Shutting Down the Pore: The Search for Small Molecule Inhibitors of the Mitochondrial Permeability Transition

Abstract

The mitochondrial permeability transition pore (PTP) is now recognized as playing a key role in a wide variety of human diseases whose common pathology may be based in mitochondrial dysfunction. Recently, PTP assays have been adapted to high-throughput screening approaches to identify small molecules specifically inhibiting the PTP. Following extensive secondary chemistry, the most potent inhibitors of the PTP described to date have been developed. This review will provide an overview of each of these screening efforts, use of resulting compounds in animal models of PTP-based diseases, and problems that will require further study.

1. Introduction

It has long been appreciated that the mitochondrial permeability transition, whose activity depends on a cyclosporin A (CsA)-sensitive channel or pore (PTP) of the inner mitochondrial membrane (IMM), can inappropriately open in a variety of human pathologies (for recent review, see¹). While the PTP opens transiently under normal physiological conditions, possibly acting as a mitochondrial Ca²⁺-release channel to help maintain cellular Ca²⁺ homeostasis, persistent pore opening appears to be a key feature of all necrotic cell death pathways. The latter results in the release of matrix Ca²⁺, cessation of oxidative phosphorylation and may result in mitochondrial swelling followed by rupture of outer mitochondrial membrane (OMM), ultimately culminating in cell death. Consequently, persistent opening of the PTP may contribute to a host of chronic and therapeutically challenging diseases (e.g., cardiac ischemic injury², stroke³, multiple sclerosis^4,5). A key pathophysiological hallmark of these diseases is that each is likely based in mitochondrial dysfunction triggered by Ca²⁺ and potentiated by oxidative stress.^6,7

While the molecular composition of the PTP is still incompletely understood, the previous longstanding belief that the PTP forms at the adjoining sites of the IMM and OMM through association of a variety of proteins in each membrane has not been supported by rigorous genetic tests; mitochondria missing these proteins still display a CsA–sensitive PTP opening¹. Other recent studies have suggested that dimers of the F_OF₁ ATP synthase (F-ATP synthase) are able to form channels with properties expected for the PTP⁸. The precise nature of the molecular transition of this complex from Mg²⁺-dependent ATP synthetic machine into a Ca²⁺-dependent pore has yet to be determined. Activation of the PTP has been described either through activation of cellular pathways that result in post-translational modification of specific proteins (e.g., see¹), or by a variety of cellular polymers⁹, and most importantly, reactive oxygen species generated by an array of cellular processes¹⁰. Yet, while progress has been made on the molecular identity of PTP, our ability to treat diseases in which inappropriate activation of the PTP plays a role has been limited to the use of CsA and its analogs that act on the pore through its matrix receptor and PTP modulator cyclophilin D (CyPD)¹¹. The restriction of cyclophilin inhibitors is that CyPD modulates the pore indirectly as shown by the fact that the PTP can still open when CyPD has been genetically ablated¹². Therefore, a number of programs have been aimed at identifying novel PTP inhibitors through the unbiased high-throughput screening (HTS) of several small molecule libraries^13,14,15. Since our recognition of the molecular components forming the PTP is only beginning to be appreciated, each represents a “phenotypic screen”; a widely-used alternative to the target-centric approaches that have dominated since the molecular biology revolution in the 1980s¹⁶. Phenotypic screens describe drug discovery based on functional biology for primary screening¹⁷. This strategy may be considered “neoclassic” since it is similar to physiology-based approaches used prior to the wide use of recombinant DNA technology but is enhanced by technology advances^18,19. Here we summarize these studies that have developed several novel series of potent inhibitors of the PTP that can be used as investigative tools, and possibly, developed into therapeutics for PTP-based diseases.

2. History of Small Molecule Inhibitors of the PTP

A modest number of small molecule inhibitors of the PTP have been reported in the literature. However, for many of these compounds, the exact mechanism of action is not known, a proof of selectivity is missing and they likely desensitize the PTP indirectly (e.g., limiting ROS production) or the mechanism of action shown to have no direct role in PTP activity (see also ^{20, 21}). A germane example is provided by TRO40303 (3,5-seco-4-nor-cholestan-5-one oxime-3-ol) (Figure 1), a compound which was advanced to Phase 2 clinical trials for cardiac ischemia^18,22. TRO40303 was initially described as a unique compound that inhibits PTP opening via an innovative mode of action – binding to the cholesterol site of an OMM protein representing a translocator protein of 18 kDa (TSPO). TSPO, a highly conserved, ubiquitous protein localized in the OMM, was also included in earlier, now discredited, models of the PTP that postulated the channel forms at the adjoining sites of the IMM and OMM (summarized in¹). Ensuing studies employing natural and synthetic ligands assessed the role of TSPO function in a number of pathological processes (e.g.,²³). Largely through the use of these compounds and biochemical associations, TSPO has been proposed to play a key role in the formation of the PTP and was associated with cell death through its participation in PTP formation in many human diseases²⁴. The role of TSPO in the function of the PTP was tested critically through the generation of mice in which the gene encoding TSPO had been conditionally eliminated²⁵. The results showed that (i) TSPO plays no role in the regulation or structure of the PTP, (ii) endogenous and synthetic ligands of TSPO do not regulate PTP activity through TSPO, (iii) OMM regulation of PTP activity occurs through a mechanism that does not require TSPO, and (iv) hearts lacking TSPO are as sensitive to ischemia-reperfusion injury as hearts from WT mice. These results call into question a wide variety of studies implicating TSPO in a number of pathological processes through its actions on the PTP and eliminate TSPO as having any role in PTP formation or regulation²⁵. Recent clinical studies have also demonstrated that TRO40303 had no clinical benefit in the treatment of human cardiac ischemia/reperfusion injury²⁶.

Figure 1.

Chemical structures of most relevant PTP inhibitors.

For all practical purposes then, relevant probes targeting the PTP are limited to CsA, a cyclic peptide from fungus Tolypocladium inflatum, and its derivatives Debio-025 (Alisporivir) and NIM811 (Figure 1). These drugs, however, have limited utility since (i) CsA is not a blocker of the PTP¹², and therefore its biological potency is limited by that of its target, CyPD, (ii) CsA binds to and inhibits the action of all members of the CyP family (16 different CyP proteins are encoded by mammalian genomes)²⁷. As a result, through interaction with another member of this family, CyPA, CsA also mediates inhibition of calcineurin resulting in immunosuppression, a major side effect of CsA-based therapy for PTP-dependent diseases²⁸, (iii) non-immunosuppressive CyPD inhibitors derived from CsA inhibit all CyPs to some extent, not just the mitochondrial isoform (e.g., ²⁹), and (iv) the use of CsA to treat PTP-based neurological diseases is challenging due to its limited blood/brain barrier penetration³⁰. Nevertheless, CsA or non-immunosuppressive analogs Debio-025 and NIM811, were demonstrated to protect dystrophic cells against mitochondria-mediated death and showed efficacy in a variety of animal models of collagen VI and Duchenne muscular dystrophies^31–32, suggesting that therapies targeting the PTP may be developed from these molecules. Recently, in order to drive CsA selectivity toward CyPD, Warne and colleagues synthesized a mitochondrially targeted CsA by tethering a quinolinium cation to the drug⁵. The resulting molecule, JW47 (Figure 1), was more potent than CsA in increasing the CRC of isolated rat liver mitochondria and protective in mouse model of experimental multiple sclerosis⁵.

Yet, because of the extremely high therapeutic potential of PTP inhibition as well as the paucity and limitations of known inhibitors, several groups have embarked on individual programs aimed at identifying and developing novel, selective, non-peptide inhibitors of the PTP through HTS strategies of several chemical libraries.

3. High-Throughput Screening (HTS)

Screening of libraries of small molecules consisting of several hundreds of thousands individual compounds requires the use of automated robot-based technology (available at pharmaceutical companies and specialized screening centers) as well as miniaturization of all assays to accommodate the volumes allowed in 1536-well microtiter plates, or ≤10 μl. Given these constraints, the assays to be used need to be robust (i.e. show good signal-to-background ratio), require a very limited number of additions, and the model system utilized should be stable within the length of the experiment. Considering the latter, most HTS screens employ isolated targets (target-based screens) or cultured mammalian cells. However, both approaches are not possible in the case of PTP. First, the molecular nature of the PTP has only recently been identified and pore closed-to-open transitions remain to be fully understood. The only “isolated” PTP assay is the recording of Ca²⁺ and oxidant-induced currents of blue-native polyacrylamide gel electrophoresis (BN-PAGE) separated F-ATP synthase dimers upon their reconstitution in planar lipid bilayers^{8, 33, 34}, which is incompatible with HTS format. Thus, a target-based approach was not feasible in the identification of PTP small molecule inhibitors. Second, attempts to establish HTS for search for PTP inhibitors using intact cells was abandoned in part due to the inherent problems associated with the available tests for the assessment of the PTP opening in situ, eliminating cellular profiling approaches. The use of isolated mitochondria (outlined below) allows phenotypic screens with higher reproducibility and a more direct measure than is possible for intact cell assays. Consequently, all HTS for small molecule inhibitors of the PTP used freshly prepared, isolated mitochondria as the basis for the primary screening, which was directly amenable to secondary and confirmatory screens. Moreover, a second compelling argument is that potentially useful but cell-impermeant compounds would be missed if intact cells were used, while these would be identified in isolated mitochondria. The shortcoming of cell-impermeability of these molecules could then be overcome by medicinal chemistry efforts.

As a result, primary screens have been based on monitoring the osmotic swelling (or lack of) of mitochondria, a process that follows Ca²⁺ triggered PTP opening in isolated energized organelles suspended in buffered isotonic sucrose-based medium³⁵. This routine assay has been modified to minimize the number of additions (therefore, the number of readings) and chemicals used (therefore, assay interference), as well as to “cast a wide net”, thereby maximizing the number of primary hits. It was easy to predict that many “hits” would turn out to be false positives because any compound interfering directly or indirectly with the Ca²⁺ uptake will score as a positive not because it inhibits PTP opening, but because it prevents Ca²⁺ uptake into mitochondrial matrix, a requirement for PTP induction. These false positives would include inhibitors of the respiratory chain, of Pi and substrate uptake, of the Ca²⁺ uniporter, and uncouplers. In order to screen out false positives, a secondary screening was established based on the fluorescence quenching of rhodamine 123 (Rh123), a fluorescent cation that is readily accumulated by energized mitochondria due to inside negative ΔΨ³⁶. As it concentrates in the matrix, rhodamine undergoes a process of fluorescence quenching. Compounds that interfere with the buildup or maintenance of the IMM potential will prevent probe accumulation, and are easily identified as false positives by this protocol (Figure 2).

Figure 2.

Assays used in HTS campaigns to identify small molecular inhibitors of the PTP. A) Mitochondrial swelling. Left panel, changes in mitochondrial volume due to PTP opening is measured as a light scattering decrease at 540 nm. Upon addition of mitochondria suspension’s turbidity, thus light scattering, increases. Ca²⁺ induces PTP opening which results in a decrease of the signal. Right panel, transmission electron micrographs of isolated mouse liver mitochondria reflecting their shape before and after PTP occurrence. B) Rhodamine 123 (Rh123) uptake assay which measures inner mitochondrial membrane potential. Rh123, a lipophilic cation, is accumulated only by respiring mitochondria due to inside negative ΔΨ resulting in the fluorescence quenching. C) Calcium Retention Capacity assay; Extra-mitochondrial Ca²⁺ fluxes are measured fluorimetrically using Calcium Green-5N, a low affinity membrane-impermeant probe that increases its fluorescence emission upon Ca²⁺ binding. Mitochondria are routinely suspended in isotonic buffer that also contains Calcium Green-5N and are subjected to train of Ca²⁺ pulses. Each addition results in a spike that represents first, the increase in fluorescence (due to extra-mitochondrial Ca²⁺ binding to Calcium Green-5N) followed by a decrease in fluorescence due to the accumulation of added Ca²⁺ into mitochondria. Mitochondrial accumulation of Ca²⁺ continues on addition of subsequent pulses until the “threshold” for PTP activation is reached and all Ca²⁺ is released from mitochondria (represented by the dramatic terminal fluorescence increase) due to opening of the PTP. Inhibitors, e.g. CsA, shift the “threshold” for PTP activation to higher Ca²⁺ loads.

On repeated trials, in our hands the above mentioned assays exhibited a mean Z score of 0.7 (Z-factor is an attempt to quantify the robustness and suitability of a particular assay for use in a full-scale HTS; HTS require Z score above 0.5³⁷), validating isolated organelles as a feasible samples for HTS and establishing the mitochondrial swelling assay as a highly effective means to identify small molecule inhibitors of the PTP. In addition, the Rh123 uptake assay was able to point out most of the false positives. The remaining class of possible false hits, i.e. Ca²⁺ uniporter and Pi carrier inhibitors, were identified by a confirmatory assay, the calcium retention capacity (CRC) (e.g., ³⁸). This test measures the amount of Ca²⁺ that mitochondria can accumulate and retain before the precipitous release that marks PTP opening (see Figure 2 for details).

Although experimental differences are present in each of the two major screening efforts reported to date, compounds that have survived all three tests have been defined as true positives with a high probability of targeting the PTP. Given these differences, the most informative are comparisons of these novel PTP inhibitors to a recognized “standard” PTP inhibitor CsA. Within each screen typically several lead compounds were selected based on their reactive functionalities and synthetic tractability as well as inhibitory activity. Their inhibitory activity was further improved by the use of standard medicinal chemistry methods through an iterative design, synthesis and evaluation approach to establish structure/activity relationships (SAR). Through this general methodology, three classes of PTP inhibitors have been identified and characterized. In each case, PTP inhibition by most potent derivative exceeds that observed for CsA (see the table in Figure 3) and actions appear to be synergistic with CsA, each likely defining a new PTP target(s) independent of CyPD. Furthermore, in the absence of structural information on their molecular target within the PTP complex, SAR studies were used to identify the structural elements of the initial hit essential for inhibition of the PTP activity. Individual publications listed below contain additional experimental details regarding each HTS effort.

Figure 3.

Chemical structures of HTS hits from cinnamic anilide, isoxazole and benzamide series and of their most potent analogs; and their respective effects on isolated mouse liver mitochondria. CsA is included for comparison.

3.1. Cinnamic Anilides

Initial attempts to identify novel PTP inhibitors were reported by Fancelli et al. who outlined the results of HTS of a commercial library¹³. The initial hit in this series (1 in Figure 3A) was active as a PTP inhibitor, yet was only 70% as effective as CsA in routine CRC assays on isolated mouse liver mitochondria. Subsequent expansion of this class of molecules through SAR studies led to the identification of cinnamic anilides (CA) with inhibitor potential 2.5-fold higher than CsA at 5 μM under the specific conditions employed (2 in Figure 3A). The most potent compounds inhibited PTP opening in response to several stimuli, including calcium overload, oxidative stress and activation by chemical cross-linkers. Subsequent data showed that most potent CAs were stable for up to 30 min following infusion into laboratory animals (mice, rabbits) allowing their assessment as potential therapies in rabbit models of acute myocardial infarction. Short-term exposure was able to induce a significant pharmacodynamic effect in the employed model of myocardial infarction, yet no significant improvement over CsA treatment was observed. By similar criterion, a variant CA was shown to cross the blood-brain barrier and significantly inhibit PTP activation in mitochondria prepared from brains of treated mice³⁹. This observation led to tests of the therapeutic potential of CA PTP inhibitors in murine models of amyotrophic lateral sclerosis, as represented by transgenic mice expressing the human form of G37R superoxide dismutase (G37R-hSOD1). These studies demonstrated that GNX-4728 (a member of CA class PTP inhibitors) slowed disease progression, as evidenced by a 2-fold extension of lifespan. Treatment was also able to prevent motor neuron and mitochondrial degeneration, attenuated spinal cord inflammation and preserved neuromuscular junction innervation in the diaphragm of G37R-hSOD1 mice.

3.2. Isoxazoles

More details are available on PTP inhibitors identified by HTS of the NIH Molecular Libraries Small Molecule Repository (MLSMR) collection developed from a variety of sources. Here, we screened over 350,000 compounds at a concentration of 10 μM in isolated mouse liver mitochondria using the screening assays outlined above^20,14. The validated hits, along with CsA and a representative CA as positive controls, were subsequently assayed using the CRC test. Among all of the validated hits, compounds from the isoxazole (IZ) chemotype exhibited comparable activity to CsA in isolated mitochondria and also increased the CRC of permeabilized murine embryonic fibroblasts and HeLa cells, thereby demonstrating that their effects were not species-specific. Based on biological activity and physicochemical properties, the IZ 3 (Figure 3B) was selected as a starting point for medicinal chemistry optimization leading to seven very potent analogues (e.g., 4 in Figure 3B)¹⁴. Among these, mitochondrial swelling was inhibited with EC₅₀ of 7.6 pM – 10,000 fold lower than CsA – and others had a CRC ratio of ~ 15 at 1.56 μM – 3 fold higher than CsA – in isolated mouse liver mitochondria. The inhibitory effect on human mitochondria was confirmed by the increased CRC ratios of permeabilized HeLa cells upon treatment with increasing concentrations of each compound. Moreover, inhibitory effect of compound 4 shown in Figure 3B was observed following treatment of both intact and permeabilized human cells, indicating that compound is able to permeate the plasma membrane and reach mitochondrial target(s). As with CA inhibitors, PTP opening activated by calcium overload, oxidative stress and chemical cross-linkers was also potently inhibited by IZs. Since it was recently suggested that the PTP is formed by a unique conformation of F-ATP synthase dimers, IZs were also tested for inhibition of F-ATP synthase activity. Thus, we measured mitochondrial respiration both in isolated mouse liver mitochondria and in intact HeLa cells in the presence or absence of IZs. No statistically significant differences in respiratory control ratios, or FCCP stimulated and oligomycin-insensitive respiration were observed in both isolated mouse liver mitochondria and HeLa cells even at 25 μM, demonstrating that IZs do not affect the respiratory chain complexes or inherent function of F-ATP synthase. Indeed, 4 was not cytotoxic up to a concentration of 12.5 μM as confirmed by the comparable number of viable HeLa cells after 24-hour treatment.

Although our true appreciation of the extent of PTP activation in human disease was fully developed following testing in murine models of each disorder, tests of IZs in these murine models were not possible since they were found to be unstable and rapidly degraded following exposure to mouse plasma. However, a zebrafish model of human Ullrich congenital muscular dystrophy, which results from the absence of the extracellular matrix protein collagen VI (ColVI) and has been reported to manifest mitochondrial dysfunction⁴⁰, has been generated by injection of antisense morpholino oligonucleotides directed to the orthologous ColVIa gene³². Severe myopathy, motor deficits and dramatic ultrastructural defects are present in morpholino-injected animals that successfully recapitulate the clinical severity of human disease⁴⁰. Consequently, the zebrafish ColVI myopathic model was used as a convenient, powerful, and easily assayed in vivo system to validate the therapeutic potential of the most potent IZ PTP-inhibitors. Mutant ColVI fish treated with an IZ simply added to the fish water (5 μM) showed a dramatic improvement in motor function and muscle structural organization when compared to controls¹⁴.

3.3. Benzamides

In addition to the IZ PTP inhibitors described above, the HTS of MLMSR library revealed several benzamide (BZ) compounds (e.g., 5 in Figure 3C) that were also chosen as starting points for the SAR studies¹⁵. When compared to the IZ compounds outlined above, biochemical characterization of one of the molecules in this class (i.e. 6 in Figure 3C) exhibited notable differences. First, the two chemotypes differ in both efficacy and potency toward attenuation of PTP opening: half-maximal effect of BZs on preventing mitochondrial swelling and increasing CRC occurred at concentrations higher than those observed for the most potent representatives of IZ class small molecule inhibitors; swelling EC₅₀s of 280 nM and 7.6 pM, and CRC EC₅₀s of 4.7μM and 0.3 μM, respectively. However, maximum CRC ratios of isolated mouse liver mitochondria treated with selected BZs were as high as 22 compared to 19, while CsA remained at 4.4. Second, BZs displayed a toxicity not observed for IZ inhibitors. While PTP opening activated by calcium overload, oxidative stress and chemical cross-linkers were also potently inhibited by BZs, a decrease in the IMM potential and respiratory control ratio in isolated mouse liver mitochondria (reflecting a decreased ability to generate ATP) and in oxygen-consumption rate in HeLa cells was observed at concentrations above 10 μM¹⁵. Likely as a result, ensuing cellular toxicity as noted in two separate cultured cell systems may be a consequence of these mitochondrial effects. Moreover, in contrast to IZs, BZs demonstrated moderate plasma and hepatic microsome stability as well as high plasma protein binding; metabolic liability was apparent after 1 h exposure, especially in mouse liver microsomes. Consequently, due to toxicity, the therapeutic potential of BZ could not be assessed neither in murine nor in zebrafish models of PTP-dependent disorders.

4. Remaining Issues

While early efforts aimed at the identification of potent inhibitors of the PTP were largely unsuccessful, two recent phenotypic HTS have resulted in the identification of a number of efficacious PTP inhibitors of high potency and selectivity. Synergistic action with CsA in each case argues strongly that the target of each inhibitor class is not CyPD. Thus, relevant probes targeting the PTP are no longer limited to CsA and its derivatives. Although selected issues remain to be addressed for each class of inhibitors (e.g., potency for CA, metabolic stability for IZ, and toxicity for BZ compounds), the target of each was, and remains, unidentified. The obvious chemical similarity of each class of compounds (see Figure 3) might argue for identical targets. Additionally, to aid in the design of more potent inhibitors, the targets of afore mentioned PTP inhibitors will need to be conclusively identified, be they different or the same. Knowledge of the protein through which the most potent compounds elicit PTP inhibition (via direct binding and modulation) is not necessarily an absolute requirement during initial drug development stages¹⁷. However, the definition of the on- and off-target space remains a crucial step toward full development of a drug candidate from the molecules outlined here¹⁹. Although a number of approaches to the identification of the target of small molecules identified in “phenotypic screens” have been defined, affinity-based approaches would seem to be the most tractable in the case of inhibitors identified in the above discussed HTS¹⁷. By whatever means, target identification will facilitate optimization of potency within a lead PTP inhibitor scaffold and the identification of alternative compounds. Moreover, it will allow assessment of safety as well as prediction and monitoring of efficacy when moving from a simplified cellular to in vivo models and ultimately the patient. Taken it all together, the molecules identified in the HTS outlined here are optimal starting points for the development of a suite of small molecules that can serve as therapeutic agents for some of the most pervasive human diseases whose etiology is based, in part, on persistent PTP opening.

Highlights.

• Need for inhibitors of the mitochondrial permeability transition pore outlined

• Methods employed in high-throughput screens

• Three classes of inhibitors compared

Abbreviations

PTP

permeability transition pore

OMM

outer mitochondrial membrane

IMM

inner mitochondrial membrane

CsA

cyclosporin A

CyPD

cyclophilin D

CyPA

cyclophilin A

F-ATP synthase

F_OF₁ ATP synthase

HTS

high-throughput screening

TRO40303

3,5-seco-4-nor-cholestan-5-one oxime-3-ol

BN-PAGE

blue-native polyacrylamide gel electrophoresis

Rh123

rhodamine 123

CRC

calcium retention capacity

SAR

structure/activity relationships

CA

cinnamic anilides

MLSMR

NIH Molecular Libraries Small Molecule Repository

IZ

isoxazole

BZ

benzamide