Mitochondrial off targets of drug therapy

Review

Mitochondrial off targets of drug therapy Kendall B. Wallace Department of Biochemistry and Molecular Biology, University of Minnesota Medical School, Duluth, MN 55812, USA

The bioenergetic features of mitochondria have long been exploited in the design of pharmacological agents suited to accomplish a desired physiological effect; uncoupling of oxidative phosphorylation to induce weight loss, for example. However, more recent experience demonstrates mitochondria to be unintended off targets of other drug therapies and responsible, at least in part, for the dose-limiting adverse events associated with a large array of pharmaceuticals. Review of the fundamentals of mitochondrial molecular biology and bioenergetics reveals a multiplicity of off targets that can be invoked to explain drug-induced mitochondrial failure. It is this redundancy of mitochondrial off targets that complicates identification of discrete mechanisms of toxicity and confounds QSAR-based design of new small molecules devoid of this potential for mitochondrial toxicity. The present review article briefly reviews the molecular biology and biophysics of mitochondrial bioenergetics, which then serves as a platform for identifying the various potential off targets for drug-induced mitochondrial toxicity. Introduction Mitochondria constitute the principal energy-producing organelles of the cell, the degree of reliance for cell sustainability varying amongst different tissue types. Organs such as cardiac and skeletal muscle as well as the nervous system rely heavily on aerobic metabolism and are thought to be particularly susceptible to agents that interfere with mitochondrial metabolism [1–6]. Besides the electron-transport complexes embedded within the inner membrane, mitochondria also contain the principal enzymes involved in both fatty acid and amino acid oxidation as well as the tricarboxylic acid cycle, each of which feeds reducing equivalents into the electron transport chain (Figure 1). Therefore, it must be realized that the scope of mitochondrial energy metabolism extends well beyond that of the respiratory (electron transport) chain (ETC) and oxidative phosphorylation (Ox/Phos). Consequently, mitochondrial toxicants include not only those chemicals that directly disrupt oxidative phosphorylation (uncouplers and inhibitors) but also those chemicals that interfere with the transport and/or oxidation of reducing substrates that deliver electrons to the respiratory chain. The multiplicity of mechanisms of mitochondrial toxicity is complicated further by the fact that mitochonCorresponding author: Wallace, K.B. ([email protected]).

dria contain their own genome, a 16.5 kb double-stranded closed loop that encodes for 13 proteins of the electron transport chain, 2 rRNA and 22 tRNA. All other proteins that make up the mitochondrion (both enzymatic and structural) are encoded by the nucleus, synthesized in the cytoplasm and imported electrophorectically across the almost 200 mV transmembrane electrical gradient into the mitochondrion, where the nuclear-encoded cationic leader sequence is cleaved by mitochondrial protease enzymes (Figure 1) [7]. Full elaboration of the genetic regulation of mitochondrial biogenesis is beyond the scope of this review but is available elsewhere [8,9]. The principal point of this review is to establish the complexity of mitochondrial biogenesis and the integration of coordinated substrate oxidation pathways, any step of which might represent a pivotal off target for druginduced mitochondrial toxicity. Further, because all these pathways converge at the level of the coordinated regulation of ATP synthesis, they share a common disease phenotype – lactic acidosis accompanied by altered lipid and carbohydrate metabolism that is expressed most often as neurologic or hematopoetic disorders and myopathies [4,5,10,11]. Each year a small percentage of registered pharmaceuticals are withdrawn from the market because of unanticipated and sometimes life-threatening adverse events that were not realized during the intensive preclinical testing and subsequent clinical trials. In many cases one basis for this is that the adverse event is subtle and without obvious histopathologic hallmarks. Such is the case for many drug-induced metabolic disorders. The past several years have witnessed the realization that numerous pharmaceuticals affect one or more of the mitochondrial off targets illustrated in Figure 1 and discussed below; the scope of the potential impact on drug development and postmarket surveillance has been reviewed by several authors [12–16]. Table 1 lists well-defined examples of classes of pharmaceutical agents that have been cited to adversely affect mitochondrial fidelity according to one or more of these distinct off targets. This article provides a review of the diverse array of pharmaceuticals that are alleged to incite dose-limiting mitochondrial toxicities and the specific molecular off targets that have been implicated in the mechanism of these toxicities. The goal of this work is to establish the broad scope of potential mitochondrial off targets for drug toxicity and to review limitations in the detection and discrimination amongst these different molecular mechanisms of drug-induced mitochondrial toxicity.

0165-6147/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2008.04.001 Available online 22 May 2008

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Figure 1. Graphic representation of the various molecular off targets that have been implicated in drug-induced mitochondrial toxicities. These mechanisms include not only direct inhibition (rotenone, antimycin, cyanide) or uncoupling (hydrophobic phenols and amides) of electron transport and oxidative phosphorylation (lower left of figure) but also inhibition of the transport or oxidation of reducing substrates (inhibition of fatty acid b-oxidation by valproic acid and salicylates or the tricarboxylic acid (TCA) cycle by fluoroacetate or fluoroacetamide) as well as molecular targets involved in mitochondrial biogenesis; examples include inhibition of mtDNA replication by nucleoside analogs, transcription and translation (mitochondrial protein synthesis by selected antibiotics) or inhibition of the import and posttranslational processing of proteins encoded by the nucleus and synthesized in the cell cytoplasm (protease inhibitors). The result is either direct interference with oxidative phosphorylation (inhibitors or uncouplers) or a failure to generate and assemble functional electron transport chains, both of which lead to ATP depletion and bioenergetic insufficiency of the cell. Abbreviations: carnitine palmitoyl transferase, CPT; a-ketoglutarate dehydrogenase, KDH; malate dehydrogenase, MDH; transporter outer membrane, TOM; transporter inner membrane, TIM.

Mitochondrial drug off targets Direct inhibitors of oxidative phosphorylation Chemicals that interfere directly with Ox/Phos include both inhibitors of the individual complexes of the ETC and uncouplers of Ox/Phos. Classic examples of inhibitors of electron transport include rotenone, antimycin A, cyanide and CO. By blocking electron transport these agents inhibit both substrate oxidation and oxygen consumption (Box 1), which leads to hyperlactic acidemia, lipid accumulation (microsteatosis) and hypoglycemia. Conversely, uncouplers of Ox/Phos collapse both the pH gradient and electrical potential across the inner mitochondrial membrane, thereby stimulating substrate oxidation and oxygen consumption (Figure 1). The result is a fatty-acid-depleting metabolic state accompanied by hypoxemia. The energy generated by uncoupling electron transport from ATP synthesis is dissipated as heat (hyperpyrexia). Although 362

there are few structural correlates for the inhibitors of the ETC, uncouplers of Ox/Phos are characterized as being hydrophobic weak acids (i.e. phenols or amides) with a pKa of 5–7 [17]. These characteristics are evident from the examples of Ox/Phos uncoupling agents presented in Table 1. A second type of uncoupling reaction that differs from the conventional protonophoric mechanism is drugs that redox cycle on the ETC, thereby diverting electrons from reducing cytochrome oxidase to alternate electron acceptors. A well-described member of this class is doxorubicin (Adriamycin), which accepts electrons from complex I of the ETC and reduces molecular oxygen to superoxide anion free radicals to complete the redox cycle [18,19]. Physical chemical characteristics of drugs capable of redox cycling on the ETC are amphiphilic chemicals sufficiently hydrophobic to diffuse through the cell to the mitochondrial

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Box 1. The ETC consists of five distinct proteinaceous respiratory complexes (Figure I), all of which are embedded in the highly impermeable inner mitochondrial membrane. Each complex is composed of four or more individual protein subunits, totaling more than 80 proteins in all. Thirteen of these proteins are encoded by the mitochondrial genome (mtDNA) and the remainder by the nucleus. Reducing equivalents, in the form of NADH, are derived either from the b-oxidation of fatty acids or from glycolysis and directly reduce complex I (NDH) of the ETC. A secondary source of reducing equivalents is FADH2 derived from the tricarboxylic acid cycle (TCA) to reduce complex II (SDH) of the ETC. Electrons then flow down the redox potential through coenzyme Q (CoQ)to reduce complex III and cytochrome c before being compiled within the terminal oxidase, cytochrome oxidase (complex IV; COX). It is at COX that molecular dioxygen (O2) undergoes a four-electron reduction to water to complete the ETC cycle. In the process of transporting electrons along the ETC, protons are actively pumped from the matrix of the mitochondrion to the intermembrane space. This separation of protons is sufficient to establish an almost full unit pH gradient (alkaline inside) and a transmembrane electrical potential that approaches 200 mV (negative inside). It is this strong pH and electrical gradient that provides the Gibb’s free energy needed to drive complex V (ATP synthase) to phosphorylate ADP to ATP. Interference with either electron transport directly or with the assembly of a functionally intact ETC comprised of all essential protein subunits results in inefficient ETC and inhibition of oxidative phosphorylation required to sustain the ATP energy requirements of the cell.

Figure I.

matrix and have a redox half-potential within the range of the respective complex of the ETC (–350 to 0 mV for complex I). Doxorubicin is primarily cardiotoxic and expresses its toxicity in the form of bioenergetic deficiency and compromised cardiac performance consistent with a dilated cardiomyopathy [19]. An example of a redox cycling drug that causes mitochondrial toxicity to the liver is menadione [20,21]. Direct-acting inhibitors of the ETC have been used in the pesticide arena and include rotenoids, cyanides and hydrogen sulfide. The lipid-lowering fibric acids and antidiabetogenic thiazolidinediones are examples of drugs suggested to express dose-limiting inhibition of the ETC [22,23]. Numerous other drugs have been implicated as direct-acting inhibitors of the ETC as well; however, one

must question the basis for some of these claims. For example, much of this evidence is based on the inhibition of the individual ETC complex enzymes in vitro by the suspected drug in isolated submitochondrial particles. Although the assays are well characterized and widely used [24], the relevance of the results are brought into question by (i) the concentrations of drug required to demonstrate statistically significant enzyme inhibition and (ii) failure to interpret the data in context of an intact mitochondria where that specific enzyme activity might not necessarily be rate limiting in determining the rate of respiration and ATP synthesis. Accordingly, these data are best interpreted to represent a ‘potential’ to cause mitochondrial toxicity. Evidence generated by using intact mitochondria or from cell or in-life studies carries more 363

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Table 1. Mechanisms of mitochondrial toxicity Example(s) Oxidative phosphorylation Uncouplers

Inhibitors

Fatty acid b-oxidation Tricarboxylic acid oxidation Cytosolic protein processing Genetic mechanisms mtDNA replication mtDNA transcription mtRNA translation/protein synthesis

2,4-dinitrophenol Pentachorophenol Lidocaine, bupivacaine Rotenone Amytal Antimycin A Malonate Cyanide Carbon monoxide Perhexiline Thiazolidinediones (glitazones) Valproic acid Salicylates Fluoroacetate Fluoroacetamide Protease inhibitors Nucleoside analog reverse transcriptase inhibitors PPAR ligands Fibrates, statins, glitazones Chloramphenicol Oxazolidinones (linezolid)

concern. What is worth noting is the scope and diversity of chemical structures that have been demonstrated to inhibit the ETC (Table 1). For this reason, inhibitors of the ETC might be the most difficult series of compounds to model on a structural or physical–chemical basis. This frustration isn’t satisfied to a significant degree by modeling the individual ETC complex because of the multiple protein subunits that comprise each complex. The reader is referred to one of several recent reviews for further detail regarding attempts to model inhibitors of each respiratory complex [17]. Inhibitors of mitochondrial substrate oxidation Inhibitors of mitochondrial substrate oxidation are, for the most part, structural mimics of the natural substrate and are, thus, competitive inhibitors of the respective reaction. Examples include the inhibition of fatty acid oxidation by valproic or salicylic acids [25–27]. Likewise, fluoroacetate and fluoroacetamide represent structural analogs that condense with oxaloacetate to form fluorocitrate, a potent inhibitor of the aconitase enzyme of the tricarboxylic acid cycle [25,27,28]. Inhibition of either pathway interferes with the generation and delivery of reducing equivalents into the mitochondrial ETC. Consequently, inhibitors of mitochondrial substrate oxidation ultimately cause the same metabolic deficit as direct inhibitors of the ETC and, like ETC inhibitors, tissues and species with high metabolic rates are most sensitive to inhibitors of substrate oxidation. It should be noted that inhibitors of mitochondrial substrate oxidation include inhibitors of both enzymes within the metabolic pathway as well as inhibitors of the transport proteins required to deliver the substrate to the mitochondrial matrix [29]. Inhibitors of mitochondrial biogenesis Mitochondrial biogenesis represents a complex process involving a series of signaling pathways that regulate 364

the coordinated expression of the nuclear and mitochondrial genomes as well as proper incorporation and assembly of individual respiratory complexes in the inner mitochondrial membrane. Mitochondrial biogenesis also involves fusion and fision, which is regulated by various cytoskeletal features. For a thorough description of the cell and molecular biology of mitochondrial biogenesis, the reader is referred to one of several recent reviews [8,30]. Perhaps the best known examples of inhibitors of mitochondrial biogenesis are the nucleoside analog antiviral drugs designed to interfere with the reverse transcription of invading viral RNA. Once phosphorylated by the respective nucleoside kinase, these analogs are recognized by the host DNA polymerases, of which there are five. Having a blocked 30 end, once the nucleotide is incorporated the replicating strand of DNA cannot be further elongated to complete the replication process. The result is a truncated and instable replicon. One hypothesis is that the toxicity of these analogs originates from the fact that mitochondria contain only a single form of DNA polymerase, Polg, whose activity is obligatory for replicating the mitochondrial genome and which has a very high affinity for many of these nucleotide analogs [31]. The hypothesis is that the truncated and unstable mitochondrial DNA (mtDNA) is responsible for the mtDNA depletion observed in some models of exposure and that this not only leads to a decrease in mitochondrial abundance but also those mitochondria that do survive lack many of the mtDNA-encoded protein subunits essential to the ETC complexes [32–34]. Lacking these key proteins, assembly of the respiratory complexes and other structures within the mitochondria is disrupted. Protease inhibitors that prevent hydrolysis of the cationic leader sequence required of cytosolic proteins imported into the mitochondria might cause a similar interference with membrane assembly and mitochondrial biogenesis. As a result the mitochondria are poorly coupled and instead of reducing oxygen to water and phosphorylating ADP, they release many of these unpaired electrons as partially reduced free radical species of molecular oxygen. Although this ‘mtDNA-depleting Polg’ hypothesis has been demonstrated in several experimental and clinical scenarios, it must be emphasized that these analogs have a much broader array of toxicities. Because of the abundance of reactions that require nucleosides, either as building blocks for pyridine nucleotide synthesis or as substrates for phosphonucleotide cofactors, selected members of this drug class are known also to interfere with mitochondrial metabolism by interrupting either NADH supply or the phosphoregulation of selected subunits of the ETC. Examples include the inhibition of phosphorylation of complex I by AZT [35], inhibition of thymidine kinase activity [36] or inhibition of nucleoside transporters associated with the cell or mitochondrial membranes [37]. Thus, although mtDNA depletion has been demonstrated by nucleoside-analog reverse-transcriptase inhibitors, the scope of mitochondrial and metabolic toxicity is far broader – lack of mtDNA depletion should by no means be taken to indicate a lack of mitochondrial or metabolic toxicity [38– 40]. A second and well-established mode of toxicity affecting mitochondrial biogenesis is drug-induced interference with

Review mitochondrial protein synthesis (mtRNA translation). Just as mitochondria contain their separate genome, they also contain all the enzymatic machinery required to translate the mtDNA-encoded message, yet the ribosomal proteins are distinct from those that decode the nuclear genome [41]. Inhibition of protein synthesis is believed to result from binding to the ribosomal protein, and it is this preferential binding affinity of the oxazolidinones (e.g, linezolid), as well as chloramphenicol and the tetracycline drugs, that is believed to account for the dose-limiting mitochondrial toxicity associated with these antibacterial drug therapies [42–47]. Regulation of mitochondrial biogenesis and bioenergetics also is influenced by several transcription factors, one of the most prominent being the peroxisome proliferator-activated receptors (PPARs), of which there are three members. Extensive investigation by a host of laboratories has established the PPARs as crucial modulators of intermediary metabolism in most cell types, controlling metabolic flux between glucose and fatty acid oxidation pathways [48–50]. This switching of metabolic flux on its own indirectly influences mitochondrial character. For example, expression of mitochondrial fatty acid b-oxidation enzymes is under the transcriptional control of PPARg and/or PPARd [51]. Of particular note is the identification of a 45 kDa protein in mitochondria that is both physically and functionally similar to PPARg2 [52]. Furthermore, these authors identified a DR2-binding sequence for this protein in the D-loop of mtDNA, which is the locale of the origin for transcription of the polycistronic mitochondrial genome. Identification of this mtPPARg might explain some of the reported off-target mitochondrial effects associated with the lipid-lowering statins and fibric acids as well as the antidiabetogenic thiazolidinediones (glitazones) [51,53–55]. A common differential In spite of the multiple distinct targets discussed above that have been invoked to explain drug-induced mitochondrial toxicities, the ultimate phenotype is fundamentally the same [10,11], with slight distinctions for uncouplers of mitochondrial Ox/Phos. Regardless of whether the drug acts directly to inhibit one or more complexes of the ETC or acts indirectly either by inhibiting substrate oxidation or the expression and assembly of the ETC unit, the ultimate result is inhibition of ATP synthesis and a change in pyridine nucleotide redox status. This then leads to failure to generate sufficient ATP to sustain normal cell and tissue function that, through the action(s) of key signaling molecules and because of NADH regulatory influences, is accompanied by changes in glucose and lipid homeostasis. Walker [16] summarizes the metabolic disorders and clinical symptoms associated with nucleoside analog antiretroviral-mediated mitochondrial toxicity, which include pleiotropic effects that span most major organ systems including neuropathies, myopathies, hepatotoxicities, nephropathies, hematotoxicities, pancreatitis and osteopenia, depending on the specific drug and exposure scenario. The scope of mitochondrial cytopathologies is equally broad for other causes of mitochondrial disease, including both inherited and acquired mitochondrial disorders

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[56,57]. In the case of drug toxicity, one might project that the specific type of organopathy might be more a function of pharmacokinetics (drug delivery) than tissue-specific differences in mitochondrial biology. Because of the convergence on the same ultimate phenotype of metabolic failure, the specific mitochondrial off target might continue to elude easy detection and the final diagnosis might be determined more by the properties of the chemical than by the characteristics of the individual. Summary Mitochondrial toxicity is a growing cause for concern for preclinical candidate failures as well as postmarket drug withdrawals. The bioenergetic fidelity of the mitochondrion is a complex and exquisitely orchestrated convergence of both nuclear and cytosolic events that control the size and number of mitochondrial units, the efficiency of electron transport and oxidative phosphorylation and the provision of substrate-derived reducing equivalents to the respiratory chain. Consequently, the common syndrome of metabolic failure is a challenging differential diagnosis and can be attributed to any one or more of numerous mitochondrial off targets of drug action. In this context attempts to model drug-induced mitochondrial toxicity must be multifaceted in scope and take into account these distinct mechanisms by which drugs and other foreign chemicals can interfere with the coordinated regulation of intermediary metabolism and energy production by the cell. Acknowledgements The author thanks J.M. Berthiaume for the original artwork of Figure 1.

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