Discovery of potent T-type calcium channel blocker

The intensive SAR study of 3,4-dihydroquinazoline series led to the most potent compound 10 (KYS05090: IC(50)=41+/-1 nM) against T-type calcium channel and its potency is nearly comparable to that of Kurtoxin. As a small organic molecule, this compound showed the highest blocking activity reported to date.     

Modulation of Cav3.2 T-type calcium channel permeability by asparagine-linked glycosylation

Low-voltage-gated T-type calcium channels are expressed throughout the nervous system where they play an essential role in shaping neuronal excitability. Defects in T-type channel expression have been linked to various neuronal disorders including neuropathic pain and epilepsy. Currently, little is known about the cellular mechanisms controlling the expression and function of T-type channels. Asparagine-linked glycosylation has recently emerged as an essential signaling pathway by which the cellular environment can control expression of T-type channels. However, the role of N-glycans in the conducting function of T-type channels remains elusive. In the present study, we used human Ca_v3.2 glycosylation-deficient channels to assess the role of N-glycosylation on the gating of the channel. Patch-clamp recordings of gating currents revealed that N-glycans attached to hCa_v3.2 channels have a minimal effect on the functioning of the channel voltage-sensor. In contrast, N-glycosylation on specific asparagine residues may have an essential role in the conducting function of the channel by enhancing the channel permeability and / or the pore opening of the channel. Our data suggest that modulation of N-linked glycosylation of hCa_v3.2 channels may play an important physiological role, and could also support the alteration of T-type currents observed in disease states.     

pH modification of human T-type calcium channel gating

External pH (pH(o)) modifies T-type calcium channel gating and permeation properties. The mechanisms of T-type channel modulation by pH remain unclear because native currents are small and are contaminated with L-type calcium currents. Heterologous expression of the human cloned T-type channel, alpha1H, enables us to determine the effect of changing pH on isolated T-type calcium currents. External acidification from pH(o) 8.2 to pH(o) 5.5 shifts the midpoint potential (V(1/2)) for steady-state inactivation by 11 mV, shifts the V(1/2) for maximal activation by 40 mV, and reduces the voltage dependence of channel activation. The alpha1H reversal potential (E(rev)) shifts from +49 mV at pH(o) 8.2 to +36 mV at pH(o) 5.5. The maximal macroscopic conductance (G(max)) of alpha1H increases at pH(o) 5.5 compared to pH(o) 8.2. The E(rev) and G(max) data taken together suggest that external protons decrease calcium/monovalent ion relative permeability. In response to a sustained depolarization alpha1H currents inactivate with a single exponential function. The macroscopic inactivation time constant is a steep function of voltage for potentials < -30 mV at pH(o) 8.2. At pH(o) 5.5 the voltage dependence of tau(inact) shifts more depolarized, and is also a more gradual function of voltage. The macroscopic deactivation time constant (tau(deact)) is a function of voltage at the potentials tested. At pH(o) 5.5 the voltage dependence of tau(deact) is simply transposed by approximately 40 mV, without a concomitant change in the voltage dependence. Similarly, the delay in recovery from inactivation at V(rec) of -80 mV in pH(o) 5.5 is similar to that with a V(rec) of -120 mV at pH(o) 8.2. We conclude that alpha1H is uniquely modified by pH(o) compared to other calcium channels. Protons do not block alpha1H current. Rather, a proton-induced change in activation gating accounts for most of the change in current magnitude with acidification.     

Synthesis and biological evaluation of pyrrolidine-based T-type calcium channel inhibitors for the treatment of neuropathic pain

The treatment of neuropathic pain is one of the urgent unmet medical needs and T-type calcium channels are promising therapeutic targets for neuropathic pain. Several potent T-type channel inhibitors showed promising in vivo efficacy in neuropathic pain animal models and are being investigated in clinical trials. Herein we report development of novel pyrrolidine-based T-type calcium channel inhibitors by pharmacophore mapping and structural hybridisation followed by evaluation of their Ca_v3.1 and Ca_v3.2 channel inhibitory activities. Among potent inhibitors against both Ca_v3.1 and Ca_v3.2 channels, a promising compound 20n based on in vitro ADME properties displayed satisfactory plasma and brain exposure in rats according to in vivo pharmacokinetic studies. We further demonstrated that 20n effectively improved the symptoms of neuropathic pain in both SNL and STZ neuropathic pain animal models, suggesting modulation of T-type calcium channels can be a promising therapeutic strategy for the treatment of neuropathic pain.     

T-type calcium channel blockers - new and notable

Since cloning of the T-type or Ca(V)3.n calcium channel family in 1998-1999 much progress was made in investigation of their regulation. Most effective metal Ca(V)3 channel blockers are trivalent cations from lanthanide group together with transition metals La(3+) and Y(3+). Divalent cations Zn(2+), Cu(2+) and Ni(2+) inhibit Ca(V)3.2 channels more efficiently than Ca(V)3.1 and Ca(V)3.3 channels via second high-affinity binding site including histidine H191 specific for the Ca(V)3.2 channel. Dihydropyridines and phenylalkylamines in addition to block of L-type calcium channel can inhibit Ca(V)3 channels in clinically relevant concentration.     

T-type calcium channel in mammalian CNS neurones.

1. In all neurones freshly isolated from various brain regions of newborn, adult and aged rats, the T-type Ca2+ currents were elicited by step depolarizations to potentials more positive than -60 mV from a holding potential of -100 mV, and reached a peak in the current-voltage relationship around -30 mV. 2. The activation and inactivation processes were highly potential-dependent, and the latter was fitted by a single exponential function. 3. It was concluded that mammalian brain neurones possess a definite class of T-type Ca2+ channel characterized by both current kinetics and ion selectivity for Ca2+, Ba2+ and Sr2+. However, the pharmacological nature of the T-type Ca2+ channel differed from that in other tissues such as cardiac and smooth muscle cells, peripheral neurones, and cultured cells.     

First pharmacophoric hypothesis for T-type calcium channel blockers

A three-dimensional pharmacophore model was developed for T-type calcium channel blockers in order to map common structural features of highly active compounds by using CATALYST program. In the absence of three dimensional structure based information like binding mode and unavailability of more number of specific T-type calcium channel blockers, this hypothesis which consists of three hydrophobic regions, one hydrogen bond acceptor and one positive ionizable regions will act as a valuable tool in designing new ligands. Further more after the withdrawal of mibefradil, the first marketed T-type calcium channel blocker, due to the drug-drug interactions, there is an urgent need for more work in this interest.     

Inhibition of cellular proliferation and induction of apoptosis in human lung adenocarcinoma A549 cells by T-type calcium channel antagonist

The anti-proliferative and apoptotic activities of new T-type calcium channel antagonist, 6e (BK10040) on human lung adenocarcinoma A549 cells were investigated. The MTT assay results indicated that BK10040 was cytotoxic against human lung adenocarcinoma (A549) and pancreatic cancer (MiaPaCa2) cells in a dose-dependent manner with IC₅₀ of 2.25 and 0.93 μM, respectively, which is ca. 2-fold more potent than lead compound KYS05090 despite of its decreased T-type calcium channel blockade. As a mode of action for cytotoxic effect of BK10040 on lung cancer (A549) cells, this cancer cell death was found to have the typical features of apoptosis, as evidenced by the accumulation of positive cells for annexin V. In addition, BK10040 triggered the activations of caspases 3 and 9, and the cleavages of poly (ADP-ribose) polymerase (PARP). Moreover, the treatment with z-VAD-fmk (a broad spectrum caspase inhibitor) significantly prevented BK10040-induced apoptosis. Based on these results, BK10040 may be used as a potential therapeutic agent for human lung cancer via the potent apoptotic activity.     

Contributions of T-type calcium channel isoforms to neuronal firing

Low voltage-activated (LVA) T-type calcium channels play critical roles in the excitability of many cell types and are a focus of research aimed both at understanding the physiological basis of calcium channel-dependent signaling and the underlying pathophysiology associated with hyperexcitability disorders such as epilepsy. These channels play a critical role towards neuronal firing in both conducting calcium ions during action potentials and also in switching neurons between distinct modes of firing. In this review the properties of the CaV3.1, CaV3.2 and CaV3.3 T-type channel isoforms is discussed in relation to their individual contributions to action potentials during burst and tonic firing states as well their roles in switching between firing states.     

Lead discovery and optimization of T-type calcium channel blockers

A series of compounds were designed as T-type calcium channel blocker containing 6 or 5 pharmacophore features from structure-based virtual screening. To optimize the suggested structure, over 130 derivatives were synthesized and their inhibitory activities on T-type calcium channel were assayed using in vitro screening system with alpha1(G) and alpha1(H) clones. For the compounds with higher activities in FDSS assay system, the efficacy was measured by patch-clamp method. Among the library with 5 features, alkaneamide derivatives (7b, 9j, 11b, 11g, 11h) with 4-arylsubstituted piperazine showed better IC(50) values than Mibefradil.     

Pyridyl amides as potent inhibitors of T-type calcium channels

A novel series of amide T-type calcium channel antagonists were prepared and evaluated using in vitro and in vivo assays. Optimization of the screening hit 3 led to identification of the potent and selective T-type antagonist 37 that displayed in vivo efficacy in rodent models of epilepsy and sleep.     

Novel T-type calcium channel blockers: dioxoquinazoline carboxamide derivatives

T-type calcium channel is one of therapeutic targets for the treatment of cardiovascular diseases and neuropathic pains. Since the withdrawal of mibefradil, a T-type calcium channel blocker, there have been a lot of efforts to develop T-type calcium channel blockers. A small molecule library of dioxoquinazoline carboxamide derivatives containing 155 compounds was designed, synthesized, and biologically evaluated for T-type calcium channel blocking activity. Among those compounds synthesized, the compound 1n shows the most potent T-type calcium current blocking activity with an IC(50) value of 1.52 microM, which is comparable to that of mibefradil.     

Mitigation of copper toxicity by DNA oligomers in green paramecia

Impact of transition metals which catalyze the generation of reactive oxygen species (ROS), on activation of cell death signaling in plant cells have been documented to date. Similarly in green paramecia (Paramecium bursaria), an aquatic protozoan species harboring symbiotic green algae in the cytoplasm, toxicities of various metallic ions have been documented. We have recently examined the effects of double-stranded GC-rich DNA fragments with copper-binding nature and ROS removal catalytic activity as novel plant cell-protecting agents, using the suspension-cultured tobacco cells. Here, we show that above DNA oligomers protect the cells of green paramecia from copper-induced cell death, suggesting that the phenomenon firstly observed in tobacco cells is not limited only within higher plants but it could be universally observable in wider range of organisms.     

Growth inhibition of human cancer cells in vitro by T-type calcium channel blockers

This paper describes the preliminary biological results that novel T-type calcium channel blockers inhibit the growth of human cancer cells by blocking calcium influx into the cell, based on unknown mechanism on the cell cycle responsible for cellular proliferation. Among the selected compounds from compound library, compound 9c (KYS05041) was identified to be nearly equipotent with Cisplatin against some human cancers in the micromolar range.     

Chemical synthesis of kurtoxin, a T-type calcium channel blocker

Kurtoxin isolated from the venom of scorpion, Parabuthus transvaalicus, is a 63-residue peptide with four intramolecular disulfide bonds which inhibits low-threshold T-type Ca²⁺channels. Kurtoxin was synthesized by native chemical ligation involving the coupling of (1--26)-thioester peptide and Cys²⁷-(28--63)-peptide. The former was synthesized by standard solid-phase peptide synthesis (SPPS) with Boc chemistry, while the latter was sequentially assembled from three protected segments onto a resin-bound C-terminal segment in a chloroform--phenol mixed solvent followed by deprotection reaction using HF. Each protected segment used for the coupling on a solid support was prepared on an N-[9-(hydroxymethyl)-2-fluorenyl] succinamic acid (HMFS) resin and detached from the resin by treatment with 20% Et ₃N in DMF to produce it in the form of an α-carboxylic acid. Synthetic kurtoxin obtained after the oxidative folding reaction was found to be identical with the natural product by means of several analytical procedures, and its disulfide structure was determined for the first time to be Cys¹²-Cys⁶¹, Cys¹⁶-Cys³⁷, Cys²³-Cys⁴⁴ and Cys²⁷-Cys⁴⁶ by peptide mapping, sequence analysis and mass measurements.     

Inhibition of recombinant human T-type calcium channels by Delta9-tetrahydrocannabinol and cannabidiol

Delta(9)-Tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most prevalent biologically active constituents of Cannabis sativa. THC is the prototypic cannabinoid CB1 receptor agonist and is psychoactive and analgesic. CBD is also analgesic, but it is not a CB1 receptor agonist. Low voltage-activated T-type calcium channels, encoded by the Ca(V)3 gene family, regulate the excitability of many cells, including neurons involved in nociceptive processing. We examined the effects of THC and CBD on human Ca(V)3 channels stably expressed in human embryonic kidney 293 cells and T-type channels in mouse sensory neurons using whole-cell, patch clamp recordings. At moderately hyperpolarized potentials, THC and CBD inhibited peak Ca(V)3.1 and Ca(V)3.2 currents with IC(50) values of approximately 1 mum but were less potent on Ca(V)3.3 channels. THC and CBD inhibited sensory neuron T-type channels by about 45% at 1 mum. However, in recordings made from a holding potential of -70 mV, 100 nm THC or CBD inhibited more than 50% of the peak Ca(V)3.1 current. THC and CBD produced a significant hyperpolarizing shift in the steady state inactivation potentials for each of the Ca(V)3 channels, which accounts for inhibition of channel currents. Additionally, THC caused a modest hyperpolarizing shift in the activation of Ca(V)3.1 and Ca(V)3.2. THC but not CBD slowed Ca(V)3.1 and Ca(V)3.2 deactivation and inactivation kinetics. Thus, THC and CBD inhibit Ca(V)3 channels at pharmacologically relevant concentrations. However, THC, but not CBD, may also increase the amount of calcium entry following T-type channel activation by stabilizing open states of the channel.     

Synthesis and biological evaluation of novel T-type calcium channel blockers

3,4-Dihydroquinazoline analogues substituted by N-methyl-N-(5-pyrrolidinopentyl)amine at the 2-position were synthesized and their blocking effects were evaluated for T- and N-type calcium channels. Compound 11b (KYS05080), compared to mibefradil (IC50=1.34+/-0.49 microM), was about 5-fold potent (IC50=0.26+/-0.01 microM) for T-type calcium channel (alpha1G) blocking and its selectivity of T/N-type was also improved (7.5 versus 1.4 of mibefradil).     

In vitro cytotoxicity on human ovarian cancer cells by T-type calcium channel blockers

The growth inhibition of human cancer cells via T-type Ca²⁺ channel blockade has been well known. Herein, a series of new 3,4-dihydroquinazoline derivatives were synthesized via a brief SAR study on KYS05090 template and evaluated for both T-type Ca²⁺ channel (Ca_v3.1) blockade and cytotoxicity on three human ovarian cancer cells (SK-OV-3, A2780 and A2780-T). Most of compounds except 6i generally exhibited more potent cytotoxicity on SK-OV-3 than mibefradil as a positive control regardless of the degree of T-type channel blockade. In particular, eight compounds (KYS05090, 6a and 6c–6h) showing strong channel blockade exhibited almost equal and more potent cytotoxicity on A2780 when compared to mibefradil. On A2780-T paclitaxel-resistant human ovarian carcinoma, two compounds (KYS05090 and 6d) were 20-fold more active than mibefradil. With respect to cell cycle arrest effect on A2780 and A2780-T cells, KYS05090 induced large proportion of sub-G₁ phase in the cell cycle progression of A2780 and A2780-T, meaning the induction of cancer cell death instead of cell cycle arrest via blocking T-type Ca²⁺ channel. Among new analogues, compounds 6g and 6h induced cell cycle arrest at G₁ phase of A2780 and A2780-T cells in dose-dependent manner and exhibited strong anti-proliferation effects of ovarian cancer cells by blocking T-type Ca²⁺ channel. Furthermore, 6g and 6h possessing strong cytotoxic effects could induce apoptosis of A2780 cells, which was detected by confocal micrographs using DAPI staining.     

Ligand-based virtual screening to identify new T-type calcium channel blockers

T-type calcium channels are involved in the generation of rhythmical firing patterns in the mammalian central nervous system and in various pathological alterations of neuronal excitability such as in epilepsy or neuropathic pain. In the search for new T-type calcium channel blockers that would help to treat these disorders, we have followed a bi-dimensional pharmacophore-based virtual screening approach to identify new inhibitors. Nineteen molecules extracted from AurSCOPE Ion Channels knowledgebase were used as query molecules to screen an external database. This in silico approach was then validated using electrophysiology. Interestingly, 16 compounds out of 38 distinct molecules tested showed more than 50% blockade of the Ca(V)3.2 mediated T-type current. Two series of compounds show chemical originality compared with known T-type calcium channel blockers.