1,4-Dihydropyridines (DHPs) are widely recognized as a highly effective class of L-type calcium channel blockers that offer significant therapeutic potential in managing cardiovascular conditions. Furthermore, their ability to target other types of calcium channels makes DHPs attractive candidates for therapeutic applications in neurological and psychiatric disorders. Close examination of the chemical structures of approved DHP-based antihypertensive drugs with a history of over forty years in the market reveals that the C-4 position is the least altered part of this privileged ring system. In the present study, we focused on this position and synthesized two novel compounds (DB1 and DB2) by carrying out chemical modifications on suitable positions of the main scaffold of DA1 (isobutyl 4-(benzo[d][1,3]dioxol-5-yl)-2,6,6-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate) that was previously identified as a DHP-based effective and selective inhibitor of T-type (Cav3.2) over L-type (Cav1.2) calcium channel. Based on whole-cell patch-clamp analysis on Cav1.2 and Cav3.2, DB1 with bromine group on the benzodioxole ring appeared to be a more effective and selective inhibitor of Cav3.2 compared to DB2 with nitro at the same locus. Molecular docking and molecular dynamics (MD) simulations were performed to investigate the binding mode of both DB1 isomers to Cav3.2. Furthermore, density functional theory (DFT) methods were employed to obtain information regarding the stability of the molecules by computing various parameters, such as electric dipole moment, band gap, electronegativity, and global chemical hardness-softness, related to their charge-transfer characteristics. According to DFT studies, DB1 also appeared to be chemically more stable than DB2. Finally, ionizing radiation-induced free radicals of gamma-irradiated DB1 and DB2 in powder form were examined utilizing the electron paramagnetic resonance (EPR) technique and the obtained data demonstrated that radiation sterilization is suitable for the dosage forms including DB1 and DB2.

Cav3.2 T-type calcium channels are important targets for pain relief in rodent models of inflammatory and neuropathic pain. Even though many T-type channel blockers have been tested in mice, only one molecule, ABT-639, has been tested in phase II clinical studies and did not produce analgesic effects over placebo. Here we examined the effects of ABT-639 on Cav3.2 channel activity in tsA-201 cells and dorsal root ganglion (DRG) neurons, in comparison with another established Cav3.2 inhibitor Z944. These experiments revealed that Z944 mediated ∼100-fold more potent inhibition of Cav3.2 currents than ABT-639, with the latter blocking channel activity by less than 15 percent when applied at a concentration of 30 μM. A slight increase in ABT-639 potency was observed at more depolarized holding potentials, suggesting that this compound may act preferentially on inactivated channels. We tested the effects of both compounds in the Complete Freund's Adjuvant (CFA) model of chronic inflammatory pain, and in partial sciatic nerve injury model of neuropathic pain in mice. In the neuropathic pain model, both Z944 and ABT-639 reversed mechanical hypersensitivity to similar degrees when delivered systemically, but remarkably, when delivered intrathecally, only Z944 was effective. In the CFA model, both compounds reversed thermal hyperalgesia upon systemic delivery, but only Z944 mediated pain relief upon intrathecal delivery, indicating that ABT-639 acts primarily at peripheral sites. ABT-639 lost its analgesic effects in CFA treated Cav3.2 null mice, indicating that these channels are essential for ABT-639-mediated pain relief despite its poor inhibition of Cav3.2 currents.

Kurarinone, the major lavandulyl flavanone identified in the roots of Sophora flavescens, has been reported to have different channel and transporter activity modulation capacities; nevertheless, its ability to block T-type channels and inflammatory pain activity have not been fully investigated. In this work, we used the whole-cell patch clamp technique to examine the ability of kurarinone to block T-type calcium channels. Kurarinone acted as a nonselective T-type channel antagonist that inhibited Cav3.2 channels expressed in tsA-201 cells with an IC50 of 1.1 +/- 0.3 mu M and blocked native T-type channels in mouse dorsal root ganglion neurons. Transiently expressed Cav2.2 channels were also blocked. Molecular docking analysis predicted that the phenyl ring, lavandulyl, and hydroxyl groups of kurarinone interact directly with the pore domains of all three T-type calcium channels via hydrogen and hydrophobic interactions. Kurarinone administered intraperitoneally (10 mg/kg/i.p.) significantly inhibited phase II of formalin-induced nocifensive responses in mice. Furthermore, kurarinone reduced thermal hyperalgesia and mechanical hypersensitivity in mice injected with Complete Freund's adjuvant (CFA) into the hind paw in an inflammatory pain mouse model. Taken together, our findings indicate that kurarinone has analgesic activity through blocking calcium channels.

Cisplatin remains the unchallenged standard therapy for NSCLC. However, it is not completely curative due to drug resistance and oxidative stress-induced toxicity. Drug resistance is linked to overexpression of matrix metalloproteinases (MMPs) and aberrant calcium signalling. We report synthesis of novel thiazole-triazole hybrids as MMP-9 inhibitors with T-type calcium channel blocking and antioxidant effects to sensitise NSCLC to cisplatin and ameliorate its toxicity. MTT and whole cell patch clamp assays revealed that 6d has a balanced profile of cytotoxicity (IC50 = 21 ± 1 nM, SI = 12.14) and T-type calcium channel blocking activity (⁓60% at 10 μM). It exhibited moderate ROS scavenging activity and nanomolar MMP-9 inhibition (IC50 = 90 ± 7 nM) surpassing NNGH with MMP-9 over −2 and MMP-10 over −13 selectivity. Docking and MDs simulated its receptor binding mode. Combination studies confirmed that 6d synergized with cisplatin (CI = 0.69 ± 0.05) lowering its IC50 by 6.89 folds. Overall, the study introduces potential lead adjuvants for NSCLC platinum-based therapy.

Migraine is a painful neurological syndrome characterized by attacks of throbbing headache, of moderate to severe intensity, which is associated with photo- and phono- sensitivity as well as nausea and vomiting. It affects about 15

Background and Purpose Cannabinoids are a promising therapeutic avenue for chronic pain. However, clinical trials often fail to report analgesic efficacy of cannabinoids. Inhibition of voltage gate calcium (Ca v ) channels is one mechanism through which cannabinoids may produce analgesia. We hypothesized that cannabinoids and cannabinoid receptor agonists target different types of Ca v channels through distinct mechanisms. Experimental Approach Electrophysiological recordings from tsA‐201 cells expressing either Ca v 3.2 or Ca v 2.2 were used to assess inhibition by HU‐210 or cannabidiol (CBD) in the absence and presence of the CB 1 receptor. Homology modelling assessed potential interaction sites for CBD in both Ca v 2.2 and Ca v 3.2. Analgesic effects of CBD were assessed in mouse models of inflammatory and neuropathic pain. Key Results HU‐210 (1 μM) inhibited Ca v 2.2 function in the presence of CB 1 receptor but had no effect on Ca v 3.2 regardless of co‐expression of CB 1 receptor. By contrast, CBD (3 μM) produced no inhibition of Ca v 2.2 and instead inhibited Ca v 3.2 independently of CB 1 receptors. Homology modelling supported these findings, indicating that CBD binds to and occludes the pore of Ca v 3.2, but not Ca v 2.2. Intrathecal CBD alleviated thermal and mechanical hypersensitivity in both male and female mice, and this effect was absent in Ca v 3.2 null mice. Conclusion and Implications Our findings reveal differential modulation of Ca v 2.2 and Ca v 3.2 channels by CB 1 receptors and CBD. This advances our understanding of how different cannabinoids produce analgesia through action at different voltage‐gated calcium channels and could influence the development of novel cannabinoid‐based therapeutics for treatment of chronic pain.

Cav3.2 channels play an important role in the afferent nociceptive pathway, which is responsible for both physiological and pathological pain transmission. Cav3.2 channels are upregulated during neuropathic pain or peripheral inflammation in part due to an increased association with the deubiquitinase USP5. In this study, we investigated nine naturally occurring flavonoid derivatives which we tested for their abilities to inhibit transiently expressed Cav3.2 channels and their interactions with USP5. Icariside II (ICA-II), one of the flavonols studied, inhibited the biochemical interactions between USP5 and Cav3.2 and concomitantly and effectively blocked Cav3.2 channels. Molecular docking analysis predicts that ICA-II binds to the cUBP domain and the Cav3.2 interaction region. In addition, ICA-II was predicted to interact with residues in close proximity to the Cav3.2 channel's fenestrations, thus accounting for the observed blocking activity. In mice with inflammatory and neuropathic pain, ICA-II inhibited both phases of the formalin-induced nocifensive responses and abolished thermal hyperalgesia induced by injection of complete Freund's adjuvant (CFA) into the hind paw. Furthermore, ICA-II produced significant and long-lasting thermal antihyperalgesia in female mice, whereas Cav3.2 null mice were resistant to the action of ICA-II. Altogether, our data show that ICA-II has analgesic activity via an action on Cav3.2 channels.

Background and PurposePostoperative pain occurs in as many as 70% of surgeries performed worldwide. Postoperative pain management still relies on opioids despite their negative consequences, resulting in a public health crisis. Therefore, it is important to develop alternative therapies to treat chronic pain. Natural products derived from medicinal plants are potential sources of novel biologically active compounds for development of safe analgesics. In this study, we screened a library of natural products to identify small molecules that target the activity of voltage-gated sodium and calcium channels that have important roles in nociceptive sensory processing. Experimental ApproachFractions derived from the Native American medicinal plant, Parthenium incanum, were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion (DRG) neurons. Further separation of these fractions yielded a cycloartane-type triterpene identified as argentatin C, which was additionally evaluated using whole-cell voltage and current-clamp electrophysiology, and behavioural analysis in a mouse model of postsurgical pain. Key ResultsArgentatin C blocked the activity of both voltage-gated sodium and low-voltage-activated (LVA) calcium channels in calcium imaging assays. Docking analysis predicted that argentatin C may bind to Na(V)1.7-1.9 and Ca(V)3.1-3.3 channels. Furthermore, argentatin C decreased Na+ and T-type Ca2+ currents as well as excitability in rat and macaque DRG neurons, and reversed mechanical allodynia in a mouse model of postsurgical pain. Conclusion and ImplicationsThese results suggest that the dual effect of argentatin C on voltage-gated sodium and calcium channels supports its potential as a novel treatment for painful conditions.

1,4-Dihydropyridines (DHPs) represent the blockbuster class of L-type calcium channel blockers that have tremendous therapeutic value against cardiovascular conditions. Due to their abilities to additionally target other subtypes of calcium channels, DHPs are also considered promising molecules for the treatment of neurological and psychiatric disorders. Having been in the market for more than forty years, DHP is one of the most modified scaffolds for the development of novel molecules acting on calcium channels. Taking the chemical structures of approved DHPs into account, it is noteworthy that C-4 position is the least modified part of the ring system. Therefore, in the present study, we focused on this location and carried out various molecular modifications to obtain twelve potential calcium channel blockers with a DHP-based hexahydroquinoline scaffold (DA1-DA12). The whole-cell patch clamp technique applied to analyze the blocking ability of the synthesized compounds on both L- (Cav1.2) and T- (Cav3.2) type calcium channels revealed five blockers with different selectivity profiles. Introducing naphthyl moiety onto the C-4 position of the main scaffold led to the identification of a selective blocker of Cav1.2 (DA8). The benzodioxole-substituted derivative (DA1) was the most potent and selective Cav3.2 inhibitor, therefore, its enantiomers were separated using HPLC on a chiral stationary phase. Retesting single isomers on Cav3.2 revealed that S-enantiomer was mainly responsible for the block. Finally, DA compounds were docked into two generated homology models of L- and T-type calcium channels. Molecular dynamics (MD) simulations and 3D pharmacophore modeling provided further insights into the detailed binding mechanism of DHPs to Cav1.2 as well as to Cav3.2.

T-type calcium channels are considered potential drug targets to combat cancer. Combining T-type calcium channel blockers with conventional chemotherapy drugs represents a promising strategy towards successful cancer treatment. From this perspective, we report in this study the design and synthesis of a novel series of N3-sustituted dihydropyrimidines (DHPMs) as anticancer adjuvants to cisplatin (Cis) and etoposide (Eto). Full spectral characterization of the new compounds was done using FT-IR, 1H NMR, 13C NMR, and HRMS. Structure elucidation was confirmed by 2D NMR 1H-H COSY, HSQC and NOESY experiments. Novel derivatives were tested for their Ca2+ channel blocking activity by employing the whole cell patch-clamp technique. Results demonstrated that most compounds were potential T-type calcium channel blockers with the triazole-based C12 and C13 being the most selective agents against CaV3.2 channel. Further electrophysiological studies demonstrated that C12 and C13 inhibited CaV3.2 currents with respective affinity of 2.26 and 1.27 µM, and induced 5 mV hyperpolarizing shifts in the half-inactivation potential. Subsequently, C12 and C13 were evaluated for their anticancer activities alone and in combination with Cis and Eto against A549 and MDA-MB 231 cancer cells. Interestingly, both compounds exhibited potential anticancer effects with IC50 values < 5 µM. Combination studies revealed that both compounds had synergistic effects (combination index CI < 1) on Cis and Eto through induction of apoptosis (p53 activation and up-regulation of BAX and p21 gene expression). Importantly, in silico physicochemical and ADMET assessment of both compounds revealed their potential drug-like properties with decreased risk of cardiac toxicity. Hence, C12 and C13 are promising anticancer adjuvants through inhibition of CaV3.2 T-type calcium channels, thereby serving as eminent leads for further modification.

T-type calcium channels are known molecular targets of certain phytocannabinoids and endocannabinoids. Here we explored the modulation of Cav3.2 T-type calcium channels by terpenes derived from cannabis plants. A screen of eight commercially available terpenes revealed that camphene and alpha-bisabolol mediated partial, but significant inhibition of Cav3.2 channels expressed in tsA-201 cells, as well as native T-type channels in mouse dorsal root ganglion neurons. Both compounds inhibited peak current amplitude with IC50s in the low micromolar range, and mediated an additional small hyperpolarizing shift in half-inactivation voltage. When delivered intrathecally, both terpenes inhibited nocifensive responses in mice that had received an intraplantar injection of formalin, with alpha-bisabolol showing greater efficacy. Both terpenes reduced thermal hyperalgesia in mice injected with Complete Freund’s adjuvant. This effect was independent of sex, and absent in Cav3.2 null mice, indicating that these compounds mediate their analgesic properties by acting on Cav3.2 channels. Both compounds also inhibited mechanical hypersensitivity in a mouse model of neuropathic pain. Hence, camphene and alpha-bisabolol have a wide spectrum of analgesic action by virtue of inhibiting Cav3.2 T-type calcium channels.

We have previously reported that cellular prion protein (PrP C ) can down-regulate NMDA receptor activity and in a copper dependent manner. Here, we employed AAV9 to introduce murine cellular prion protein into mouse hippocampal neurons in primary cultures from PrP null mice to determine the role of the six copper binding motifs located within the N-terminal domain of PrP C . The results demonstrate that viral expression of wild type PrP C lowers NMDAR activity in PrP null mouse hippocampal neurons by reducing the magnitude of non-desensitizing currents. Elimination of the last two copper binding sites alone, or in combination with the remaining four attenuates this protective effect. Thus our data suggest that copper ion interactions with specific binding sites on PrP C are critical for PrP C dependent modulation of NMDA receptor function.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive loss of cortical, brain stem and spinal motor neurons that leads to muscle weakness and death. A previous study implicated CACNA1H encoding for Cav3.2 calcium channels as a susceptibility gene in ALS. In the present study, two heterozygous CACNA1H variants were identified by whole genome sequencing in a small cohort of ALS patients. These variants were functionally characterized using patch clamp electrophysiology, biochemistry assays, and molecular modeling. A previously unreported c.454GTAC > G variant produced an inframe deletion of a highly conserved isoleucine residue in Cav3.2 (p.ΔI153) and caused a complete loss-of-function of the channel, with an additional dominant-negative effect on the wild-type channel when expressed in trans. In contrast, the c.3629C > T variant caused a missense substitution of a proline with a leucine (p.P1210L) and produced a comparatively mild alteration of Cav3.2 channel activity. The newly identified ΔI153 variant is the first to be reported to cause a complete loss of Cav3.2 channel function. These findings add to the notion that loss-of-function of Cav3.2 channels associated with rare CACNA1H variants may be risk factors in the complex etiology of ALS.

(The American Journal of Human Genetics 103, 666–678; November 1, 2018) In the version of this article originally published online, Qinghe Xing's name was misspelled as Qinghe Xin. Also, Azita Sadeghpour, Erica E. Davis, and Nicholas Katsanis (all at Center for Human Disease Modeling, Duke University Medical Center, Durham, NC 27701, USA) and the Task Force for Neonatal Genomics were omitted from the author list. The members of the Task Force for Neonatal Genomics are as follows: Alexander Allori, Misha Angrist, Patricia Ashley, Margarita Bidegain, Brita Boyd, Eileen Chambers, Heidi Cope, C. Michael Cotten, Theresa Curington, Erica E. Davis, Sarah Ellestad, Kimberley Fisher, Amanda French, William Gallentine, Ronald Goldberg, Kevin Hill, Sujay Kansagra, Nicholas Katsanis, Sara Katsanis, Joanne Kurtzberg, Jeffrey Marcus, Marie McDonald, Mohammed Mikati, Stephen Miller, Amy Murtha, Yezmin Perilla, Carolyn Pizoli, Todd Purves, Sherry Ross, Azita Sadeghpour, Edward Smith, and John Wiener. The authors apologize for these omissions.

µ-Conotoxin PIIIA, in the sub-picomolar, range inhibits the archetypal bacterial sodium channel NaChBac (NavBh) in a voltage- and use-dependent manner. Peptide µ-conotoxins were first recognized as potent components of the venoms of fish-hunting cone snails that selectively inhibit voltage-gated skeletal muscle sodium channels, thus preventing muscle contraction. Intriguingly, computer simulations predicted that PIIIA binds to prokaryotic channel NavAb with much higher affinity than to fish (and other vertebrates) skeletal muscle sodium channel (Nav 1.4). Here, using whole-cell voltage clamp, we demonstrate that PIIIA inhibits NavBac mediated currents even more potently than predicted. From concentration-response data, with [PIIIA] varying more than 6 orders of magnitude (10-12 to 10-5 M), we estimated an IC50 = ~5 pM, maximal block of 0.95 and a Hill coefficient of 0.81 for the inhibition of peak currents. Inhibition was stronger at depolarized holding potentials and was modulated by the frequency and duration of the stimulation pulses. An important feature of the PIIIA action was acceleration of macroscopic inactivation. Docking of PIIIA in a NaChBac (NavBh) model revealed two interconvertible binding modes. In one mode, PIIIA sterically and electrostatically blocks the permeation pathway. In a second mode, apparent stabilization of the inactivated state was achieved by PIIIA binding between P2 helices and trans-membrane S5s from adjacent channel subunits, partially occluding the outer pore. Together, our experimental and computational results suggest that, besides blocking the channel-mediated currents by directly occluding the conducting pathway, PIIIA may also change the relative populations of conducting (activated) and non-conducting (inactivated) states.

Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on EEG, and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the alpha(1)-subunit of the voltage-gated Ca(V)2.3 channel, which conducts high voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all four S6 segments, which form the presumed Ca(V)2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the anti-epileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders.

N-Methyl-D-aspartate receptors (NMDARs) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are two major types of ionotropic glutamate receptors involved in synaptic transmission. However, excessive activity of these receptors can be cytotoxic and thus their function must be precisely controlled. We have previously reported that NMDA receptor activity is dysregulated following genetic knockout of cellular prion protein (PrPC), and that PrPC regulation of NMDA receptors is copper-dependent. Here, we employed electrophysiological methods to study NMDAR and AMPAR currents of cultured hippocampal neurons from PrPC overexpresser mice. We show that NMDA receptor current amplitude and kinetics are differentially modulated by overexpression of human or mouse PrPC. By contrast, AMPA receptor activity was unaffected. Nonetheless, AMPA receptor activity was modulated by copper ions in a manner similar to what we previously reported for NMDA receptors. Taken together, our findings reveal that AMPA and NMDA receptors are differentially regulated by PrPC, but share common modulation by copper ions.

The prokaryotic, voltage-gated sodium channel, NaChBac, has features common to both eukaryotic Nav and Cav channels, but also differs in important details. NaChBac assembles as a homo-tetramer, and it achieves Na+-selective conduction with a pseudo-symmetric selectivity filter possessing a 4-glutamate motif (EEEE), which is common also to eukaryotic Cav channels, but is distinctly different from the asymmetric selectivity filter (DEKA) of eukaryotic Nav channels. Block by impermeant, or weakly permeant, ions offers functional clues as to the nature and location of ion-binding sites within the channel's conducting pathway. Here, we examine block by a transition metal (Cd2+), or a lanthanide (La3+), in the extracellular solution. Whole-cell patch-clamp experiments were performed using tsA201 cells transiently expressing NaChBac. Onset of block and washout were observed using repeated depolarizations to −10 mV, from a holding potential of −120mV (IC50s (mM, mean±SEM): Cd, 2.0±0.06; La, 0.81±0.03. Two analyses examined voltage dependence of block and provided estimates of the "apparent electrical distance", δ, from external solution to the blocking site based on the analysis of Woodhull (J. Gen. Physiol., 1973). First, peak current-voltage relations were determined in the presence and absence of the blocker. Second, instantaneous I-V relations allowed calculation of fractional block of the conductance activated by a fixed, activating prepulse. In each analysis, a fit of fractional block, fb, vs V yielded an estimate of δ, and the IC50(V=0). IC50 values were similar to those obtained directly from dose-response data (see above). Values for δ appeared to vary slightly with the method, but indicated only weak V dependence of block: Cd, 0.02 - 0.08; La, 0.02 - 0.13. These data are consistent with a superficial binding site, and only weak coupling of block to movements of permeant ions within the channel. Supported by: MOP-10053 (CIHR).