suggest that the acyl chains in 2,3-dipalmitoylcyclopentano-1-phosphocholine (n = 2) bilayers have restricted rotation below the main phase transition temperature.

Synthesis of polar head group homologs of all-trans-cyclopentano-phosphatidylcholine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylethanolamine.

The polar head group region of a conformationally restricted analog of phosphatidic acid (diacylglycero-phosphate) has been systematically modified to give analogs of phosphatidylcholine, phosphatidylethanolamine, and phosphatidyl-N,N-dimethylethanolamine. These analogs differ from their natural counterpart in both the backbone region and in the polar head region, respectively, as follows: the diacylglyceryl moiety has been replaced by an all-trans diacylcyclopentane-1,2,3-triol moiety and the phosphorus-nitrogen separation has been increased incrementally from two to nine methylene units. The synthesis of these homologous series involved phosphorylation of (1,3/2)-2,3-dipalmitoylcyclopentane-1,2,3-triol with each of a series of homologous bromoalkylphosphoric acid dichlorides, which were themselves obtained by phosphorus oxychloride treatment of the homologous bromoalkanols. The resulting bromoalkyl esters of 2,3-dipalmitoylcyclopentane-1,2,3-triol-1-phosphoric acid were reacted with trimethylamine, dimethylamine, or ammonia to give the cyclopentano-phosphatidylcholines, cyclopentano-N,N-dimethylethanolamines, and cyclopentano-phosphatidylethanolamine, respectively. All the compounds were obtained as stable microcrystalline solids. The yields of cyclopentano-phosphatidylethanolamines and of cyclopentano-N,N-dimethylethanolamines were reduced by the formation of compounds which analyzed as monoacyl (lyso) derivatives.

Synthesis of cyclopentano-N-methylphosphatidylethanolamines: aminolysis during the use of methylamine.

Monomethylamine and N-benzyl-N-methylamine were used as nucleophiles in the amination of the bromoethylester of cyclopentano-phosphatidic acid. The former reagent led to extensive aminolysis and the quantitative formation of N-methylpalmitamide, rather than the desired cyclopentano-phosphatidyl-N-methylethanolamine. However, the method of Shapiro and Rabinsohn (1964. Biochemistry. 3:603-605), in which N-benzyl-N-methylamine was used as a nucleophile, was adapted for a successful synthesis.

Synthesis of conformationally restricted acidic lipids. I. Cyclopentanoid analogs of phosphatidylserine.

A series of six analogs of phosphatidylserine (PS) was synthesized in which the conformational mobility of the backbone was limited. Each analog was a derivative of one of the three diastereoisomeric cyclopentane-1,2,3-triols, so that the glycerol moiety of the PS was, in effect, replaced by each cyclopentane-1,2,3-triol. Four of the members of the series were vicinal dipalmitates [1,2/3-(1P); 1,2/3-(3P); 1,2,3/0-(1P); 1,3/2-(1P)] while the other two members were "unnatural" in that they contained a 2-phosphate group. The two 2-phosphate derivatives were meso-forms and each of the other four derivatives was a DL-pair. Each PS analog was obtained as a stable microcrystalline precipitate analyzing for the monohydrate of a mixture of mono- and di-sodium salts. The infrared spectra, melting behavior, and chromatographic mobility of each sodium salt mixture resembled those obtained for bovine (glycero-)-phosphatidyl-L-serine; the stereochemical differences in the ring caused only marginal variation in these properties. The optical rotation values of the compounds varied with the stereochemistry of the ring. The all-trans-2-phosphate isomer 8d exhibited a negative rotation value, in contrast to each of the other isomers. The all-trans isomer [1,3/2-(1P)] was shown to undergo diazometholysis with diazomethane to give the dimethyl ester of cyclopentano-phosphatidic acid.