Ongoing movement controls sensory integration in the dorsolateral striatum.

The dorsolateral striatum (DLS) receives excitatory inputs from both sensory and motor cortical regions. In the neocortex, sensory responses are affected by motor activity, however, it is not known whether such sensorimotor interactions occur in the striatum and how they are shaped by dopamine. To determine the impact of motor activity on striatal sensory processing, we performed in vivo whole-cell recordings in the DLS of awake mice during the presentation of tactile stimuli. Striatal medium spiny neurons (MSNs) were activated by both whisker stimulation and spontaneous whisking, however, their responses to whisker deflection during ongoing whisking were attenuated. Dopamine depletion reduced the representation of whisking in direct-pathway MSNs, but not in those of the indirect-pathway. Furthermore, dopamine depletion impaired the discrimination between ipsilateral and contralateral sensory stimulation in both direct and indirect pathway MSNs. Our results show that whisking affects sensory responses in DLS and that striatal representation of both processes is dopamine- and cell type-dependent.

ß-Lactam TRPM8 Antagonist RGM8-51 Displays Antinociceptive Activity in Different Animal Models.

Transient receptor potential melastatin subtype 8 (TRPM8) is a cation channel extensively expressed in sensory neurons and implicated in different painful states. However, the effectiveness of TRPM8 modulators for pain relief is still a matter of discussion, since structurally diverse modulators lead to different results, depending on the animal pain model. In this work, we described the antinociceptive activity of a ß-lactam derivative, RGM8-51, showing good TRPM8 antagonist activity, and selectivity against related thermoTRP channels and other pain-mediating receptors. In primary cultures of rat dorsal root ganglion (DRG) neurons, RGM8-51 potently reduced menthol-evoked neuronal firing without affecting the major ion conductances responsible for action potential generation. This compound has in vivo antinociceptive activity in response to cold, in a mouse model of oxaliplatin-induced peripheral neuropathy. In addition, it reduces cold, mechanical and heat hypersensitivity in a rat model of neuropathic pain arising after chronic constriction of the sciatic nerve. Furthermore, RGM8-51 exhibits mechanical hypersensitivity-relieving activity, in a mouse model of NTG-induced hyperesthesia. Taken together, these preclinical results substantiate that this TRPM8 antagonist is a promising pharmacological tool to study TRPM8-related diseases.

Paclitaxel in vitro reversibly sensitizes the excitability of IB4(-) and IB4(+) sensory neurons from male and female rats.

BACKGROUND AND PURPOSE: Paclitaxel produces a chemotherapy-induced peripheral neuropathy that persists in 50-60% of cancer patients upon treatment. Evidence from animal models suggests an axonopathy of peripheral A- and C-type fibres that affects their excitability. However, direct effects of paclitaxel on sensory neuron excitability and sexual dimorphism remain poorly understood. EXPERIMENTAL APPROACH: We used a long-lasting (10 days in vitro) primary culture of rat dorsal root ganglion (DRG) neurons to investigate the time course effect of paclitaxel on the electrical activity of IB4(-) and IB4(+) sensory neurons of female and male adult Wistar rats. KEY RESULTS: Paclitaxel strongly and reversibly stimulated spontaneous activity and augmented action potential tonic firing in IB4(-) and IB4(+) neurons in both sexes, peaking at 48 h post-treatment and virtually disappearing at 96 h. Paclitaxel decreased the current rheobase for action potential firing by reducing and accelerating the after-hyperpolarization phase. Molecularly, paclitaxel modulated Na+ and K+ ion currents. Particularly, the drug significantly augmented the function of Nav 1.8, TRPV1 and TRPM8 channels. Furthermore, paclitaxel increased Nav 1.8 and TRPV1 expression at 48 h post-treatment. Notably, we observed that female DRG neurons appear more sensitive to paclitaxel sensitization than their male counterparts. CONCLUSIONS AND IMPLICATIONS: Our data indicate that paclitaxel similarly potentiated IB4(-) and IB4(+) electrogenicity and uncover a potential sex dimorphism in paclitaxel-induced chemotherapy-induced peripheral neuropathy. Our in vitro, pre-clinical, chemotherapy-induced peripheral neuropathy paradigm provides a tool for studying the dynamics and underlying molecular mechanisms contributing to nociceptor sensitization in peripheral neuropathies and for testing desensitizing compounds.

Phenylalanine-Derived ß-Lactam TRPM8 Modulators. Configuration Effect on the Antagonist Activity.

Transient receptor potential cation channel subfamily M member 8 (TRPM8) is a Ca2+ non-selective ion channel implicated in a variety of pathological conditions, including cancer, inflammatory and neuropathic pain. In previous works we identified a family of chiral, highly hydrophobic ß-lactam derivatives, and began to intuit a possible effect of the stereogenic centers on the antagonist activity. To investigate the influence of configuration on the TRPM8 antagonist properties, here we prepare and characterize four possible diastereoisomeric derivatives of 4-benzyl-1-[(3'-phenyl-2'-dibenzylamino)prop-1'-yl]-4-benzyloxycarbonyl-3-methyl-2-oxoazetidine. In microfluorography assays, all isomers were able to reduce the menthol-induced cell Ca2+ entry to larger or lesser extent. Potency follows the order 3R,4R,2'R > 3S,4S,2'R ≅ 3R,4R,2'S > 3S,4S,2'S, with the most potent diastereoisomer showing a half inhibitory concentration (IC50) in the low nanomolar range, confirmed by Patch-Clamp electrophysiology experiments. All four compounds display high receptor selectivity against other members of the TRP family. Furthermore, in primary cultures of rat dorsal root ganglion (DRG) neurons, the most potent diastereoisomers do not produce any alteration in neuronal excitability, indicating their high specificity for TRPM8 channels. Docking studies positioned these ß-lactams at different subsites by the pore zone, suggesting a different mechanism than the known N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide (AMTB) antagonist.

Highly functionalized ß-lactams and 2-ketopiperazines as TRPM8 antagonists with antiallodynic activity.

The cool sensor transient receptor potential melastatin channel 8 (TRPM8) is highly expressed in trigeminal and dorsal root ganglia, playing a key role in cold hypersensitivity associated to different peripheral neuropathies. Moreover, these channels are aberrantly expressed in different cancers, and seem to participate in tumor progression, survival and invasion. Accordingly, the search for potent and selective TRPM8 modulators attracted great interest in recent years. We describe new heterocyclic TRPM8 antagonist chemotypes derived from N-cloroalkyl phenylalaninol-Phe conjugates. The cyclization of these conjugates afforded highly substituted ß-lactams and/or 2-ketopiperazine (KP) derivatives, with regioselectivity depending on the N-chloroalkyl group and the configuration. These derivatives behave as TRPM8 antagonists in the Ca2+ microfluorometry assay, and confirmed electrophysiologically for the best enantiopure ß-lactams 24a and 29a (IC50, 1.4 and 0.8 µM). Two putative binding sites by the pore zone, different from those found for typical agonists and antagonists, were identified by in silico studies for both ß-lactams and KPs. ß-Lactams 24a and 29a display antitumor activity in different human tumor cell lines (micromolar potencies, A549, HT29, PSN1), but correlation with TRPM8 expression could not be established. Additionally, compound 24a significantly reduced cold allodynia in a mice model of oxaliplatin-induced peripheral neuropathy.

1-(2',5'-Dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (RGM079): A Positive Allosteric Modulator of α7 Nicotinic Receptors with Analgesic and Neuroprotective Activity.

Acetylcholine α7 nicotinic receptors are widely expressed in the brain, where they are involved in the central processing of pain as well as in neuropsychiatric, neurodegenerative, and inflammatory processes. Positive allosteric modulators (PAMs) show the advantage of allowing the selective regulation of different subtypes of acetylcholine receptors without directly interacting with the agonist binding site. Here, we report the preparation and biological activity of a fluoro-containing compound, 1-(2',5'-dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (8, RGM079), that behaves as a potent PAM of the α7 receptors and has a balanced pharmacokinetic profile and antioxidant properties comparable or even higher than well-known natural polyphenols. In addition, compound RGM079 shows neuroprotective properties in Alzheimer's disease (AD)-toxicity related models. Thus, it causes a concentration-dependent neuroprotective effect against the toxicity induced by okadaic acid (OA) in the human neuroblastoma cell line SH-SY5Y. Similarly, in primary cultures of rat cortical neurons, RGM079 is able to restore the cellular viability after exposure to OA and amyloid peptide Aß1-42, with cell death almost completely prevented at 10 and 30 µM, respectively. Finally, compound RGM079 shows in vivo analgesic activity in the complete Freund's adjuvant (CFA)-induced paw inflammation model after intraperitoneal administration.

Amino acid and peptide prodrugs of diphenylpropanones positive allosteric modulators of α7 nicotinic receptors with analgesic activity.

α7 Nicotinic acetylcholine receptors (nAChRs) are ion channels implicated in a number of CNS pathological processes, including pain and psychiatric, cognitive and inflammatory diseases. Comparing with orthosteric agonism, positive allosteric modulation of these channels constitutes an interesting approach to achieve selectivity versus other nicotinic receptors. We have recently described new chalcones and 1,3-diphenylpropanones as positive allosteric modulators (PAMs) of α7 nAChRs, which proved to have good analgesic activities but poor pharmacokinetic properties. Here we report the preparation of amino acid and peptide derivatives as prodrugs of these modulators with the aim of improving their in vivo biological activity. While the valine derivative showed very short half life in aqueous solutions to be considered a prodrug, Val-Val and Val-Pro-Val are suitable precursors of the parent 1,3-diphenylpropanones, via chemical and enzymatic transformation, respectively. Compounds 19 (Val-Val) and 21 (Val-Pro-Val), prodrugs of the 2',5',4-trihydroxy-1,3-diphenylpropan-1-one 3, showed significant antinociceptive activity in in vivo assays. The best compound, 21, displayed a better profile in the analgesia test than its parent compound 3, exhibiting about the same potency but long-lasting effects.

Synthesis, high-throughput screening and pharmacological characterization of ß-lactam derivatives as TRPM8 antagonists.

The mammalian transient receptor potential melastatin channel 8 (TRPM8), highly expressed in trigeminal and dorsal root ganglia, mediates the cooling sensation and plays an important role in the cold hypersensitivity characteristic of some types of neuropathic pain, as well as in cancer. Consequently, the identification of selective and potent ligands for TRPM8 is of great interest. Here, a series of compounds, having a ß-lactam central scaffold, were prepared to explore the pharmacophore requirements for TRPM8 modulation. Structure-activity studies indicate that the minimal requirements for potent ß-lactam-based TRPM8 blockers are hydrophobic groups (benzyl preferentially or t Bu) on R1, R2, R3 and R5 and a short N-alkyl chain (≤3 carbons). The best compounds in the focused library (41 and 45) showed IC50 values of 46 nM and 83 nM, respectively, in electrophysiology assays. These compounds selectively blocked all modalities of TRPM8 activation, i.e. menthol, voltage, and temperature. Molecular modelling studies using a homology model of TRPM8 identified two putative binding sites, involving networks of hydrophobic interactions, and suggesting a negative allosteric modulation through the stabilization of the closed state. Thus, these ß-lactams provide a novel pharmacophore scaffold to evolve TRPM8 allosteric modulators to treat TRPM8 channel dysfunction.

1,3-diphenylpropan-1-ones as allosteric modulators of α7 nACh receptors with analgesic and antioxidant properties.

UNLABELLED: Nicotine acethylcholine receptors (nAChRs) play critical roles in cognitive processes, neuroprotection and inflammation. RESULTS: According to their substituents, 1,3-diphenylpropan-1-one derivatives act as α7 nAChRs negative allosteric modulators (NAM, OMe) or Type I positive allosteric modulators (PAMs, OH). Compounds 7 and 31 were the most effective (989 and 666% enhancement of ACh-induced currents) and potent (EC50: 12.9 and 6.85 µM) PAMs. They exhibited strong radical scavenging values. Compound 31, selective over other neuronal nAChR subtypes and with acceptable pharmacokinetic profile, showed antinociceptive effects in a model of inflammatory pain. CONCLUSION: Compound 31 is a novel, potent and selective α7 nAChR PAM, displaying antioxidant and analgesic activities. The 1,3-diphenylpropan-1-one scaffold could be the base toward more advanced type I PAMs for the treatment of nAChR-mediated diseases.

Tryptamine-Based Derivatives as Transient Receptor Potential Melastatin Type 8 (TRPM8) Channel Modulators.

Pharmacological modulation of the transient receptor potential melastatin type 8 (TRPM8) is currently under investigation as a new approach for the treatment of pain and other diseases. In this study, a series of N-substituted tryptamines was prepared to explore the structural requirements determining TRPM8 modulation. Using a fluorescence-based screening assay, we identified two compounds acting as an activator (2-(1H-indol-3-yl)-N-(4-phenoxybenzyl)ethanamine, 21) or an inhibitor (N,N-dibenzyl-2-(1H-indol-3-yl)ethanamine, 12) of calcium influx in HEK293 cells. In patch-clamp recordings, compound 21 displayed a significantly higher potency (EC50 = 40 ± 4 µM) and a similar efficacy when compared to menthol; by contrast, compound 12 produced a concentration-dependent inhibition of menthol-induced TRPM8 currents (IC50 = 367 ± 24 nM). Molecular modeling studies using a homology model of a single rat TRPM8 subunit identified a putative binding site located between the VSD and the TRP box, disclosing differences in the binding modes for the agonist and the antagonist.

Adamantyl analogues of paracetamol as potent analgesic drugs via inhibition of TRPA1.

Paracetamol also known as acetaminophen, is a widely used analgesic and antipyretic agent. We report the synthesis and biological evaluation of adamantyl analogues of paracetamol with important analgesic properties. The mechanism of nociception of compound 6a/b, an analog of paracetamol, is not exerted through direct interaction with cannabinoid receptors, nor by inhibiting COX. It behaves as an interesting selective TRPA1 channel antagonist, which may be responsible for its analgesic properties, whereas it has no effect on the TRPM8 nor TRPV1 channels. The possibility of replacing a phenyl ring by an adamantyl ring opens new avenues in other fields of medicinal chemistry.

Chalcones as positive allosteric modulators of α7 nicotinic acetylcholine receptors: a new target for a privileged structure.

The α7 acetylcholine nicotine receptor is a ligand-gated ion channel that is involved in cognition disorders, schizophrenia, pain and inflammation among other diseases. Therefore, the development of new agents that target this receptor has great significance. Positive allosteric modulators might be advantageous, since they facilitate receptor responses without directly interacting with the agonist binding site. Here we report the search for and further design of new positive allosteric modulators having the relatively simple chalcone structure. From the natural product isoliquiritigenin as starting point, chalcones substituted with hydroxyl groups at defined locations were identified as optimal and specific promoters of α7 nicotinic function. The most potent compound (2,4,2',5'-tetrahydroxychalcone, 111) was further characterized showing its potential as neuroprotective, analgesic and cognitive enhancer, opening the way for future developments around the chalcone structure.

De novo designed library of linear helical peptides: an exploratory tool in the discovery of protein-protein interaction modulators.

Protein-protein interactions (PPIs) have emerged as important targets for pharmaceutical intervention because of their essential role in numerous physiological and pathological processes, but screening efforts using small-molecules have led to very low hit rates. Linear peptides could represent a quick and effective approach to discover initial PPI hits, particularly if they have inherent ability to adopt specific peptide secondary structures. Here, we address this hypothesis through a linear helical peptide library, composed of four sublibraries, which was designed by theoretical predictions of helicity (Agadir software). The 13-mer peptides of this collection fixes either a combination of three aromatic or two aromatic and one aliphatic residues on one face of the helix (Ac-SSEEX(5)ARNX(9)AAX(12)N-NH2), since these are structural features quite common at PPIs interfaces. The 81 designed peptides were conveniently synthesized by parallel solid-phase methodologies, and the tendency of some representative library components to adopt the intended secondary structure was corroborated through CD and NMR experiments. As proof of concept in the search for PPI modulators, the usefulness of this library was verified on the widely studied p53-MDM2 interaction and on the communication between VEGF and its receptor Flt-1, two PPIs for which a hydrophobic α-helix is essential for the interaction. We have demonstrated here that, in both cases, selected peptides from the library, containing the right hydrophobic sequence of the hot-spot in one of the protein partners, are able to interact with the complementary protein. Moreover, we have discover some new, quite potent inhibitors of the VEGF-Flt-1 interaction, just by replacing one of the aromatic residues of the initial F(5)Y(9)Y(12) peptide by W, in agreement with previous results on related antiangiogenic peptides. Finally, the HTS evaluation of the full collection on thermoTRPs has led to a few antagonists of TRPV1 and TRPA1 channels, which open new avenues on the way to innovative modulators of these channels.