Benzothiazepine Research Articles

Effects of selective calcium channel blockers on ions’ permeation through the human Cav1.2 ion channel: A computational study

Selective calcium channel antagonists are widely used in the treatment of cardiovascular disorders. They are mainly classified into 1,4-dihydropyridine (1,4-DHPs) and non-DHPs. The non-DHPs class is further classified into phenylalkylamines (PAAs) and benzothiazepines (BZTs) derivatives. These blockers are used for the treatment of hypertension, angina pectoris, and cardiac arrhythmias. Despite their well-established efficiency, the structural basis behind their activity is not very clear. Here we report the use of a near-open confirmation (NOC) model of the Cav1.2 cardiac ion channel to examine the mode of binding of these antagonists within the pore domain as well as the fenestration of the pore-forming domains. Effects of calcium ion permeation in the presence of drug molecules were assessed using steered molecular dynamics (SMD) simulations. These studies reveal that nicardipine, a DHP derivative, shows a strong Cav1.2 blocking activity, requiring more 2500 pN force to pull calcium ion towards the channel’s pore in the presence of the compound. Similar blocking activity was observed for verapamil, a PAA derivative, requiring almost 2300 pN of force. The least blocking activity was observed for Diltiazem, a BZT derivative. Our results explain the structural basis and the binding details of 1,4-DHPs, PAAs and BZTs at their distinct Cav1.2 sites and offer detailed insights into their mechanism of action in modulating the Cav1.2 channel.

Pharmacological Targeting of SERCA in Breast Cancer

About 12% of U.S. woman develop breast cancer at some time during their lifetime. In addition to surgery and radiation therapy, chemotherapy is a key part of treatment following diagnosis. However, current drugs have variable success due to challenging disease subtypes that exhibit drug resistance. As a result, over 40,000 women die every year from intractable metastatic breast cancer. Thus, various new targets against breast cancer cells are currently under study. Aberrant intracellular Ca2+ homeostasis is observed in most cancer cells, hence, proteins that modulate Ca2+ levels may serve as a potential therapeutic target. Thapsigargin (TG) and cyclopiazonic acid (CPA) are agents known to potently inhibit the endoplasmic/sarcoplasmic reticulum (ER/SR) Ca2+‐ATPase (SERCA) and have shown high efficacy for impairing cancer cell growth. However, TG and CPZ are highly cytotoxic for normal cells. In recent years, our lab used spectrometry UV‐visible Ca2+ loading and ATPase activity assays in SR microsomes to identify novel drugs that target SERCA in striated muscle via an inhibitory, calcium‐dependent mechanism. These include drugs of the benzothiazepine (BZT) family (K201, CGP, PH00095) and antiepileptic drugs (sipatrigin, pimozide). The action of these new agents would cause partial SR depletion in cells by affecting Ca2+ sequestration at low cytosolic Ca2+. Interestingly, some of these drugs have been found to be cytoprotective under various stressful conditions. It is possible that these drugs prevent damaging SR Ca2+ overload under pathological conditions, including ischemia, via its action on SERCA. These findings contrast with the cytotoxicity associated with TG and CPZ. We compared the efficacy of these new SERCA inhibitors versus TG/CPZ on cell viability of breast cancer cell lines MCF‐7 and MDA‐MB‐231. MTS assay revealed TG and CPZ were more efficacious than our new compounds to decrease MCF‐7 viability. However, none of the agents tested significantly affected MDA‐MB‐231, a more metastatic breast cancer cell line. When paired with chemotherapeutics, K201, TG and CPZ synergistically reduce MCF‐7 viability. Still, the effects of chemotherapeutics on MDA‐MD‐231 was not significantly changed. Based on our studies, targeting intracellular Ca2+ homeostasis may serve as a potential therapeutic target for some breast cancer cell types but not all. Here, we find that drugs of the BZT family, as well as some antiepileptics, reduce MCF‐7 cell viability and potentially synergize with the action of chemotherapeutics. However, MDA‐MB‐231 remain largely unaffected by these agents. In principle, we thought that MDA‐MB‐231 may not have significant intracellular Ca2+ stores; yet immunoblot analysis reveal comparable levels of SERCA1/2 protein expression in both cell lines. Still, MDA‐MB‐231 seems to preferentially utilize Ca2+ entry from the extracellular space for cell survival. Further studies of plasma membrane Ca2+ transporters are required to elucidate the dependence of intracellular Ca2+ signaling among these breast cancer cell lines.Support or Funding InformationCT Moy Endowed Fund, Eskridge Foundation Grant, SIUSOM ‐ Team Development Grant

Molecular Basis for Ligand Modulation of a Mammalian Voltage-Gated Ca2+ Channel

The L-type voltage-gated Ca2+ (Cav) channels are modulated by various compounds exemplified by 1,4-dihydropyridines (DHP), benzothiazepines (BTZ), and phenylalkylamines (PAA), many of which have been used for characterizing channel properties and for treatment of hypertension and other disorders. Here, we report the cryoelectron microscopy (cryo-EM) structures of Cav1.1 in complex with archetypal antagonistic drugs, nifedipine, diltiazem, and verapamil, at resolutions of 2.9Å, 3.0Å, and 2.7Å, respectively, and with a DHP agonist Bay K 8644 at 2.8Å. Diltiazem and verapamil traverse the central cavity of the pore domain, directly blocking ion permeation. Although nifedipine and Bay K 8644 occupy the same fenestration site at the interface of repeats III and IV, the coordination details support previous functional observations that Bay K 8644 is less favored in the inactivated state. These structures elucidate the modes of action of different Cav ligands and establish a framework for structure-guided drug discovery.

Pharmacological Targeting of Serca May Have Potential for Cellular Protection

We previously reported that CGP-37157 and K201, two benzothiazepines (BZT) with cardioprotective action, inhibit the Ca2+ ATPase of sarcoplasmic reticulum (SR) intracellular Ca2+ stores (SERCA). We tested if SERCA block could be part of the mechanism by which drugs protect cells from ischemic damage. We also screened structural characteristics in BZT that could affect their drug potency as SERCA inhibitors. SR microsomes isolated from rabbit skeletal muscle (SkM) and pig heart ventricle were utilized to measure modulation of SR Ca2+ loading and SERCA-mediated ATPase activity by drugs. Seven out of twenty cell-protective drugs tested (including pimozide, EGCG, KN-93 and carvedilol) inhibited, at least partially, SR Ca2+ loading and Ca2+-stimulated ATPase activity in SkM and heart SR microsomes. We also screened ten novel BZT derivatives and seven FDA-approved benzodiazepines (BZD; including bromazepam and clonazepam), which have close homology to CGP37157. Ca2+-dependent block of SERCA was found in four BZT's, which displayed higher (PH000995, PH000902) and similar potency (PH000902, PH006796) compared to CGP. BZD were all ineffective, which suggest that the sulfur atom in the BZT ring (substituted by nitrogen in BZD) is crucial for their SERCA blocking ability. All compounds above, were tested on ryanodine receptor (RyR) activity (planar bilayers, SR leak, [3H] ryanodine binding). None of these agents directly inhibited RyR function in heart and muscle. In contrast, some agents (BZT and BZD) had mild agonistic action on channel function. We think that SERCA block by these drugs, which persists at pH ∼6.5, may benefit ischemic cells by preventing SR Ca2+ overload, known to trigger, upon reperfusion, abnormal RyR-mediated Ca2+ leak associated with cell death and tissue injury. BZT have potential as templates for therapeutic targeting of SERCA (Supported by AHA and Eskridge Foundation).

Design, synthesis, structural elucidation and antimicrobial screening of novel 1, 5-benzothiazepine derivatives derived from thieno [3, 2-d] pyrimidine nucleus

1, 5-Benzo Thiazepine is the Main Seven membered Heterocyclic ring systems reported for their cardiac and Psychotherapeutic activities.1, 5 Benzo Thiazepines were highlighted as important Biologically active Scaffolds. In This Article New series of benzo [b] [1, 5]-thiazepine derivatives 6 a-j were synthesized by applying the cyclo condensation of (E)-3-(thieno [3, 2-d] pyrimidin-6-yl)-1-p-Substituted prop-2-en-1-one derivatives 4a-j with 2amino thio phenol (5) in DMF. The new intermediate Chalcone derivatives 4a-j were obtained from interaction of various P-substituted Acetophenone & Heterocyclic Acetyl derivatives 3(a-j) and thieno[3, 2-d]pyrimidine-7carbaldehyde. The synthesized 1, 5-benzothiazepines 6a-j have been screened for their antimicrobial activity. From anti-bacterial and anti-fungal activity screening results, it has been observed that compounds 6h, 6e, 6i and 6d possess good activity.

Modulation by CGP-37157 (CGP) Analogs of the Sarcoplasmic Reticulum Calcium ATPase SERCA)

We have reported that CGP, a 4-1 benzothiazepine (BZT) associated with cardioprotection in ischemia, is a SERCA inhibitor. CGP blocking action differs from that of thapsigargin because is Ca2+ dependent; i.e., CGP blocking potency increases when Ca2+ levels decrease. We utilized purified sarcoplasmic reticulum microsomal membranes (SRM; from rabbit skeletal muscle and pig ventricle) to test the inhibitory actions of various novel CGP analogs on ATPase function (estimated by measurements of Ca2+ loading and ATPase activity). The 4,1-benzothiazepin-2-one structural arrangement seems to be important for SERCA blocking action since function is lost within compounds where: the Sulfur is substituted by Nitrogen (as seen in benzodiazepines); the Oxygen is replaced by an alkyl group; or the arrangement between Nitrogen, Oxygen, and Sulfur in the BZT ring is reorganized (as seen in Diltiazem). Removal of Chlorine from position 7 in the BZT ring did not affect SERCA blocking potency. A combination of substitutions: 3H (benzylaminomethyl-carboxymethyl), alkylation of the Nitrogen (neopentyl) and replacement of the position 1 of benzyl group (naphtyl), significantly increased potency 10-fold (IC50 ∼ 1.5 μM, in presence of 2 μM free Ca2+). We determined (using SRM Ca2+ leak assay, [3H] ryanodine binding and channel studies in bilayers) CGP analogs can also activate ryanodine receptors (RyRs), albeit with low potency. In summary, we have identified structural features that may confer Ca2+-dependent SERCA blocking in 4,1 BZT by utilizing CGP-like molecules. We also identified ph000995, a potent blocker, which may have potential for assessing SERCA roles in cells as well as being a potential drug-template for therapeutic intervention in diseases of abnormal Ca2+ homeostasis, such as ischemia. (Supported by American Heart Association, Eskridge Foundation and APS Frontiers in Physiology Research Program).

Benzodiazepines and Benzothiazepines as Modulators of the Sarcoplasmic Reticulum Calcium ATP-ase and Ryanodine Receptors in Striated Muscle

We have reported that CGP-37157, a benzothiazepine (BZT) derivative of clonazepam utilized as a blocker of the mitochondrial Na+/Ca2+ exchanger, also activates ryanodine receptors (RyRs) and inhibits the sarcoplasmic reticulum (SR) Ca2+-stimulated ATPase (SERCA). We extended the studies to other BZT (e.g., clonazepan, diltiazem) as well as to benzodiazepines (BZD; e.g. diazepan, lorazepan). We aimed to determine if these drug classes have as a common trait the ability to modulate RyRs and/or SERCA. The effects of BZD and BZT on RyRs activity were tested in SR microsomes with a Ca2+ leak assay as well as after reconstituting RyRs into lipid bilayers. The agents tested had variable potency to increase RyR-mediated Ca2+ leak from skeletal SR microsomes. As an example, while diazepam significantly increased RyR activity, most of the others (clonazepam, lorazepam) had minor effects at high doses. In contrast, diltiazem produced moderate inhibition. Planar bilayer studies confirmed the leak observations with both cardiac and skeletal RyR. In the presence of ruthenium red, most agents decreased the rate of loading which indicates inhibitory effects on SERCA activity. The effects of BZT and BZD on loading correlated with a decrease in ATPase activity of SERCA-enriched skeletal SR fractions. In summary, most BZT and BZD utilized in therapeutics or as pharmacological tools have an inhibitory action on SERCA. In contrast, they show a variety of effects on RyRs ranging from inhibition to activation. Hence, the pharmacological action of BZT and BZD on cellular Ca2+ homeostasis reported in the literature of cardiac and skeletal muscle as well as other non-muscle systems may require taking into consideration the contributions of all drug-sensitive intracellular Ca2+ transporters. (Supported by NIH-GM078665 and AHA-MWA 12180038).

Identification and Validation of Tetracyclic Benzothiazepines as Plasmodium falciparum Cytochrome bc1 Inhibitors

Here we report the discovery of tetracyclic benzothiazepines (BTZs) as highly potent and selective antimalarials along with the identification of the Plasmodium falciparum cytochrome bc(1) complex as the primary functional target of this novel compound class. Investigation of the structure activity relationship within this previously unexplored chemical scaffold has yielded inhibitors with low nanomolar activity. A combined approach employing genetically modified parasites, biochemical profiling, and resistance selection validated inhibition of cytochrome bc(1) activity, an essential component of the parasite respiratory chain and target of the widely used antimalarial drug atovaquone, as the mode of action of this novel compound class. Resistance to atovaquone is eroding the efficacy of this widely used antimalarial drug. Intriguingly, BTZ-based inhibitors retain activity against atovaquone resistant parasites, suggesting this chemical class may provide an alternative to atovaquone in combination therapy.

Molecular Modeling of Benzothiazepine Binding in the L-type Calcium Channel

Benz(othi)azepine (BTZ) derivatives constitute one of three major classes of L-type Ca(2+) channel ligands. Despite intensive experimental studies, no three-dimensional model of BTZ binding is available. Here we have built KvAP- and KcsA-based models of the Ca(v)1.2 pore domain in the open and closed states and used multiple Monte Carlo minimizations to dock representative ligands. In our open channel model, key functional groups of BTZs interact with BTZ-sensing residues, which were identified in previous mutational experiments. The bulky tricyclic moiety occupies interface between domains III and IV, while the ammonium group protrudes into the inner pore, where it is stabilized by nucleophilic C-ends of the pore helices. In the closed channel model, contacts with several ligand-sensing residues in the inner helices are lost, which weakens ligand-channel interactions. An important feature of the ligand-binding mode in both open and closed channels is an interaction between the BTZ carbonyl group and a Ca(2+) ion chelated by the selectivity filter glutamates in domains III and IV. In the absence of Ca(2+), the tricyclic BTZ moiety remains in the domain interface, while the ammonium group directly interacts with a glutamate residue in the selectivity filter. Our model suggests that the Ca(2+) potentiation involves a direct electrostatic interaction between aCa(2+) ion and the ligand rather than an allosteric mechanism. Energy profiles indicate that BTZs can reach the binding site from the domain interface, whereas access through the open activation gate is unlikely, because reorientation of the bulky molecule in the pore is hindered.

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 ω-conotoxin (ω-CgTx) MVIIC; and by the T-type VDCC antagonist amiloride. The N-type antagonist ω-CgTx GVIA is the most potent in inhibiting the high-K +-stimulated JHA release. No inhibitory effect is shown by the P-type antagonist ω-agatoxin TK (ω-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 ω-CgTx MVIIC and by the T-type antagonist amiloride, and is totally inhibited by the N-type antagonist ω-CgTx GVIA. No inhibitory effect is shown by the P-type antagonist ω-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.

Identification of Benz(othi)azepine-binding Regions within L-type Calcium Channel α1 Subunits

To identify the binding domain for diltiazem-like Ca2+ antagonists on L-type Ca2+ channel alpha1 subunits we synthesized the benzazepine [3H]benziazem as a novel photoaffinity probe. [3H]Benziazem reversibly labeled the benzothiazepine (BTZ)-binding domain of partially purified skeletal muscle Ca2+ channels with high affinity (Kd = 12 nM) and photoincorporated into its binding domain with high yield (>66%). Antibody mapping of proteolytic labeled fragments revealed specific labeling of regions associated with transmembrane segments S6 in repeats III and IV. More than 50% of the labeling was found in the tryptic fragment alanine 1023-lysine 1077 containing IIIS6 together with extracellular and intracellular amino acid residues. The remaining labeling was identified in a second site comprising segment S6 in repeat IV and adjacent residues. Unlike for dihydropyridines, no labeling was observed in the connecting IIIS5-IIIS6 linker. The [3H]benziazem photolabeled regions must be in close contact to the drug molecule when bound to the channel. We propose that the determinants for high affinity BTZ binding are located within or in close proximity to segments IIIS6 and/or IVS6. Therefore the binding domain for BTZs, like for the other main classes of Ca2+ antagonists, must be located in close proximity to pore-forming regions of the channel.

Dihydropyridines, phenylalkylamines and benzothiazepines block N-, P/Q- and R-type calcium currents.

We compared the effects of representative members of three major classes of cardiac L-type channel antagonists, i.e. dihydropyridines (DHPs), phenylalkylamines (PAAs) and benzothiazepines (BTZs) on high-voltage-activated (HVA) Ca2+ channel currents recorded from a holding potential of -100 mV in rat ventricular cells, mouse sensory neurons and rat motoneurons. Nimodipine (DHP), verapamil (PAA) and diltiazem (BTZ) block the cardiac L-type Ca2+ channel current (EC50: 1 microM, 4 microM and 40 microM, respectively). At these concentrations, the drugs could also inhibit HVA Ca2+ channel currents in both sensory and motor neurons. Large blocking effects (> 50%) could be observed at 2-10 times these concentrations. The omega -conotoxin-GVIA-sensitive (omega -CTx-GVIA, N-type), omega -agatoxin-IVA-sensitive (omega -Aga-IVA, P- and Q-types) and non-L-type omega -CTx-GVIA-, omega -Aga-IVA-insensitive (R-types) currents accounted for more than 90% of the global current. Furthermore, our data showed that omega -CTx-GVIA and omega -Aga-IVA spare L-type currents and have only additive blocking effects on neuronal HVA currents. We conclude that DHPs, PAAs and BTZs have substantial inhibitory effects on neuronal non-L-type Ca2+ channels. Inhibitions occur at concentrations that are not maximally active on cardiac L-type Ca2+ channels.

Allosteric interaction of semotiadil fumarate, a novel benzothiazine, with 1,4-dihydropyridines, phenylalkylamines, and 1,5-benzothiazepines at the Ca(2+)-channel antagonist binding sites in canine skeletal muscle membranes.

We assessed the binding characteristics of a benzothiazine Ca(2+)-channel antagonist, semotiadil, in canine skeletal muscle membranes. Semotiadil inhibited binding of (+)-[3H]PN 200-110 (maximum inhibition 80%), and almost completely inhibited binding of both (-)-[3H]desmethoxyverapamil and D-cis-[3H]diltiazem to their specific binding sites with an IC50 value of 0.2-2 microM and a Hill slope of 0.6-0.9. Saturation isotherm and dissociation kinetic studies suggest that semotiadil acts as a noncompetitive inhibitor at the 1,4-dihydropyridine, phenylalkylamine, and benzothiazepine (BTZ) recognition sites in the L-type Ca2+ channel: (a) Scatchard analysis showed that semotiadil decreased maximum binding (Bmax) of the three classes of Ca2+ channel antagonists, while causing a slight increase in the equilibrium dissociation constant (Kd) in the case of (+)-[3H]PN 200-110 binding or no significant change in Kd values for binding of (-)-[3H]desmethoxyverapamil and D-cis-[3H]diltiazem to their specific binding sites; and (b) dissociation kinetics of the (+)-[3H]PN 200-110 and D-cis-[3H]diltiazem bindings were accelerated by semotiadil. These results suggest that semotiadil has a strong negative allosteric interaction with three classes of Ca2+ channel antagonists, including 1,4-dihydropyridines, phenylalkylamines, and BTZ at their specific binding sites.

Ca2+ antagonists as tools in the analysis of excitation-contraction coupling in skeletal muscle fibres.

Organic Ca2+ antagonists are specific substances which interfere with the gating of Ca2+ channels. They are of great interest as they are effective drugs in the therapy of different heart diseases. At present one distinguishes between three main classes of Ca2+ antagonists: dihydropyridines (DHPs), phenylalkylamines (PAAs) and benzothiazepines (BTZs). These classes appear to have different binding sites at the Ca2+ channel which show a reciprocal allosteric interaction among each other and with additional Ca2+ binding sites (Glossmann et al., 1985).