Structural dependency of the inhibitory action of benzodiazepines and related compounds on the mitochondrial Na+-Ca2+ exchanger

doi:10.1016/0006-2952(88)90623-5

Biochemical Pharmacology

Volume 37, Issue 22, 15 November 1988, Pages 4399-4403

https://doi.org/10.1016/0006-2952(88)90623-5 Get rights and content

Abstract

Na⁺-induced Ca²⁺-release from guinea-pig heart mitochondria is inhibited by benzodiazepines such as clonazepam (compound II, ic₅₀: 12 μM). The capacity of various related compounds to inhibit the rapid Ca²⁺-efflux induced by 20 mM Na⁺ was examined. The potency of inhibition was found to depend on several factors, such as a 2'-halogen substitution and the preence of a secondary amido group. Very effective inhibitors were identified among the triazolo derivatives of benzodiazepines or obtained by replacing the diazepine ring by an oxazepine or a thiazepine. Some of these favourable structural modifications were compounded in the benzothiazepine 7-chloro-3,5-dihydro-5-phenyl-1H- 4,1-benzothiazepine-2-on (compound XVI), which proved to be about 20 times more potent than the related compounds clonazepam and diltiazem. Compound XVI, which has an ic₅₀ in the submumolar range, is the most potent selective inhibitor of the mitochondrial exchanger so far reported. The structural requirements found for the inhibition of the mitochondrial Na⁺-Ca²⁺ exchanger were quite distinct from those described for the binding of benzodiazepines to their central-type and peripheral- type sites.

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Calcium signals between the ryanodine receptor- and mitochondria critically regulate the effects of arsenite on mitochondrial superoxide formation and on the ensuing survival vs apoptotic signaling
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Citation Excerpt :
formation. For this purpose, we used CGP-37157, an inhibitor of mitochondrial Na+/Ca2+ exchanger (mNCX) [46], and an exposure protocol (3 µM for 4 h) failing to produce detectable toxicity, as measured by either the trypan blue exclusion assay or the release of lactate dehydrogenase (not shown). An additional advantage of this exposure protocol is represented by the fact that, under these conditions, arsenite produces weak responses in terms of mitochondrial Ca2+ accumulation and mitoO2.-
A low concentration of arsenite (6 h), selectively stimulating the intraluminal crosstalk between the inositol-1, 4, 5-triphosphate receptor and the ryanodine receptor (RyR), increased the mitochondrial transport of RyR-derived Ca²⁺ through the mitochondrial Ca²⁺ uniporter. This event was characterized in intact and permeabilized cells, and was shown to be critical for mitochondrial superoxide (mitoO₂^.-) formation. Inhibition of mitochondrial Ca²⁺ accumulation therefore prevented the effects of arsenite, in both the mitochondrial (e.g., cardiolipin oxidation) and extramitochondrial (e.g., DNA single- strand breakage) compartments, and suppressed the Nrf2/GSH survival signaling. The effects of arsenite on Ca²⁺ homeostasis and mitoO₂^.- formation were reversible, as determined after an additional 10 h incubation in fresh culture medium and by measuring long-term viability. A 16 h continuous exposure to arsenite instead produced a sustained increase in the cytosolic and mitochondrial Ca²⁺ concentrations, a further increased mitoO₂^.- formation and mitochondrial permeability transition. These events, followed by delayed apoptosis (48 h), were sensitive to treatments/manipulations preventing mitochondrial Ca²⁺ accumulation. Interestingly, cells remained viable under conditions in which the deregulated Ca²⁺ homeostasis was not accompanied by mitoO₂^.-formation.
In conclusion, we report that the fraction of Ca²⁺ taken up by the mitochondria in response to arsenite derives from the RyR. Mitochondrial Ca²⁺ appears critical for mitoO₂^.- formation and for the triggering of both the cytoprotective and apoptotic signaling. The effects of arsenite were reversible, whereas its prolonged exposure caused a sustained increase in mitochondrial Ca²⁺ and mitoO₂^.- formation, and the prevalence of the apoptotic vs survival signaling.
Function, regulation and physiological role of the mitochondrial Na+/Ca2+ exchanger, NCLX
2018, Current Opinion in Physiology
Mitochondrial Ca²⁺ signaling is linking global cellular Ca²⁺ signals to energy production and controls local Ca²⁺ concentrations in the ER and cell membrane. On the other hand, aberrant change in mitochondrial Ca²⁺ is a hallmark of ischemic and neurodegenerative syndromes. Mitochondrial Ca²⁺ transients are mediated by a Ca²⁺ channel, MCU (mitochondrial Ca²⁺ uniporter), and by a much slower, rate-limiting, mitochondrial Na⁺/Ca²⁺ exchanger, NCLX. This review describes recent studies on the essential role of NCLX in cardiac survival and in diverse physiological activities. It further focuses on the regulatory and transport sites that are controlling NCLX function.
Neuroprotective profile of pyridothiazepines with blocking activity of the mitochondrial Na+/Ca2+ exchanger
2016, European Journal of Medicinal Chemistry
Citation Excerpt :
In the frame of this Ca2+ set-point hypothesis, we found that a mild sustained elevation of [Ca2+]c elicited by the Ca2+ promoter ITH4012 is associated with neuroprotection [9,10]. We also explored the possibility that the mitigation of the rate of mitochondrial Ca2+ efflux by the mNCX blocker CGP37157 [11] (1, Fig. 1) could afford neuroprotection against neurotoxicity elicited by cell Ca2+ overload; we confirm this firstly in chromaffin cells [12] and, subsequently, in hippocampal slices [13,14]. However, 1 also blocks voltage-gated Na+ channels [12], voltage-gated Ca2+ channels (VGCC) [12,15], the plasmalemmal NCX [16], and the recently discovered channel Ca2+ homeostasis modulator 1 (CALHM1) [17], among other targets.
The mitochondrial Na⁺/Ca²⁺ exchanger plays an important role in the control of cytosolic Ca²⁺ cycling in excitable cells, essential for the regulation of a plethora of Ca²⁺-dependent physio-pathological events, such as apoptosis in the presence of a Ca²⁺ overload. There are very few pharmacological tools available to study both physiological and pathological implications of the mitochondrial Na⁺/Ca²⁺ exchanger, where the benzothiazepine CGP37157 is the best-known ligand, used since the 1980s. However, it is not an efficient blocker and lacks of selectivity, as also blocks several other cellular Ca²⁺ transporters. Moreover, CGP37157 is a very lipophilic drug, showing very poor water solubility, what has hindered its therapeutic use. Attempting to improve its pharmacokinetic profile as well as its potency and selectivity, we herein describe the synthesis of new CGP37157 analogs, where the benzene-fused ring has been replaced by a pyridine. On top of a better water solubility and lower log P value, some of these new pyridothiazepine derivatives also presented a higher capacity to regulate the mitochondrial Ca²⁺ clearance, while keeping the neuroprotective properties presented in the head compound CGP37157.
Benzothiazepine CGP37157 and its 2′-isopropyl analogue modulate Ca2+ entry through CALHM1
2015, Neuropharmacology
CALHM1 is a Ca²⁺ channel discovered in 2008, which plays a key role in the neuronal electrical activity, among other functions. However, there are no known efficient blockers able to modulate its Ca²⁺ handling ability. We herein describe that benzothiazepine CGP37157 and its newly synthesized analogue ITH12575 reduced Ca²⁺ influx through CALHM1 at low micromolar concentrations. These results could serve as a starting point for the development of more selective CALHM1 ligands using CGP37157 as a hit compound, which would help to study the physiological role of CALHM1 in the control of [Ca²⁺]_cyt in excitable cells, as well as its implication in CNS diseases.
Mitochondrial calcium transport in trypanosomes
2014, Molecular and Biochemical Parasitology
The biochemical peculiarities of trypanosomes were fundamental for the recent molecular identification of the long-sought channel involved in mitochondrial Ca²⁺ uptake, the mitochondrial Ca²⁺ uniporter or MCU. This discovery led to the finding of numerous regulators of the channel, which form a high molecular weight complex with MCU. Some of these regulators have been bioinformatically identified in trypanosomes, which are the first eukaryotic organisms described for which MCU is essential. In trypanosomes MCU is important for buffering cytosolic Ca²⁺ changes and for activation of the bioenergetics of the cells. Future work on this pathway in trypanosomes promises further insight into the biology of these fascinating eukaryotes, as well as the potential for novel target discovery.
Molecular identity and functional properties of the mitochondrial Na +/Ca2+ exchanger
2012, Journal of Biological Chemistry
Citation Excerpt :
Among them are CGP-37157 (24, 25), tetraphenylphosphonium (21), trifluoperazine (15), diltiazem (24), verapamil (26), clonazepam (24), amiloride (27), and cyclosporin A (28). The most selective and effective inhibitor of mitochondrial Na+/Ca2+ exchange is the CGP-37157 compound (24, 25). The affinity of CGP-37157 is at least 10-fold higher than that of any other blocker, and when tested in intact cells, CGP-37157 did not interfere with contraction and did not affect Ca2+ fluxes through L-type Ca2+ channels or the activity of the ER Ca2+ pump sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), in isolated mitochondria and cardiomyocytes (29, 30).
The mitochondrial membrane potential that powers the generation of ATP also facilitates mitochondrial Ca²⁺ shuttling. This process is fundamental to a wide range of cellular activities, as it regulates ATP production, shapes cytosolic and endoplasmic recticulum Ca²⁺ signaling, and determines cell fate. Mitochondrial Ca²⁺ transport is mediated primarily by two major transporters: a Ca²⁺ uniporter that mediates Ca²⁺ uptake and a Na⁺/Ca²⁺ exchanger that subsequently extrudes mitochondrial Ca²⁺. In this minireview, we focus on the specific role of the mitochondrial Na⁺/Ca²⁺ exchanger and describe its ion exchange mechanism, regulation by ions, and putative partner proteins. We discuss the recent molecular identification of the mitochondrial exchanger and how its activity is linked to physiological and pathophysiological processes.

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Biochemical Pharmacology

Abstract

References (16)

Mitochondrial calcium transport

Biochim Biophys Acta

Inhibition of Na-dependent Ca-efflux from heart mitochondria by amiloride analogs

FEBS Lett

The peripheral type benzodiazepine receptor

J Biol Chem

Mitochondrial Ca-transport and Ca-binding proteins

The sodium- induced efflux of calcium from heart mitochondria

Eur J Biochem

The interrelations between the transport of sodium and calcium in mitochondria of various mammalian tissues

Eur J Biochem

Selective effects of dilti- azem, a benzothiazepine calcium channel blocker, and diazepam, and other benzodiazepines on the Na/Ca exchange carrier system of heart and brain mitochondria

Life Sci

Mechanisms for intracellular Ca regulation in heart

J Gen Physiol

Cited by (63)

Calcium signals between the ryanodine receptor- and mitochondria critically regulate the effects of arsenite on mitochondrial superoxide formation and on the ensuing survival vs apoptotic signaling

Function, regulation and physiological role of the mitochondrial Na<sup>+</sup>/Ca<sup>2+</sup> exchanger, NCLX

Neuroprotective profile of pyridothiazepines with blocking activity of the mitochondrial Na<sup>+</sup>/Ca<sup>2+</sup> exchanger

Benzothiazepine CGP37157 and its 2′-isopropyl analogue modulate Ca<sup>2+</sup> entry through CALHM1

Mitochondrial calcium transport in trypanosomes

Molecular identity and functional properties of the mitochondrial Na <sup>+</sup>/Ca<sup>2+</sup> exchanger