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Mitochondrial Diseases: Shortcuts to Therapies and Therapeutic Shortcuts
Molecular Cell
Previews Mitochondrial Diseases: Shortcuts to Therapies and Therapeutic Shortcuts Michael P. Murphy1,* 1MRC Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, UK *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2016.09.022
In this issue of Molecular Cell, Barrow et al. (2016) use two complementary approaches—one an assessment of a chemical library, and the other a genome-wide CRISPR screen—that both identify bromodomaincontaining protein 4 (Brd4) as a therapeutic target for mtDNA diseases affecting complex I. I am always on the lookout for shortcuts to the development of new therapies— sadly, my experience is that they rarely work. This has certainly been the case in the hunt for treatments for mtDNA diseases. These disorders arise due to defects in mitochondrial oxidative phosphorylation due to mutations in mtDNA, which in mammals encodes 37 genes necessary for assembly of the respiratory chain and the FoF1-ATP synthase (Wallace, 2010). Unsurprisingly, considering the role of mitochondria in energy metabolism, these disorders typically present as neuromuscular syndromes (Wallace, 2010). There are no therapies, in part because of the complications associated with mtDNA, which include inaccessibility, heteroplasmy, coordination with the nuclear genome, and our inability to manipulate mtDNA (Pfeffer et al., 2012; Smith et al., 2012). These challenges have been addressed in ingenious ways—for example, by replacing defective mtDNA in the eggs of affected mothers in conjunction with in vitro fertilization (Hyslop et al., 2016). Even so, there is a major unmet need for drugs that can help patients with mtDNA diseases. In this issue of Molecular Cell, Barrow et al. (2016) present an elegant shortcut to identify both potential therapeutic targets and promising drug candidates. To do this, the authors assessed cybrid cells containing a mtDNA mutation that led to defective complex I, which is the entry point for electrons from NADH into the respiratory chain and which is frequently affected in mtDNA disorders. They screened these complex I-defective cells against a chemical library to select compounds that induced a compensatory increase in respiratory chain content. In
parallel, they carried out a genome-wide CRISPR screen to find genes that, upon inactivation, enabled cells to grow when galactose was the major energy source. Galactose forces cells to use oxidative phosphorylation to make ATP; hence, cells with the complex I defect die unless gene loss facilitates a compensatory mechanism. What was fascinating was the fact that both screens identified a common target. One of the hits from the chemical library, I-BET 525762A, is an inhibitor of bromodomain proteins, while the CRISPR screen identified the gene that encodes the bromodomain-containing protein 4 (Brd4). Thus, inhibiting the activity of Brd4 helps cells compensate for the complex I defect. What is the mechanism? Bromodomain proteins such as Bdr4 bind acetylated lysines on histones to coordinate and alter gene expression. It seems that blocking Brd4 increases expression of genes that allow mitochondria to compensate for complex I defects by rewiring metabolism so as to bypass this respiratory chain blockade (Figure 1). Interestingly, the inhibition of Bdr4 only compensated for defects in complex I, not for those in complexes III or IV, consistent with selective rewiring of metabolism to bypass complex I rather than a general protection of mitochondria. This encouraging specificity suggests that the screens can be extended to find therapies for defects in other complexes and emphasizes that therapies for mtDNA diseases are not generic, but need to be tailored to the affected complex. The finding of Brd4 as a druggable therapeutic target joins the list of approaches, such as the activation by small molecules of the transcriptional coactivator PGC-1a (Viscomi et al., 2011), to altering gene
expression and thereby enabling the cell to cope with defective mtDNA. There are also resonances with the recent finding from the Mootha lab that hypoxia triggers activation of a suite of genes under the regulation of HIF-1a that can suppress phenotypes associated with mutated mtDNA (Jain et al., 2016). More generally, these studies highlight the potential of small-molecule therapies that intervene upstream of mitochondrial biogenesis to compensate for mitochondrial defects (Smith et al., 2012). Intriguingly, these findings also point to how mitochondrial function itself is altered by the shift in gene expression, and thus suggest more direct interventions in mitochondria. It seems that the inhibition of Brd4 works by bypassing complex I, so that electrons which would otherwise build up on NADH go by a ‘‘rewired’’ metabolism to the CoQ pool (Figure 1). This shortcut for electrons should bypass the bottleneck of a defective complex I, thereby allowing the rest of the respiratory chain to carry out proton pumping at complexes III and IV and thus produce ATP, albeit less efficiently. In addition, it also allows oxidation of the NADH pool by providing a pathway to oxygen and avoiding the metabolic inhibition caused by an increase in the NADH/NAD+ ratio (Titov et al., 2016). Similar therapeutic bypassing of complex I has been achieved by the ectopic expression of the NADH dehydrogenase I from yeast, which takes electrons from NADH to CoQ without pumping protons across the inner membrane (Sanz et al., 2010). Thus, the mode of action of Brd4 inhibition shows the therapeutic potential of introducing shortcuts into the respiratory chain to bypass pathological blockades. Perhaps this may lead
Molecular Cell 64, October 6, 2016 ª 2016 Elsevier Inc. 5
Molecular Cell
Previews Therapy Nuclear genome
Bdr4
cuts to be an elegant screening approach, which may lead on to therapies that act by bypassing defects in the respiratory chain. Occasionally, shortcuts can work out well!
Conventional REFERENCES
to complex I Rewiring
Barrow, J.J., Balsa, E., Verdeguer, F., Tavares, C.D.J., Soustek, M.S., Hollingsworth, L.R., IV, Jedrychowski, M., Vogel, R., Paulo, J.A., Smeitink, J., et al. (2016). Mol. Cell 64, this issue, 163–175.
to the CoQ pool NADH NAD+ 2H+ 4H+ Q QH2
Defective complex I
SDH
mtDNA
4H+
Cyt c
2H+
Complex IV
mtDNA mutation
Figure 1. Rewiring of Respiration from NADH to the CoQ Pool by Inhibting Brd4 The mitochondrial respiratory chain complexes I, II (SDH, succinate dehydrogenase), III, and IV are shown, with the mobile electron carriers CoEnzyme Q (Q) and cytochrome c (cyt c) also illustrated. A mutation in mtDNA of one of the genes encoding a complex I subunit causes a defect in respiration, preventing oxidation of NADH. Inhibition of the nuclear protein Brd4 removes suppression of an alternative pathway that allows electrons from NADH to bypass complex I and go to the CoEnzyme Q pool. This shortcut enables complexes III and IV to pump protons across the mitochondrial inner membrane, thereby building up a proton motive force so that mitochondria can still make ATP despite the complex I defect.
onward to other small molecule therapies that could act directly on the mitochondria as shortcuts to bypass respiratory defects? Such approaches have been piloted in the past (Eleff et al., 1984), and extended more recently (Ehinger et al., 2016), while some current approaches such as idebenone may act in this way.
6 Molecular Cell 64, October 6, 2016
Ehinger, J.K., Piel, S., Ford, R., Karlsson, M., Sjo¨vall, F., Frostner, E.A., Morota, S., Taylor, R.W., Turnbull, D.M., Cornell, C., et al. (2016). Nat. Commun. 7, 12317. Eleff, S., Kennaway, N.G., Buist, N.R., DarleyUsmar, V.M., Capaldi, R.A., Bank, W.J., and Chance, B. (1984). Proc. Natl. Acad. Sci. USA 81, 3529–3533.
Q QH2
Complex III
4H+
4H+ 1/2 O H2 O 2
More work is required, however, to be sure that these compounds are effective metabolic shortcuts in vivo. Even so, the results shown here suggest that it is well worth developing further small molecules to facilitate metabolic shortcuts in vivo. In summary, this work by Barrow et al. (2016) has shown these heuristic short-
Hyslop, L.A., Blakeley, P., Craven, L., Richardson, J., Fogarty, N.M., Fragouli, E., Lamb, M., Wamaitha, S.E., Prathalingam, N., Zhang, Q., et al. (2016). Nature 534, 383–386. Jain, I.H., Zazzeron, L., Goli, R., Alexa, K., Schatzman-Bone, S., Dhillon, H., Goldberger, O., Peng, J., Shalem, O., Sanjana, N.E., et al. (2016). Science 352, 54–61. Pfeffer, G., Majamaa, K., Turnbull, D.M., Thorburn, D., and Chinnery, P.F. (2012). Cochrane Database Syst. Rev. 4, CD004426. Sanz, A., Soikkeli, M., Portero-Otı´n, M., Wilson, A., Kemppainen, E., McIlroy, G., Ellila¨, S., Kemppainen, K.K., Tuomela, T., Lakanmaa, M., et al. (2010). Proc. Natl. Acad. Sci. USA 107, 9105–9110. Smith, R.A., Hartley, R.C., Cocheme´, H.M., and Murphy, M.P. (2012). Trends Pharmacol. Sci. 33, 341–352. Titov, D.V., Cracan, V., Goodman, R.P., Peng, J., Grabarek, Z., and Mootha, V.K. (2016). Science 352, 231–235. Viscomi, C., Bottani, E., Civiletto, G., Cerutti, R., Moggio, M., Fagiolari, G., Schon, E.A., Lamperti, C., and Zeviani, M. (2011). Cell Metab. 14, 80–90. Wallace, D.C. (2010). Environ. Mol. Mutagen. 51, 440–450.
Previews Mitochondrial Diseases: Shortcuts to Therapies and Therapeutic Shortcuts Michael P. Murphy1,* 1MRC Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, UK *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2016.09.022
In this issue of Molecular Cell, Barrow et al. (2016) use two complementary approaches—one an assessment of a chemical library, and the other a genome-wide CRISPR screen—that both identify bromodomaincontaining protein 4 (Brd4) as a therapeutic target for mtDNA diseases affecting complex I. I am always on the lookout for shortcuts to the development of new therapies— sadly, my experience is that they rarely work. This has certainly been the case in the hunt for treatments for mtDNA diseases. These disorders arise due to defects in mitochondrial oxidative phosphorylation due to mutations in mtDNA, which in mammals encodes 37 genes necessary for assembly of the respiratory chain and the FoF1-ATP synthase (Wallace, 2010). Unsurprisingly, considering the role of mitochondria in energy metabolism, these disorders typically present as neuromuscular syndromes (Wallace, 2010). There are no therapies, in part because of the complications associated with mtDNA, which include inaccessibility, heteroplasmy, coordination with the nuclear genome, and our inability to manipulate mtDNA (Pfeffer et al., 2012; Smith et al., 2012). These challenges have been addressed in ingenious ways—for example, by replacing defective mtDNA in the eggs of affected mothers in conjunction with in vitro fertilization (Hyslop et al., 2016). Even so, there is a major unmet need for drugs that can help patients with mtDNA diseases. In this issue of Molecular Cell, Barrow et al. (2016) present an elegant shortcut to identify both potential therapeutic targets and promising drug candidates. To do this, the authors assessed cybrid cells containing a mtDNA mutation that led to defective complex I, which is the entry point for electrons from NADH into the respiratory chain and which is frequently affected in mtDNA disorders. They screened these complex I-defective cells against a chemical library to select compounds that induced a compensatory increase in respiratory chain content. In
parallel, they carried out a genome-wide CRISPR screen to find genes that, upon inactivation, enabled cells to grow when galactose was the major energy source. Galactose forces cells to use oxidative phosphorylation to make ATP; hence, cells with the complex I defect die unless gene loss facilitates a compensatory mechanism. What was fascinating was the fact that both screens identified a common target. One of the hits from the chemical library, I-BET 525762A, is an inhibitor of bromodomain proteins, while the CRISPR screen identified the gene that encodes the bromodomain-containing protein 4 (Brd4). Thus, inhibiting the activity of Brd4 helps cells compensate for the complex I defect. What is the mechanism? Bromodomain proteins such as Bdr4 bind acetylated lysines on histones to coordinate and alter gene expression. It seems that blocking Brd4 increases expression of genes that allow mitochondria to compensate for complex I defects by rewiring metabolism so as to bypass this respiratory chain blockade (Figure 1). Interestingly, the inhibition of Bdr4 only compensated for defects in complex I, not for those in complexes III or IV, consistent with selective rewiring of metabolism to bypass complex I rather than a general protection of mitochondria. This encouraging specificity suggests that the screens can be extended to find therapies for defects in other complexes and emphasizes that therapies for mtDNA diseases are not generic, but need to be tailored to the affected complex. The finding of Brd4 as a druggable therapeutic target joins the list of approaches, such as the activation by small molecules of the transcriptional coactivator PGC-1a (Viscomi et al., 2011), to altering gene
expression and thereby enabling the cell to cope with defective mtDNA. There are also resonances with the recent finding from the Mootha lab that hypoxia triggers activation of a suite of genes under the regulation of HIF-1a that can suppress phenotypes associated with mutated mtDNA (Jain et al., 2016). More generally, these studies highlight the potential of small-molecule therapies that intervene upstream of mitochondrial biogenesis to compensate for mitochondrial defects (Smith et al., 2012). Intriguingly, these findings also point to how mitochondrial function itself is altered by the shift in gene expression, and thus suggest more direct interventions in mitochondria. It seems that the inhibition of Brd4 works by bypassing complex I, so that electrons which would otherwise build up on NADH go by a ‘‘rewired’’ metabolism to the CoQ pool (Figure 1). This shortcut for electrons should bypass the bottleneck of a defective complex I, thereby allowing the rest of the respiratory chain to carry out proton pumping at complexes III and IV and thus produce ATP, albeit less efficiently. In addition, it also allows oxidation of the NADH pool by providing a pathway to oxygen and avoiding the metabolic inhibition caused by an increase in the NADH/NAD+ ratio (Titov et al., 2016). Similar therapeutic bypassing of complex I has been achieved by the ectopic expression of the NADH dehydrogenase I from yeast, which takes electrons from NADH to CoQ without pumping protons across the inner membrane (Sanz et al., 2010). Thus, the mode of action of Brd4 inhibition shows the therapeutic potential of introducing shortcuts into the respiratory chain to bypass pathological blockades. Perhaps this may lead
Molecular Cell 64, October 6, 2016 ª 2016 Elsevier Inc. 5
Molecular Cell
Previews Therapy Nuclear genome
Bdr4
cuts to be an elegant screening approach, which may lead on to therapies that act by bypassing defects in the respiratory chain. Occasionally, shortcuts can work out well!
Conventional REFERENCES
to complex I Rewiring
Barrow, J.J., Balsa, E., Verdeguer, F., Tavares, C.D.J., Soustek, M.S., Hollingsworth, L.R., IV, Jedrychowski, M., Vogel, R., Paulo, J.A., Smeitink, J., et al. (2016). Mol. Cell 64, this issue, 163–175.
to the CoQ pool NADH NAD+ 2H+ 4H+ Q QH2
Defective complex I
SDH
mtDNA
4H+
Cyt c
2H+
Complex IV
mtDNA mutation
Figure 1. Rewiring of Respiration from NADH to the CoQ Pool by Inhibting Brd4 The mitochondrial respiratory chain complexes I, II (SDH, succinate dehydrogenase), III, and IV are shown, with the mobile electron carriers CoEnzyme Q (Q) and cytochrome c (cyt c) also illustrated. A mutation in mtDNA of one of the genes encoding a complex I subunit causes a defect in respiration, preventing oxidation of NADH. Inhibition of the nuclear protein Brd4 removes suppression of an alternative pathway that allows electrons from NADH to bypass complex I and go to the CoEnzyme Q pool. This shortcut enables complexes III and IV to pump protons across the mitochondrial inner membrane, thereby building up a proton motive force so that mitochondria can still make ATP despite the complex I defect.
onward to other small molecule therapies that could act directly on the mitochondria as shortcuts to bypass respiratory defects? Such approaches have been piloted in the past (Eleff et al., 1984), and extended more recently (Ehinger et al., 2016), while some current approaches such as idebenone may act in this way.
6 Molecular Cell 64, October 6, 2016
Ehinger, J.K., Piel, S., Ford, R., Karlsson, M., Sjo¨vall, F., Frostner, E.A., Morota, S., Taylor, R.W., Turnbull, D.M., Cornell, C., et al. (2016). Nat. Commun. 7, 12317. Eleff, S., Kennaway, N.G., Buist, N.R., DarleyUsmar, V.M., Capaldi, R.A., Bank, W.J., and Chance, B. (1984). Proc. Natl. Acad. Sci. USA 81, 3529–3533.
Q QH2
Complex III
4H+
4H+ 1/2 O H2 O 2
More work is required, however, to be sure that these compounds are effective metabolic shortcuts in vivo. Even so, the results shown here suggest that it is well worth developing further small molecules to facilitate metabolic shortcuts in vivo. In summary, this work by Barrow et al. (2016) has shown these heuristic short-
Hyslop, L.A., Blakeley, P., Craven, L., Richardson, J., Fogarty, N.M., Fragouli, E., Lamb, M., Wamaitha, S.E., Prathalingam, N., Zhang, Q., et al. (2016). Nature 534, 383–386. Jain, I.H., Zazzeron, L., Goli, R., Alexa, K., Schatzman-Bone, S., Dhillon, H., Goldberger, O., Peng, J., Shalem, O., Sanjana, N.E., et al. (2016). Science 352, 54–61. Pfeffer, G., Majamaa, K., Turnbull, D.M., Thorburn, D., and Chinnery, P.F. (2012). Cochrane Database Syst. Rev. 4, CD004426. Sanz, A., Soikkeli, M., Portero-Otı´n, M., Wilson, A., Kemppainen, E., McIlroy, G., Ellila¨, S., Kemppainen, K.K., Tuomela, T., Lakanmaa, M., et al. (2010). Proc. Natl. Acad. Sci. USA 107, 9105–9110. Smith, R.A., Hartley, R.C., Cocheme´, H.M., and Murphy, M.P. (2012). Trends Pharmacol. Sci. 33, 341–352. Titov, D.V., Cracan, V., Goodman, R.P., Peng, J., Grabarek, Z., and Mootha, V.K. (2016). Science 352, 231–235. Viscomi, C., Bottani, E., Civiletto, G., Cerutti, R., Moggio, M., Fagiolari, G., Schon, E.A., Lamperti, C., and Zeviani, M. (2011). Cell Metab. 14, 80–90. Wallace, D.C. (2010). Environ. Mol. Mutagen. 51, 440–450.