key: cord-0709328-g71mr447
authors: Singh, Aarti; Faccenda, Danilo; Campanella, Michelangelo
title: Pharmacological advances in mitochondrial therapy
date: 2021-02-26
journal: EBioMedicine
DOI: 10.1016/j.ebiom.2021.103244
sha: 595b437a383df0f6687aee07b0ae7e0fdf4d6703
doc_id: 709328
cord_uid: g71mr447

Mitochondria play a vital role in cellular metabolism and are central mediator of intracellular signalling, cell differentiation, morphogenesis and demise. An increasingly higher number of pathologies is linked with mitochondrial dysfunction, which can arise from either genetic defects affecting core mitochondrial components or malfunctioning pathways impairing mitochondrial homeostasis. As such, mitochondria are considered an important target in several pathologies spanning from neoplastic to neurodegenerative diseases as well as metabolic syndromes. In this review we provide an overview of the state-of-the-art in mitochondrial pharmacology, focusing on the novel compounds that have been generated in the bid to correct mitochondrial aberrations. Our work aims to serve the scientific community working on translational medical science by highlighting the most promising pharmacological approaches to target mitochondrial dysfunction in disease.

Mitochondria are core regulators of cellular homeostasis by contributing to a variety of cellular functions including metabolism, apoptosis, intracellular signalling and immunity [1] .

Due to the vast number of conditions linked with mitochondrial dysfunction, there is growing interest in the development of pharmacological tools to restore mitochondrial integrity. Here, we will describe the most promising therapeutic strategies, which can be divided into three major classes: (i) targeted and (ii)untargeted mitochondrial drugs, and inhibitors of mitochondrial translation (iii).

Primary mitochondrial diseases (PMD) are a group of rare genetic metabolic disorders characterized by the presence of malfunctioning mitochondria due defects in oxidative phosphorylation (OXPHOS) [2] . Mitochondria contain their own genetic material (mtDNA), which ensures the synthesis of 13 subunits of the respiratory chain. All other mitochondrial components are encoded by the nuclear DNA (nDNA). While mutations in the mtDNA only affect the integrity of the respiratory chain [3] , nuclear gene defects disrupt OXPHOS by also impairing pathways involved in mitochondrial protein import, translation and assembly [4] . PMD manifest with diverse clinical symptoms, age of onset and progression [5, 6] , and the most affected tissues are those that highly depend on oxidative metabolism, such as heart, skeletal muscle, brain and retina [2] . Symptoms often affect multiple organs and range from muscle weakness to exercise intolerance, cardiomyopathy, cognitive disabilities, metabolic deficiencies, vision and/or hearing loss. Differently from mitochondrial impairments caused by primary OXPHOS impairments, secondary mitochondrial dysfunctions (SMD) arise when other pathological processes negatively impact on mitochondrial homeostasis. SMD is often involved in the development of age-related diseases, such as neurodegeneration, cancer, diabetes and cardiovascular diseases. In these pathologies, SMD can result from defects in (i) mitochondrial dynamics, (ii) biogenesis, (iii) quality control and (iv) metabolism.

The heterogenic aetiology of mitochondrial diseases demands for alternative pharmacological strategies to target mitochondrial defects and many compounds have therefore been devised. Here, we will focus on the ones that are already in clinical development and thereby bear promise as tools to treat mitochondrial diseases and other pathological conditions highly affected by SMD.

One of the widest class of mitochondria-targeted therapeutic agents comprises compounds that exert their function through interaction with respiratory chain components. Among these, some of the most promising ones are the antioxidant molecules idebenone and OP2113, and the insulin sensitizer imeglimin.

Idebenone (also known as Raxone, Catena, Sovrima, Puldysa, CV-2619) is a synthetic short chain analogue of coenzyme Q 10 with improved solubility and pharmacokinetics [7, 8] . Originally described as a lipophilic electron carrier with antioxidant properties, idebenone is predicted to act as an antioxidant in the transfer of electrons from respiratory complex II to complex III [9] . Due to its antioxidant properties and safety profile, idebenone has been evaluated in several clinical trials for a variety of mitochondria-related disorders ( Table 1) . The trials generated inconclusive results for many of the tested diseases, including Friederich's ataxia (FRDA) and Alzheimer's disease (AD). In spite of this, the drug retains promising potential for the treatment of Leber's hereditary optic neuropathy (LHON) [10] and Duchenne muscular dystrophy (DMD) [11] . At this regard, idebenone has received fast track and orphan drug designation by the United States Food and Drug Administration (FDA) for the treatment of DMD [12] . Marketed as Raxone, the drug is being reviewed by the European Medicines Agency (EMA) for the treatment of visual impairments in patients with LHON [13] . Under the trade name Puldysa, idebenone has also been granted orphan drug designation by the EMA, which is currently reviewing its marketing authorization application in the treatment of DMD patients who are not using glucocorticoids, with decision likely to be made this year [14] . The successful applications of this small molecule have warranted further studies aimed at improving bioavailability and effectiveness [15] .

Another compound targeting mitochondrial ROS production is OP2113 [(5-(4-methoxyphenyl)dithiole-3-thione], also known as anethole trithione and marketed as Sulfarlem. Two independent studies demonstrated that OP2113 inhibits up to 80% superoxide and hydrogen peroxide production by respiratory complex I (I Q site), without disrupting OXPHOS, thereby exerting cardioprotecting function [16, 17] . Though there are currently no clinical trials to document the efficacy of OP2113 for targeting mitochondrial dysfunction, the drug is already available for alternative therapies given its safety profile in humans. Hence, any new clinical trials could emerge and proceed quickly.

Mitochondrial dysfunction has recently emerged as a key component of diabetes physiopathology, promoting both hepatic and skeletal muscle insulin resistance and insulin secretory defects in pancreatic b-cells [18, 19] . Despite this, there are no known therapies for type 2 diabetes (T2D) that aim at preserving mitochondrial function. Imeglimin is a recently developed antidiabetic compound targeting and correcting mitochondrial dysfunction in all affected organs of T2D patients, namely pancreas, liver and skeletal muscle [20] . However, the precise mitochondrial mechanism of action of the compound remains unknown. Studies conducted on rat hepatocytes and isolated liver mitochondria found that imeglimin is a competitive inhibitor of respiratory complex I and restores the hyperglycemiainduced reduction in complex III content and activity, lowering reverse electron flow-associated ROS production and improving mitochondrial respiration [21] . Amelioration of mitochondrial function by imeglimin was also confirmed by following studies demonstrating prevention of mitochondrial permeability transition-induced cell death in endothelial and islet cells [22, 23] . The compound has so far successfully passed several multi-country phase 1 and 2 clinical trials assessing the efficacy, safety and tolerability profiles in patients with T2D, both as monotherapy and add-on therapy in patients inadequately controlled with the glucose-lowering medications metformin or sitagliptin (Table 1) . Recently concluded phase 3 clinical trials showed significant improvement in patients with T2D without causing severe hypoglycemia events [24] . The compound is being prepared for regulatory submission and prospective 2021 approval, which would result in the release of this innovative first oral antidiabetic agent in the upcoming year.

Another potential mitochondrial target to treat metabolic disorders is the mitochondrial pyruvate carrier (MPC), that mediates the import of pyruvate into the mitochondrial matrix across the mitochondrial inner membrane thereby linking glycolysis to OXPHOS [25] . Aberrant mitochondrial pyruvate uptake, which is linked to mitochondrial dysfunction and metabolic rewiring, plays an important role in the pathogenesis of metabolic diseases [26, 27] , including non-alcoholic fatty liver disease (NAFLD). Hepatic mitochondrial dysfunction, characterized by excessive oxidative metabolism and predisposing cells to oxidative damage, plays a key role in the pathophysiology of NAFLD [28] . Therefore, pharmacological inhibition of MPC is being explored as potential strategy to correct mitochondrial dysfunction in NAFLD, especially in patients suffering from its most severe form, called non-alcoholic steatohepatitis (NASH), for which there are no approved pharmacological therapies.

Thiazolidinediones (TZDs), also called glitazones, are a class of peroxisomal proliferator-activated receptor (PPAR)-g agonists with insulin-sensitizing activity that also exert an inhibitory effect on MPC [29] . Pilot studies provided evidence for mild beneficial effects of glitazone compounds in the treatment of NAFLD-NASH (Table 1) . Therefore, PPAR-g-sparing glitazone derivatives have been developed to improve the therapeutic effect of glitazones in NASH patients specifically through inhibition of MPC. Among these are MSDC-0602K and PXL065, both of which are now being tested at phase 2 clinical trials ( Table 1) .

The promising therapeutic potential of the next-generation glitazone MSDC-0602K in NASH treatment was suggested in a pre-clinical study conducted in a murine model of the disease [30] . However, subsequent clinical trials indicate that the compound has little effect on reversing either liver steatosis or fibrosis in patients with NASH [31] . The compound might therefore be better suited as a therapeutic agent for T2D associated with NASH, with a phase 3 clinical trial currently ongoing (Table 1) .

PXL065 (deuterium-stabilized R-pioglitazone, formerly known as DRX065) is another valuable promising molecule for the treatment of NASH. Encouraging results showing symptom reduction come from pre-clinical studies in a mouse model of NASH [32] , while safety and tolerability in NASH patients were assessed in a phase 1 clinical study [33] . PXL065 is advancing into a phase 2 clinical trial assessing doseefficacy in subjects with NASH (Table 1) .

Pharmacological inhibition of MCP is also explored for treating neurodegenerative diseases [26, 27] . The MSDC-0602K analogue MSDC-0160 is being investigated in AD and Parkinson's disease (PD) therapies. A phase 2 clinical trial showed improved cerebral glucose metabolism and reduced brain damage in AD patients (Table 1) , while a pre-clinical study in experimental models of PD confirmed the neuroprotective and anti-inflammatory properties of the compound [34] .

Considering the large number of trials with positive outcomes and the clinical success of idebenone, direct modulation of the mitochondrial respiratory chain components represents one of the most promising approaches to tackle mitochondrial dysfunction in human disease.

Along with OXPHOS modulators and MPC blockers, other mitochondria-targeted compounds are currently being tested. Two pharmacological strategies that will not be discussed in this review due to inconsistent results comprise inhibitors of the mitochondrial permeability transition pore (mPTP), such as (i) cyclosporine A (CsA), and (ii) lipid-binding molecules that regulate mitochondrial function and structure through modulation of mitochondrial membrane properties, including the cardiolipin (CL)-binding peptide elamipretide (formerly known as Bendavia, MTP-131 and SS-31). Patients that had previously participated in a 12-month randomised clinical trial were asked to assess their treatment assignment. The study concluded that none of the patients were accurately able to ascertain their treatment assignment, though a significant difference in ICARS score was measured when ambulatory patients alone were considered [38] . DMD NCT00654784 21 150 mg tid Treatment with idebenone in patients with DMD was shown to be safe and well-tolerated. Treated patients showed significant increase in peak systolic radial strain in the region of the heart that is early and severely affected by DMD. Peak expiratory flow (PEF) was also quantified to show a significant treatment effect [39] Though best recovery in visual acuity post-treatment was not observed, treatment with idebenone appeared to be beneficial in patienbts with discordant visual acuities [41] . 39 Treatment with idebenone improved tritan and protan color vision, most prominently in patients with discordant visual acuity [42] . NCT01421381 85 900 mg/day Visual acuity was preserved only in patients treated with idebenone. This effect was maintained at least 2.5 years post-treatment, supporting the therapeutic potential of idebenone in recovery of vision and attenuating vision loss [43] . [44] . AD Private trial 102 45 mg bid Treatment was well-tolerated. Significant improvements in patients treated with idebenone were measured using the Randt memory test of global mesic capability. Similar improvements with treatment were observed in scoring of "intellectual impairment" and "emotional impairment", as well as measurements of reaction time. The placebo group deteriorated progressively [45] .

(continued)

An encouraging therapeutic approach to treat mitochondrial diseases is represented by the pharmacological induction of mitochondrial biogenesis to overcome inherent OXPHOS deficiency. Mitochondrial biogenesis refers to the processes that increment mitochondrial mass through import and membrane integration of newly synthesized proteins and division of pre-existing mitochondria [53] . In the past years, considerable research focused on the development of molecules able to potentiate the transcription of nuclear and mitochondrial genes encoding respiratory chain subunits to improve OXPHOS activity. A promising strategy consists of targeting peroxisomal proliferator-activated receptors (PPARs), a group of transcription factors governing a multitude of cellular functions, including mitochondrial metabolism and energy homeostasis. Among the downstream targets of PPARs is PPAR-g coactivator-1a (PGC-1a) that, once activated, binds to and stimulates transcription factors involved in mitochondrial biogenesis [54] . To date, several agonists of PPARs are being tested as potential treatments for mitochondrial disorders and neurodegenerative syndromes.

Among them, TZDs have shown the highest clinical significance. TZDs are PPAR-g activators with insulin-sensitizing and hypolipidemic effects currently used for management of T2D in lieu of or in combination with metformin. One of the most promising TZDs for the treatment of mitochondrial dysfunction is the FDA-approved drug pioglitazone, which has been shown to promote mitochondrial biogenesis and function in diabetic patients [55, 56] . Pre-clinical studies provided auspicious results suggesting its applicability to the treatment of mitochondria-related neurological disorders [57À59]. A phase 2 clinical trial is ongoing to assess the effectiveness of pioglitazone in ameliorating neurological symptoms in patients suffering from X-linked adrenoleukodystrophy (X-ALD), a severe chronic neurodegenerative disorder characterized by peroxisomal and mitochondrial dysfunction. Moreover, a recently developed pioglitazone metabolite with optimal brain penetration, MIN-102 (leriglitazone), has entered phase 2/3 clinical trials to test its efficacy and safety on X-ALD and FRDA (Table 2) .

Other PPAR agonists that entered clinical trials for mitochondriarelated diseases are bezafibrate and REN001. Bezafibrate is a pan-PPAR activator [60] currently used as antilipemic agent (not FDAapproved). The repurposing of bezafibrate to correct metabolic defects in mitochondrial myopathies through induction of mitochondrial biogenesis has been the subject of a recently completed phase 2 clinical trial ( Table 2) . Although the beneficial effects of bezafibrate on patients suffering from mitochondrial disorders were confirmed Patients with either pre-diabetes or T2DM and NASH were treated with pioglitazone. 58% of patients showed at least a 2 point reduction in NAFLD score when compared to placebo (statistically significant). Histologic scores also showed improvements in fibrosis and adipose tissue, hepatic and muscle insulin sensitivity. Resolution of NASH was demonstrated in 44% of patients with T2DM compared to 26% in patients without diabetes. Significant reduction in fibrosis and insulin sensitivity in adipose tissue was observed in patients with diabetes when compared to those without diabetes ( [105] , [106] for short term usage, the results of the trial argued against the safety of long-term use due to the development of mitochondrial toxicity.

REN001 is a recently developed PPAR b/d agonist that, like bezafibrate, is under investigation as potential treatment for severe mitochondrial diseases including primary mitochondrial myopathies and fatty acid oxidation disorders (FAOD). Phase 1 clinical trials are ongoing to test its safety and tolerability (Table 2) . Other potential targets to modulate PGC-1a activity and restore mitochondrial function are sirtuin 1 (Sirt1) and AMP-protein activated kinase (AMPK). Several activating compounds have been identified for both proteins, with some already in clinical trials for mitochondria-related diseases.

Sirt1 is a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase that plays a prominent role in metabolic tissues, promoting mitochondrial biogenesis and turnover [61] . Sirt1 activity can be pharmacologically regulated by means of allosteric modulators, often referred to as Sirt1-activating compounds (STACs). Alternatively, Sirt1 activation can be promoted by using NAD+ precursors, also called NAD+ boosting molecules (NBMs), which target enzymes promoting the synthesis of NAD+ or inhibiting its degradation. Studies conducted on murine models suggest the applicability of STACs and NBMs as potential therapeutic agents for mitochondrial diseases and neurodegenerative disorders linked with mitochondrial dysfunction [62, 63] .

One of the most studied STAC is resveratrol, a naturally occurring antioxidant compound, the use of which as a dietary supplement has been linked to prevention of various aging-associated diseases [64] . Resveratrol is currently undergoing clinical trials to evaluate its beneficial effects on diseases associated with mitochondrial dysfunction. However, based on the available results, no definitive conclusions can be drawn on its efficacy. Two independent phase 2 clinical trials conducted on patients with AD had opposite outcomes, with one reporting no differences between control and treatment groups, and the other proposing reduced neuro-inflammation and cognitive decline associated with the use of resveratrol (Table 2) . Similarly, a study conducted on subjects suffering from FRDA revealed that highdose resveratrol ameliorates symptoms, despite having no effects on mitochondrial function and causing development of gastrointestinal adverse reactions (Table 2) . Regarding primary mitochondrial syndromes, a trial to study the therapeutic effects of resveratrol on subjects suffering from mitochondrial myopathy or fatty acid oxidation defects has recently been completed, but the results have not yet been disclosed (Table 2 ). An example of NBMs is instead the niacin derivative acipimox (also known as Olbetam), a NAD+ precursor that is not yet approved by FDA and currently used only as additional or alternative treatment for hyperlipidaemia and insulin resistance. The use of acipimox to improve mitochondrial function has been studied in several clinical trials, evaluating efficacy of treatment in obesity, diabetes and mitochondrial myopathy ( Table 2 ). The results of these studies are contradictory. While some studies confirmed the positive effect of acipimox on skeletal muscle mitochondrial function in diabetic and obese patients, including increased respiration and ATP synthesis, others reported that the insulin-sensitizing and lipid-lowering effects of the drug were not matched by alterations in mitochondrial function between treated and control groups ( Table 2) . To explore whether acipimox is an alternative to treat mitochondrial disease, a clinical trial is currently underway testing the ability of the compound to reduce symptoms in patients with mitochondrial myopathy (Table 2) .

Together with modulation of PPARs and Sirt1 activities, activation of AMPK is recognized as another therapeutic strategy to restore mitochondrial function [65] . Several direct and indirect AMPK activators have been identified so far, the majority of which were mainly developed to treat metabolic diseases such as diabetes and non-alcoholic liver conditions, independently of their effect on mitochondrial function. Among these, the direct AMPK activator PXL770 is instead under investigation as potential treatment for mitochondria-related diseases [66] . After successful completion of phase 1 clinical trials to assess safety and tolerability on diabetic subjects [67] , PXL770 demonstrated promising therapeutic potential in a preclinical study involving a murine model of NASH [68] . PXL770 is currently in phase 2 trials to evaluate its effectiveness in NASH patients and already demonstrated good efficacy, safety and tolerability profiles [69] .

Oxidative stress is unanimously acknowledged as a main driving factor for the onset and progression of many age-related, chronic human diseases [70] . Mitochondria are a major intracellular source of reactive oxygen species (ROS) [71] , the toxic potential of which affects many cellular and mitochondrial components. Among the diseases whose pathogenesis depends on ROS-induced cellular damage are mitochondrial disorders and neurodegenerative diseases ( [72] , [73] ) . Mitochondrial redox balance is therefore a target to treat diseases dominated by mitochondrial dysfunction. Among the antioxidant agents with highest therapeutic potential are those targeting the glutathione (GSH) and thioredoxin (Trx) redox systems.

Two compounds known to increase GSH levels and optimizing GSH detoxification pathways and the effectiveness of which in the treatment of mitochondrial conditions is currently being tested are the vitamin E analogue EPI-743 [74] (also called vincerinone, vatiquinone or a-tocotrienol quinone) and RP103 [75] (cysteamine bitartrate delayed-release). Since the applicability of both compounds to treat mitochondrial dysfunction has been extensively covered in recent publications [76 , 77] , we will focus on Sonlicromanol (KH176), a novel antioxidant agent targeting the Trx system [78] . The drug interacts with peroxiredoxins (Prxs), increasing their peroxidase activity and reducing ROS-induced cell death [78] . The safety, efficacy and tolerability of Sonlicromanol in patients with mitochondrial diseases including MELAS, LHON and Leigh syndrome, were tested in phase 1 and 2 clinical trials, confirming mild positive effects in m.3243A>G patients ( Table 2) . Sonlicromanol was recently granted orphan drug designation from the European Commission for maternally inherited diabetes and deafness (MIDD) and larger trials are being implemented to establish the clinical relevance of the compound [79] .

Mitochondria are highly dynamic organelles that constantly change their shape and organizational complexity, ranging from isolated, discrete entities to connected, highly branched networks that allow spatial and temporal synchronization. Mitochondrial dynamics is an essential physiological process that,through coordinated fusion and fission events, orchestrates proper intracellular positioning of mitochondria at areas of high-energy requirements [80] . Alterations in mitochondrial dynamics are associated with cancer development and progression of cardiovascular and neurodegenerative conditions [81, 82] , besides representing a causative event in some mitochondria-related neuropathies, such as Charcot-Marie-Tooth type 2 subtype A (CMT2A) and autosomal dominant optic atrophy (ADOA) [83, 84] . There is therefore a growing interest in the development of compounds to restore mitochondrial dynamics. Recent studies focused on targeting the pro-fission protein dynamin related protein 1 (Drp1) and the pro-fusion protein mitofusin 2 (Mfn2) as potential strategies to treat neurodegenerative conditions. The quinazolinone derivative mdivi-1 (mitochondrial division inhibitor) was the first inhibitor of mitochondrial fission discovered [85] ; it attenuates mitochondrial fission by interfering with the Drp1 filament assembly at mitochondrial constriction sites protecting from apoptosis via inhibition of their mitochondrial outer membrane permeabilization (MOMP) [85] . More recent studies have suggested that mdivi-1-mediated repression of mitochondrial fragmentation and improvement in mitochondrial function might be independent of Drp1 and might be linked to inhibition of complex I-mediated ROS production [86] or regulation of mitochondrial Ca 2+ uptake [87] . The clinical potential of mdivi-1 in the treatment of neurodegenerative diseases has been evaluated in a few pre-clinical studies. A study in an AD mouse model found that the compound reduces mitochondrial fragmentation and energy imbalance thereby improving learning and memory deficits as well as preventing deposition of amyloid-b (Ab) plaques in the brain [88] . Mdivi-1 has also shown protective effects against the development of MS. In EAE mice, mdivi-1 reduced infiltration of inflammatory cells in the spinal cords and inflammationmediated demyelination [89] . Additionally, studies conducted on murine models of heart diseases, including myocardial infarction, ischaemia/reperfusion injury and diabetic cardiomyopathy revealed that the Drp1 inhibitor preserved cardiac function and reduced severity of symptoms [90À93]. Mdivi-1 represents therefore a valuable candidate compound for the development of cardioprotective drugs.

In the search for molecules reverting pathological mitochondrial fragmentation, a novel Drp1-derived peptide, P110, has been designed [94] . Differently from mdivi-1, P110 is selective for Drp1 and inhibits both Drp1 enzymatic activity and interaction with the mitochondrial adaptor protein mitochondrial fission 1 (Fis1). Subsequently, P110 administration in animal models of neurodegeneration and myocardial infarction results in improved mitochondrial function and decreased cell damage [94] . Administration of P110 has also been shown to exert a neuroprotective effect in murine models of PD [95] , AD [96] and ALS [97] , preventing locomotor deficits, cognitive decline and muscle atrophy, respectively.

Another promising approach to correct defective mitochondrial dynamics consists in promoting mitochondrial fusion through stimulation of MFNs fusogenic activity. A recent study showed that treatment of neuronal cells expressing CMT2A Mfn2 mutant clones with a chimeric MFN2 agonist (B/A-l) restores mitochondrial fusion along with improved mitochondrial function and motility [98] . More importantly, the use of the Mfn2 agonist in the Mfn2 T105M murine model of CMT2A reverted axonal mitochondrial dysmotility [98] , thus suggesting its potential application to treat neurodegenerative conditions caused by defective axonal mitochondrial transport.

Even though the safety and efficacy of P110 in humans is to be yet tested, all of the above supports the viability of Drp1 and Mfn2 modulators as therapeutic agents in mitochondria-related diseases.

In addition to strategies aimed at restoring mitochondrial function in diseased tissues, other compounds are currently being tested to target mitochondrial protein synthesis in rapidly proliferating cancerous cells. This approach seems to be particularly promising to selectively kill cancer cells under chemotherapy, irradiation and during metastasis, when they are highly reliant on mitochondrial metabolism [115] .

Like their bacterial progenitors, not only do mitochondria contain their own genome, but also possess a dedicated machinery for protein synthesis. This comprises 22 transfer ribonucleic acids (tRNAs) and two ribosomes that allow the synthesis of all the mitochondrially encoded protein subunits of the OXPHOS system [116] . Pharmacological interference with the mitochondrial translation apparatus would lead to mitochondrial dysfunction without impairing cytosolic protein synthesis, thereby ensuring selectivity of targeting. This assumption has made the repurposing of antimicrobial drugs an elected strategy to target cancer cells.

Currently, only the FDA-approved antibiotic tigecycline has entered clinical trials for cancer treatment. Tigecycline belongs to the glycylcycline class of antibiotics and was developed to treat tetracycline-resistant bacterial infections. Like tetracyclines, tigecycline binds the 30S subunit of elongating ribosomes and halt bacterial protein synthesis through inhibition of tRNA entry [117] . To date, tigecycline is being tested for both acute and chronic myeloid leukaemia (Table 3) , but several studies also suggest its applicability to other hematologic and solid tumours, including lymphomas [118] , ovarian cancer [119] and nonsmall lung cancer [120] . Other FDA-approved tetracycline derivatives, such as doxycycline and tetracycline-3 (COL-3), have also been enrolled in trials to treat solid and hematologic tumours as well as metastatic cancers (Table 3) . Although their repurposing was initially based on their inhibitory activity on metalloproteases [121] , the direct link with suppression of mitochondrial translation was proposed at the basis of their anticancer activity [122] .

Another molecule whose anticancer effect has been linked to repression of mitochondrial translation is RK-33 (diimidazo[4,5d:4 0 ,5 0 -f-1,3]diazepine), an inhibitor of the DEAD-Box RNA Helicase DDX3 [123] . RK-33 showed promising results in preclinical studies as radiosensitizing agent for the treatment of DDX3-overexpressing tumours [124, 125] . Differently from tigecycline, RK-33-mediated inhibition of mitochondrial translation causes cancer cell death through OXPHOS impairment and ROS burst [124] and the compound is due to enter clinical trials for treatment of Ewing sarcoma and breast cancer [126] .

Mitochondria represent a critical therapeutic target for a variety of common debilitating pathologies and significant progresses have recently been made in the development of therapeutic strategies to restore mitochondrial homeostasis in affected tissues. Nevertheless, drug development for mitochondrial medicine still faces many challenges. Despite the large number of mitochondria-specific agents that have been tested in clinical studies, only a few compounds have been approved so far for the treatment of rare mitochondrial diseases. This review detailed the recent achievements in mitochondrial pharmacology, which derived from multi-disciplinary advances in the fields of mitochondrial biology and molecular targeting. Attention should now be focused on mitochondria-controlled processes and mitochondrial interactions with the surrounding organelles.

Recent advancements have unveiled that a critical aspect in the biology of mitochondria is represented by their interaction through membrane contact sites with the endoplasmic reticulum (ER), lysosomes and the nucleus. As such, can interorganellar contacts represent a novel target to amend mitochondrial defects? And is the signalling between mitochondria and their interacting organelles involved in PMD or SMD? The study was designed to evaluate the maximum tolerate dose of COL-3 in patients with recurrent high-grade glioma in combination with enzyme-inducing anti-seizure drugs. It showed that administration of COL-3 alone only had a partial response and didn't warrant further studies as a standalone therapy [127] . Kaposi's sarcoma N/A 18 25, 50 or 70 mg/m 2 /day Patients with AIDS-related Kaposi's sarcoma were assessed for changes in blood levels of metalloproteinases (MMP)-2, -9 and vascular endothelial growth factor (VEGF). The treatment was well tolerated and induced both a decline in MMP-2 serum levels and tumour regression [128] . NCT00020683 75 50 or 100 mg Patients with AIDS-related Kaposi's sarcoma were given one of two doses of COL-3. The lower daily dosage of COL-3 was shown to have a greater response rate compared to the higher dose group. In both dose groups a significant decrease in MMP-2 and MMP-9 serum levels was observed, along with antitumour activity [129] . Soft tissue sarcomas N/A 15 50 mg/m 2 /day Patients with soft tissue sarcomas were treated with COL-3 to assess the effects of the drug on disease progression and survival. Treatment did not yield an attenuation of disease progression [130] . Impaired mitochondrial quality control has been demonstrated to promote age-related pathologies. Nevertheless, drugs that effectively modulate the process in vitro have shown little effect in vivo. Does the pharmacological induction of mitochondrial clearance still represent a viable strategy to treat mitochondria-related pathologies or is it accompanied by further cell damage?

To gather data for this review, references were identified using the MEDLINE/PubMed and Google Scholar databases from relevant articles preferentially published in the last ten years applying the search terms "mitochondrial diseases" or "mitochondrial myopathy" or "Barth syndrome" or "Leigh syndrome" or "LHON" or "MELAS" or "Pearson syndrome" or "neurodegenerative diseases" or "AD" or "AMD" or "DMD" or "FRDA" or "HD" or "multiple sclerosis" "PD" or "diabetes" or "NASH" or "cardiovascular diseases" or "obesity" or "cancer" AND "targeting mitochondria" or "mitochondrial therapies" or "mitochondrial dysfunction". Clinical trials were searched using ClinicalTrials.gov and ClinicalTrialsRegister.eu databases. All studies were considered regardless of date published with preference for the most recently published articles relevant to compounds included in clinical trials.

All authors declare that they have no competing interests.

AS, DF and MC conceived the original draft. AS and DF wrote the original manuscript. AS, DF and MC edited the final manuscript.

Mitochondria and cell signalling

Mitochondrial DNA mutations in human disease

Nuclear gene defects in mitochondrial disorders

Clinical spectrum and diagnosis of mitochondrial disorders

The broadening spectrum of mitochondrial disease: shifts in the diagnostic paradigm

Pharmacokinetic evaluation of idebenone

Features of idebenone and related short-chain quinones that rescue ATP levels under conditions of impaired mitochondrial complex I

The effects of idebenone on mitochondrial bioenergetics

Idebenone is effective and well tolerated in Leber's Hereditary Optic Neuropathy (LHON): results of a 3-year expanded access program (S48.005)

Longterm data with idebenone on respiratory function outcomes in patients with Duchenne muscular dystrophy

Santhera receives FDA Fast Track Designation for Raxone Ò / Catena Ò (idebenone) for the Treatment of Duchenne Muscular Dystrophy (DMD)

Summary of the European public assessment report (EPAR) for Raxone

EMA Validates Santhera's Marketing Authorization Application for Puldysa Ò in Duchenne Muscular Dystrophy

Idebenone: novel strategies to improve its systemic and local efficacy

An old medicine as a new drug to prevent mitochondrial complex I from producing oxygen radicals

P4607A specific complex I-induced ROS modulator, OP2113, is a new cardioproctective agent against acute myocardial infarction injuries during reperfusion

Mitochondrial dysfunction in pancreatic b cells

Antidiabetic potential of saffron and its active constituents

Imeglimin -a new oral anti-diabetic that targets the three key defects of type 2 diabetes

Unique Mitochondria-targeting Mechanism of Action Confirmed for Poxel's Oral Agent Imeglimin

Imeglimin prevents human endothelial cell death by inhibiting mitochondrial permeability transition without inhibiting mitochondrial respiration

Imeglimin protects INS-1 cells and human islets against high glucoseÀand high fructoseÀinduced cell death by inhibiting the mitochondrial PTP opening

Poxel Announces Detailed Imeglimin Phase 3 TIMES 1 Results Presented at 55th Annual Meeting of the European Association for the Study of Diabetes

Mitochondrial pyruvate import and its effects on homeostasis

The mitochondrial pyruvate carrier in health and disease: to carry or not to carry?

Targeting energy metabolism via the mitochondrial pyruvate carrier as a novel approach to attenuate neurodegeneration

Excessive hepatic mitochondrial TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease

Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier

Targeting the mitochondrial pyruvate carrier attenuates fibrosis in a mouse model of nonalcoholic steatohepatitis

Insulin sensitizer MSDC-0602K in non-alcoholic steatohepatitis: a randomized, doubleblind, placebo-controlled phase IIb study

Efficacy of DRX-065, the stabilized R-enantiomer of pioglitazone (pio), in choline-deficient (CD) and methionine/choline-deficient (MCD) diet mouse models of nonalcoholic steatohepatitis (NASH)

Phase 1 Study of PXL065 Confirms Dose-Proportionality & Stabilization of the Preferred Stereoisomer (R-Pioglitazone) for the Treatment of NASH

Mitochondrial pyruvate carrier regulates autophagy, inflammation, and neurodegeneration in experimental models of Parkinson's disease

Neurological effects of highdose idebenone in patients with Friedreich's ataxia: a randomised, placebo-controlled trial

A phase 3, double-blind, placebo-controlled trial of idebenone in friedreich ataxia

Assessment of neurological efficacy of idebenone in pediatric patients with Friedreich's ataxia: data from a 6-month controlled study followed by a 12-month open-label extension study

Patient-reported outcomes in Friedreich's ataxia after withdrawal from idebenone

Idebenone as a novel, therapeutic approach for Duchenne muscular dystrophy: results from a 12 month, double-blind, randomized placebo-controlled trial

Efficacy of idebenone on respiratory function in patients with Duchenne muscular dystrophy not using glucocorticoids (DELOS): a double-blind randomised placebo-controlled phase 3 trial

A randomized placebo-controlled trial of idebenone in Leber's hereditary optic neuropathy

Effects of idebenone on color vision in patients with leber hereditary optic neuropathy

Persistence of the treatment effect of idebenone in Leber's hereditary optic neuropathy

A controlled trial of idebenone in Huntington's disease

Idebenone in senile dementia of Alzheimer type: a multicentre study

Idebenone treatment fails to slow cognitive decline in Alzheimer's disease

Sustained efficacy and safety of idebenone in the treatment of Alzheimer's disease: update on a 2-year double-blind multicentre study

Imeglimin, a novel glimin oral antidiabetic, exhibits a good efficacy and safety profile in type 2 diabetic patients

The Efficacy and Safety of Imeglimin as Add-on Therapy in Patients With Type 2 Diabetes Inadequately Controlled With Metformin Monotherapy

The efficacy and safety of imeglimin as add-on therapy in patients with type 2 diabetes inadequately controlled with sitagliptin monotherapy

An evaluation of MSDC-0160, a prototype mTOT modulating insulin sensitizer, in patients with mild Alzheimer's disease

Insulin sensitizers in 2013: new insights for the development of novel therapeutic agents to treat metabolic diseases

Mitochondrial biogenesis and clearance: a balancing act

Metabolic control of mitochondrial biogenesis through the PGC-1 family regulatory network

Pioglitazone induces mitochondrial biogenesis in human subcutaneous adipose tissue in vivo

Pioglitazone stimulates AMP-activated protein kinase signalling and increases the expression of genes involved in adiponectin signalling, mitochondrial function and fat oxidation in human skeletal muscle in vivo: a randomised trial

Effects of long-term treatment with pioglitazone on cognition and glucose metabolism of PS1-KI, 3xTg-AD, and wild-type mice

Pioglitazone halts axonal degeneration in a mouse model of X-linked adrenoleukodystrophy

Mitochondrial dysfunction plays a key role in the development of neurodegenerative diseases in diabetes

Dual and pan-peroxisome proliferator-activated receptors (PPAR) co-agonism: the bezafibrate lessons

Sirt1 and the Mitochondria

NAD(+)-dependent activation of Sirt1 corrects the phenotype in a mouse model of mitochondrial disease

Activation of sirtuin 1 as therapy for the peroxisomal disease adrenoleukodystrophy

Resveratrol: A Double-Edged Sword in Health Benefits

AMPK: guardian of metabolism and mitochondrial homeostasis

PXL770 Demonstrates Therapeutic Potential as a New Direct AMP Kinase Activator

Poxel Announces PXL770 Advances into a Phase 1b Multiple Ascending Dose Trial

Poxel Presents New Preclinical Proof-of-Concept Results for PXL770 at the 3rd Annual Global NASH Congress

Poxel Announces Favorable Results for PXL770 Phase 1b Multiple Ascending Dose Trial and Drug-Drug Interaction Study

Oxidative stress, aging, and diseases

How mitochondria produce reactive oxygen species

Oxidative stress in inherited mitochondrial diseases

Role of mitochondrial ROS in the brain: from physiology to neurodegeneration

a-Tocotrienol quinone modulates oxidative stress response and the biochemistry of aging

Cysteamine restores glutathione redox status in cultured cystinotic proximal tubular epithelial cells

Clin Trials Mitochondrial Dis:An Update on EPI-743 and RP103

Therapies for mitochondrial diseases and current clinical trials

KH176 Safeguards mitochondrial diseased cells from redox stress-induced cell death by interacting with the Thioredoxin System

Khondrion granted Orphan Drug Designation for KH176 for the treatment of MIDD from European Commission

Mito-Morphosis: mitochondrial fusion, fission, and cristae remodeling as key mediators of cellular function

Mitochondrial dynamics in cancer and neurodegenerative and neuroinflammatory diseases

Mitochondrial dynamics in cardiovascular health and disease

Role of mitofusin 2 mutations in the physiopathology of Charcot-Marie-Tooth disease type 2A

Effects of OPA1 mutations on mitochondrial morphology and apoptosis: Relevance to ADOA pathogenesis

Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bakdependent mitochondrial outer membrane permeabilization

The Putative Drp1 Inhibitor mdivi-1 Is a reversible mitochondrial complex I inhibitor that modulates reactive oxygen species

Mitochondrial Division Inhibitor 1 (mdivi-1) Protects Neurons against Excitotoxicity through the Modulation of Mitochondrial Function and Intracellular Ca2+ Signaling

Inhibition of Drp1 Ameliorates Synaptic Depression, Ab Deposition, and Cognitive Impairment in an Alzheimer's Disease Model

Mdivi-1, a mitochondrial fission inhibitor, modulates T helper cells and suppresses the development of experimental autoimmune encephalomyelitis

Inhibiting mitochondrial fission protects the heart against ischemia/reperfusion injury

Mdivi-1 induced acute changes in the angiogenic profile after ischemia-reperfusion injury in female mice

Differential temporal inhibition of mitochondrial fission by Mdivi-1 exerts effective cardioprotection in cardiac ischemia/reperfusion injury

Mitochondrial dynamic modulation exerts cardiometabolic protection in obese insulin-resistant rats

Mochly-Rosen D. A novel Drp1 inhibitor diminishes aberrant mitochondrial fission and neurotoxicity

Inhibition of Drp1 mitochondrial translocation provides neural protection in dopaminergic system in a Parkinson's disease model induced by

Drp1/Fis1 interaction mediates mitochondrial dysfunction, bioenergetic failure and cognitive decline in Alzheimer's disease

Inhibition of Drp1/Fis1 interaction slows progression of amyotrophic lateral sclerosis

MFN2 agonists reverse mitochondrial defects in preclinical models of Charcot-Marie-Tooth disease type 2A

Reduction of non-esterified fatty acids improves insulin sensitivity and lowers oxidative stress, but fails to restore oxidative capacity in type 2 diabetes: a randomised clinical trial

Evidence for a direct effect of the NAD+ precursor acipimox on muscle mitochondrial function in humans

Chronic reduction of plasma free fatty acid improves mitochondrial function and wholebody insulin sensitivity in obese and type 2 diabetic individuals

Metabolic effects of bezafibrate in mitochondrial disease

Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis

Randomized, placebo-controlled trial of pioglitazone in nondiabetic subjects with nonalcoholic steatohepatitis

Long-term pioglitazone treatment for patients with nonalcoholic steatohepatitis and prediabetes or Type 2 diabetes mellitus: a randomized trial

Response to pioglitazone in patients with nonalcoholic steatohepatitis with vs without Type 2 diabetes

A pilot study of pioglitazone treatment for nonalcoholic steatohepatitis

Pioglitazone in early Parkinson's disease: a phase 2, multicentre, double-blind, randomised trial

Glitazone use associated with reduced risk of Parkinson's disease

A randomized, double-blind, placebo-controlled trial of resveratrol with glucose and malate (RGM) to slow the progression of Alzheimer's disease: A pilot study

Resveratrol regulates neuro-inflammation and induces adaptive immunity in Alzheimer's disease

An open-label trial in Friedreich ataxia suggests clinical benefit with high-dose resveratrol, without effect on frataxin levels

KH176 under development for rare mitochondrial disease: a first in man randomized controlled clinical trial in healthy male volunteers

The KHENERGY Study: Safety and Efficacy of KH176 in Mitochondrial m.3243A>G Spectrum Disorders

Mitochondrial metabolism and cancer

The process of mammalian mitochondrial protein synthesis

Structural basis for potent inhibitory activity of the antibiotic tigecycline during protein synthesis

Differential contribution of the mitochondrial translation pathway to the survival of diffuse large B-cell lymphoma subsets

Inhibition of mitochondrial translation as a therapeutic strategy for human ovarian cancer to overcome chemoresistance

Tigecycline targets nonsmall cell lung cancer through inhibition of mitochondrial function

Development of matrix metalloproteinase inhibitors in cancer therapy

Mitochondria as oncotarget: a comparison between the tetracycline analogs doxycycline and COL-3

Novel, broad spectrum anti-cancer agents containing the Tricyclic 5:7:5-Fused diimidazodiazepine ring system

Targeting mitochondrial translation by inhibiting DDX3: a novel radiosensitization strategy for cancer treatment

RNA helicase DDX3: a novel therapeutic target in Ewing sarcoma

Clinical Development of RK-33

Phase I and pharmacokinetic study of COL-3 in patients with recurrent high-grade gliomas

Matrix metalloproteinase inhibitor COL-3 in the treatment of AIDS-related Kaposi's sarcoma: a phase I AIDS malignancy consortium study

Randomized phase II trial of matrix metalloproteinase inhibitor COL-3 in AIDS-related Kaposi's sarcoma: an AIDS Malignancy Consortium Study

A phase II and pharmacological study of the matrix metalloproteinase inhibitor (MMPI) COL-3 in patients with advanced soft tissue sarcomas