Synthesis and biological activity of 3,4-dihydroquinazolines for selective T-type Ca2+ channel blockers

We have synthesized 3,4-dihydroquinazoline derivatives for the potent and selective T-type Ca(2+) channel blockers and evaluated for their inhibitory activities against two subtypes T-type Ca(2+) channels and N-type Ca(2+) channels. Among them, 5b (KYS05044, IC(50)=0.56+/-0.10 microM) was identified as potent T-type Ca(2+) channel blocker with in vitro selectivity profile at meaningful level (T/N-type, SI=>100).     

Morpholin-2-one derivatives as novel selective T-type Ca2+ channel blockers

Morpholin-2-one-5-carboxamide derivatives were prepared by using the one-pot Ugi multicomponent reaction and evaluated for blocking effects on T- and N-type Ca(2+) channels. Among them, compound 5i produced the highest potency (IC(50)=0.45+/-0.02 microM), while compounds 5d, 5f, 5k, 5n, 5o, and 6m produced relatively high potency as well as selectivity on T-type Ca(2+) channels. These novel scaffolds showed potent and selective T-type Ca(2+) channel blocking activities.     

Effects of calcium channel antagonists on the corpora allata of adult male loreyi leafworm Mythimna loreyi: juvenile hormone acids release and intracellular calcium level

The effects of voltage-dependent calcium channel (VDCC) antagonists and the non-specific calcium channel antagonists on both juvenile hormone acids (JHA) release and cytosolic free calcium concentration ([Ca2+]i) are investigated in the corpora allata (CA) of the adult males loreyi leafworm Mythimna loreyi. The VDCC antagonists used in this study are: the L-type antagonists diltiazem, nifedipine, and verapamil, the N-type antagonist omega-Conotoxin (CgTx) GVIA, the N- and P/Q-type antagonist omega-CgTx MVIIC, and the T-type antagonist amiloride. The non-specific calcium channel antagonists used in this study were cadmium (Cd2+), cobalt (Co2+), nickle (Ni2+), and lanthanum (La3+). The results show that both the DHPs-sensitive L-type antagonist nifedipine and the N-type antagonist omega-CgTx GVIA were able to inhibit JHA release, but only omega-CgTx GVIA was able to reduce the [Ca2+]i. Among the non-specific calcium channel antagonists, Cd2+ is the most potent in reducing JHA release but without obvious effect on the [Ca2+]i, La3+ significantly increases the [Ca2+]i but without effect on JHA release.     

Clostridium perfringens alpha-toxin-induced hemolysis of horse erythrocytes is dependent on Ca2+ uptake

Clostridium perfringens alpha-toxin is able to lyse various erythrocytes. Exposure of horse erythrocytes to alpha-toxin simultaneously induced hot-cold hemolysis and stimulated production of diacylglycerol and phosphorylcholine. When A23187-treated erythrocytes were treated with the toxin, these events were dependent on the concentration of extracellular Ca2+ . Incubation with the toxin of BAPTA-AM-treated horse erythrocytes caused no hemolysis or production of phosphorylcholine, but that of the BAPTA-treated erythrocytes did. When Quin 2-AM-treated erythrocytes were incubated with the toxin in the presence of 45Ca2+, the cells accumulated 45Ca2+ in a dose- and a time-dependent manner. These results suggest that the toxin-induced hemolysis and hydrolysis of phosphatidylcholine are closely related to the presence of Ca2+ in the cells. Flunarizine, a T-type Ca2+ channel blocker, and tetrandrine, an L- and T-type Ca2+ channel blocker, inhibited the toxin-induced hemolysis and Ca2+ uptake. However, L-type Ca2+ channel blockers, nifedipine, verpamil and diltiazem, an N-type blocker, omega-conotoxin SVIB, P-type blockers, omega-agatoxin TK and omega-agatoxin IVA, and a Q-type blocker, omega-conotoxin MVII C, had no such inhibitory effect. The observation suggests that Ca2+ taken up through T-type Ca2+ channels activated by the toxin plays an important role in hemolysis induced by the toxin.     

Frequency-dependent blockade of T-type Ca2+ current by efonidipine in cardiomyocytes

Efonidipine is a dihydropyridine Ca2+ antagonist with inhibitory effects on both L-type and T-type Ca2+ channels and potent bradycardiac activity especially in patients with high heart rate. In the present study, we examined the frequency dependence of efonidipine action on the T-type Ca2+ channel in isolated guinea-pig ventricular myocytes. The potency of efonidipine to inhibit the T-type Ca2+ current was higher under higher stimulation frequencies. The IC50 values were 1.3 x 10(-8), 2.0 x 10(-6) and 6.3 x 10(-6) M under stimulation frequencies of 1, 0.2 and 0.05 Hz, respectively. The reduction of T-type Ca2+ current amplitude was not accompanied by change in the time course of current decay. Efonidipine (10 microM) inhibited T-type Ca2+ current elicited by depolarization from holding potentials ranging from -90 to -30 mV by about 30%; the voltage-dependence of steady-state inactivation was not changed by the drug. Efonidipine slowed the recovery from inactivation following an inactivating prepulse. In conclusion, efonidipine was shown to have frequency-dependent inhibitory effects on the T-type Ca2+ channel, which could be explained by slow dissociation of the drug from the inactivated state of the channel.     

T-type calcium channels and tumor proliferation

The role of T-type Ca2+ channels in proliferation of tumor cells is reviewed. Intracellular Ca2+ is important in controlling proliferation as evidenced by pulses, or oscillations, of intracellular Ca2+ which occur in a cell cycle-dependent manner in many tumor cells. Voltage-gated calcium channels, such as the T-type Ca2+ channel, are well suited to participate in such oscillations due to their unique activation/inactivation properties. Expression of the T-type Ca2+ channels has been reported in numerous types of tumors, and has been shown to be cell cycle-dependent. Overexpression of the alpha1 subunit of T-type Ca2+ channels in human astrocytoma, neuroblastoma and renal tumor cell lines enhanced proliferation of these cells. In contrast, targeting of the alpha1 subunit of the T-type calcium channel via siRNA decreased proliferation of these cells. A Ca2+ oscillatory model is proposed involving potassium channels, Ca2+ stores and Ca2+ exchangers/transporters. A review of T-type channel blockers is presented, with a focus on mibefradil-induced inhibition of proliferation. The development of newer blockers with higher selectivity and less potential side effects are discussed. The conclusion reached is that calcium channel blockers serve as a potential therapeutic approach for tumors whose proliferation depends on T-type calcium channel expression.     

Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release

Electrophysiological studies of neurons reveal different Ca2+ currents designated L-, N-, P-, Q-, R-, and T-type. High-voltage-activated neuronal Ca2+ channels are complexes of a pore-forming alpha 1 subunit of about 190-250 kDa, a transmembrane, disulfide-linked complex of alpha 2 and delta subunits, and an intracellular beta subunit, similar to the alpha 1, alpha 2 delta, and beta subunits previously described for skeletal muscle Ca2+ channels. The primary structures of these subunits have all been determined by homology cDNA cloning using the corresponding subunits of skeletal muscle Ca2+ channels as probes. In most neurons, L-type channels contain alpha 1C or alpha 1D subunits, N-type contain alpha 1B subunits, P- and Q-types contain alternatively spliced forms of alpha 1A subunits, R-type contain alpha 1E subunits, and T-type contain alpha 1G or alpha 1H subunits. Association with different beta subunits also influences Ca2+ channel gating substantially, yielding a remarkable diversity of functionally distinct molecular species of Ca2+ channels in neurons.     

3,4-Dihydroquinazoline derivatives as novel selective T-type Ca2+ channel blockers

For LVA T-type Ca2+ channel blockers, 3,4-dihydroquinazoline derivatives as new scaffolds were prepared and evaluated for the inhibitory activity against two members of the recombinant T-type Ca2+ channel family. Among them, 8a (KYS05001, IC50=0.9 microM) was nearly equipotent with mibefradil (IC50=0.84 microM) and inhibited LVA T-type Ca2+ channel with greater efficacy than HVA Ca2+ channel.     

Both T- and L-type Ca2+ channels can contribute to excitation-contraction coupling in cardiac Purkinje cells.

Although L-type Ca2+ channels have been shown to play a central role in cardiac excitation-contraction (E-C) coupling, little is known about the role of T-type Ca2+ channels in this process. We used the amphotericin B perforated patch method to study the possible role of T-type Ca2+ current in E-C coupling in isolated canine Purkinje myocytes where both Ca2+ currents are large. T-type Ca2+ current was separated from L-type Ca2+ current using protocols employing the different voltage dependencies of the channel types and their different sensitivities to pharmacological blockade. We showed that Ca2+ admitted through either T- or L-type Ca2+ channels is capable of initiating contraction and that the contractions depended on Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR). The contractions, however, had different properties. Those initiated by Ca2+ entry through T-type Ca2+ channels had a longer delay to the onset of shortening, slower rates of shortening and relaxation, lower peak shortening, and longer time to peak shortening. These differences were present even when L-type Ca2+ current amplitude, or charge entry, was less than that of T-type Ca2+ current, suggesting that Ca2+ entry through the T-type Ca2+ channel is a less effective signal transduction mechanism to the SR than is Ca2+ entry through the L-type Ca2+ channel. We conclude that under our experimental conditions in cardiac Purkinje cells Ca2+ entry through the T-type Ca2+ channel can activate cell contraction. However, Ca2+ entry through the L-type Ca2+ channel is a more effective signal transduction mechanism. Our findings support the concept that different structural relationships exist between these channel types and the SR Ca2+ release mechanism.     

T-type Ca2+ channel blockers suppress the growth of human cancer cells

In order to further clarify the role of T-type Ca²⁺ channels in cell proliferation, we have measured the growth inhibition of human cancer cells by using our potent T-type Ca²⁺ channel blockers. As a result, KYS05090, a most potent T-type Ca²⁺ channel blocker, was found to be as potent as doxorubicin against some human cancer cells without acute toxicity. Therefore, this letter provides the biological results that T-type calcium channel is important in regulating the important cellular phenotype transition leading to cell proliferation, and thus novel T-type Ca²⁺ channel blocker presents new prospects for cancer treatment.     

Effect of mibefradil on sodium and calcium currents

Interstitial cells of Cajal (ICC) generate the electrical slow wave. The ionic conductances that contribute to the slow wave appear to vary among species. In humans, a tetrodotoxin-resistant Na+ current (Na(V)1.5) encoded by SCN5A contributes to the rising phase of the slow wave, whereas T-type Ca2+ currents have been reported from cultured mouse intestine ICC and also from canine colonic ICC. Mibefradil has a higher affinity for T-type over L-type Ca2+ channels, and the drug has been used in the gastrointestinal tract to identify T-type currents. However, the selectivity of mibefradil for T-type Ca2+ channels over ICC and smooth muscle Na+ channels has not been clearly demonstrated. The aim of this study was to determine the effect of mibefradil on T-type and L-type Ca2+ and Na+ currents. Whole cell currents were recorded from HEK-293 cells coexpressing green fluorescent protein with either the rat brain T-type Ca2+ channel alpha(1)3.3b + beta(2), the human intestinal L-type Ca2+ channel subunits alpha(1C) + beta(2), or Na(V)1.5. Mibefradil significantly reduced expressed T-type Ca2+ current at concentrations > or = 0.1 microM (IC(50) = 0.29 microM), L-type Ca2+ current at > 1 microM (IC(50) = 2.7 microM), and Na+ current at > or = 0.3 microM (IC(50) = 0.98 microM). In conclusion, mibefradil inhibits the human intestinal tetrodotoxin-resistant Na+ channel at submicromolar concentrations. Caution must be used in the interpretation of the effects of mibefradil when several ion channel classes are coexpressed.     

ZD7288 inhibits low-threshold Ca(2+) channel activity and regulates sperm function

In this study, ZD7288, a blocker of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, has been found to inhibit the mouse sperm acrosome reaction (AR). HCN channels have not yet been either recorded or implicated in mouse sperm AR, but low-threshold (T-type) Ca(2+) channels have. Interestingly, ZD7288 blocked native T-type Ca(2+) currents in mouse spermatogenic cells with an IC(50) of about 100 microM. This blockade was more effective at voltages producing low levels of inactivation, suggesting a differential affinity of ZD7288 for different channel conformations. Furthermore, ZD7288 inhibited all cloned T-type but not high-threshold N-type channels heterologously expressed in HEK-293 cells. Our results further support the role of T-type Ca(2+) channels in the mouse sperm AR.     

Cardiac calcium channels in planar lipid bilayers. L-type channels and calcium-permeable channels open at negative membrane potentials

Planar lipid bilayer recordings were used to study Ca channels from bovine cardiac sarcolemmal membranes. Ca channel activity was recorded in the absence of nucleotides or soluble enzymes, over a range of membrane potentials and ionic conditions that cannot be achieved in intact cells. The dihydropyridine-sensitive L-type Ca channel, studied in the presence of Bay K 8644, was identified by a detailed comparison of its properties in artificial membranes and in intact cells. L-type Ca channels in bilayers showed voltage dependence of channel activation and inactivation, open and closed times, and single-channel conductances in Ba2+ and Ca2+ very similar to those found in cell-attached patch recordings. Open channels were blocked by micromolar concentrations of external Cd2+. In this cell-free system, channel activity tended to decrease during the course of an experiment, reminiscent of Ca2+ channel "rundown" in whole-cell and excised-patch recordings. A purely voltage-dependent component of inactivation was observed in the absence of Ca2+ stores or changes in intracellular Ca2+. Millimolar internal Ca2+ reduced unitary Ba2+ influx but did not greatly increase the rate or extent of inactivation or the rate of channel rundown. In symmetrical Ba2+ solutions, unitary conductance saturated as the Ba2+ concentration was increased up to 500 mM. The bilayer recordings also revealed activity of a novel Ca2+-permeable channel, termed "B-type" because it may contribute a steady background current at negative membrane potentials, which is distinct from L-type or T-type Ca channels previously reported. Unlike L-type channels, B-type channels have a small unitary Ba2+ conductance (7 pS), but do not discriminate between Ba2+ and Ca2+, show no obvious sensitivity to Bay K 8644, and do not run down. Unlike either L- or T-type channels, B-type channels did not require a depolarization for activation and displayed mean open times of greater than 100 ms.     

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).     

T-type Ca2+ channels in vascular smooth muscle: multiple functions

Vascular smooth muscle is a major constituent of the blood vessel wall, and its many functions depend on type and location of the vessel, developmental or pathological state, and environmental and chemical factors. Vascular smooth muscle cells (VSMCs) use calcium as a signal molecule for multiple functions. An important component of calcium signaling pathways is the entry of extracellular calcium via voltage-gated Ca2+ channels, which in vascular smooth muscle cells (VSMCs) are of two main types, the high voltage-activated (HVA) L-type and low voltage-activated (LVA) T-type channels. Whereas L-type channels function primarily to regulate Ca2+ entry for contraction, it is generally accepted that T-type Ca2+ channels do not contribute significantly to arterial vasoconstriction, with the possible exception of the renal microcirculation. T-type Ca2+ channels are also present in some veins that display spontaneous contractile activity, where they likely generate pacemaker activity. T-type Ca2+ channel expression has also been associated with normal and pathological proliferation of VSMCs, often stimulated by external cues in response to insult or injury. Expression of T-type channels has been linked to the G1 and S phases of the cell cycle, a period important for the signaling of gene expression necessary for cell growth, progression of the cell cycle and ultimately cell division. To better understand T-type Ca2+ channel functions in VSM, it will be necessary to develop new approaches that are specifically targeted to this class of Ca2+ channels and its individual members.     

Voltage-dependent calcium channels in the corpora allata of the adult male loreyi leafworm, Mythimna loreyi

In the corpora allata (CA) of the adult male loreyi leafworm, Mythimna loreyi, juvenile hormone acid (JHA) biosynthesis and release show a dose dependence on extracellular Ca(2+) concentration. Maxima are obtained with Ca(2+) concentrations of 2-10 mM, and synthesis and release are significantly inhibited under a Ca(2+)-free condition. The Ca(2+)-free inhibition of JHA release can be reversed by returning the glands to medium at 5 mM Ca(2+). The cytosolic free Ca(2+) concentration ([Ca(2+)](i)), which was measured with fura-2, in individual CA cells also shows a dose dependence on extracellular Ca(2+) concentration, with significant [Ca(2+)](i) depression being observed in the absence of extracellular Ca(2+).High K(+) significantly increases the JHA release and causes a transient [Ca(2+)](i) increase within seconds in CA cells. High-K(+)-stimulated JHA release is partially inhibited by the benzothiazepine (BTZ)-, dihydropyridine (DHP)- and phenylalkylamine (PAA)-sensitive L-type voltage-dependent calcium channel (VDCC) antagonists diltiazem, nifedipine and verapamil, respectively; by the N- and P/Q-type VDCC antagonist omega-conotoxin (omega-CgTx) MVIIC; and by the T-type VDCC antagonist amiloride. The N-type antagonist omega-CgTx GVIA is the most potent in inhibiting the high-K(+)-stimulated JHA release. No inhibitory effect is shown by the P-type antagonist omega-agatoxin TK (omega-Aga TK). The high-K(+)-induced transient [Ca(2+)](i) increase is largely inhibited by the L-type antagonists (diltiazem, nifedipine, verapamil), by the N- and P/Q-type antagonist omega-CgTx MVIIC and by the T-type antagonist amiloride, and is totally inhibited by the N-type antagonist omega-CgTx GVIA. No inhibitory effect is shown by the P-type antagonist omega-Aga TK.We hypothesize that L-type, N-type and T-type VDCCs may be involved to different degrees in the high-K(+)-stimulated JHA release and transient [Ca(2+)](i) increase in the individual CA cells of the adult male M. loreyi, and that the N-type VDCCs may play important roles in these cellular events.     

Morphological transformation induced by activation of the mitogen-activated protein kinase pathway requires suppression of the T-type Ca2+ channel.

Transformation of fibroblasts by various oncogenes, including ras, mos, and src accompanies with characteristic morphological changes from flat to round (or spindle) shapes. Such morphological change is believed to play an important role in establishing malignant characteristics of cancer cells. Activation of the mitogen-activated protein kinase (MAPK) pathway is a converging downstream event of transforming activities of many oncogene products commonly found in human cancers. Intracellular calcium is known to regulate cellular morphology. In fibroblasts, Ca2+ influx is primarily controlled by two types of Ca2+ channels (T- and L-types). Here, we report that the T-type current was specifically inhibited in cells expressing oncogenically activated Ras as well as gain-of-function mutant MEK (MAPK/extracellular signal-regulated kinase (ERK) kinase, a direct activator of MAPK), whereas treatment of ras-transformed cells with a MEK-specific inhibitor restored T-type Ca2+ channel activity. Using a T-type Ca2+ channel antagonist, we further found that suppression of the T-type Ca2+ channel by the activated MAPK pathway is a prerequisite event for the induction and/or maintenance of transformation-associated morphological changes.     

Ligand-activated signal transduction in the 2-cell embryo

Platelet-activating factor (PAF) is an autocrine trophic/survival factor for the preimplantation embryo. PAF induced an increase in intracellular calcium concentration ([Ca2+]i) in the 2-cell embryo that had an absolute requirement for external calcium. L-type calcium channel blockers (diltiazem, verapamil, and nimodipine) significantly inhibited PAF-induced Ca2+ transients, but inhibitors of P/Q type (omega-agatoxin; omega-conotoxin MVIIC), N-type (omega-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the alpha1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (+/-) BAY K 8644 induced [Ca2+]i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each other's responses. Depolarization of the embryo (75 mM KCl) induced a [Ca2+]i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca2+ with Mn2+ allowed Mn2+ influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.     

T-type Ca2+ channels in non-vascular smooth muscles

T-type Ca2+ current has been recorded in smooth muscle myocytes, and associated interstitial cells, isolated from the gastro-intestinal tract, urinary bladder, urethra, prostate gland, myometrium, vas deferens, lymphatic vessels and airways smooth muscle. By contrast, current through such channels has not been recorded from other tissues, such as the ureter. Whilst the properties of this Ca2+ current are similar in most of these cells, with respect to their voltage-dependence, ion selectivity and response to channel modulators, some differences have been recorded, most notably in the gastro-intestinal tract, and may demand a reappraisal of how a T-type Ca2+ current is characterised. The functions of such a current in different tissues remains uncertain. In most of smooth muscles discussed in this review, it is hypothesised that it underlies rhythmic or spontaneous electrical activity, especially in concert with other current-carrying systems, such as Ca2+-activated outward currents. Of equal interest is that the T-type Ca2+ channel may be a target for agents that modulate tissue function, especially in pathological conditions, or are the site of secondary effects of agents used in clinical medicine. For example, T-type Ca2+ channel modulators have been proposed to reduce overactive muscular activity in the gastro-intestinal or urinary tract, or function as tocolytic agents: and the action of volatile anaesthetics on them in airways smooth muscle requires consideration in their overall action.