WO2023104159A1

WO2023104159A1 - Calcium channel blocker and screening method thereof

Info

Publication number: WO2023104159A1
Application number: PCT/CN2022/137596
Authority: WO
Inventors: Mingqi XIE; Hui Wang; Martin Fussenegger
Original assignee: Xie Mingqi; Hui Wang
Priority date: 2021-12-09
Filing date: 2022-12-08
Publication date: 2023-06-15

Abstract

The present invention relates to compounds as calcium channel blocker, a composition comprising thereof, use of said compounds and a method for treating diseases or disorders associated with calcium ion channel using said compounds. The present invention also relates to an in vitro screening method of calcium channel blocker compounds.

Description

CALCIUM CHANNEL BLOCKER AND SCREENING METHOD THEREOF

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Calcium channel blockers (CCBs) are defined as a class of clinically used molecules, peptides or proteins that selectively block the entry of Ca ²⁺ into cells through inhibition of calcium-permeable ion channels, resulting in decrease of intracellular Ca ²⁺ concentration. Currently known calcium channel blockers include phenylalkylamines such as verapamil, or dihydropyridines (DHP) such as amlodipine, felodipine, isradipine, lacidipine, nicardipine, nifedipine, niguldipine, niludipine, nimodipine, nisoldipine, nitrendipine, nivaldipine, and the like. In general, CCBs are used alone or in combiantion with other drugs such as angiotensin receptor blocker (ARB) and/or diuretics, for the treatment of hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , heart failure such as congestive heart failure, left ventricular dysfunction and hypertrophic cardiomyopathy, diabetic cardiac myopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter, detrimental vascular remodeling, myocardial infarction, atherosclerosis, renal insufficiency (diabetic and non-diabetic) , diabetes, primary and secondary pulmonary hypertension, renal failure, diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria of primary renal disease, and also renal vascular hypertension, diabetic retinopathy, the management of other vascular disorders, such as migraine, peripheral vascular disease, Raynaud's disease, luminal hyperplasia, cognitive dysfunction (such as Alzheimer's diseases) , glaucoma and stroke (see, e.g. WO03097045) .

Parkinson’s disease (PD) is an age-related neurodegenerative disorder characterized by progressive motor impairments such as tremors, rigidity and bradykinesia (Daniels et al. 2019, Gao et al. 2003, Sarkar, Raymick and Imam 2016) . These symptoms are primarily driven by the selective loss of mesencephalic dopamine-producing neurons in the pars compacta of the substantia nigra (SNc) of the midbrain (Daniels et al. 2019, Gao et al. 2003, Izumi et al. 2007, Sarkar et al. 2016, Singh et al. 2016) . Defective dopaminergic (DA) neurons are characterized by the formation of Lewy bodies consisting of ubiquitin and α-synuclein aggregates, are highly sensitive to stress, and show markedly impaired mitochondrial functions, which lead to reduced ATP production and poor calcium homeostasis (Crocker et al. 2003, Sarkar et al. 2016) . Excessive-activity-related Ca ²⁺ oscillations contribute to the generation of reactive oxygen species (ROS) , resulting in excitotoxicity and apoptosis (Ilijic, Guzman and Surmeier 2011, Singh et al. 2016) . Although a plethora of putative therapeutics have been proposed to relieve PD symptoms, including DA receptor agonists, anti-inflammatory drugs, inhibitors of α-synuclein aggregates, neurotrophic growth factors and calpain inhibitors, none of them affords fully effective neuroprotection –a term that, strictly defined, refers to any treatment strategy that protects the integrity of DA neurons (Sarkar et al. 2016) .

In recent years, strategies based on inhibition of L-type voltage-gated calcium channels (LTCC) with dihydropyridine (DHP) blockers have gained increased attention for neuroprotective therapy of PD (Biglan et al. 2017, Ortner et al. 2017) . Specifically, Ca _V1.3 is a LTCC isoform primarily expressed in neurons and pancreatic endocrine cells, and opens at subthreshold membrane potentials due to its negative activation voltage range (Xu and Lipscombe 2001, Yang and Berggren 2006) . DA neurons exhibit Ca _V1.3-dependent pacemaking activity, which is a major contributor to excitotoxicity during PD progression (Ortner et al. 2017, Singh et al. 2016) . However, DHP channel blockers are generally non-selective, blocking both Ca _V1.2 and Ca _V1.3 channel isoforms in most cases (Ortner and Striessnig 2016) . Because Ca _V1.2 channels are expressed at very high levels in cardiac tissues, cross-antagonism to these channels severely limits the dose of DHPs that can be used for neuroprotective purposes (Ilijic et al. 2011, Ortner and Striessnig 2016) . Therefore, Ca _V1.3-selective blockers without Ca _V1.2-mediated cardiovascular side effects are currently considered elusive candidates for PD drug discovery (Ortner et al. 2017) . Hence, there is still need for blockers that have high selectivity towards Ca _V1.3 channel with minimal crosstalk to Ca _V1.2 channel to solve the above-mentioned problem.

DISCLOSURE OF THE INVENTION

Ion channel drug discovery is primarily limited by the scarcity of technologies that allow high-throughput screening of candidate compounds. The state-of-the art technology for high-throughput screening of ion channel modulators is the fluorescent imaging plate reader (FLIPR) assay, which is based on calcium-activated fluorescent dyes (Zamponi 2016) . However, FLIPR experiments require proprietary instruments, is inherently prone to signal fluctuation (Inglese et al. 2007, Heusinkveld and Westerink 2011) , and therefore necessitates integrated patch-clamp platforms to provide secondary validation (Dunlop et al. 2008) . Because FLIPR is also limited to snapshot-recordings of single ion-influx events of cells harvested in non-physiological buffer solutions, it is not applicable to the identification of use-dependent CCBs such as antiarrhythmics that require methods for assessing repetitive channel activation (Zamponi 2016) . Conventional patch clamp platforms are more accurate and most information-rich, but are primarily constrained by low throughput and poor cost-efficiency in a drug screening context. Furthermore, an ideal asset of high-throughput drug discovery is multiplexed drug screening, allowing simultaneous assessment of multiple disease-specific drug targets within a single experiment (Bachovchin et al. 2014) .

By capitalizing on synthetic biology-inspired gene circuits that can flexibly program cells to perform application-specific biological tasks with high robustness and precision (Xie and Fussenegger 2018) , the inventors have custom-designed a mammalian cell-based drug discovery platform for high-throughput screening of isoform-specific calcium channel blockers (CCB) . Specifically, deflection of Ca _V-dependent NFAT-activation to the repression of reporter protein translation allowed for the engineering of an antagonist-inducible reporter system (CaB-A assay) that effectively reduced the susceptibility to false-negatives associated to cytotoxicity-mediated signal decrease. After validating this technology with a selection of clinically approved CCB drugs, the inventors identified five plant-derived essential oils that could effectively block Ca _v1.2 and Ca _v1.3. Further integration of in silico virtual screening and deep-learning technology eventually enabled the identification of sclareol and gingerol as natural bioactive compounds that inhibits Ca _v1.3 stronger than Ca _v1.2. Finally, using whole cell patch-clamp recordings and a PD mouse model, the inventors revealed strong neuroprotective activities of sclareol both in vitro and in vivo with comparable efficacy but higher safety than currently known CCB drugs in the clinics, rendering sclareol and gingerol promising candidate drugs for neuroprotection in PD patients. Furthermore, the inventors believe the combined application of synthetic-biology-inspired technology, advanced computational methods and molecular medicine, as exemplified here, represents an efficient platform that could help to set the stage for next-generation drug discovery in a variety of contexts.

In a first aspect, the present invention provides compounds as CCB. In particular, the present invention provides the compound of formula (I) , having the following structure:

wherein X is C _1-2 alkylene, and each of R ₁ and R ₂ is independently selected from C _1-4 alkyl, C _2-4 alkenyl, or C _2-4 alkynyl, which is optionally substituted with one or more halogen atoms,

or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof.

In a preferred embodiment, the compound of formula (I) has the structure of formula (Ia) :

wherein X is C _1-2 alkylene, and R ₁ is independently selected from C _1-4 alkyl, C _2-4 alkenyl, or C _2-4 alkynyl, which is optionally substituted with one or more halogen atoms,

In a further preferred embodiment, the compound of formula (I) has the structure of formula (Ib) :

In a particular embodiment, the compound of formula (Ib) is either (1R, 2R, 4aS, 8aS) -1- [ (3R) -3-hydroxy-3-methylpent-4-enyl] -2, 5, 5, 8a-tetramethyl-3, 4, 4a, 6, 7, 8-hexahydro-1H-naphthalen-2-ol or (1R, 2R, 4aS, 8aS) -1- ( (R) -3-hydroxy-3-methylpent-4-enyl) -2, 5, 5, 8a-tetramethyl-decahydronaphthalen-2-ol, and any enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof.

The present invention also provides the compound of formula (II) as CCB, having the following structure:

wherein R ₃ is C _1-10 alkyl, which is optionally substituted with one or more hydroxy groups, preferably is substituted with one hydroxy group,

In a preferred embodiment, the compound of formula (II) has the structure of formula (IIa) :

wherein R ₄ is C _1-8 alkyl, preferably C _1-6 alkyl, more preferably C _1-4 alkyl,

In a further preferred embodiment, the compound of formula (II) has the following structures:

In a particular embodiment, the compound of formula (IIb-1) is (5S) -5-hydroxy-1- (4-hydroxy-3-methoxyphenyl) octan-3-one, or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof.

In another particular embodiment, the compound of formula (IIb-2) is (5S) -5-hydroxy-1- (4-hydroxy-3-methoxyphenyl) decan-3-one, or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof.

In another particular embodiment, the compound of formula (IIb-3) is (5S) -5-hydroxy-1- (4-hydroxy-3-methoxyphenyl) dodecan-3-one, or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof.

In another particular embodiment, the compound of formula (IIb-4) is (5S) -5-hydroxy-1- (4-hydroxy-3-methoxyphenyl) tetradecan-3-one, or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof.

In a second aspect, the present invention provides an essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) . The present invention also provides a composition comprising an essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) . Preferably, the composition is a pharmaceutical composition comprising the essential oil and pharmaceutically acceptable carrier or diluent.

In a third aspect, the present invention provides a pharmaceutical composition comprising an effective amount of the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof, or the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) .

In a preferred embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof, or the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , for use in the treatment of diseases or disorders selected from hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , heart failure such as congestive heart failure, left ventricular dysfunction and hypertrophic cardiomyopathy, diabetic cardiac myopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter, detrimental vascular remodeling, myocardial infarction, atherosclerosis, renal insufficiency (diabetic and non-diabetic) , diabetes, primary and secondary pulmonary hypertension, renal failure, diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria of primary renal disease, and also renal vascular hypertension, diabetic retinopathy, the management of other vascular disorders, such as migraine, peripheral vascular disease, Raynaud's disease, luminal hyperplasia, cognitive dysfunction (such as Alzheimer's diseases) , glaucoma, stroke and Parkinson disease.

Preferably, the disease or disorder is selected from hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , and Parkinson disease.

In a fourth aspect, the present invention provides a method of treating diseases or disorders, comprising the administration of effective amount of the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof, or the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or the composition comprising the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , to a subject, wherein the diseases or disorders is the disease or disorder which can be benefited from blockade of calcium channel, preferably the disease or disorder which can be benefited from selective blockade of Ca _V1.3 channel compared with Ca _V1.2 channel, more preferably the disease or disorder is selected from hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , heart failure such as congestive heart failure, left ventricular dysfunction and hypertrophic cardiomyopathy, diabetic cardiac myopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter, detrimental vascular remodeling, myocardial infarction, atherosclerosis, renal insufficiency (diabetic and non-diabetic) , diabetes, primary and secondary pulmonary hypertension, renal failure, diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria of primary renal disease, and also renal vascular hypertension, diabetic retinopathy, the management of other vascular disorders, such as migraine, peripheral vascular disease, Raynaud's disease, luminal hyperplasia, cognitive dysfunction (such as Alzheimer's diseases) , glaucoma, stroke and Parkinson disease.

More preferably, the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof has high selectivity to Ca _V1.3 channel compared with Ca _V1.2 channel.

In a fifth aspect, the present invention provides use of the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof as calcium channel blocker (CCB) . Preferably, the present invention provides use of the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof as selective calcium channel blockers. Particularly, the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof has high selectivity to Ca _V1.3 channel compared with Ca _V1.2 channel. More particularly, the present invention provides use of the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof, or the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or the composition comprising the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , for the treatment of diseases or disorders selected from hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , heart failure such as congestive heart failure, left ventricular dysfunction and hypertrophic cardiomyopathy, diabetic cardiac myopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter, detrimental vascular remodeling, myocardial infarction, atherosclerosis, renal insufficiency (diabetic and non-diabetic) , diabetes, primary and secondary pulmonary hypertension, renal failure, diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria of primary renal disease, and also renal vascular hypertension, diabetic retinopathy, the management of other vascular disorders, such as migraine, peripheral vascular disease, Raynaud's disease, luminal hyperplasia, cognitive dysfunction (such as Alzheimer's diseases) , glaucoma, stroke and Parkinson disease.

In a sixth aspect, the present invention provides use of the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof in manufacture of a medicament for the treatment of diseases or disorders which can be benefited from blockade of calcium channels. Preferably, the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof is used as selective calcium channel blockers. Particularly, the present invention provides use of the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof in manufacture of a medicament for the treatment of the disease or disorder which can be benefited from selective blockade of Ca _V1.3 channel compared with Ca _V1.2 channel. More particularly, the present invention provides use of the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof, or the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or the composition comprising the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , in manufacture of a medicament for the treatment of diseases or disorders selected from hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , heart failure such as congestive heart failure, left ventricular dysfunction and hypertrophic cardiomyopathy, diabetic cardiac myopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter, detrimental vascular remodeling, myocardial infarction, atherosclerosis, renal insufficiency (diabetic and non-diabetic) , diabetes, primary and secondary pulmonary hypertension, renal failure, diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria of primary renal disease, and also renal vascular hypertension, diabetic retinopathy, the management of other vascular disorders, such as migraine, peripheral vascular disease, Raynaud's disease, luminal hyperplasia, cognitive dysfunction (such as Alzheimer's diseases) , glaucoma, stroke and Parkinson disease.

In a seventh aspect, the present invention provides a method of screening compound as calcium channel blocker, especially having high selectivity to Ca _V1.3 channel compared with Ca _V1.2 channel, said method comprising the following steps:

(1) selection of a suitable host cell that weakly expresses or does not express Ca _V1.3 or Ca _V1.2 channel endogenously;

(2) delivery of (i) nucleic acid segments that encode one or more calcium channel subunits alongside (ii) any calcium-responsive reporter system into the host cell described in (1) ;

(3) depolarization of cells described in (1-2) through electrical stimulation or by treatment of aqueous salt solutions such as potassium chloride;

(4) incubation of depolarized cells described in (1-3) with candidate calcium channel blocker compounds for at least 1 minute; and

(5) quantification of signals produced by the calcium-responsive reporter system described in (1) .

In a preferred embodiment, the suitable host cell is of mammalian or amphibian origin. In a further preferred embodiment, the selected host cell is derived from human embryonic kidney cells, patient-specific tumors or Chinese hamster ovary cells, such as HEK-293, HeLa or CHO-K1 cells. In another preferred embodiment, the selected host cell is derived from Xenopus oocytes.

In another embodiment, the nucleic acid segments that encode one or more calcium channel subunits are one or more nucleic acid sequences comprising a mRNA sequence that can translate into any one of the protein subunits selected from α1, α2, β1, β2, β3, β4, γ, δ or α2δdomains of voltage-gated calcium channels. In another embodiment, the nucleic acid segments that encode one or more calcium channel subunits are one or more nucleic acid sequences comprising a DNA sequence that can transcribe into a RNA sequence that produces any one of the protein subunits selected from α1, α2, β1, β2, β3, β4, γ, δ or α2δ domains of voltage-gated calcium channels. In a preferred embodiment, the nucleic acid segment (s) that encode (s) one or more calcium channel subunits is (are) a nucleic acid sequence comprising one or more gene sequences selected from CACNA1S, CACNA1C, CACNA1D, CACNA1F, CACNA1A, CACNA1B, CACNA1E, CACNA1G, CACNA1H, CACNA1I, CACNA2D1, CACNA2D2, CACNA2D3, CACNA2D4, CACNB1, CACNB2, CACNB3, CACNB4, CACNG1, CACNG2, CACNG3, CACNG4, CACNG5, CACNG6, CACNG7 or CACNG8 or any homologue thereof.

In another embodiment, the calcium-responsive reporter system is a protein whose activity and/or integrity is naturally evolved or engineered to depend on local concentrations of calcium ions. In another embodiment, the calcium-responsive reporter system is an RNA molecule whose stability and/or translation is naturally evolved or engineered to depend on local concentrations of calcium ions. In another embodiment, the calcium-responsive reporter system is a natural or synthetic promoter whose activation is naturally evolved or engineered to depend on intracellular concentrations of calcium ions. In another embodiment, the calcium-responsive reporter system is any combination of a calcium-dependent protein, calcium-dependent RNA and calcium-dependent promoter described above. In a preferred embodiment, the calcium-responsive reporter system consists of a synthetic promoter that contains DNA segments bound by the calcium-responsive transcription factor NFAT (nuclear factor of activated T cells) .

DEFINITIONS

As used herein, the singular forms “a” , “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

As used herein, the term “subject” refers to a mammal, for example, a human.

As used herein, the term “halogen” signifies fluorine, chlorine, bromine or iodine, particularly fluorine or chlorine.

“Preventing” or “prevention” refers to a reduction in risk of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease) . In some embodiments, “preventing” or “prevention” refers to reducing symptoms of the disease by taking the compound in a preventative fashion.

“Treating” or “treatment” of a disease or disorder refers to arresting or ameliorating a disease or at least one of the clinical symptoms of a disease or disorder, reducing the risk of acquiring a disease or at least one of the clinical symptoms of a disease, reducing the development of a disease or at least one of the clinical symptoms of the disease or reducing the risk of developing a disease or at least one of the clinical symptoms of a disease. “Treating” or “treatment” also refers to inhibiting the disease, either physically, (e.g., stabilization of a discernible symptom) , physiologically, (e.g., stabilization of a physical parameter) , or both, and to inhibiting at least one physical parameter or manifestation that may or may not be discernible to the subject. “Treating” or “treatment” also refers to delaying the onset of the disease, or at least one or more symptoms thereof in a subject who may be exposed to or predisposed to a disease or disorder even though that subject does not yet experience or display symptoms of the disease.

The term “effective amount” as used herein refers to an amount of the compound of the present invention effective for “treating” or “preventing” a disease or disorder in a subject. The effective amount may cause any changes observable or measurable in a subject as described in the definition of “treating” , “treatment” , “preventing” , or “prevention” above. The “effective amount” may vary depending, for example, on the compound, the disease and/or symptoms of the disease, severity of the disease and/or symptoms of the disease or disorder, the age, weight, and/or health of the subject to be treated, and the judgment of the prescribing physician. An appropriate amount in any given instance may be ascertained by those skilled in the art or capable of determination by routine experimentation.

As used herein, “alkyl” means a straight or branched chain saturated hydrocarbon moieties, such as those containing from 1 to 10 carbon atoms (C _1-10) , 1 to 8 carbon atoms (C _1-8) , 1 to 6 carbon atoms (C _1-6) , 1-4 carbon atoms (C _1-4) or 1-3 carbon atoms (C _1-3) . For example, “C _1-6alkyl” refers to the alkyl having 1-6 (including 1, 2, 3, 4, 5 or 6) carbon atoms. “C _1-4alkyl” refers to the alkyl having 1-4 (including 1, 2, 3, or 4) carbon atoms. Representative C _1-6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl and the like.

As used herein, “alkenyl” means a straight or branched chain saturated hydrocarbon moieties comprising at least one double bond, such as those containing from 2 to 6 carbon atoms (C _2-6) , 2-4 carbon atoms (C _2-4) or 2-3 carbon atoms (C _2-3) . For example, “C _2-4 alkenyl” refers to the alkenyl having 2-4 (including 2, 3, or 4) carbon atoms. Representative C _2-4 alkenyl groups include ethenyl, propenyl, allyl, butenyl, and the like.

As used herein, “alkynyl” means a straight or branched chain saturated hydrocarbon moieties comprising at least one triple bond, such as those containing from 2 to 6 carbon atoms (C _2-6) , 2-4 carbon atoms (C _2-4) or 2-3 carbon atoms (C _2-3) . For example, “C _2-4 alkynyl” refers to the alkynyl having 2-4 (including 2, 3, or 4) carbon atoms. Representative C _2-4 alkynyl groups include ethynyl, propynyl, propargyl, butynyl, and the like.

As used herein, “alkylene” means a straight or branched chain saturated divalent hydrocarbon moieties, such as those containing from 1 to 4 carbon atoms (C _1-4) , 1-3 carbon atoms (C _1-3) or 1-2 carbon atoms (C _1-2) . For example, “C _1-2alkylene” refers to the alkyl having 1 or 2 carbon atoms. Representative C _1-2 alkylene groups include methylene or ethylene.

The term “diastereomer” denotes a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another.

The term “pharmaceutically acceptable salt” refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of formula I and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Acid-addition salts include for example those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethyl ammonium hydroxide. The chemical modification of a pharmaceutical compound into a salt is a technique well known to pharmaceutical chemists in order to obtain improved physical and chemical stability, hygroscopicity, flow ability and solubility of compounds. It is for example described in Bastin R.J., et al., Organic

Process Research &Development

2000, 4, 427-435; or in Ansel, H., et al., In: Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995) , pp. 196 and 1456-1457.

As used herein, the term “essential oil” refers to volatile oil naturally produced by plants as defensive metabolites for the protection against diseases, parasite, fungal, bacterial and viral infections, extreme temperature fluctuations and dehydration. In many countries, essential oils isolated from plants are used as additives in cosmetics, drugs, detergents and foods in order to ameliorate the aromatic, antioxidant and antimicrobial effects of the respective products [Chamorro et al. 2012; Gas Chromatography in Plant Science, Wine Technology, Toxicology and Some Specific Applications; Chapter 15] . In France and Germany, essential oils are also used as major components of medical formulations within the field of aromatherapy, which is a clinically approved branch of phytotherapy. Common extraction methods of essential oil include distillation, freezing compression, chemical solvent extraction, oil separation, carbon dioxide extraction, soaking and so on. Steam distillation extraction is the most commonly used method. Preferably, essential oil is derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) .

BRIEF DESCRIPTION OF FIGURES

Figure 1. Development of a cell-based assay for multiplexed drug screening.

(A) Experimental setup. For multiplexed screening of anti-Parkinson drugs, Ca _V1.3 (target) -specific and Ca _V1.2 (anti-target) -specific cell populations –each controlling the expression of a flexibly chosen reporter protein –are placed in the same reaction well to enable simultaneous assessment of inhibitory potency and specificity in potassium chloride (KCl) -mediated cell depolarization. (B) Design of a calcium channel blocker-activated (CaB-A) reporter assay. Synthetic NFAT-specific promoters control the production of L7Ae, which inhibits the translation of reporter mRNA by binding to specific C/D-box aptamers in the 5’-UTR. Calcium channel blockers (CCBs) activate reporter protein expression by inhibiting depolarization-dependent L7Ae expression. (C, D) Optimization of CaB-A for use-dependent CCB analysis. (C) Ca _V1.2 (pCa _V1.2/pKK56) -or (D) Ca _V1.3 (pCa _V1.3/pKK56) -transgenic HEK-293 cells were co-transfected with a NFAT-controlled L7Ae expression vector (pMX125; P _NFAT4-L7Ae-pA) and different reporter vectors containing one (pMX195; P _SV40- (C/D-box) ₁-SEAP-pA) or two tandem C/D-box aptamer repeats (pMX199; P _SV40- (C/D-box) ₂-SEAP-pA) . The cells were depolarized with different levels of KCl (0, 20, 30 mM) and cultivated for 48 h in the absence or presence (10 μM) of nicardipine. SEAP levels in culture supernatants were scored. Data points are presented as mean ± SD (n = 3 independent experiments) . Numeric values displayed on top of each column-group represent induction-folds as calculated by SEAP values resulting from 10μM nicardipine divided by SEAP values resulting from 0μM nicardipine. (E, F) Validation of CaB-A with clinically approved CCBs. (E) HEK-293 cells transfected with Ca _V1.2 (pCa _V1.2/pKK56/pMX125/pMX199) -or (F) Ca _V1.3 (pCa _V1.3/pKK56/pMX125/pMX199) -dependent CaB-A systems were depolarized with 20 mM KCl and immediately seeded into culture wells containing different concentrations of CCBs. Data are mean ± SD of SEAP levels scored at 48 h after exposure to CCBs (n = 3 independent experiments) .

Figure 2. Identification of putative Ca _V1.2-and Ca _V1.3-antagonizing essential oil products.

(A) High-throughput analysis. Independent Ca _V1.2-and Ca _V1.3-specific CaB-A systems (pCa _V/pKK56/pMX125/pMX199) were depolarized with 20 mM KCl and immediately seeded into culture wells supplemented with 4x10 ^-5 v/v of different plant essential oils. Data are mean FOC (fold of DMSO control) ± SD of SEAP levels scored at 48 h after exposure to essential oils (n = 3 independent experiments) . (B, C) Dose-dependent validation of the most active essential oil hits. (B) Ca _V1.2-and (C) Ca _V1.3-specific CaB-A systems were depolarized with 20 mM KCl and added to culture wells containing different essential oil dilutions (v/v) . Data in (B-C) are mean ± SD of SEAP levels scored at 48 h after exposure to essential oils (n = 3 independent experiments) . DMSO (solvent) levels in cell culture medium were kept below 0.4%.

Figure 3. Identification of active constituents that selectively inhibit Ca _V1.3. (A) Ligand-based virtual screening. Representative pharmacophore model for Ca _V1.3 inhibitors created with LigandScout using the positive and negative reference compounds listed in Table S3. This illustration exemplifies the alignment of (6) -gingerol to the Ca _V1.3-blocking pharmacophore. (B) Structure clustering analysis of candidate compounds. All 13 hits from the virtual screening experiment using 198 candidate molecules derived from GC-MS data of essential oils (Table S3) were clustered based on structure similarity using the ChemMine tool. Right panel: chemical structures of the five compounds selected as representatives of the clusters. (C) Assessment of putative Ca _V1.3 antagonism by the PD drug candidates. HEK-293 cells transfected with the Ca _V1.3-dependent CaB-A system were depolarized with 30 mM KCl and immediately seeded into culture wells containing different drug candidates supplemented at 10 μM or 100 μM. Data points are presented as mean FOC (fold of DMSO control) of SEAP levels scored at 48 h after exposure to nicardipine (n = 3 independent experiments) . (D) Quantification of Ca _V1 antagonism by sclareol using CaB-R. HEK-293 cells transfected with Ca _V1.2-or Ca _V1.3-dependent CaB-R were depolarized with 30 mM KCl and immediately seeded into culture wells containing different concentrations of sclareol. HEK-293 cells transfected with a constitutive SEAP-expression vector (pSEAP2-Control; P _SV40-SEAP-pA) were used as a reference for putative cytotoxicity caused by drug exposure. HEK-293 cells transfected with a bacterial expression vector (pViM41; P _T7-mCherry-MCS) was used as negative control indicating Ca _V-unrelated assay readouts. Data are mean ± SD of SEAP levels scored at 48 h after drug exposure (n = 3 independent experiments) . (E) Quantification of Ca _V1 antagonism by sclareol and nifedipine on different Ca _V1.3 mutants. HEK-293 cells transfected with CaB-R regulated by different synthetic Ca _V1.3 mutants (WT, pCa _V1.3/pKK56/pMX57; Ca _V1.3 ^Y1048A, pWH154/pKK56/pMX57; Ca _V1.3 ^{Y1365A, A1369S, I1372A}, pWH155/pKK56/pMX57) were depolarized with 30 mM KCl and immediately seeded into culture wells containing different concentrations of sclareol or nifedipine. HEK-293 cells transfected with a constitutive SEAP-expression vector (pSEAP2-Control; P _SV40-SEAP-pA) were used as a reference for putative cytotoxicity caused by drug exposure. Data are mean ± SD of SEAP levels scored at 48 h after drug exposure (n = 3 independent experiments) .

Fig. S1. Principles of Ca _V1-specific designer cell-based screening assays.

(A) Synthetic excitation-transcription coupling. In human embryonic kidney (HEK-293) cells, membrane depolarization activates L-type voltage-gated calcium channels (Ca _V1) and triggers Ca ²⁺ influx, activation of endogenous calcium-responsive transcriptional factors (CTFs) and initiation of reporter gene transcription from synthetic cognate calcium-specific promoters (CSPs) containing CTF-specific response elements. Inhibition of Ca _V1 by specific calcium channel blockers (CCBs) antagonizes membrane depolarization and attenuates Ca ²⁺-dependent gene regulation. (B, C) Selection of synthetic CSPs to quantify depolarization-dependent Ca _V1 signaling. (B) Ca _V1.2 (pCa _V1.2/pKK56) -or (C) Ca _V1.3 (pCa _V1.3: Δ42/pKK56) -transgenic HEK-293 cells were co-transfected with pWH31 (P _cFOS-SEAP-pA) , pHY30 (P _NFAT1-SEAP-pA) , pMX57 (P _NFAT3-SEAP-pA) or pWH90 (P _CRE2-SEAP-pA) and cultivated in the presence or absence of 60 mM potassium chloride (KCl) . SEAP levels in culture supernatants were scored after 48 h. Data are shown as mean ± SD (n=3 independent experiments) .

Fig. S2. Depolarization-inducible reporter gene expression.

(A) KCl-triggered SEAP expression. HEK-293 cells were co-transfected with pMX57 (P _NFAT3-SEAP-pA) , pKK56 (P _hEF1α-Cacna2d1-P2A-Cacnb3-pA) and either Ca _V1.2 (pCa _V1.2; P _hCMV-Cacna1c-pA) , Ca _V1.3 (pCa _V1.3: Δ42; P _hCMV-Cacna1d: Δ42-pA) or no Ca _V (pcDNA3.1 (+) ; P _hCMV-MCS-pA) , and depolarized with KCl (0-70 mM) . SEAP levels in culture supernatants were scored after 48 h. (B) KCl-triggered GLuc expression. Isogenic HEK-293 cells to (A) were transfected with a GLuc-expressing reporter vector (pWH29; P _NFAT3-Gluc-pA) instead of pMX57 and cultivated in cell culture medium containing different levels of KCl (0-60 mM) . GLuc levels in culture supernatants were scored after 48 h. (C) KCl-triggered expression of GFP and DsRed. Isogenic HEK-293 cells to (A) were transfected with reporter vectors expressing DsRed (pWH37; P _NFAT3-DsRed-Express-pA) or tGFP (pFS119; P _NFAT3-TurboGFP: dest1-pA) instead of pMX57, and depolarized with KCl (0-60 mM) . Fluorescence levels were recorded after 48 h by quantitative microplate reading (left; a. u., arbitrary unit) or microscopy (right) . All data are presented as mean± SD (n=3 independent experiments) .

Fig. S3. Design and validation of a calcium channel blocker-repressible (CaB-R) reporter assay. (A) Design principle. The presence of calcium channel blockers (CCBs) prevents reporter protein expression by inhibiting depolarization-dependent activation of endogenous NFAT (nuclear factor of activated T-cells) signaling. (B, C) Validation of dose-dependent CaB-R with clinically approved CCBs. (B) Ca _V1.2 (pCa _V1.2/pKK56/pMX57) -or (C) Ca _V1.3 (pCa _V1.3: Δ42/pKK56/pMX57) -transgenic HEK-293 cells were depolarized with 40 mM KCl and immediately placed in culture wells containing different concentrations of CCBs. (D, E) Validation of CaB-R for analysis of use-dependent CCBs. (D) Ca _V1.2 (pCa _V1.2/pKK56/pMX57) -or (E) Ca _V1.3 (pCa _V1.3: Δ42/pKK56/pMX57) -transgenic HEK-293 cells were depolarized with different levels of KCl (0, 20, 40 mM) and immediately added to culture wells containing different concentrations of nicardipine. SEAP levels in (B-E) were scored at 48 h after exposure to CCBs. Data are shown as mean percentage of relative blocking activity, normalized to maximal depolarization-dependent SEAP levels (0%; 40 mM KCl, no CCB) and maximal CCB blocking activity (100%; 10 μM nicardipine) . Statistics of (D-E) were analyzed by means of an extra-sum-of-squares F test (n=3 independent experiments) .

Fig. S4. Design and validation of a calcium channel blocker-activated (CaB-A) reporter assay. (A) Depolarization-repressible reporter protein expression. HEK-293 cells were co-transfected with pWH5 (P _PMS-SEAP-pA) , pWH75 (P _NFAT4-PMS-pA) , pKK56 (P _hEF1α-Cacna2d1-P2A-Cacnb3-pA) and either Ca _V1.2 (pCa _V1.2; P _hCMV-Cacna1c-pA) , Ca _V1.3 (pCa _V1.3; P _hCMV-Cacna1d-pA) or no Ca _V (pcDNA3.1 (+) ; P _hCMV-MCS-pA) , and depolarized with KCl (0-70 mM) and SEAP levels in culture supernatants were scored after 48 h. Data are presented as mean ± SD (n=3 independent experiments) . (B) Transcription-based CaB-A. Synthetic NFAT-promoters control the production of the synthetic mammalian trans-silencer (PMS) , which silences reporter gene transcription controlled by binding to synthetic cognate promoters (P _PMS) . Calcium channel blockers (CCBs) activate reporter protein expression by inhibiting depolarization-dependent PMS-expression. (C, D) Dose-dependent CaB-A activity. (C) Ca _V1.2 (pCa _V1.2/pKK56/pWH75/pWH5) -or (D) Ca _V1.3 (pCa _V1.3/pKK56/pWH75/pWH5) -transgenic HEK-293 cells were depolarized with 30 mM KCl and immediately placed in culture wells containing different concentrations of CCBs. Data are shown as mean ± SD of SEAP levels scored at 48 h after exposure to CCBs (n=3 independent experiments) . (E) Cytotoxicity control of CCBs. HEK-293 cells were transfected with pSEAP2-Control (P _SV40-SEAP-pA) and cultivated for 48 h in cell culture medium containing 1 μM or 10 μM CCBs. Data are shown as mean ± SD (n=3 independent experiments) . (F, G) Splice variant-dependent CaB-A activity. (F) Ca _V1.3: Δ42-transgenic HEK-293 cells (pCa _V1.3: Δ42/pKK56/pWH75/pWH5) were depolarized with 30 mM KCl and immediately placed in culture wells containing different concentrations of CCBs. (G) HEK-293 cells expressing Ca _V1.3 (pCa _V1.3/pKK56/pWH75/pWH5) , Ca _V1.3: Δ42 (pCa _V1.3: Δ42/pKK56/pWH75/pWH5) or no Ca _V (pcDNA3.1 (+) ; P _hCMV-MCS-pA) were cultivated in cell culture medium containing different KCl levels (0-70 mM) . Data in (F, G) are shown as mean ± SD of SEAP levels scored at 48 h after addition of KCl (n=3 independent experiments) . (H) Impact of CCBs on PMS activity. HEK-293 cells were co-transfected with pWH9 (P _SV40-PMS-pA) and pWH5 (P _PMS-SEAP-pA) , and cultivated in culture medium containing different concentration of nicardipine or benidipine. Data are shown as mean ± SD of SEAP levels scored at 48 h after exposure to CCBs (n=3 independent experiments) .

Fig. S5. Control experiments of the translation-based (CaB-A) reporter assay.

(A) Promoter optimization for NFAT-dependent L7Ae expression. HEK-293 cells were co-transfected with pCa _V1.2, pKK56, pMX199 and different L7Ae expression vectors containing five (pMX124; P _NFAT3-L7Ae-pA) , seven (pMX125; P _NFAT4-L7Ae-pA) or nine (pMX126; P _NFAT5-L7Ae-pA) tandem repeats of NFAT response elements, depolarized with 20 mM KCl and immediately placed in culture wells containing 0 or 10 μM nicardipine. Data points are presented as mean FOC (fold of control, DMSO) of SEAP levels scored at 48 h after exposure to nicardipine (n=3 independent experiments) . (B-D) Impact of CCBs on NFAT-controlled repressors. (B) Transcription- (PMS-P _PMS; pWH9/pWH5) or translation-based (L7Ae-C/D box; pWH145/pMX199; pWH145, P _hCMV-L7Ae-pA) control systems were cultivated for 48 h in cell culture medium containing 0 μM (DMSO control) or 10 μM CCBs. (C) pWH145/pMX199-transgenic HEK-293 cells were cultivated for 48 h in cell culture medium containing different concentrations of CCBs. (D) pMX125/pMX199-transgenic HEK-293 cells were cultivated for 48 h in cell culture medium containing 1 μM or 10 μM CCBs. Data in (B-D) are shown as mean ±SD of SEAP levels scored at 48 h after exposure to CCBs (n=3 independent experiments) .

Fig. S6. Identification of putative anti-Parkinson drug candidates.

(A) Impact of plant essential oils on L7Ae-dependent control systems. pWH145/pMX199 (P _hCMV-L7Ae) -or pMX125/pMX199 (P _NFAT4-L7Ae) -transgenic HEK-293 cells were depolarized with 20 mM KCl and cultivated for 48 h in cell culture medium containing different levels of plant essential oils (4 x 10 ^-5 or 8 x 10 ^-5 v/v) . (B) Cytotoxicity control of plant essential oils. HEK-293 cells were transfected with pSEAP2-Control (P _SV40-SEAP-pA) and cultivated for 48 h in cell culture medium containing different levels of plant essential oils (4 x 10 ^-5 or 8 x 10 ^-5 v/v) . All data are shown as mean ± SD of SEAP levels scored at 48 h after exposure to essential oils (n=3 independent experiments) . (C) Validation of CaB-A for multiplexed drug screening. Independent Ca _V1.2-specific CaB-R (pCa _V1.2/pKK56/pWH29) and Ca _V1.3-specific CaB-A (pCa _V1.3/pKK56/pMX125/pMX199) systems were mixed, depolarized with 30 mM KCl, and added to culture wells supplemented with different plant essential oils (4x10 ^-5 v/v) or CCBs (10 μM) . Data are shown as mean ± SD of reporter proteins scored at 48 h after exposure to antagonists (n=3 independent experiments) . Levels of the DMSO solvent (control) in cell culture medium were kept below 0.4%. (D) Quantification of Ca _V1 antagonism by (6) -gingerol using CaB-A. HEK-293 cells transfected with Ca _V1.2-or Ca _V1.3-dependent CaB-A were depolarized with 30 mM KCl and immediately seeded into culture wells containing different concentrations of (6) -gingerol. Data are shown as mean ± SD of SEAP levels scored at 48 h after drug exposure (n = 3 independent experiments) . (E) Quantification of Ca _V1 antagonism by (6) -gingerol using CaB-R. HEK-293 cells transfected with Ca _V1.2-or Ca _V1.3-dependent CaB-R were depolarized with 30 mM KCl and immediately seeded into culture wells containing different concentrations of (6) -gingerol. HEK-293 cells transfected with a constitutive SEAP-expression vector (pSEAP2-Control; P _SV40-SEAP-pA) were used as a reference for putative cytotoxicity caused by drug exposure. Data are mean ± SD of SEAP levels scored at 48 h after drug exposure (n = 3 independent experiments) . (F) Quantification of Ca _V1 antagonism by sclareol using CaB-A. HEK-293 cells transfected with Ca _V1.2-or Ca _V1.3-dependent CaB-A were depolarized with 30 mM KCl and immediately seeded into culture wells containing different concentrations of sclareol. HEK-293 cells transfected with a bacterial expression vector (pViM41; P _T7-mCherry-MCS) or Ca _V2.2 (pCa _V2.2; P _hCMV-Cacna1b-pA) instead of Ca _V1.2-or Ca _V1.3 were used as negative controls indicating Ca _V-unrelated or L-type Ca _V-unrelated assay readouts, respectively. Data are shown as mean ± SD of SEAP levels scored at 48 h after drug exposure (n = 3 independent experiments) .

Fig. S7 describes IC ₅₀ values of FDA-approved calcium channel blockers (CCBs) on Ca _v1.2 and Ca _v1.3.

Fig. S8 describes essential oils used in this study.

Fig. S9 describes reference compounds for the generation of Ca _V1.3-inhibiting pharmacophores. Fig. S10 describes FDA-approved drugs used in this study.

EXAMPLES

Methods

Vector Design. Comprehensive design and construction details for all expression plasmids are provided in Table 2. Mouse Ca _V1.2 α1C (GenBank accession number AY728090, Addgene plasmid #26572) , rat Ca _V1.3α1D (GenBank accession number: AF370009, Addgene plasmid #49333) , rat Ca _V1.3α1D: Δ42a (GenBank accession number: AF370010, Addgene plasmid # 49332) , rat Ca _Vβ3 (GenBank accession number: M88751, Addgene plasmid #26574) and rat Ca _Vα2δ-1 (GenBank accession number: AF286488, Addgene plasmid #26575) were provided by Prof. Diane Lipscombe (Brown University) .

Cell culture and transfection. Human embryonic kidney cells (HEK-293T, ATCC: CRL-11268) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Basel, Switzerland; cat. no. 52100–39) supplemented with 10% (v/v) fetal bovine serum (FBS; Sigma-Aldrich, Buchs, Switzerland; cat. no. F7524, lot no. 022M3395) and 1% (v/v) penicillin/streptomycin solution (PenStrep; Biowest, Nuaillé, France; cat. no. L0022-100) . Cells were cultured at 37℃ in a humidified atmosphere of 5%CO ₂ in air. For passaging, cells of pre-confluent cultures were detached by incubation in 0.05%trypsin-EDTA (Life Technologies, CA, USA; cat. no. 25300-054) for 3 min at 37℃, collected in 10 ml of cell culture medium, centrifuged for 3 min at 290 g, and resuspended in fresh culture medium at 1.5 x 10 ⁵ cells/mL, before seeding into new tissue culture plates. Cell number and viability were quantified using an electric field multichannel cell counting device (Casy Cell Counter and Analyzer Model TT, Roche Diagnostics GmbH) . HEK-293T cells were transfected using a polyethyleneimine (PEI) -based protocol. In brief, 2 x 10 ⁵ cells/ml cells were seeded into 48-well plates (225 μl medium per well) , 6-well plates (1800 μl medium per well) , or 10 cm petri dishes (9 mL medium) . At 24 h after seeding, cells were incubated for 12 h with 50 μl, 100 μl or 400 μl of a 10: 3 PEI: DNA mixture (w/w) , containing 0.3-0.4 μg (48-well plate) , 2.4-3 μg (6-well plate) , or 11-13 μg of total DNA (10 cm petri dish) , respectively. For experiments performed in 24-well plates, the culture medium was exchanged to PEI-free medium containing defined concentrations of control compounds after 12 h. For experiments performed in 6-well plates or 10-cm dishes, transfected cells were detached with 0.05%trypsin-EDTA, re-seeded into 96-well plates (15000-40000 cells/well) , and cultured in medium containing defined concentrations of control compounds. Analytical reporter assays were performed at 48 h after medium exchange.

Chemicals and drugs. Ethanol (EtOH; cat. no. 02860) , sclareol (cat. no. 357995) , linalool oxide (cat. no. 62141) , zingerone (cat. no. 88787) , Cremophor (cat. no. C5135) and dimethyl sulfoxide (DMSO; cat. no. D8418) were purchased from Sigma-Aldrich (Buchs, Switzerland) . Polyethyleneimine (PEI; cat. no. 24765-2; stock solution 1 mg/ml in ddH ₂O) was purchased from Polysciences (Eppelheim, Germany) . (6) -Gingerol (cat. no. sc-201519) and diethyl phthalate (cat. no.sc-239738) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany) . Potassium chloride (KCl; cat. no. A3582; stock solution 4 M in ddH ₂O) was purchased from AppliChem (Darmstadt, Germany) . Calcium channel blocker (CCB) drugs (Fig. S10) and plant essential oils (Fig. S8) were stored in DMSO and diluted with DMEM to final working concentrations. Final DMSO levels in the cell culture medium were kept below 0.4%.

Quantification of target gene expression. Expression levels of human placental secreted alkaline phosphatase (SEAP) in culture supernatants were quantified based on p-nitrophenyl phosphate-derived light absorbance at 415 nm (Wang et al. 2015) . Gaussia Luciferase (GLuc) levels in culture supernatants were profiled using the

Gaussia luciferase assay kit (New England Biolabs, Ipswich, MA; cat. no. E3300) . Fluorescence levels of HEK-293T cells grown in a black 96-well plate with a transparent bottom (Greiner Bio-one; cat. no. 655090) and cultivated in phenol red-free DMEM (ThermoFisher Scientific, cat. no. 21063029) were quantified with a microplate reader (Infinite M1000 PRO plate reader, Tecan Group Ltd) . TurboGFP levels were recorded at excitation 460/9 nm, emission 502/20 nm. dsRed-Express levels were recorded at excitation 554/9 nm, emission 586/20 nm.

Fluorescence imaging. Fluorescence microscopy was performed with an inverted fluorescence microscope (Nikon Ti-E; Nikon) equipped with an incubation chamber, an Orca Flash-4 digital camera (Hamamatsu) , a pE-100-LED (CoolLED) as the transmission light source, a Spectra X (Lumencor) as the fluorescent light source and a 4× objective, an excitation and emission filter set (TurboGFP: 475/525 nm; dsRed-Express 549/593 nm) and NIS Elements AR software (version 4.3.0) .

Data Analysis. CCB-activity in CaB-R assays was calculated as “percentage of control” , with reporter protein levels normalized to maximum average counts (100%; 40 mM KCl addition) and minimum average counts (0%; 10 μM nicardipine) . Normalization calculations and nonlinear regression curve-fittings (log (inhibitor) normalized response–variable slope) , and statistical analysis were all conducted in Prism 7.0 (Graph Pad Software, San Diego, CA, USA) . For statistical analysis, an extra sum-of-squares F test was performed to determine the significance of differences in Log (IC ₅₀) among the data sets of Fig. 2D, E. Fold of control (FOC) values were calculated as FOC = Xi/avg (c ₊) ×100, where Xi is the measurement of the i ^th compound and avg(c ₊) is the average measurement of the DMSO-treated samples. The Z’ value was calculated between the positive (10 μM nicardipine) and negative (0.1%DMSO) controls according to the reported equation (Zhang et al., 1999) . All values for in vitro experiments are expressed as the mean ± SD.

Computer-aided drug screening. Representative pharmacophore models were created with LigandScout software (Wolber and Langer 2005) based on the reference ion channel blockers listed in Fig. S9. To perform alignments to the pharmacophore, all constituents of rose flower, cistus ladanifer, pinus sylvestris, ginger and clary sage obtained from GC-MS data kindly provided by Welfine Beijing Science &Technology Development Co. Ltd (Beijing, China) were assigned with a chemical SMILES (Simplified Molecular-Input Line-Entry System) language. Suggested hits of virtual screening were refined by structure similarity analysis using the free ChemMine software (http: //chemmine. ucr. edu/) (Backman, Cao and Girke 2011) . Drug-likeness properties of candidate Ca _V-blocker drugs, including pharmacokinetics, Lipinski’s Rule of Five criteria (Lipinski et al. 2001) , and blood-brain barrier permeability, were evaluated using the ADME toxicity predictor SwissADME ( https: //www. click2drug. org/directory_ADMET. html) .

Results

Engineering of a cell-based drug screening platform for multiplexed and use-dependent analysis of Ca _V1 channel blockers. PD drug discovery would greatly benefit from multiplexed drug screening, allowing simultaneous assessment of multiple disease-specific drug targets within a single experiment (Bachovchin et al. 2014) (Fig. 1A) . In cell-based assay designs, secreted reporter proteins such as human placental secreted alkaline phosphatase (SEAP) or Gaussia princeps luciferase (GLuc) are advantageous for qualitative, non-disruptive and high-throughput recording of gene expression, while on the other hand, intracellular reporter systems such as fluorescent proteins facilitate resource-efficient and simple experimental setups (Muller et al. 2012) . To design a CCB-regulated reporter protein assay, we created a synthetic excitation-transcription coupling system (Xie et al. 2016) . Activation of Ca _V1 channels by membrane depolarization triggers a surge in cytosolic [Ca ²⁺] _i, initiating different signal-transduction pathways that modulate endogenous calcium-specific promoters (D'Arco and Dolphin 2012) (Fig. S1A) . Among different calcium-specific promoters (CSP) known to respond to Ca _V1-dependent cell signaling (Chawla 2002, Doerks et al. 2002, Dolmetsch 2003) , the synthetic NFAT promoter P _NFAT3 (pMX57, P _NFAT3-SEAP-pA; P _NFAT3, (NFAT _IL4) ₅-P _min) showed the most suitable Ca _V1.2-and Ca _V1.3-dependent SEAP induction profiles triggered by potassium chloride (KCl) -mediated depolarization (Figs. S1B and S1C) . After validating dose-dependent excitation-transcription coupling with different secreted (Figs. S2A and S2B) and fluorescent reporter systems (Fig. S2C) , we tested the potential of the cell-based SEAP assay for CCB drug discovery. In a genetic configuration enabling CCB-repressible reporter expression (CaB-R assay) (Fig. S3A) , the presence of CCBs blocking Ca _V1.2 and Ca _V1.3 inhibits NFAT signalling and causes a dose-dependent decrease of SEAP production (Figs. S3B and S3C) . When we validated CaB-R with a selection of clinically approved CCB drugs, the IC ₅₀ values determined in this study generally lay within the reference ranges reported for both Ca _V1-channel isoforms (Fig. S7) . In addition, CaB-R allowed for use-dependent analysis of repetitive CCB-mediated channel inhibition and activation, which is a critical but often elusive screening requirement in ion channel drug discovery (Zamponi 2016) . Following prolonged depolarization of cells loaded with Ca _V1.2 or Ca _V1.3-dependent CaB-R systems, the representative CCB nicardipine showed a typical use-dependent channel antagonism profile characterized by stronger inhibition at hyperactive channel states (KCl = 20, 40 mM) as compared to the degree of inhibition at baseline channel activities (KCl = 0 mM) (Figs. S3D and S3E) (Colella et al. 2008, He et al. 2011) .

Engineering of an antagonist-inducible reporter assay to reduce false-positive results. In many drug-screening studies that involve the use of living cells, cytotoxicity-mediated signal decrease often interferes with antagonist-associated reporter signals, thus generating false-positives (Didiot et al. 2011, Zhang and Xie 2012) . To overcome this limitation, we engineered a CCB-activated reporter assay (CaB-A) that operates in a reversed configuration, allowing depolarization-dependent NFAT signaling to repress reporter protein expression (Fig. S4A) . The presence of CCBs antagonizes Ca _V1-mediated NFAT activation and triggers de-repression of reporter gene transcription (Fig. S4B) . Not only did CaB-A reveal all CCB-mediated drug effects in the expected dose-dependent manner (Figs. S4C and S4D) , but it also effectively reduced the risk of obtaining false-positives, as expected. For example, cytotoxicity control experiments might have led to classification of the CCB-repressible effect of flunarizine as a false positive in CaB-R (Fig. S4E) , but the potency of flunarizine in activating gene expression in CaB-A corroborated the true channel-blocking efficacy of this drug (Figs. S4C and S4D) . Baseline signal levels of CaB-A assays could be further fine-tuned by choosing different splice-variants of each channel isoform (Fig. S4F) , as we demonstrated with two alternatively spliced Ca _V1.3 α1-domains characterized by different basal channel activities (Xu and Lipscombe 2001) (Fig. S4G) .

In CaB-A (Fig. S4B) , CCB-activated gene expression results from inhibition of NFAT-repressible gene expression of a synthetic transcription factor, which binds to and silences synthetic cognate promoters driving constitutive expression of the reporter gene. However, most synthetic transcription factors –especially those having a TetR-family repressor domain –are inherently under allosteric control by particular ligands (Cuthbertson and Nodwell 2013, Vargas et al. 2011) . Indeed, when we used a paraben-dependent mammalian trans-silencer (PMS, PmeR-KRAB (Wang et al. 2015) ) as the NFAT-driven repressor, we found that nicardipine and benidipine interfered with de-repression of PMS-specific promoters at high concentrations (>1 μM) (Fig. S4H) , which would likely cause erroneous interpretation of the CaB-A results (Figs. S4C and S4D) . To improve screening accuracy, we designed an optimized CaB-A configuration in which the synthetic NFAT promoter controls the expression of L7Ae (an archaeal ribosome-derived RNA-binding protein with high affinity for a C/D box-aptamer motif) (Auslander et al. 2014, Saito et al. 2010) (Fig. 1B) . The presence of CCBs prevents NFAT-dependent L7Ae expression (pMX125, P _NFAT4-L7Ae-pA; P _NFAT4, (NFAT _IL4) _7-P _min; Fig. S5A) and de-represses translation of reporter mRNA engineered to contain cognate C/D-box motifs in the 5’-UTR (Fig. 1B) . Depolarization-dependent production of L7Ae could knock down translation of SEAP mRNA harboring either one (pMX195, P _SV40- (C/D box) ₁-SEAP-pA) or two C/D-box repeats (pMX199, P _SV40- (C/D box) ₂-SEAP-pA) , with the vector combination of pMX125/pMX199 affording optimal nicardipine-inducible SEAP expression characterized by low background signals and high induction profiles for use-dependent Ca _V1-inhibition (Fig. 1, C and D) . Importantly, this modified CaB-A assay is no longer influenced by potential crosstalk between CCBs and the L7Ae-C/D box interaction (Figs. S5B-S5D) , and thus it enables accurate assessment of dose-dependent CCB-channel antagonism (Fig. 1, E and F) .

Multiplexed and high-throughput screen of plant essential oils to identify Ca _V1.2 and Ca _V1.3 antagonism. Next, we used CaB-A and performed a pilot test of high-throughput screening with a random selection of plant essential oils. Each essential oil is a complex mixture of volatile and aromatic organic compounds produced as part of a plant’s protective system, and can be regarded as a naturally selected package of biocompatible, bioavailable and bioactive substances (Reichling et al. 2009) . Among 42 essential oil products tested (Fig. S8) , the CaB-A assay identified five oils (i.e.; rose flower, cistrus ladanifer, pinus sylvestris, ginger, clary sage) that most effectively inhibited Ca _V1.2 and Ca _V1.3 (Fig. 2A) . All five essential oils dose-dependently activated SEAP expression in the CaB-A assay (Fig. 2, B and C) , and control experiments confirmed that none of these essential oils interfered with L7Ae activity or intracellular calcium signaling (Fig. S6A) . Notably, the results obtained with clary sage (Salvia sclarea) essential oil corroborated the advantage of CaB-A; although high concentrations of this oil were cytotoxic according to a reporter-based assay determining protein production capacity (Fig. S6B) , the unique antagonism-inducible gene expression readout of CaB-A (Fig. 2, B and C) ensured that the clary sage data was not excluded as false-negative. In terms of assay quality, both CaB-R and CaB-A assays have excellent Z’ screening windows (Z’ factor (CaB-R) = 0.68 ±0.14; Z’ factor (CaB-A) = 0.73 ± 0.07, n = 12 independent experiments) , and therefore should be suitable for rapid, robust and resource-efficient high-throughput screening (HTS) . As already mentioned, treatment of Parkinson’s disease (PD) requires a compound that can maximally inhibit Ca _V1.3 but not Ca _V1.2 (Ilijic et al. 2011) . To quantify the antagonistic activities towards Ca _V1.3 (PD drug target) and Ca _V1.2 (PD anti-target) simultaneously (i.e.; in a multiplexed screening configuration; Fig. 1A) , we mixed individual cell populations of Ca _V1.2-specific CaB-R and Ca _V1.3-specific CaB-A systems, each driving a different reporter protein. This cell mixture was exposed to clinically approved CCB drugs (positive control) , negative control compounds (i.e.; amitriptyline, tetracaine, lidocaine) and the five essential oil hits, in order to determine their impact on the depolarization-dependent expression of SEAP (reflecting Ca _V1.3 activity) and GLuc (reflecting Ca _V1.2 activity) (Fig. S6C) . The experimental results confirmed the multiplexed screening capability of our system. All five essential oils showed the required basic selectivity profile of maximal Ca _V1.3 inhibition (highest CaB-A score vs. control) and minimal Ca _v1.2 inhibition (closest CaB-R score to the control) .

Integration of in silico virtual screening and deep learning enables the discovery of (6) -gingerol and sclareol as novel Ca _V1.3-antagonists. To identify the putative active constituents of the five essential oils accounting for inhibition of Ca _V1.3, we used the LigandScout software to perform ligand-based virtual screening (Barreca et al. 2007, Wolber and Langer 2005) . First, we generated 3D pharmacophore models of putative Ca _V1.3 blockers (Fig. 3A) based on a series of Ca _V1 blockers (Kang et al. 2012) (positive reference) , as well as Ca _V-independent ion channel modulators found in the multiplexed screening experiment (negative reference; fig. S6C) (Fig. S9) . Using these pharmacophores, we performed in silico analysis of all constituents of rose flower, cistus ladanifer, pinus sylvestris, ginger, and clary sage essential oils by computing the similarity of each chemical structure to a theoretically ideal pharmacologically active moiety. From a total of 198 different candidate molecules, this virtual screening experiment suggested 13 hits as the most promising Ca _V1.3-antagonists (Table 1) , and structure clustering analysis enabled us to select the five chemicals diethyl phthalate, linalool oxide, zingerone, (6) -gingerol and sclareol as representative structures (Fig. 3B) . Experimental testing of these 5 candidate compounds with the CaB-A assay confirmed that (6) -gingerol and sclareol showed Ca _V1.3-antagonistic activity (Fig. 3C) . Notably, both compounds showed a stronger inhibitory effect on Ca _V1.3-mediated reporter gene expression than on the Ca _V1.2-dependent CaB system (Fig. 3, D and E; Figs. S6D and S6E) , and were also predicted to have optimal drug-likeness properties according to Lipinski’s Rule of Five criteria (Lipinski et al. 2001) . Importantly, sclareol (8.8 ± 1.0 μM; Figure 3D) had a more than three-fold lower IC ₅₀ value for Ca _V1.3 than (6) -gingerol (30.5 ±6.3 μM; Figure S6E) and is also structurally divergent to all currently known CCB compounds (Fig. S9, Fig. S10) , such as dihydropyridines (DHP) represented by nifedipine (Figure 3B) . Indeed, when we created putative DHP-insensitive Ca _V1.3 mutants based on amino acid alterations that were previously shown to be critical for CCB-sensitivity of the related L-type Ca _V1.1 channel (Zhao et al. 2019, Peterson, Tanada and Catterall 1996) , we found that our synthetic Ca _V1.3 ^{Y1365A, A1369S, I1372A} mutant was no longer inhibited by nifedipine but still retained full sensitivity to sclareol (Figure 3E) , indicating potential differences in the binding modes between sclareol and DHPs to Ca _V1.3. These features render sclareol a promising lead compound for PD pharmacotherapy.

Table 1. Virtual Screening Experiment.

(A) 309 compounds from 5 essential oils.

(B) LigandScout result.

Screening Results:
CC (=O) CCC1=CC (=C (C=C1) O) OC	VANILLYLACETONE
CCCC (CC (=O) CCC1=CC (=C (C=C1) O) OC) O	(4) -Gingerol
CCCCCC=CC (=O) CCC1=CC (=C (C=C1) O) OC	6-Shogaol
CCCCCC (CC (=O) CCC1=CC (=C (C=C1) O) OC) O	6-Gingerol
CCCCCCCC (CC (=O) CCC1=CC (=C (C=C1) O) OC) O	(8) -Gingerol
CCOC (=O) C1=CC=CC=C1C (=O) OCC	DIETHYL PHTHALATE
CCCCCCCCCC (CC (=O) CCC1=CC (=C (C=C1) O) OC) O	(10) -Gingerol
CC1 (CCCC2 (C1CCC (C2CCC (C) (C=C) O) (C) O) C) C	SCLAREOL
CC1=C (C=CC (=C1) C (=O) C) O	4-Hydroxy-3-methylacetophenone
CC1 (CCC (O1) C (C) (C) O) C=C	LINALOOL OXIDE
CC=CC1=CC (=C (C=C1) OC) OC	METHYL ISOEUGENOL
COC1=C (C=CC (=C1) CC=C) O	EUGENOL
COC1=C (C=C (C=C1) CC=C) OC	METHYLEUGENOL

Table 2. Plasmids and oligonucleotides designed and used in this study

Oligonucleotides: Restriction endonuclease-recognition sites are underlined and annealing nucleotides are shown in capital letters.

Abbreviations: BFP, blue fluorescent protein; Ca _V1.2, member 2 of the Ca _V1 family of L-type voltage-gated Ca ²⁺ channels; Cacna1c, α1-subunit of mouse Ca _V1.2 (NCBI Gene ID: 12288) ; Cav1.3, member 3 of the Cav1 family of L-type voltage-gated Ca ²⁺ channels; Cav1.3 ^AXB, synthetic Cav1.3 mutant where amino acid A at position X of the α1-subunit was exchanged to amino acid B; Cav2.2, N-type voltage-gated Ca ²⁺ channel; Cacna1d, α1-subunit of rat Cav1.3 (NCBI Gene ID: 29716) ; Cacna1d ^AXB, mutant α1-subunit of Cav1.3 where amino acid A at position X was exchanged to amino acid B; Cacna1d: Δ42a, Cacna1d-isoform lacking exon 42;Cacna1b, α1-subunit of rat Cav2.2 (NCBI Gene ID: 257648) ; Cacnb3, β3-subunit of rat Cav1.3 (NCBI Gene ID: 25297) ; Cacna2d1, α2δ-subunit of rat Cav1.3 (NCBI Gene ID: 25399) ; C/D-box, L7Ae-specific RNA aptamer (sequence: 5’-GGGCGUGAUCCGAAAGGUGACCC-3’) ; c-fos, human proto-oncogene from the Fos family of transcription factors; CRE, cAMP-response element (CREB1 binding site) ; d2YFP, destabilized variant of yellow fluorescent protein; DsRed-Express, destabilized variant of Discosoma sp. red fluorescent protein; GLuc, Gaussia princeps luciferase; IL4, murine interleukin 4; KRAB, Krueppel-associated box protein of the human kox-1 gene; L7Ae, archaeal ribosomal protein; MCS, multiple cloning site; NFAT, nuclear factor of activated T-cells; O _PmeR2, tandem PmeR-specific operator; P2A, picornavirus-derived ribosome skipping sequence (N'-GATNFSLLKQAGDVEENPGP-C') optimized for bicistronic expression in mammalian cells; pA, polyadenylation signal; PCR, polymerase chain reaction; P _CRE2, synthetic mammalian promoter containing six CRE repeats; P _CREm, modified P _CRE variant; P _cFOS, synthetic mammalian promoter containing three c-fos tandem repeats; PEST, peptide sequence rich in proline, glutamic acid, serine and threonine; P _ETR2, erythromycin-responsive promoter; P _hCMV, human cytomegalovirus immediate early promoter; P _hCMV*-1, tetracycline-responsive promoter (tetO ₇-P _hCMVmin) ; P _hEF1α, human elongation factor 1αpromoter; PmeR, Pseudomonas syringae pathovar tomato DC3000-derived multidrug efflux pump repressor; PMS, PmeR-derived paraben-mediated mammalian transsilencer (PmeR-KRAB) ; P _PMS, paraben-inducible promoter (P _SV40-O _PmeR2) ; P _NFAT1, synthetic mammalian promoter containing three tandem repeats of a human IL2 NFAT-binding site ( (NFAT _IL2) ₃-P _min; P _NFAT3, synthetic mammalian promoter containing five tandem repeats of a IL4-derived NFAT-binding site; P _NFAT4, synthetic mammalian promoter containing seven tandem repeats of a IL4-derived NFAT-binding site P _NFAT5, synthetic mammalian promoter containing nine tandem repeats of a IL4-derived NFAT-binding site; P _SV40, simian virus 40 promoter; P _T7, promoter activated by the T7 bacteriophage RNA polymerase; PuroR, gene conferring puromycin resistance; SB100X, optimized Sleeping Beauty transposase; SEAP, human placental secreted alkaline phosphatase; TetR, Escherichia coli Tn10-derived tetracycline-dependent repressor of the tetracycline resistance gene; tetO ₇, TetR-specific heptameric operator sequence; TurboGFP: dest1, PEST-tagged TurboGFP variant (Evrogen)

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Claims

A compound of formula (I) , having the following structure:

wherein X is C _1-2 alkylene, and each of R ₁ and R ₂ is independently selected from C _1-4 alkyl, C _1-4 alkenyl, or C _1-4 alkynyl, which is optionally substituted with one or more halogen atoms,

or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof.
The compound of formula (I) according to claim 1, which has the following structure:

wherein X is C _1-2 alkylene, and R ₁ is independently selected from C _1-4 alkyl, C _1-4 alkenyl, or C _1-4 alkynyl, which is optionally substituted with one or more halogen atoms,

or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof.
The compound of formula (I) according to claim 1, which has the following structure:

or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof.
A compound of formula (II) , having the following structure:

wherein R ₃ is C _1-10 alkyl, which is optionally substituted with one or more hydroxy groups, preferably is substituted with one hydroxy group,

or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof.
The compound of formula (II) according to claim 4, having the structure of formula (IIa) :

wherein R ₄ is C _1-8 alkyl, preferably C _1-6 alkyl, more preferably C _1-4 alkyl,

or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof.
The compound of formula (II) according to claim 4, having the following structures:

or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof.
A essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) .
A composition comprising the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) .
A pharmaceutical composition comprising an effective amount of the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to any one of claims 1-6, or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof, or the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to claim 7, or the composition comprising the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to claim 8.
A pharmaceutical composition comprising an effective amount of the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to any one of claims 1-6, or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof, or the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to claim 7, or the composition comprising the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to claim 8, for use in the treatment of diseases or disorders selected from hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , heart failure such as congestive heart failure, left ventricular dysfunction and hypertrophic cardiomyopathy, diabetic cardiac myopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter, detrimental vascular remodeling, myocardial infarction, atherosclerosis, renal insufficiency (diabetic and non-diabetic) , diabetes, primary and secondary pulmonary hypertension, renal failure, diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria of primary renal disease, and also renal vascular hypertension, diabetic retinopathy, the management of other vascular disorders, such as migraine, peripheral vascular disease, Raynaud's disease, luminal hyperplasia, cognitive dysfunction (such as Alzheimer's diseases) , glaucoma, stroke and Parkinson disease.
The pharmaceutical composition according to claim 10, wherein the disease or disorder is selected from hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , and Parkinson disease.
A method of treating diseases or disorders, comprising the administration of effective amount of the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to any one of claims 1-6, or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof, or the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to claim 7, or the composition comprising the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to claim 8, to a subject, wherein the diseases or disorders is the disease or disorder which can be benefited from blockade of calcium channel, preferably the disease or disorder which can be benefited from selective blockade of Ca _V1.3 channel compared with Ca _V1.2 channel.
The method according to claim 12, wherein the diseases or disorders is selected from hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , heart failure such as congestive heart failure, left ventricular dysfunction and hypertrophic cardiomyopathy, diabetic cardiac myopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter, detrimental vascular remodeling, myocardial infarction, atherosclerosis, renal insufficiency (diabetic and non-diabetic) , diabetes, primary and secondary pulmonary hypertension, renal failure, diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria of primary renal disease, and also renal vascular hypertension, diabetic retinopathy, the management of other vascular disorders, such as migraine, peripheral vascular disease, Raynaud's disease, luminal hyperplasia, cognitive dysfunction (such as Alzheimer's diseases) , glaucoma, stroke and Parkinson disease.
The method according to claim 13, wherein the disease or disorder is selected from hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , and Parkinson disease.
Use of the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to any one of claims 1-6, or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof as calcium channel blocker (CCB) .
Use according to claim 15, wherein the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof is used as selective calcium channel blockers, preferably having high selectivity to Ca _V1.3 channel compared with Ca _V1.2 channel.
Use according to claim 15, wherein the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) , or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof is used for the treatment of diseases or disorders selected from hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , heart failure such as congestive heart failure, left ventricular dysfunction and hypertrophic cardiomyopathy, diabetic cardiac myopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter, detrimental vascular remodeling, myocardial infarction, atherosclerosis, renal insufficiency (diabetic and non-diabetic) , diabetes, primary and secondary pulmonary hypertension, renal failure, diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria of primary renal disease, and also renal vascular hypertension, diabetic retinopathy, the management of other vascular disorders, such as migraine, peripheral vascular disease, Raynaud's disease, luminal hyperplasia, cognitive dysfunction (such as Alzheimer's diseases) , glaucoma, stroke and Parkinson disease.
Use of the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to claim 7, or the composition comprising the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to claim 8, for the treatment of diseases or disorders selected from hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , heart failure such as congestive heart failure, left ventricular dysfunction and hypertrophic cardiomyopathy, diabetic cardiac myopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter, detrimental vascular remodeling, myocardial infarction, atherosclerosis, renal insufficiency (diabetic and non-diabetic) , diabetes, primary and secondary pulmonary hypertension, renal failure, diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria of primary renal disease, and also renal vascular hypertension, diabetic retinopathy, the management of other vascular disorders, such as migraine, peripheral vascular disease, Raynaud's disease, luminal hyperplasia, cognitive dysfunction (such as Alzheimer's diseases) , glaucoma, stroke and Parkinson disease.
The use according to claim 17 or 18, wherein the disease or disorder is selected from hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , and Parkinson disease.
Use of the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to any one of claims 1-6, or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof in manufacture of a medicament for the treatment of diseases or disorders which can be benefited from blockade of calcium channel, preferably the disease or disorder which can be benefited from selective blockade of Ca _V1.3 channel compared with Ca _V1.2 channel.
Use according to claim 20, wherein the compound of formula (I) , (Ia) , (Ib) , (II) , (IIa) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to any one of claims 1-6, or its enantiomer, diastereomer, tautomer or a mixture thereof, or pharmaceutically acceptable salt thereof is used in manufacture of a medicament for the treatment of diseases or disorders selected from hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , heart failure such as congestive heart failure, left ventricular dysfunction and hypertrophic cardiomyopathy, diabetic cardiac myopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter, detrimental vascular remodeling, myocardial infarction, atherosclerosis, renal insufficiency (diabetic and non-diabetic) , diabetes, primary and secondary pulmonary hypertension, renal failure, diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria of primary renal disease, and also renal vascular hypertension, diabetic retinopathy, the management of other vascular disorders, such as migraine, peripheral vascular disease, Raynaud's disease, luminal hyperplasia, cognitive dysfunction (such as Alzheimer's diseases) , glaucoma, stroke and Parkinson disease.
Use of the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to claim 7, or the composition comprising the essential oil derived from ginger (Zingiber officinale) or clary sage (Salvia sclarea) which comprises the compound of formula (Ib) , (IIb-1) , (IIb-2) , (IIb-3) or (IIb-4) according to claim 8, in manufacture of a medicament for the treatment of diseases or disorders selected from hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , heart failure such as congestive heart failure, left ventricular dysfunction and hypertrophic cardiomyopathy, diabetic cardiac myopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter, detrimental vascular remodeling, myocardial infarction, atherosclerosis, renal insufficiency (diabetic and non-diabetic) , diabetes, primary and secondary pulmonary hypertension, renal failure, diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria of primary renal disease, and also renal vascular hypertension, diabetic retinopathy, the management of other vascular disorders, such as migraine, peripheral vascular disease, Raynaud's disease, luminal hyperplasia, cognitive dysfunction (such as Alzheimer's diseases) , glaucoma, stroke and Parkinson disease
The use according to claim 21 or 22, wherein the disease or disorder is selected from hypertension, coronary heart disease (CAD) , angina pectoris (whether unstable or stable) , and Parkinson disease.
A method of screening compound as calcium channel blocker, especially having high selectivity to Ca _V1.3 channel compared with Ca _V1.2 channel, said method comprising the following steps:

(1) selection of a suitable host cell that weakly expresses or does not express Ca _V1.3 or Ca _V1.2 channel endogenously;

(2) delivery of (i) nucleic acid segments that encode one or more calcium channel subunits alongside (ii) any calcium-responsive reporter system into the host cell described in (1) ;

(3) depolarization of cells described in (1-2) through electrical stimulation or by treatment of aqueous salt solutions such as potassium chloride;

(4) incubation of depolarized cells described in (1-3) with candidate calcium channel blocker compounds for at least 1 minute; and

(5) quantification of signals produced by the calcium-responsive reporter system described in (1) .
The method according to claim 24, wherein the suitable host cell is of mammalian or amphibian origin, preferably, the host cell is derived from human embryonic kidney cells, patient-specific tumors or Chinese hamster ovary cells, such as HEK-293, HeLa or CHO-K1 cells, or the host cell is derived from Xenopus oocytes.
The method according to claim 24, wherein the nucleic acid segments that encode one or more calcium channel subunits are one or more nucleic acid sequences comprising a mRNA sequence that can translate into any one of the protein subunits selected from α1, α2, β1, β2, β3, β4, γ, δ or α2δ domains of voltage-gated calcium channels, or the nucleic acid segments that encode one or more calcium channel subunits are one or more nucleic acid sequences comprising a DNA sequence that can transcribe into a RNA sequence that produces any one of the protein subunits selected from α1, α2, β1, β2, β3, β4, γ, δ or α2δ domains of voltage-gated calcium channels, or the nucleic acid segment (s) that encode (s) one or more calcium channel subunits is (are) a nucleic acid sequence comprising one or more gene sequences selected from CACNA1S, CACNA1C, CACNA1D, CACNA1F, CACNA1A, CACNA1B, CACNA1E, CACNA1G, CACNA1H, CACNA1I, CACNA2D1, CACNA2D2, CACNA2D3, CACNA2D4, CACNB1, CACNB2, CACNB3, CACNB4, CACNG1, CACNG2, CACNG3, CACNG4, CACNG5, CACNG6, CACNG7 or CACNG8 or any homologue thereof.
The method according to claim 24, wherein the calcium-responsive reporter system is a protein whose activity and/or integrity is naturally evolved or engineered to depend on local concentrations of calcium ions, or the calcium-responsive reporter system is an RNA molecule whose stability and/or translation is naturally evolved or engineered to depend on local concentrations of calcium ions, or the calcium-responsive reporter system is a natural or synthetic promoter whose activation is naturally evolved or engineered to depend on intracellular concentrations of calcium ions, or the calcium-responsive reporter system is any combination of a calcium-dependent protein, calcium-dependent RNA and calcium-dependent promoter described above, or the calcium-responsive reporter system consists of a synthetic promoter that contains DNA segments bound by the calcium-responsive transcription factor NFAT (nuclear factor of activated T cells) .