Background and Purpose: The melastatin‐related transient receptor potential TRPM3 is a calcium‐permeable nonselective cation channel that can be activated by the neurosteroid pregnenolone sulphate (PregS) and heat. TRPM3‐deficient mice show an impaired perception of noxious heat. Hence, drugs inhibiting TRPM3 possibly get in focus of analgesic therapy.Experimental Approach: Fluorometric methods were used to identify novel TRPM3‐blocking compounds and to characterize their potency and selectivity to block TRPM3 but not other sensory TRP channels. Biophysical properties of the block were assessed using electrophysiological methods. Single cell calcium measurements confirmed the block of endogenously expressed TRPM3 channels in rat and mouse dorsal root ganglion (DRG) neurones.Key Results: By screening a compound library, we identified three natural compounds as potent blockers of TRPM3. Naringenin and hesperetin belong to the citrus fruit flavanones, and ononetin is a deoxybenzoin. Eriodictyol, a metabolite of naringenin and hesperetin, was still biologically active as a TRPM3 blocker. The compounds exhibited a marked specificity for recombinant TRPM3 and blocked PregS‐induced [Ca²⁺]_i signals in freshly isolated DRG neurones.Conclusion and Implications: The data indicate that citrus fruit flavonoids are potent and selective blockers of TRPM3. Their potencies ranged from upper nanomolar to lower micromolar concentrations. Since physiological functions of TRPM3 channels are still poorly defined, the development and validation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo. Considering the involvement of TRPM3 in nociception, TRPM3 blockers may represent a novel concept for analgesic treatment.

The melastatin-related transient receptor potential (TRP) channel TRPM3 is a nonselective cation channel expressed in nociceptive neurons and activated by heat. Because TRPM3-deficient mice show inflammatory thermal hyperalgesia, pharmacological inhibition of TRPM3 may exert antinociceptive properties. Fluorometric Ca²⁺influx assays and a compound library containing approved or clinically tested drugs were used to identify TRPM3 inhibitors. Biophysical properties of channel inhibition were assessed using electrophysiological methods. The nonsteroidal anti-inflammatory drug diclofenac, the tetracyclic antidepressant maprotiline, and the anticonvulsant primidone were identified as highly efficient TRPM3 blockers with half-maximal inhibition at 0.6 to 6 μM and marked specificity for TRPM3. Most prominently, primidone was biologically active to suppress TRPM3 activation by pregnenolone sulfate (PregS) and heat at concentrations markedly lower than plasma concentrations commonly used in antiepileptic therapy. Primidone blocked PregS-induced Ca²⁺_iinflux through TRPM3 by allosteric modulation and reversibly inhibited atypical inwardly rectifying TRPM3 currents induced by coapplication of PregS and clotrimazole. In vivo, analgesic effects of low doses of primidone were demonstrated in mice, applying PregS- and heat-induced pain models, including inflammatory hyperalgesia. Thus, applying the approved drug at concentrations that are lower than those needed to induce anticonvulsive effects offers a shortcut for studying physiological and pathophysiological roles of TRPM3 in vivo.

P2X7 receptors have emerged as potential drug targets for the treatment of medical conditions such as e.g. rheumatoid arthritis and neuropathic pain. To assess the impact of pharmaceuticals on P2X7, we screened a compound library comprising approved or clinically tested drugs and identified several compounds that augment the ATP-triggered P2X7 activity in a stably transfected HEK293 cell line. Of these, clemastine markedly sensitized Ca(2+) entry through P2X7 to lower ATP concentrations. Extracellularly but not intracellularly applied clemastine rapidly and reversibly augmented P2X7-mediated whole-cell currents evoked by non-saturating ATP concentrations. Clemastine also accelerated the ATP-induced pore formation and Yo-Pro-1 uptake, increased the fractional NMDG(+) permeability, and stabilized the open channel conformation of P2X7. Thus, clemastine is an extracellularly binding allosteric modulator of P2X7 that sensitizes P2X7 to lower ATP concentrations and facilitates its pore dilation. The activity of clemastine on native P2X7 receptors, Ca(2+) entry, and whole-cell currents was confirmed in human monocyte-derived macrophages. Similar effects were observed in murine bone marrow-derived macrophages. Consistent with the data on recombinant P2X7, clemastine augmented the ATP-induced cation entry and Yo-Pro-1 uptake. In accordance with the observation that P2X7 controls the cytokine release from LPS-primed macrophages, we found that clemastine augmented the IL-1β release from LPS-primed human macrophages. Collectively, these data point to a sensitization of the recombinantly or natively expressed human P2X7 receptor toward its physiological activator, ATP, possibly leading to a modulation of macrophage-dependent immune responses.

Classical or canonical transient receptor potential 6 (TRPC6), a nonselective and Ca²⁺-permeable cation channel, mediates pathophysiological responses within pulmonary and renal diseases that are still poorly controlled by current medication. Thus, controlling TRPC6 activity may provide a promising and challenging pharmacological approach. Recently identified chemical entities have demonstrated that TRPC6 is pharmacologically targetable. However, isotype-selectivity with regard to its closest relative, TRPC3, is difficult to achieve. Reasoning that balsams, essential oils, or incense materials that are traditionally used for inhalation may contain biologic activities to block TRPC6 activity, we embarked on a natural compound strategy to identify new TRPC6-blocking chemical entities. Within several preparations of plant extracts, a strong TRPC6-inhibitory activity was found in conifer balsams. The biologic activity was associated with nonvolatile resins, but not with essential oils. Of various conifers, the larch balsam was unique in displaying a marked TRPC6-prevalent mode of action. By testing the main constituents of larch resin, we identified larixol and larixyl acetate as blockers of Ca²⁺ entry and ionic currents through diacylglycerol- or receptor-activated recombinant TRPC6 channels, exhibiting approximately 12- and 5-fold selectivity compared with its closest relatives TRPC3 and TRPC7, respectively. No significant inhibition of more distantly related TRPV or TRPM channels was seen. The potent inhibition of recombinant TRPC6 by larixyl acetate (IC₅₀ = 0.1–0.6 µM) was confirmed for native TRPC6-like [Ca²⁺]_i signals in diacylglycerol-stimulated rat pulmonary artery smooth muscle cells. In isolated mouse lungs, larix-6-yl monoacetate (CAS 4608-49-5; larixyl acetate; 5 µM) prevented acute hypoxia-induced vasoconstriction. We conclude that larch-derived labdane-type diterpenes are TRPC6-selective inhibitors and may represent a starting point for pharmacological TRPC6 modulation within experimental therapies.