A chemical screen for inhibitors of mitochondrial division
The growth phenotypes of mitochondrial division- and mitochondrial fusion-defective mutants formed the basis of our screen for small molecules that inhibit mitochondrial division. Specifically, exposure of yeast cells harboring the temperature-sensitive fzo1-1 allele to the non-permissive temperature causes mitochondrial membranes to fragment and as a consequence, cells quantitatively lose mtDNA and are unable to grow on the non-fermentable carbon source glycerol (, fzo1-1, YPEG, 37°C). These cells can still be propagated if grown using a fermentable carbon source, such as glucose (, fzo1-1, YPD, 37°C). Mutations in components required for division, such as DNM1, which encodes the mitochondrial division dynamin, suppress division-mediated mitochondrial fragmentation and also mitochondrial DNA loss in fzo1-1 cells and thus also suppress the glycerol growth defect at the non-permissive temperature (, fzo1-1 Δdnm1). In yeast, loss of mitochondrial division has virtually no associated growth phenotype under laboratory conditions (, Δdnm1, YPD and YPEG).
Chemical screen for mitochondrial division inhibitors
Thus, to identify mitochondrial division inhibitors, we performed a straightforward growth-based screen to identify small molecules that suppress the glycerol growth defect of fzo1-1
cells. To enhance the steady state intracellular concentration of the drugs in yeast cells, null mutations in the PDR1
genes, which encode for transcriptional regulatory proteins that positively control the expression of multi-drug resistance ABC transporters, were created in the strains used in the screen and in the characterization of the small molecules (Rogers et al., 2001
). These additional mutations had no effect on mitochondrial division and fusion in cells (not shown). Initially, small molecules were screened at single concentrations between 10-100 μM in primary and secondary assays due to the limited amount of the compounds obtained. When tested alone, DMSO, the solvent used to solubilize the small molecules, had no significant effects in any of the assays described.
We screened approximately 23,000 compounds, representative of several commercially available libraries, using the primary growth assay-based screen (, 1° screen). All compounds identified were further tested in a secondary analysis for their effects on steady state mitochondrial morphology in yeast (, 2° screen). The steady state structure of mitochondria in yeast and mammalian cells is an indicator of the relative rates of mitochondrial division and fusion in cells (Bleazard et al., 1999
; Hermann et al., 1998
; Nunnari et al., 1997
; Sesaki and Jensen, 1999
). Specifically, the presence of fragmented mitochondrial structures indicates that mitochondrial fusion is selectively attenuated. In contrast, the presence of net-like mitochondrial structures indicates that mitochondrial division is selectively attenuated. We assayed for these morphological phenotypes using a mitochondrially targeted GFP that is efficiently localized to both wild type and respiratory deficient mitochondria. In this secondary assay, small molecules were judged to be positive if they produced a mutant phenotype in greater than 20% of the cell population. As summarized in , the overall frequency of division inhibitor hits (total of 3) identified using our primary and secondary assays was extremely low, indicating our screening strategy was selective.
Mitochondrial Division Inhibitor Screen
Characterization of the mitochondrial division inhibitor, mdivi-1
We identified three potential mitochondrial division inhibitors and pursued the most efficacious, which is a derivative of quinazolinone, termed mdivi-1 (for mitochondrial division inhibitor, ). As expected, we observed that mdivi-1 suppresses the glycerol growth defects in fzo1-1 cells (). Significantly, mdivi-1 also suppressed the glycerol growth defects observed in other mutants defective in the mitochondrial fusion pathway, such as mgm1-5 cells, which contain a mutated copy of the gene encoding the mitochondrial inner membrane fusion dynamin, Mgm1 (mgm1-5, ). In addition, we observed that mdivi-1 causes the rapid (≤ 5 min), reversible and dose-dependent formation of net-like mitochondria in wild type cells, with an IC50 of approximately 10 μM (). These observations indicate that mdivi-1 acts as a general suppressor of mitochondrial fusion defects by selectively inhibiting mitochondrial division.
We also directly measured the rates of division and fusion events in yeast by time-lapse fluorescence microscopy after the addition mdivi-1. Time-lapse analysis of mitochondria in mdivi-1 treated cells indicates that no detectable division events occurred, but that fusion events were observed (not shown). In addition, mdivi-1 did not change the net-like morphology of mitochondria in Δdnm1 cells, further suggesting that it blocks division by acting in the Dnm1-dependent division pathway (not shown). Taken together, our results indicate that mdivi-1 is a selective inhibitor of mitochondrial division.
To address the specificity of mdivi-1 effects on mitochondrial division, we examined its effect on two cellular structures that, when perturbed, can cause indirect changes in mitochondrial morphology: the actin cytoskeleton and the peripheral ER network. These structures are routinely examined in yeast mitochondrial morphology mutants as a test for the specificity of the mitochondrial phenotype (McConnell et al., 1990
). Treatment of cells with 100 μM mdivi-1 caused the formation of mitochondrial net-like structures, but did not result in significant changes in either the actin cytoskeleton (, 100%, n=100, left panel) or the peripheral ER network (not shown, 100%, n=50), as compared to control DMSO-treated cells. In contrast, addition of the F-actin depolymerizing compound Latrunculin-A after mdivi-1 treatment caused disassembly of actin cables and patches and caused mitochondrial nets to collapse and aggregate, consistent with published observations (Bleazard et al., 1999
)(, right panel). These observations indicate that the effect of mdivi-1 on mitochondrial morphology is not the result of secondary changes in either the actin cytoskeleton or ER network and are consistent with our data indicating that mdivi-1 produces net-like structures by directly attenuating mitochondrial division.
Structure-activity analysis of mdivi-1
To determine which structural features are important for the effects of mdivi-1 on mitochondrial division, we used ChemNavigator to search available compound databases for small molecules that uniquely represented key structural features of mdivi-1. We tested a total of over 30 mdivi-1 like molecules for their effects on mitochondrial morphology in yeast. A summary of representative compounds, termed A-H, and their efficacy is shown in and . In no case did we identify a compound that was more efficacious than mdivi-1 (compound A); rather most compounds had the same (, compound B), moderate (, compounds C-E), or poor/no efficacy (, compounds F-H) when examined in our assay for mitochondrial morphology (). We utilized these derivatives as tools to help determine the target and the specificity of mdivi-1.
Compounds related to mdivi-1 have different efficacies
mdivi-1 structure-activity analysis
Analysis of our structure-function results indicates that at least two structural features are important for the efficacy of mdivi-1 (, shown in red): an unblocked sulfhydryl moiety on the 2-position of the quinazolinone and limited rotation about the 3-position nitrogen-phenyl bond. Indeed, the bulky ortho chloro substituent of the phenyl ring attached at the N-3 of mdivi-1 predicts that mdivi-1 is a mixture of two atropisomers: isomers that in this case are distinct because rotation about the nitrogen-phenyl bond is prevented or greatly slowed by the rotational energy barrier created by the bulky ortho chloro substituent. Consistent with this, mdivi-1 can be resolved into two distinct species by chiral chromatography (not shown). Assuming that one mdivi-1 isomer is selectively active in attenuating mitochondrial division, the efficacy of mdivi-1 would be two fold greater than our experimental data indicate. Taken together, our preliminary structure-activity analysis indicates that the ability of mdivi-1 to inhibit mitochondrial division is dependent upon stringent structural requirements, consistent with it being a selective inhibitor.
mdivi-1 is a selective inhibitor of the mitochondrial division dynamin
Using a coupled assay for GTPase activity and EM analysis, we previously characterized the kinetic and structural properties of recombinant Dnm1 (Ingerman et al., 2005
)). Our analysis indicates that Dnm1 self-assembly greatly stimulates GTP hydrolysis and in the presence of non-hydrolyzable GTP analogs, Dnm1 forms spiral structures, whose diameters correspond to those of mitochondrial constriction sites in vivo. These data strongly suggest a model where the self-assembly of Dnm1 drives mitochondrial constriction during division in vivo.
To test whether mdivi-1 targets Dnm1, we tested its effects on self-assembly stimulated Dnm1 GTPase activity. As shown in , mdivi-1 inhibited Dnm1 GTPase activity in a dose dependent manner, with an estimated IC50 of 1-10 μM, which is lower, but consistent with the IC50 observed for the effects of mdivi-1 on the formation of mitochondrial net-like structure in vivo. These observations suggest that mdivi-1 attenuates mitochondrial division in vivo by inhibiting Dnm1.
The target of mdivi-1 is the mitochondrial division dynamin, Dnm1
Our observations raise the question of whether mdivi-1 is simply a general inhibitor of GTPase super family members and/or DRPs. Thus, we examined the effects of mdivi-1 on Dnm1 1-338, the monomeric GTPase domain of Dnm1, which lacks other DRP-specific regions (Ingerman et al., 2005
). As shown in , mdivi-1 had no effect on Dnm1 1-338 GTP hydrolysis, indicating that it is not a general inhibitor of GTPases and suggesting that mdivi-1 inhibits Dnm1 by binding to other DRP regions or, more interestingly, to a region dependent on multiple domains. We also tested whether mdivi-1 targets other DRP family members by testing its effects on dynamin-1, which functions during endocytosis in the scission of clathrin coated pits from the plasma membrane. Significantly, mdivi-1 had no effect on either basal (not shown) or assembly-stimulated rates of GTP hydrolysis for dynamin-1 (). Together these results suggest that mdivi-1 is a selective inhibitor of the mitochondrial division DRP.
To further test the hypothesis that mdivi-1 blocks mitochondrial division in vivo by inhibiting Dnm1 GTPase activity, we tested mdivi-1 like molecules of variable efficacy for their ability to inhibit division in vivo ( and ). The results from multiple independent double-blinded experiments demonstrated that there is a tight correlation between the efficacy of a given derivative to block mitochondrial division in vivo and Dnm1 GTPase activity in vitro ( and ). This observation further supports the conclusion that the mitochondrial division DRP, Dnm1, is the target of mdivi-1 in vivo.
To gain insight into the mechanism of mdivi-1 inhibition of Dnm1, we performed a detailed kinetic analysis of the effects of mdivi-1 on Dnm1 GTPase activity (). From our analysis, we estimate that the Ki of mdivi-1 for Dnm1 is 1-50 μM, which is in the range of the IC50
for mdivi-1in vivo. Our kinetic data fit well to the concerted transition model of Monod, Wyman, and Changeux, which describes the behavior of allosteric proteins that can form oligomers of identical subunits (Fig. S1
). Thus, based on this model, the Dnm1 dimer, which we have previously shown to be the building block for assembled Dnm1, is predicted to exist in either one of two states: R (relaxed and assembled) or T (taut and unassembled), that are in equilibrium with one another, where the R state has a relatively high affinity for GTP and T has a relatively low affinity for GTP. This model predicts that mdivi-1 is an allosteric inhibitor with relatively high affinity for the T (unassembled) state and relatively low affinity for the R (assembled) state of Dnm1 and thus implies that mdivi-1 inhibits GTP hydrolysis by blocking the self-assembly of Dnm1. Indeed, the kinetic effects that mdivi-1 has on Dnm1 mimic those observed under high ionic conditions, which antagonize assembly (Ingerman et al., 2005
). Specifically, we observed that mdivi-1 increases the apparent K0.5
for GTP, lowers the apparent Vmax for GTP hydrolysis, and causes an increase in the Hill coefficient observed for GTP in the Dnm1 GTP hydrolysis reaction ().
We directly tested the hypothesis that mdivi-1 inhibits Dnm1 self-assembly by examining its effects by EM on Dnm1 spirals formed in the presence of the non-hydrolyzable GTP analog, GMPPCP. As shown in , when present at the start of the self-assembly reaction, mdivi-1 quantitatively blocked GMPPCP-dependent Dnm1 self-assembly in a concentration range similar to its effects on both Dnm1 GTPase activity in vitro and mitochondrial division in vivo. Interestingly, when mdivi-1 was added after the formation of GMPPCP-Dnm1 spirals, the compound had no discernable effect, i.e. failed to promote their disassembly (not shown). Given that Dnm1 spirals formed in the presence of non-hydrolyzable GMPPCP are likely stable, not dynamic structures, our observations suggest that mdivi-1 blocks self-assembly by inhibiting Dnm1 polymerization and not by promoting disassembly.
Consistent with this, we observed that mdivi-1 inhibits GTP hydrolysis of the dimeric middle domain mutant, Dnm1G385D, which is defective for self-assembly (Ingerman et al) (Fig. S2A and B
). Analysis of the kinetic data for Dnm1G385D indicated that, in contrast to the concerted transition mechanism for Dnm1, mdivi-1 likely functions as a mixed type inhibitor of Dnm1G385D with a Ki of 1-4 μM, which significantly lowers the affinity of Dnm1 for GTP. These observations indicate that mdivi-1 targets the fundamental building block of the Dnm1 spiral structure to block polymerization. This mechanism is similar to the action of latrunculin A, which alters the actin monomer subunit interface, alters nucleotide binding, and prevents polymerization of F-actin filaments (Morton et al., 2000
). Taken together, our analysis of the mechanism of mdivi-1 effects on Dnm1 activity suggests that it inhibits division by binding to an allosteric site that blocks or retards a conformational change required for Dnm1 self-assembly and GTP hydrolysis.
mdivi-1 attenuates mammalian mitochondrial division
Drp1, the mammalian mitochondrial division DRP, has a high degree of identity to its yeast ortholog, Dnm1 (Labrousse et al., 1999
). This encouraged us to exploit the chemical genetic approach and examine the effects of mdivi-1 on mitochondrial morphology in mammalian cells. In mammalian cells, when mitochondrial division is retarded by expression of dominant-negative Drp1 or by RNAi of mitochondrial division proteins, tubular mitochondria become progressively more interconnected to form net-like structures, and also collapse into degenerate perinuclear structures (Smirnova et al., 2001
; Smirnova et al., 1998
The addition of mdivi-1 to mammalian cells (COS) in culture caused a rapid and reversible formation of mitochondrial net-like and degenerate perinuclear structures, consistent with an attenuation in mitochondrial division ( and Supplemental Table 1
). The IC50
of mdivi-1 for its effects on mitochondrial morphology in mammalian cells (IC50
≈ 50 μM) is comparable to that observed for the effect of mdivi-1 on mitochondrial morphology in yeast (IC50
≈ 10 μM). In addition, we observed that the mdivi-1 structural derivatives that do not affect mitochondrial morphology in yeast and do not inhibit Dnm1 GTPase activity, also do not affect mitochondrial morphology in COS cells (Supplemental Table 4
). Thus, the characteristics of mdivi-1’s effect on mitochondria in mammalian cells are similar to those observed in yeast cells and, by extension, suggest that mdivi-1 inhibits mitochondrial division in mammalian cells by inhibiting Drp1 activity.
mdivi-1 inhibits mitochondrial division in mammalian cells by attenuating Drp1 self-assembly
To test whether mdivi-1 targets Drp1, we examined its effects on recombinant Drp1 GTPase activity in vitro. In contrast to its effects on Dnm1, mdivi-1 had no effect on Drp1 GTP hydrolysis. However, the maximal GTP hydrolysis rate of Drp1 was relatively low (2.1 min-1) and we failed to detect the formation of GMPPCP-dependent Drp1 spirals in vitro by EM, even under molecular crowding conditions (not shown), indicating that recombinant Drp1 is not capable of self-assembly and thus is not fully functional.
Thus, we asked whether over expression of Drp1 in mammalian cells could rescue the effects mdivi-1 on mitochondrial morphology. As shown in , the concentration of mdivi-1 required to observe either net-like or collapsed/degenerate perinuclear mitochondrial structures in cells was significantly higher in the population of cells over expressing Drp1 (IC50 between 75-100 μM) as compared to those transfected with a control empty vector (IC50 between 10-50 μM). In addition, in agreement with published work, depletion of Drp1 by RNAi also caused the formation of net-like or collapsed perinuclear mitochondrial structures in cells and treatment of these cells with mdivi-1 did not produce any additional changes to mitochondrial morphology (not shown). These observations substantiate our conclusion that the mitochondrial division dynamin is the target of mdivi-1 in both yeast and mammalian cells.
mdivi-1 attenuates mammalian mitochondrial division and Drp1 self-assembly during apoptosis
Drp1-mediated mitochondrial division in mammalian cells is stimulated by apoptotic signals, such as staurosporine (STS), which promote intrinsic apoptotic cell death via Bcl-2 proteins (Frank et al., 2001
). We examined the effects of mdivi-1 on mitochondrial fragmentation caused by STS in mammalian COS cells ( and Table 5). As shown previously, STS stimulation caused a significant increase in mitochondrial fragmentation in cells ((Frank et al., 2001
), and Supplemental Table 2
). In comparison, mitochondrial fragmentation was significantly reduced in cells treated with both STS and mdivi-1, ( and Table 5). As a control, we observed that expression of dominant-negative Drp1 or RNAi mediated depletion of Drp1 also inhibited STS-induced mitochondrial fragmentation to a similar degree as mdivi-1, which is in agreement with published observations (not shown) (Frank et al., 2001
). These observations indicate that mdivi-1 inhibits apoptosis-stimulated Drp1-dependent mitochondrial division.
To gain insight into the mechanistic basis of mdivi-1 inhibition of Drp1-mediated mitochondrial division, we exploited the fact that during apoptosis, Drp1 self-assembly and recruitment to mitochondria are significantly increased and these events can easily be assayed by monitoring the behavior of GFP-Drp1 in cells (Frank et al. 2001
). As shown in , addition of the apoptotic stimulant STS caused a decrease in diffusely localized GFP-Drp1 and a concomitant increase in the number of GFP-Drp1 clusters and GFP-Drp1 clusters associated with mitochondria (compare green fluorescence in - STS/DMSO with +STS/DMSO cells). In contrast, in cells treated with STS and mdivi-1 (compound A) or the active compound B, the majority of GFP-Drp1 was diffusely distributed in the cytoplasm, in a manner similar to that observed for GFP-Drp1 in cells that were not treated with STS (, compare -STS/DMSO and +STS/DMSO with +STS/A and +STS/B). Consistent with our previous observation () and as expected, mitochondria in cells treated with STS and mdivi-1 (compound A) or the active compound B were tubular as compared to fragmented mitochondria present in cells treated with STS only (, in red). In addition, in cells treated with STS and either of the inactive mdivi-1 compounds, G and H, the localization pattern of GFP-Drp1 and the mitochondrial morphology was similar to cells that were treated only with STS (, compare +STS/DMSO with +STS/G and +STS/H). These observations indicate that mdivi-1 attenuates mitochondrial division during apoptosis by blocking Drp1 self-assembly and the recruitment of Drp1 assembled structures to mitochondria and are consistent with our biochemical data indicating that mdivi-1 inhibits the yeast ortholog, Dnm1, by blocking polymerization. Together, these observations indicate that mdivi-1 targets the mitochondrial division dynamin and acts in a mechanistically conserved manner in yeast and mammalian cells
mdivi-1 attenuates apoptosis by inhibiting mitochondrial outer membrane permeabilization
Given the inhibitory effect of mdivi-1 on STS-induced mitochondrial division, we tested whether mdivi-1 also retards apoptosis in mammalian HeLa cells. We initially examined the effects of mdivi-1 on the externalization of plasma membrane phosphatidylserine (PS), which is a relatively late event in apoptotic cell death (Fadok and Henson, 2003
). We quantified this event using fluorescently labeled (FITC)-annexin V, which is a Ca2+
-dependent phospholipid binding protein with a high affinity for PS, in conjunction with established fluorescent-activated cell sorter (FACS) methodology. As shown in , we observed that mdivi-1, but not the inactive mdivi-1 derivative, G, significantly inhibits STS-induced annexin V staining of non-necrotic cells as assessed by FACS analysis, indicating that mdivi-1 inhibits apoptosis. Significantly, although the mdivi-1 inhibition of STS-induced apoptosis was fractional, the extent of inhibition was comparable to that observed when the dominant negative Drp1K38A mutant is over expressed in HeLa cells and is consistent with mdivi-1 targeting Drp1 in vivo (Frank et al. 2001
and STS-induced apoptotic cells in Drp1: Drp1 K38A = 0.8 by annexin V analysis).
mdivi-1 and its active analogs attenuate apoptosis
To resolve the point at which mdivi-1 inhibits apoptosis, we examined its effect on the early MOMP event, measured by cytochrome c release. To directly stimulate MOMP and cytochrome c release, HeLa cells were microinjected with caspase-8 cleaved recombinant Bid (C8-Bid). C8-Bid directly (Kuwana et al., 2002
; Walensky et al., 2006
) or indirectly (Willis et al., 2007
) activates Bax/Bak, causing MOMP and cytochrome c release. As expected, cytochrome c release, as monitored by a cytochrome c-GFP fusion, was not stimulated in control cells injected with the marker Texas Red with or without the active mdivi-1 derivative, B ( and Supplemental Table 3
). In contrast, but also as expected, cytochrome c-GFP release was greatly stimulated in cells injected with C8-Bid, (, DMSO, cells stained with Texas red and Supplemental Table 3
). Significantly, the active mdivi-1 derivative, B, dramatically inhibited C8-Bid stimulated cytochrome c-GFP release, whereas the inactive mdivi-1 derivative, F had virtually no effect on C8-Bid stimulated cytochrome c-GFP release ( and Supplemental Table 3
). Similar effects of mdivi-1 on STS-induced cytochrome c release were also observed (Fig. S3
). Together our results indicate that mdivi-1 inhibits the activity of the mitochondrial division dynamin Drp1 and as a result impedes apoptosis early in the intrinsic pathway by blocking Bax/Bak dependent MOMP.
To further test this conclusion, we examined the effect of mdivi-1 on C8-Bid induced, BAK dependent cytochrome c release from isolated wild-type murine liver mitochondria (). As assessed by western blot analysis of mitochondrial derived supernatant and pellet fractions with anti-cytochrome c, mdivi-1 derivatives had no effect on cytochrome c release in vitro when tested alone (). In contrast and as expected, cytochrome c release was greatly stimulated by the addition of recombinant active C8-Bid (). Significantly, the addition of mdivi-1, as well as the active mdivi-1 like compounds B and C, appreciably blocked C8-Bid induced cytochrome c release, whereas mdivi-1 derivatives lacking efficacy, either failed or had relatively minor effects on cytochrome c release (, compare compounds A-C to D-H). In addition, the ability of mdivi-1 and active mdivi-1 compounds to inhibit C8-BID induced, BAX dependent cytochrome c release was examined in bak/bax double knockout mitochondria from MxCre bak-/-bax-/-livers () supplemented with monomeric full-length BAX.
mdivi-1 and its active analogs inhibit activated Bax/Bak-dependent MOMP in vitro
These findings indicated that mdivi-1 and active derivatives inhibit cytochrome c release by preventing the full Bid-dependent activation of Bax and Bak. Finally, as a control, we examined whether mdivi-1 derivatives directly affect Bid-dependent Bax induced membrane permeabilization using an established large unilamellar vesicle (LUV) release assay in which LUVs are preloaded with fluorescein dextran (Kuwana et al., 2002
). As shown in , the amount of fluorescein dextran released from LUVs by C8-Bid/Bax induced permeabilization was not affected by the addition of mdivi-1 and mdivi-1 derivatives, nor did the mdivi-1 compounds cause significant permeabilization on their own. These observations indicate that mdivi-1 does not block MOMP by directly inhibiting C-8 Bid activated Bax. Thus, together our data suggest that mdivi-1 attenuates MOMP by inhibiting the self-assembly of the mitochondrial division dynamin, which functions upstream, together with Bcl-2 proteins to directly stimulate Bcl-2 mediated outer membrane permeabilization. Consistent with this conclusion, we observed that a significant level of Drp1 is present and cofractionates with mitochondrial membranes in vitro (not shown).