The endocannabinoid anandamide (AEA) is an antinociceptive lipid that is inactivated through cellular uptake and subsequent catabolism by fatty acid amide hydrolase (FAAH). Fatty acid binding proteins (FABPs) are intracellular carriers that deliver AEA and related N-acylethanolamines (NAEs) to FAAH for hydrolysis. The mammalian brain expresses three FABP subtypes: FABP3, FABP5, and FABP7. Recent work from our group has revealed that pharmacological inhibition of FABPs reduces inflammatory pain in mice. The goal of the current work was to explore the effects of FABP inhibition upon nociception in diverse models of pain. We developed inhibitors with differential affinities for FABPs to elucidate the subtype(s) that contributes to the antinociceptive effects of FABP inhibitors.Inhibition of FABPs reduced nociception associated with inflammatory, visceral, and neuropathic pain. The antinociceptive effects of FABP inhibitors mirrored their affinities for FABP5, while binding to FABP3 and FABP7 was not a predictor of in vivo efficacy. The antinociceptive effects of FABP inhibitors were mediated by cannabinoid receptor 1 (CB1) and peroxisome proliferator-activated receptor alpha (PPARα) and FABP inhibition elevated brain levels of AEA, providing the first direct evidence that FABPs regulate brain endocannabinoid tone. These results highlight FABPs as novel targets for the development of analgesic and anti-inflammatory therapeutics.

BackgroundFatty-acid-binding proteins (FABPs) are intracellular carriers for endocannabinoids, N-acylethanolamines, and related lipids. Previous work indicates that systemically administered FABP5 inhibitors produce analgesia in models of inflammatory pain. It is currently not known whether FABP inhibitors exert their effects through peripheral or central mechanisms. Here, we examined FABP5 distribution in dorsal root ganglia and spinal cord and examined the analgesic effects of peripherally and centrally administered FABP5 inhibitors.ResultsImmunofluorescence revealed robust expression of FABP5 in lumbar dorsal root ganglia. FABP5 was distributed in peptidergic calcitonin gene-related peptide-expressing dorsal root ganglia and non-peptidergic isolectin B4-expressing dorsal root ganglia. In addition, the majority of dorsal root ganglia expressing FABP5 also expressed transient receptor potential vanilloid 1 (TRPV1) and peripherin, a marker of nociceptive fibers. Intraplantar administration of FABP5 inhibitors reduced thermal and mechanical hyperalgesia in the complete Freund’s adjuvant model of chronic inflammatory pain. In contrast to its robust expression in dorsal root ganglia, FABP5 was sparsely distributed in the lumbar spinal cord and intrathecal administration of FABP inhibitor did not confer analgesic effects. Administration of FABP inhibitor via the intracerebroventricular (i.c.v.) route reduced thermal hyperalgesia. Antagonists of peroxisome proliferator-activated receptor alpha blocked the analgesic effects of peripherally and i.c.v. administered FABP inhibitor while antagonism of cannabinoid receptor 1 blocked the effects of peripheral FABP inhibition and a TRPV1 antagonist blocked the effects of i.c.v. administered inhibitor. Although FABP5 and TRPV1 were co-expressed in the periaqueductal gray region of the brain, which is known to modulate pain, knockdown of FABP5 in the periaqueductal gray using adeno-associated viruses and pharmacological FABP5 inhibition did not produce analgesic effects.ConclusionsThis study demonstrates that FABP5 is highly expressed in nociceptive dorsal root ganglia neurons and FABP inhibitors exert peripheral and supraspinal analgesic effects. This indicates that peripherally restricted FABP inhibitors may serve as a new class of analgesic and anti-inflammatory agents.

Botulinum neurotoxins (BoNT) are among the most poisonous substances known, and of the 7 serotypes (A-G) identified thus far at least 4 can cause death in humans. The goal of this work was identification of inhibitors that specifically target the light chain catalytic site of the highly pathogenic but lesser-studied E serotype (BoNT/E). Large-scale computational screening, employing the program DOCK, was used to perform atomic-level docking of 1.4 million small molecules to prioritize those making favorable interactions with the BoNT/E site. In particular, 'footprint similarity' (FPS) scoring was used to identify compounds that could potentially mimic features on the known substrate tetrapeptide RIME. Among 92 compounds purchased and experimentally tested, compound C562-1101 emerged as the most promising hit with an apparent IC value three-fold more potent than that of the first reported BoNT/E small molecule inhibitor NSC-77053. Additional analysis showed the predicted binding pose of C562-1101 was geometrically and energetically stable over an ensemble of structures generated by molecular dynamic simulations and that many of the intended interactions seen with RIME were maintained. Several analogs were also computationally designed and predicted to have further molecular mimicry thereby demonstrating the potential utility of footprint-based scoring protocols to help guide hit refinement.

The secondary amine moieties of polyamines prepared by the reactions of aliphatic diamines, and 1,5-difluoro-2,4-dinitrobenzene, were nitrosated using sodium nitrite and aqueous concentrated sulfuric acid at low temperature. Released NO from these polymers, suspended in water or phosphate buffer solutions (PBS), was measured at increasing time intervals by colorimetric techniques using Griess Reagent. Similar tests were also conducted with model compounds. In general, both the model compounds and the corresponding polymers exhibited similar NO release profiles, with apparent half lives up to 88 h and 81 h, respectively. They were strongly dependent on the length of the alkyl chains derived from the primary amines used for the preparation of these NO-releasing materials. Results from in vitro studies using low molecular weight NO-releasing compounds with PC-12 cell are also reported. High concentration of NO induced neuronal cell death. On the other hand, low concentration of NO inhibited cell death induced by oxidative stress. This suggests that cell survival effect is NO-dependent.KEY WORDS: Nitrosated Polyamines / Nitric Oxide / Controlled Release / Cell Culture / Nitrogen monoxide, NO, more commonly known as nitric oxide, plays a critical role in a variety of biological functions.1-3 Towards the end of 1987, the discovery that mammalian cells synthesize nitric oxide, which regulates virtually every critical cellular function, has led to an explosion of research activities.4,5 For example, NO, which is produced from the amino acid L-Arginine in vivo, is involved in the regulation of neurotransmissions, blood pressure, and immune response.6 Nitric oxide can exist as a radical, NO , as a positively charged ion, NO þ (nitrosonium ion), and as nitroxyl anion (NO À ). 7 This allows NO to participate in numerous important biological functions mentioned above as well as others. Diseased states ensue when the bioavailability of NO, for a variety of reasons, becomes impaired. 8,9 Exogenous NO, derived from various families of NO-donors, has been used to ameliorate debilitating symptoms of a few of these diseases. 10A vast majority of NO-donors are low molecular weight compounds including: nitrates, nitrites, N-nitroso, C-nitroso, certain heterocycles, metal-NO complexes, and diazeniumdiolates. 10 Depending on the chemical nature of these compounds, NO is released spontaneously either in the presence or the absence of a catalyst. In the case of diazeniumdiolates, half-life of NO generation can be varied between 2 s to 56 h by changing the nature of the backbone to which the diazenuimdiolate moiety is attached. 11In contrast to the low molecular weight NO-donors, which have been investigated extensively, polymeric NO donors are new arrivals in this field. Since NO is known to prevent platelet aggregation, 12 NO generating polymers can be used as biomaterials, which are likely to come in contact with blood. In addition, the biocompatibility of implanted chemical sensors can be enhanced significantly by using NO-releasing...

Botulinum neurotoxins (BoNTs) are among the most potent biological toxin known to humans, and are classified as Category A bioterrorism agents by the Centers for Disease Control and prevention (CDC). There are seven known BoNT serotypes (A-G) which have been thus far identified in literature. BoNTs have been shown to block neurotransmitter release by cleaving proteins of the soluble NSF attachment protein receptor (SNARE) complex. Disruption of the SNARE complex precludes motor neuron failure which ultimately results in flaccid paralysis in humans and animals. Currently, there are no effective therapeutic treatments against the neurotoxin light chain (LC) after translocation into the cytosols of motor neurons. In this work, high-throughput in silico screening was employed to screen a library of commercially available compounds from ZINC database against BoNT/A-LC. Among the hit compounds from the in silico screening, two lead compounds were identified and found to have potent inhibitory activity against BoNT/A-LC in vitro, as well as in Neuro-2a cells. A few analogs of the lead compounds were synthesized and their potency examined. One of these analogs showed an enhanced activity than the lead compounds.