Effects of novel 17β-hydroxysteroid dehydrogenase type 10 inhibitors on mitochondrial respiration
Graphical abstract
Introduction
Alzheimer’s disease (AD) is the most common cause of dementia in elderly. The formation of senile plaques, which are composed of amyloid beta (Aβ) oligomers, and intraneuronal neurofibrillary tangles comprising hyperphosphorylated tau proteins, is thought to contribute to the etiology of AD. According to the amyloid cascade hypothesis, the primary cause of Alzheimer’s disease is an imbalance in the production and clearance of the Aβ oligomers (Bayer and Wirths, 2014; De Strooper and Karran, 2016). However, a growing body of evidence suggests that other factors, including perturbations in cellular energy metabolism and mitochondrial dysfunction, participate in the pathophysiology of AD (Bonda et al., 2014; Hroudová et al., 2014; Chaturvedi and Beal, 2013; Lopez Sanchez et al., 2017). The mitochondrial cascade hypothesis of sporadic AD (Swerdlow et al., 2014; Swerdlow and Khan, 2004) assumes that mitochondrial dysfunctions are critical to the development of AD, whereas gene-induced disturbances of mitochondrial enzyme functions affect Aβ peptide accumulation and determine the rate of the mitochondrial damage and the progression of AD. It is accepted that soluble Aβ protein exerts toxicity, at least partially, by inducing mitochondrial dysfunction (Benek et al., 2015).
There is no causal treatment for AD to date. The current pharmacotherapy for AD only relieves some symptoms of slows disease progression and it is based on the cholinergic support of brain functions by the use of acetylcholinesterase inhibitors (donepezil, rivastigmine, or galantamine) and the regulation of glutamate neurotransmission by an N-methyl-d-aspartate receptor antagonist (memantine). Novel drugs for the treatment of AD may affect multiple targets, including e.g. cholinesterase activity, monoamine oxidase (MAO) activity, Aβ aggregation, α-, β-, or γ-secretase activity, serotonin transporter activity, calcium channel function, fusion or fission of mitochondria, and mitochondrial functions that include ATP production, reactive oxygen species (ROS) production, and mitochondrial permeability transition pore (mPTP) opening (Bolea et al., 2013; Elkamhawy et al., 2018; Hroudová et al., 2016; Mohamed et al., 2016; Schon et al., 2010; Zhang et al., 2019).
Mitochondrial enzymes seem to be appropriate targets for drugs being developed for the treatment of neurodegenerative disorders, including AD. The enzyme 17β-hydroxysteroid dehydrogenase type 10 (HSD10, 17β-HSD10, formerly known as Aβ binding alcohol dehydrogenase or ABAD) is a mitochondrial multifunctional enzyme that catalyzes the oxidation of neuroactive steroids and the degradation of isoleucine (Vinklarova et al., 2020). Moreover, it binds to other peptides, such as Aβ. It is assumed that the intracellular Aβ protein may contribute to the mitochondrial and neuronal dysfunctions associated with AD by inhibiting HSD10 (He et al., 2018; Lustbader et al., 2004; Yan et al., 1997, 1999). It can be assumed that the inhibition of Aβ interactions with HSD10 can eliminate the mitochondrial toxicity of Aβ (Aβ-induced apoptosis and ROS generation) and may be the basis for the therapeutic effects of new drugs (Lim et al., 2011; Xie et al., 2006). The crystal structure of human HSD10 in complex with an inhibitory small molecule has been determined (Kissinger et al., 2004), and new HSD10 or HSD10-Aβ inhibitors have been synthetized (Ayan et al., 2012; Benek et al., 2017, 2018; Hroch et al., 2016, 2017; Xie et al., 2006).
Compound AG18051 (1-(azepan-1-yl)-2-phenyl-2-(4-sulfanylidene-1H-pyrazolo[3,4-d]pyrimidin-5-yl)ethanone) (Fig. 1), a small molecule inhibitor of HSD10, has been used to determine whether Aβ-induced mitochondrial toxicity is caused by HSD10 inhibition. It was found that AG18051 partially blocks the interaction between Aβ42 and HSD10 and may confer neuroprotective effects by preventing Aβ42-induced mitochondrial toxicity (Lim et al., 2011). Further, riluzole (6-(trifluoromethoxy)-1,3-benzothiazol-2-amine) (Fig. 1) is a glutamate antagonist used as an anticonvulsant and for the treatment of patients with amyotrophic lateral sclerosis. Riluzole serves as a neuroprotector (Cheah et al., 2010), may mitigate Aβ-induced ionic imbalance, and has been shown to reduce Aβ toxicity in a mouse model of AD (Okamoto et al., 2018). Its analogs were studied as potential HSD10 modulators for the treatment of AD as well (Benek et al., 2018; Hroch et al., 2016). And frentizole (1-(6-methoxy-1,3-benzothiazol-2-yl)-3-phenylurea) (Fig. 1) was identified as a weak inhibitor of the HSD10-Aβ interaction (Xie et al., 2006). Frentizole, an antiviral and immunosuppressive drug, is used in the treatment of rheumatoid arthritis and systemic lupus erythematosus. Its analogs, which had increased inhibitory potency on HSD10-Aβ interactions or towards HSD10, have been tested as potential drugs for the treatment of AD (Hroch et al., 2015; Xie et al., 2006).
Routine assays for mitochondrial functions are used during the process of drug development and include those used to study mitochondrial impairment in isolated mitochondria, cell lines, isolated organ models, and animal models. In vitro mechanism-based assays were used to analyze the effect of drugs on the mitochondrial respiration rates; activities of the respiratory complexes; membrane potential and mPTP; production of ATP and ROS; and mitochondrial DNA depletion and replication (Dykens and Will, 2007; Nadanaciva and Will, 2011).
A common approach to study drug-induced changes in mitochondrial function is based on determination of the activity of individual mitochondrial respiration complexes or tricarboxylic acid cycle enzymes. However, it is possible that the system of oxidative phosphorylation (OXPHOS) compensates any abnormalities in the activity of individual enzymes. The molecular targets that are implicated in drug-induced mitochondrial toxicity have been described (Wallace, 2015). And it has been found that measuring overall functional parameters, such as oxygen consumption kinetics and OXPHOS uncoupling, allow accurate recognition of mitochondrial compensatory and regulatory mechanisms and can better quantify the effect of a drug on mitochondrial function (Brand and Nicholls, 2011). Some drugs may not have direct effects on mitochondrial functions but may have protective effects against the impairment of electron transfer system (ETS) activity (Fišar et al., 2016).
In addition, mitochondrial MAO may be a target of drugs used in the modulation of brain function and in the treatment of various mental diseases, including mood disorders (Shulman et al., 2013), schizophrenia (Samson et al., 1995; Siever and Coursey, 1985), anxiety (Tyrer and Shawcross, 1988), and attention deficit hyperactivity disorder (Wargelius et al., 2012); sexual maturation (Moreno et al., 1992); migraine (Filic et al., 2005; Merikangas and Merikangas, 1995); and neurodegenerative disorders (Cai, 2014; Fišar, 2016; Youdim et al., 2004). MAO inhibition is considered during the development of multitarget-directed drugs that have neuroprotective effects and are intended to treat Parkinson's or Alzheimer's disease (Benek et al., 2015; Cai, 2014; Hroch et al., 2017; Huang et al., 2012; Youdim et al., 2004; Zheng et al., 2012). An abundance of evidence suggests that the therapeutic potential of MAO type A (MAO-A) inhibitors are related to their antidepressant effects and that MAO type B (MAO-B) inhibitors may confer neuroprotective properties in the treatment of neurodegenerative diseases. This description of the MAO types represents a simplified view because the inhibitory effects of drugs on MAO-A and MAO-B could be significantly overlapped (Fišar, 2016). Both MAO-A and MAO-B inhibition lower the metabolism rate of the monoamine neurotransmitters and decrease the production of hydrogen peroxide and are understood to be involved in the principal processes related to the therapeutic effects of the MAO inhibitors.
A total of 42 analogs of frentizole and riluzole (inhibitors of HSD10) were synthesized. All tested molecules were previously assayed for their HSD10 inhibitory ability (Benek et al., 2017, 2018; Hroch et al., 2016, 2017). For the tested compounds, a drug-induced decrease in HSD10 activity was observed in the range of 50 % or less. The key structure-activity relationships required for HSD10 inhibition were evaluated (Aitken et al., 2019; Schmidt et al., 2020). In this study, we chose a respiratory rate to measure the effect of test substances on the overall function of mitochondria. In addition, measurement of complex I activity was performed because complex I is known to play a major role in OXPHOS control and is the most sensitive structure of ETS to drug-induced effects (Cikánková et al., 2020; Hroudová and Fišar, 2010; Hroudová et al., 2020; Singh et al., 2017). We report the in vitro effects of frentizole, riluzole, AG18051, and 42 novel HSD10 inhibitors on the following mitochondrial parameters: (1) complex I- and complex II-linked mitochondrial respiration rates, (2) citrate synthase (CS) activity, (3) mitochondrial complex I activity, and (4) MAO-A and MAO-B activity. We have proposed criteria based on the drug-induced changes in mitochondrial enzymes for the selection of new drugs suitable for further in vivo testing.
Section snippets
Material and methods
Drug-induced changes in mitochondrial respiration and mitochondrial enzyme activity (CS, complex I, MAO-A, and MAO-B) were measured in mitochondria isolated from gray matter of pig brain. 42 new HSD10 modulators and 3 standards (riluzole, frentizole, and AG18051) were selected for measurement (Fig. 1, Table S1).
Drug effects on mitochondrial respiration
A series of 45 compounds, including standards riluzole, frentizole, and AG18051, was screened to determine the effects of each drug on the complex I activity, CS activity, complex I- and complex II-linked respiration rates, and MAO-A and MAO-B activities. The complete results are summarized in the Supplementary materials (Tables S2, S3, and S4) and illustrated in Figs. S1-S6.
The activity of CS at a drug concentration of 50 μmol/L was not significantly affected by riluzole, AG18051, and 18 new
Discussion
Drug-induced mitochondrial toxicity manifestation is predominant in the brain due to the high-energy consumption of this tissue. Even a small disruption in mitochondrial respiration can aggravate neurobehavioral and cognitive deficits in AD. The evaluation of novel compounds potentially usable for AD therapy must therefore involve careful testing of their effect on mitochondrial respiration. Standard mitochondrial toxicity screening does not take into account some of the inhibitory effects on
Conclusion
Neurodegenerative diseases and the effects of many drugs are associated with changes in cellular bioenergetics. Therefore, it is necessary to test the effects of new and current drugs on mitochondrial dysfunction. The 42 selected novel HSD10 inhibitors were tested for effects on mitochondrial respiration. Our in vitro assay enabled the selection of six novel compounds, which were considered as safe in terms of mitochondrial respiration. One of the novel molecules met the criteria for partial
Author contributions
The manuscript was written through contributions of all named authors. All authors make substantial contributions to conception and design, acquisition of data, analysis and interpretation of data, and drafting the article.
CRediT authorship contribution statement
Zdeněk Fišar: Conceptualization, Data curation, Formal analysis, Methodology, Writing - original draft. Kamil Musílek: Conceptualization, Data curation, Formal analysis, Methodology, Writing - review & editing. Ondřej Benek: Data curation, Formal analysis. Lukáš Hroch: Data curation, Formal analysis. Lucie Vinklářová: Data curation. Monika Schmidt: Data curation. Jana Hroudová: Data curation, Writing - review & editing. Jiří Raboch: Supervision, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no conflict of interests
Acknowledgments
This work was supported by the Ministry of Health of the Czech Republic (grant number 15-28967A), Charles University (Progress Project Q27/LF1), Czech Science Foundation (grant numbers 17-05292S and 17-07585Y), Ministry of Education, Youth and Sports of Czech Republic (project ESF no. CZ.02.1.01/0.0/0.0/18_069/0010054), and University of Hradec Kralove (No. SV2105-2020, VT2019-2021).
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