Opioids are first-line drugs for moderate to severe acute pain and cancer pain. However, these medications are associated with severe side effects, and whether they are efficacious in treatment of chronic nonmalignant pain remains controversial. Medications that act through alternative molecular mechanisms are critically needed. Antagonists of α9α10 nicotinic acetylcholine receptors (nAChRs) have been proposed as an important nonopioid mechanism based on studies demonstrating prevention of neuropathology after trauma-induced nerve injury. However, the key α9α10 ligands characterized to date are at least two orders of magnitude less potent on human vs. rodent nAChRs, limiting their translational application. Furthermore, an alternative proposal that these ligands achieve their beneficial effects by acting as agonists of GABA B receptors has caused confusion over whether blockade of α9α10 nAChRs is the fundamental underlying mechanism. To address these issues definitively, we developed RgIA4, a peptide that exhibits high potency for both human and rodent α9α10 nAChRs, and was at least 1,000-fold more selective for α9α10 nAChRs vs. all other molecular targets tested, including opioid and GABA B receptors. A daily s.c. dose of RgIA4 prevented chemotherapy-induced neuropathic pain in rats. In wild-type mice, oxaliplatin treatment produced cold allodynia that could be prevented by RgIA4. Additionally, in α9 KO mice, chemotherapy-induced development of cold allodynia was attenuated and the milder, temporary cold allodynia was not relieved by RgIA4. These findings establish blockade of α9-containing nAChRs as the basis for the efficacy of RgIA4, and that α9-containing nAChRs are a critical target for prevention of chronic cancer chemotherapyinduced neuropathic pain.pain | chemotherapy | alpha9 | nicotinic

Voltage-gated sodium channels (VGSCs) are important for action potentials. There are seven major isoforms of the pore-forming and gate-bearing α-subunit (Na V 1) of VGSCs in mammalian neurons, and a given neuron can express more than one isoform. Five of the neuronal isoforms, Na V 1.1, 1.2, 1.3, 1.6, and 1.7, are exquisitely sensitive to tetrodotoxin (TTX), and a functional differentiation of these presents a serious challenge. Here, we examined a panel of 11 μ-conopeptides for their ability to block rodent Na V 1.1 through 1.8 expressed in Xenopus oocytes. Although none blocked Na V 1.8, a TTX-resistant isoform, the resulting "activity matrix" revealed that the panel could readily discriminate between the members of all pair-wise combinations of the tested isoforms. To examine the identities of endogenous VGSCs, a subset of the panel was tested on A-and C-compound action potentials recorded from isolated preparations of rat sciatic nerve. The results show that the major subtypes in the corresponding A-and C-fibers were Na V 1.6 and 1.7, respectively. Ruled out as major players in both fiber types were Na V 1.1, 1.2, and 1.3. These results are consistent with immunohistochemical findings of others. To our awareness this is the first report describing a qualitative pharmacological survey of TTX-sensitive Na V 1 isoforms responsible for propagating action potentials in peripheral nerve. The panel of μ-conopeptides should be useful in identifying the functional contributions of Na V 1 isoforms in other preparations. Hodgkin and Huxley developed a quantitative theory for the ionic basis of the action potential (1)-the molecular correlates of their pioneering efforts are the voltage-gated sodium channel (VGSC) and the voltage-gated potassium channel. Critical for the appreciation of the discrete biochemical nature of VGSCs were the investigations of the mechanism of action of the alkaloids tetrodotoxin (TTX) and saxitoxin (2-4). In the half century since these classic experiments, our understanding of the molecular structure of VGSCs has advanced dramatically (5). We now recognize that mammals have nine isoforms of the α-subunit of VGSCs, or Na V 1, the subunit containing both the Na + -conducting pore and voltage-sensing gate (5-7). Meanwhile, progress in the characterization of ligands that target VGSCs has also progressed (8, 9), although TTX remains a major pharmacological investigative tool for VGSCs.The nine mammalian Na V 1 isoforms can be categorized into those that are TTX-sensitive versus those that are resistant, with K d or IC 50 values in the nM versus μM ranges, respectively (7). Two, Na V 1.8 and Na V 1.9, are found in primary sensory neurons and are highly resistant to TTX (10-12). One, Na V 1.5, found in cardiac muscle, is moderately TTX resistant (13), and the remaining six Na V 1 isoforms are exquisitely sensitive to TTX. One of these, Na V 1.4 is found exclusively in skeletal muscle. Presently, the five neuronal subtypes that are TTX-sensitive (i.e., Na V 1.1, 1.2, 1.3, 1.6, and 1.7) cannot be re...

Signal transduction modifiers that modulate the lysophosphatidic acid (LPA) pathway have potential as anticancer agents. Herein, we describe metabolically stabilized LPA analogues that reduce cell migration and invasion and cause regression of orthotopic breast tumors in vivo. Two diastereoisomeric α-bromophosphonates (BrP-LPA) were synthesized, and the pharmacology was determined for five LPA G protein–coupled receptors (GPCRs). The syn and anti diastereomers of BrP-LPA are pan-LPA GPCR antagonists and are also nanomolar inhibitors of the lysophospholipase D activity of autotaxin, the dominant biosynthetic source of LPA. Computational models correctly predicted the diastereoselectivity of antagonism for three GPCR isoforms. The anti isomer of BrP-LPA was more effective than syn isomer in reducing migration of MDA-MB-231 cells, and the anti isomer was superior in reducing invasion of these cells. Finally, orthotopic breast cancer xenografts were established in nude mice by injection of MB-231 cells in an in situ cross-linkable extracellular matrix. After 2 weeks, mice were treated with the BrP-LPA alone (10 mg/kg), Taxol alone (10 mg/kg), or Taxol followed by BrP-LPA. All treatments significantly reduced tumor burden, and BrP-LPA was superior to Taxol in reducing blood vessel density in tumors. Moreover, both the anti- and syn-BrP-LPA significantly reduced tumors at 3 mg/kg.

More than 100 species of venomous cone snails (genus Conus) are highly effective predators of fish. The vast majority of venom components identified and functionally characterized to date are neurotoxins specifically targeted to receptors, ion channels, and transporters in the nervous system of prey, predators, or competitors. Here we describe a venom component targeting energy metabolism, a radically different mechanism. Two fish-hunting cone snails, Conus geographus and Conus tulipa, have evolved specialized insulins that are expressed as major components of their venoms. These insulins are distinctive in having much greater similarity to fish insulins than to the molluscan hormone and are unique in that posttranslational modifications characteristic of conotoxins (hydroxyproline, γ-carboxyglutamate) are present. When injected into fish, the venom insulin elicits hypoglycemic shock, a condition characterized by dangerously low blood glucose. Our evidence suggests that insulin is specifically used as a weapon for prey capture by a subset of fish-hunting cone snails that use a net strategy to capture prey. Insulin appears to be a component of the nirvana cabal, a toxin combination in these venoms that is released into the water to disorient schools of small fish, making them easier to engulf with the snail's distended false mouth, which functions as a net. If an entire school of fish simultaneously experiences hypoglycemic shock, this should directly facilitate capture by the predatory snail.insulin shock | cone snails | conotoxins | nirvana cabal | venom

Isoform-selective agonists and antagonists of the lysophosphatidic acid (LPA) G-protein-coupled receptors (GPCRs) have important potential applications in cell biology and therapy. LPA GPCRs regulate cancer cell proliferation, invasion, angiogenesis, and biochemical resistance to chemotherapy-and radiotherapy-induced apoptosis. LPA and its analogues are also feedback inhibitors of the enzyme lysophospholipase D (lysoPLD, also known as autotaxin), a central regulator of invasion and metastasis. For cancer therapy, the ideal therapeutic profile would be a metabolically stabilized pan-LPA receptor antagonist that also inhibits lysoPLD. Herein we describe the synthesis of a series of novel α-substituted methylene phosphonate analogues of LPA. Each of these analogues contains a hydrolysis-resistant phosphonate mimic of the labile monophosphate of natural LPA. The pharmacological properties of these phosphono-LPA analogues were characterized in terms of LPA receptor subtype-specific agonist and antagonist activity using Ca 2+ mobilization assays in RH7777 and CHO cells expressing the individual LPA GPCRs. In particular, the methylene phosphonate LPA analogue is a selective LPA 2 agonist, whereas the corresponding α-hydroxymethylene phosphonate is a selective LPA 3 agonist. Most importantly, the α-bromomethylene and α-chloromethylene phosphonates show pan-LPA receptor subtype antagonist activity. The α-bromomethylene phosphonates are the first reported antagonists for the LPA 4 GPCR. Each of the α-substituted methylene phosphonates inhibits lysoPLD, with the unsubstituted methylene phosphonate showing the most potent inhibition. Finally, unlike many LPA analogues, none of these compounds activate the intracellular LPA receptor PPARγ.