Didier Morin
email: <didier.morin@inserm.fr

Rana Assaly

Stéphanie Paradis

Alain Berdeaux

St Phanie Paradis

Inhibition of mitochondrial

Keywords: Adenosine Triphosphatases, metabolism, Apoptosis, Mitochondrial Membrane Transport Proteins, antagonists & inhibitors, metabolism, Mitochondrial Membranes, drug effects, metabolism, Myocardial Reperfusion Injury, drug therapy, prevention & control, Permeability, bcl-2 Homologous Antagonist-Killer Protein, metabolism

Introduction

Myocardial ischemia is a leading cause of death in developed countries. After an acute myocardial infarction, the only available treatment to reduce infarct size and to improve the clinical outcome is to restore blood flow (reperfusion) to the ischemic myocardium by thrombolysis, primary percutaneous coronary intervention or cardiac surgery. It is now well-known that reperfusion can induce injury and is at least as deleterious as the oxygen deficit (for a complete review on the mechanisms of the disease see

).

[1 ]

Cell death during myocardial ischemia-reperfusion has been assumed to occur primarily by necrosis. However, several studies indicate now that cell death following myocardial ischemia has features of apoptosis , ; autophagy has also been suggested to play a role in [2 3 ] myocardial ischemia-reperfusion injury , . Given that recent data indicate that the different forms of cell death are regulated and are [4 5 ] probably interrelated , , the better strategy to develop cardioprotective agents is not to define the precise mode of cell death and its [6 7 ] proportion occuring during ischemia-reperfusion, but to identify mediators active in all forms of cell death. In this context, the mitochondrion has emerged as a relevant candidate. More importantly, the change in mitochondrial membrane permeability appears to be a major regulator of both necrotic and apoptotic cell death; the mode of death likely depends on the severity of the insult and on the ability of the cell to maintain ATP synthesis.

In this review, we will examine the role of mitochondria during myocardial ischemia-reperfusion and we will show that pharmacological inhibition of mitochondrial membrane permeability represents a relevant strategy to protect the myocardium from ischemia-reperfusion. We will address this new pharmacological concept of cardioprotection using to two approaches:

The pharmacological strategies acting directly on the mitochondrial membrane by targeting mitochondrial channels (section 3).

The use of pharmacological agents acting indirectly on mitochondrial membrane permeability, by preventing conditions promoting membrane permeabilization (e.g., oxidative stress or by stimulation of the survival pathways referred to as the reperfusion injury salvage " kinases (RISK) pathways ) (section 4).

" [START_REF] Hausenloy | The reperfusion injury salvage kinase pathway: a common target for both ischemic preconditioning and postconditioning[END_REF] Mitochondrial dysfunctions are at the crossroad of the deleterious events caused by myocardial ischemia-reperfusion

Ischemia results in insufficient oxygen availability for mitochondrial respiration and oxidative phosphorylation and, rapidly leads to interruption of aerobic ATP synthesis ( and for extensive information concerning the cellular and mitochondrial events occurring Fig. [START_REF] Yellon | Myocardial reperfusion injury[END_REF] during cardiac ischemia see reviews

). The rapid depletion of intracellular creatine kinase and the concomitant increase in inorganic [9 -12 ] phosphates (Pi) stimulate anaerobic ATP generation with an increase in glycolysis and lactate production. This temporarily supplies the deficiency of the aerobic pathway in the first period of the ischemic process. When hypoxic conditions continue, the intracellular acidification induced by lactate production and the hydrolysis ATP give rise to activation of the Na /H exchanger (as the cell tries to + + restore intracellular pH), Na /K ATPase inhibition (because of the drop in ATP levels) and activation of the Na /Ca exchanger (due to

+ + + 2 +
the increase in intracellular Na concentration). This results in a profound ionic imbalance, cell membrane depolarization and + accumulation of cytosolic Ca (with depletion of the sarcoplasmic reticulum Ca ). Elevated cytosolic Ca concentrations may

2 + 2 + 2 +
contribute to cellular damage by activation of degrading enzymes such as phospholipases, proteases and nucleases, cumulating in the destruction of the membrane integrity and leading to cell death if the ischemic period is of sufficient duration.

Mitochondria are initially able to buffer cytosolic Ca by storing the excess, but this process is limited since mitochondria become at 2 + least partly de-energized, inhibiting the Ca uniporter, the main route of entry of Ca into mitochondria. However, during prolonged

2 + 2 +
ischemia, Ca entry may occur via reversal of the Na /Ca exchanger in the heart . Temporally, the maintenance of a low 2 + + 2 + [START_REF] Suleiman | Mitochondria: a target for myocardial protection[END_REF] mitochondrial Ca concentration associated with the cytosolic acidosis and a high concentration of ADP prevents the opening of the 2 + mitochondrial transition pore, and thus protects mitochondrial membrane impermeability and potential. If the ischemic event continues, it may contribute to myocardial damage during reperfusion, since the electron transfer chain is altered, resulting in the increased production of superoxide anions from complexes I and III. This sets the stage for an increase in reactive oxygen species (ROS) production during reperfusion . In addition, the F F -ATPsynthase now hydrolyses ATP. reperfusion.

Some reports have also observed decreases in NADH dehydrogenase and adenine translocase activities, a reduction of the protective mechanisms against oxygen toxicity, and decreases in superoxide dismutase (SOD) activity and glutathione content. The final extent of the damages depends on the duration and the severity of ischemic event, which emphasises the crucial time for the onset of the reperfusion (which can be combined with a pharmacological intervention). This is a critical juncture which may limit or even reverse at least some of the changes in mitochondrial functions.

Ischemic damage can paradoxically be amplified by reperfusion that corresponds to the restoration of oxygenated blood flow to the ischemic tissue. This is mainly due to the generation of ROS, which can result from the reactivation of respiratory complexes blocked in a reduced state without functional coordination (inefficient transfer of electrons generating superoxide anions). ROS have direct effects on the respiratory chain components, resulting in decreased efficiency of oxidative phosphorylation; complex I appears to be especially sensitive to ischemic injury , . Changes in complexes III and IV were also observed, but this would occur later during the [13 15 ] deleterious process . They also cause inhibition of the enzymes of the Krebs cycle (i.e. aconitase) , . ROS cause non-specific [START_REF] Lesnefsky | Mitochondrial dysfunction in cardiac disease: ischemia--reperfusion, aging, and heart failure[END_REF] [ 16 17 ] damages to lipids, proteins and mitochondrial DNA, and induce peroxidation of cardiolipin, a major constituent of the inner membrane, which increases the inhibition of oxidative phosphorylation , .

[18 19 ]
ROS production is exacerbated by the high concentrations of Ca which is driven from the cytosol into the mitochondria by 2 ,

+

restoration of the mitochondrial membrane potential. These conditions favour an increase in mitochondrial membrane permeability and the induction of the mitochondrial permeability transition pore (mPTP), which cause swelling, collapse of membrane potential, and ultimately total inhibition of mitochondrial functions . This phenomenon is now considered to play a central role in different kinds of cell death;

[10 ]

Crompton et al.

were the first to report that mPTP opening plays a crucial role in myocardial ischemia-reperfusion injury. It is [START_REF] Crompton | Evidence for the presence of a reversible Ca2 -dependent pore activated by oxidative stress in heart mitochondria +[END_REF] generally accepted that mPTP opening is associated with postischemic reperfusion but some studies have suggested that mPTP [START_REF] Griffiths | Mitochondrial non-specific pores remain closed during cardiac ischaemia, but open upon reperfusion[END_REF] opening might also occur during ischemia . It should be noted that mPTP is not the only mechanism by which mitochondrial [START_REF] Borutaite | Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction, cytochrome c release and apoptosis induced by heart ischemia[END_REF] membranes can be permeabilized, and the search for cardioprotective agents must consider all the mitochondrial targets that can prevent mitochondrial membrane permeabilization.

Pharmacological strategies acting by direct inhibition of mitochondrial membrane permeability by targeting mitochondrial channels

During ischemia-reperfusion, cell death occurs by necrosis and apoptosis, both being induced by the change in mitochondrial membrane permeability. Mitochondrial membrane permeability is regulated by stress signals originating in mitochondria but also those of cytosolic origin such as proteins of the Bcl-2 family. Two classes of mechanisms ( ), which are not mutually exclusive, have been Fig. [START_REF] Olivetti | Apoptosis in the failing human heart[END_REF] proposed to explain mitochondrial membrane permeabilization (for review see

).

[23 ]

The first one involves the participation of both the inner and the outer membranes and corresponds to the mPTP. The second mechanism involves only outer mitochondrial membrane and the formation of channels across the membrane.

Although there is a controversy concerning the structure, the regulation and the definite role of these different channels, strong evidences indicate that proteins of the Bcl-2 family contribute to both mechanisms , .

[ 24 25 ] Triggering mitochondrial membrane permeabilization induces the release of cell death effectors and the loss of mitochondrial functions which are fundamental for cell survival. Thus, drugs able to block or to limit mitochondrial membrane permeabilization might be cytoprotective during ischemia-reperfusion and this pharmacological approach is reviewed in this section (

). Fig. (3)

The mitochondrial transition pore as a pharmacological target

The mitochondrial respiratory chain catalyzes the oxidation of substrates by oxygen and couples electron flux to H pumping to + establish an H electrochemical gradient across the inner membrane. ATP is synthesized by the enzyme F F -ATPsynthase which utilizes

+ 1 0
the free energy released by the H backflow from intermembrane to mitochondrial matrix. Therefore, the maintenance of the H gradient

+ +
and the impermeability of the inner membrane to ions, especially to H , is vital to the cell. However, under stress conditions, the + permeability of the membrane may increase with the formation of a voltage-dependent nonspecific pore, known as the mPTP, which allows the influx of water and molecules up to 1.5 kD and therefore induces swelling of the organelle matrix. An extensive swelling results in the rupture of the outer membrane and in the release of proapoptotic proteins into the cytosol. The exact composition of this pore remains uncertain although it was formerly thought to include adenine nucleotide translocase (ANT) modulated by cyclophilin D (CyP-D) in the inner membane and a voltage-dependent anion channel (VDAC) in the outer membrane , . CyP-D is a member of the [10 26 ] cyclophilin family which displays peptidyl-prolyl cis-trans isomerase activity and resides in the mitochondrial matrix. During mPTP formation, CyP-D interacts with the inner membrane and is thought to induce a conformational change of ANT, leading to an increase in inner membrane permeability ( ). Fig. [START_REF] Olivetti | Apoptosis in the failing human heart[END_REF] In vivo experiments confirm the role of CyP-D-dependent mitochondrial mPTP for mediating Ca -and oxidative damage-induced 2 + cell death: CyP-D-deficient mice are protected from ischaemia-reperfusion-induced cell death , whereas CyP-D-overexpressing in vivo mice show mitochondrial swelling and spontaneous cell death . These genetic experiments also provided significant information [27 -29 ] about the mechanism of cell death triggered by mPTP. CyP-D-deficient cells responded to various apoptotic stimuli, but showed resistance to necrotic cell death induced by ROS and Ca overload. This led to the conclusion that CyP-D and thus mPTP do not play a major role in 2 +

the apoptotic process, but rather are major components of the necrotic death pathway.

Studies on mitochondria lacking these putative components of the mPTP did not permit definit conclusions about the identity of its membrane components. Indeed, a functional mPTP can form in the absence of ANT in mice , and it was suggested that ANT may not [START_REF] Kokoszka | The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore[END_REF] be the protein which binds CyP-D . Juhaszova et al. suggest that ANT may be a non-essential structural component of the mPTP [START_REF] Leung | The cyclophilin-D binding protein of the mitochondrial permeability transition pore may not be the adenine nucleotide translocase[END_REF] [32 ]

but may have a regulatory role in mPTP induction (see ). VDAC also may not be an essential component of the mPTP as the Fig. (2A) pore can be induced in mitochondria devoid of all VDAC isoforms . It should be added that other proteins such as hexokinase, creatine [START_REF] Baines | Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death[END_REF] kinase or the mitochondrial translocator protein (TSPO), have been proposed to be involved in pore formation, but to date it is not known whether these proteins are structural or regulatory components of the pore.

Taken together, these data show that the molecular composition of mPTP remains a source of debate, but nevertheless, strong evidence suggests that mPTP contributes to cellular injury during myocardial ischemia-reperfusion: (1) mPTP opening is favored by conditions prevailing during ischemia-reperfusion (2) mPTP might control cell death by releasing apoptogenic factors , , ,

mPTP is [10 34 35 ] regulated by the pro-and the anti-apoptotic members of the Bcl-2 family , , (4) increasing lines of evidence suggest that mPTP is [36 37 ] involved in the cardioprotective effect of ischemic pre-and post-conditioning (see section 4.1.).

Therefore, targeting mPTP appears as a promising pharmacological approach in ischemia-reperfusion and numerous studies show that almost any procedure that reduces mPTP opening affords protection against ischemia-reperfusion injury.

The first demonstration of the relevance of this approach was provided by the immunosuppressant agent cyclosporin A (CsA) which binds to CyP-D with high affinity and precludes the interaction of this protein with a component of the mPTP, thus inhibiting its opening.

CsA protected against hypoxia-reoxygenation-mediated cell death in isolated cardiomyocytes , and reduced infarct size in ex-vivo [ CsA at the onset of reoxygenation protected the human myocardium against lethal hypoxia-reoxygenation injury. The clinical use of CsA is hampered by pharmacokinetic and pharmacodynamic parameters and by the fact that CsA fails to inhibit pore opening when [START_REF] Morin | Mitochondria as target for antiischemic drugs[END_REF] mitochondria are exposed to strong stimuli . However, a recent clinical study showed that an intravenous bolus of 2.5 mg/kg CsA just [START_REF] Halestrap | Mitochondrial permeability transition pore opening during myocardial reperfusion--a target for cardioprotection[END_REF] before reperfusion reduced infarct size in patients suffering from acute myocardial infarction .

[45 ]

Similar cardioprotective effects were obtained with non-immunosuppressant derivatives of CsA, such as N-methyl-ala CsA, It should be added that the inhibition of mPTP opening by CsA was also effective in a particular case of ischemia-reperfusion, the hypothermic preservation of the heart before transplantation. Indeed, CsA improved functional recovery after long-time hypothermic heart preservation .

[50 ]

Although the molecular structure of mPTP remains unknown, other approaches have been developed to find new mPTP inhibitors.

Binding sites involved in the regulation of mPTP were described . Clinically approved drugs, including tricyclic antidepressants, [START_REF] Morin | Evidence for the existence of 3H -trimetazidine binding sites involved in the regulation [ ] of the mitochondrial permeability transition pore[END_REF] antipsychotics and antiarythmic agents were found to bind to these sites and to inhibit mPTP in vitro , . Some of these drugs [51 52 ] reduced neurological impairment in mice subjected to middle cerebral artery occlusion/reperfusion , but, to our knowledge, have not [START_REF] Stavrovskaya | Clinically approved heterocyclics act on a mitochondrial target and reduce stroke-induced pathology[END_REF] been tested in myocardial models of ischemia-reperfusion. Most of these drugs are amphiphilic cations which are known to interact with biological membranes which could change mitochondrial membrane potential and thus possibly induces closure of the pore .

[53 ]

Another approach to design new pore inhibitors was provided by the results of Bernardi and colleagues who demonstrated that the coenzyme Q family in addition to its antioxidant effects and its role in the regulation of electron transfer modulates mPTP opening by acting on a specific site . Exogenous coenzyme Q (ubiquinone 0) and coenzyme Q (decylubiquinone) are very potent inhibitors of [START_REF] Fontaine | A ubiquinone-binding site regulates the mitochondrial permeability transition pore[END_REF] 0 10 mPTP opening induced by Ca overload, whereas coenzyme Q (ubiquinone 5), which did not inhibit pore opening per se, counteracted 2 + 1 the effects of coenzyme Q and decylubiquinone . As a putative component of mPTP, VDAC was suggested to be one of the target of 0 [START_REF] Fontaine | A ubiquinone-binding site regulates the mitochondrial permeability transition pore[END_REF] ubiquinone analogues , but since liver mitochondria from mice lacking VDAC1 exhibit normal mPTP opening and inhibition by [START_REF] Cesura | The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore[END_REF] ubiquinone analogues , this hypothesis seems unlikely. It should be noticed that the design of new mPTP inhibitors from coenzyme Q [START_REF] Krauskopf | Properties of the permeability transition in VDAC1( / ) mitochondria[END_REF] may be difficult since minor structural changes profoundly modify the effects of quinones on the mPTP .

[57 ]

To our knowledge, it has not been established whether this is what is responsible for the antiischemic effect of coenzymes Q and

[58 ]
data indicate that coenzyme Q even impairs myocardial performance following ischemia-reperfusion . However, the antioxidant 0 [START_REF] Halestrap | Mitochondrial permeability transition pore opening during myocardial reperfusion--a target for cardioprotection[END_REF] properties of coenzyme Q may contribute to mPTP inhibition as decylubiquinone was shown to inhibit redox-activated permeability transition .

[59 ]

Taken together, these data suggest that the direct inhibition of mPTP opening constitutes a relevant objective in cardioprotection. This requires the search for new direct mPTP inhibitors , with good specificity and selectivity and the identification of the molecular site [55 60 ] of action of these compounds to further elucidate mPTP structure.

The MAC/Bak channel

It is now well-established that mPTP is not the only mechanism by which the mitochondrial membrane can be permeabilized .

[61 ]

Indeed, cytochrome c release was observed in the absence of mitochondrial depolarization and without loss of mitochondrial outer [START_REF] Bossy-Wetzel | Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization[END_REF] membrane integrity (i.e., independently of mPTP opening), indicating that permeabilization of the outer membrane is a regulated phenomenon occurring via the formation of a pore . This type of release has been observed during cardiac and liver ischemia .

[24 ] [63 -65 ] Mitochondrial membrane permeabilization is under the control of the Bcl-2 family of proteins (for a recent review see ).

[66 ]

Proapoptotic members (e.g. Bax, Bak or the BH3-only subfamily proteins Bid, Bad, Bnip3) facilitate membrane permeabilization and promote the release of cytochrome c and other intermembrane space components. Cytochrome c triggers formation of the apoptosome complex and activation of caspase-9 . This is not the only pathway which can be activated by outer membrane permeabilization, since [START_REF] Spierings | Connected to death: the (unexpurgated) mitochondrial pathway of apoptosis[END_REF] two other proteins, apoptosis inducing factor (AIF) and endonuclease G, which are also released after outer membrane permeabilization, can induce apoptosis in a caspase-independent manner , . Two main possibilities have been proposed to be responsible for [ 67 68 ] governing this increase in permeability. Bax resides in the cytosol in normal, healthy cells but, once activated, translocates to mitochondria, where 1) it incorporates into the outer membranes and oligomerizes to form permeable pores , and/or 2) interacts with [3 61 ] existing channels such as VDAC , . The activation of Bax is regulated by the BH3-only proteins, which facilitate Bax channel [3 69 ] formation. It should be pointed out that the Bax channel appears to be identical to the mitochondrial apoptosis-induced channel (MAC channel;

), which was first detected on mitochondria in patch-clamp experiments. The anti-apoptotic members Bcl-2 and Bcl-x are [START_REF] Kinnally | A tale of two mitochondrial channels, MAC and PTP, in apoptosis[END_REF] L mainly localized to the mitochondrial outer membrane where they antagonize the pro-apoptotic effect of Bax and Bak .

[70 ]

The importance of the Bcl-2 family proteins in myocardial ischemia-reperfusion injury was demonstrated in mouse models. Indeed, Bax deletion in mice, interference with Bax activation, or over-expression of Bcl-2 levels have been shown to attenuate apoptosis and reduce infarct size, while reduction of Bcl-2 levels (via antisense oligonucleotides) suppressed protection against injury .

[72 -76 ] Therefore, anti-apoptotic interventions targeting Bcl-2 family proteins provide opportunities for possible antiischemic therapies , [START_REF] Reed | Apoptosis-regulating proteins as targets for drug discovery[END_REF] either through a gain of antiapoptotic function or loss of proapoptotic function. Polster et al. demonstrated that the well-known drugs [START_REF] Polster | Inhibition of Bax-induced cytochrome c release from neural cell and brain mitochondria by dibucaine and propranolol[END_REF] propranolol and dibucaine inhibited Bax-induced permeability changes and cytochrome c release from mitochondria through a direct interaction with the lipid membrane. A similar approach allowed others to identify 3,6-dibromocarbazole piperazine derivatives of 2-propanol as inhibitors of cytochrome c release triggered by induction of the Bax channel . In the same way, a pentapeptide derived [START_REF] Bombrun | 3,6-dibromocarbazole piperazine derivatives of 2-propanol as first inhibitors of cytochrome c release via Bax channel modulation[END_REF] from the Bax-modulating Ku70 protein inhibited Bax-dependent apoptosis . More recently, Hetz et al. identified two blockers of [START_REF] Sawada | Cytoprotective membrane-permeable peptides designed from the Bax-binding domain of Ku70[END_REF] [81 ]

Bax channel activity, bci1 and bci2 ( ), which prevented cytchrome c release in vitro and in vivo, and protected neurons in a gerbil Fig. [START_REF] Yan | Autophagy in chronically ischemic myocardium[END_REF] model of global brain ischemia at reperfusion. It is a promising pharmacological strategy, but to our knowledge, these compounds have not been evaluated during myocardial ischemia.

BH3-only Bcl-2 proteins

The activation of Bax and Bak is regulated by the class of Bcl-2 proteins that contain single BH3 domains. These proteins control apoptotic signals upstream of mitochondria and transmit them directly or indirectly to Bax and Bak , thus they represent specific [START_REF] Gustafsson | Bcl-2 family members and apoptosis, taken to heart[END_REF] targets for preventing myocardial ischemia-reperfusion injury. This appears to be an excellent point of intervention because only activation of Bax and Bak would be affected, minimizing cell apoptosis. This approach would also facilitate maintenance of mitochondrial integrity.

Among these proteins Bid (BH3-Interacting Domain death agonist) is one of the most abundant in mammalian tissues, including the heart . Bid is subjected to a protein cleavage by caspase-8, granzyme, or calpain, and truncated Bid (tBid) translocates to the [START_REF] Esposti | The roles of Bid[END_REF] mitochondria where it is involved in Bax/Bak activation and/or Bax/Bak channel formation . Thus, Bid is at the crossroad of the [START_REF] Gustafsson | Bcl-2 family members and apoptosis, taken to heart[END_REF] intrinsic and the extrinsic death pathway. Bid-mediated apoptosis has been shown to contribute to myocardial ischemic injury and [START_REF] Chen | Bid is cleaved by calpain to an active fragment in vitro and during myocardial ischemia/reperfusion[END_REF] Bid-deficient mice have reduced infarct size and improved cardiac functions after ischemia-reperfusion . In addition, calpain inhibitors [START_REF] Crow | The mitochondrial death pathway and cardiac myocyte apoptosis[END_REF] reduce infarct size in animal models of isolated perfused heart and slow the progression of heart failure in rats , . These data [START_REF] Chen | Calpain and mitochondria in ischemia/reperfusion injury[END_REF] [ 85 86 ] suggest that Bid is an attractive target during myocardial ischemia-reperfusion, and pharmacological inhibitors which are effective in vitro assays have already been designed .

[87 ]

Other BH3-only Bcl-2 proteins have been implicated in cardiac ischemia-reperfusion injury . Bnip3 (Bcl-2/adenovirus E1B 19kDa [START_REF] Webster | Redox stress and the contributions of BH3-only proteins to infarction[END_REF] interacting protein 3) has been shown to mediate mitochondrial dysfunction and cell death of ventricular myocytes subjected to hypoxia [

. It was also reported to contribute to myocardial ischemia-reperfusion injury through a mitochondrial death pathway . 89 -90 ] [ 91 -92 ] Interestingly, a TAT-fusion protein encoding a carboxyl terminal transmembrane deletion mutation in of Bnip3 conferred protection against myocardial ischemia-reperfusion injury, improved cardiac function, and protected mitochondrial integrity , confirming that [START_REF] Hamacher-Brady | Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy[END_REF] Bnip3 could constitute a relevant therapeutic target.

In the same way, Puma (p53-Upregulated Modulator of Apoptosis) was upregulated in response to hypoxia/reoxygenation in isolated cardiomyocytes and Puma-deficient mice have reduced infarct size and improved cardiac function after myocardial ischemia-reperfusion [ . Until now, pharmacological strategies targeting selectively Puma have not been described.

]

The pharmacological strategy inhibiting mitochondrial outer membrane permeability, and thus apoptosis, by manipulation of the Bcl-2 family proteins to protect the myocardium against ischemia-reperfusion is very recent. It has already provided interesting results, which support the idea that a clinical benefit might be obtained in the near future.

The voltage-dependent-anion channel (VDAC) as a pharmacological target

Another potential mechanism for outer mitochondrial membrane permeabilization might involve VDAC which is the major permeability pathway for metabolites through the mitochondrial outer membrane . Different isoforms of VDAC have been identified , and the role of VDAC in cell death induced by mitochondria seems to be isoform specific. Indeed, VDAC1 has been implicated in 95 96 ] the mitochondrial release of proapototic protein whereas VDAC2 displays antiapoptotic properties , .

[97 98 ]
It is important to note that much of the evidence involving VDAC opening and closing in proapototic protein release has arisen from in vitro experiments, and it is not clearly established whether this can be observed in vivo . However, VDAC is a hypothetical [START_REF] Blachly-Dyson | VDAC channels[END_REF] component of mPTP, interacts with pro-and anti-apoptotic proteins of the Bcl-2 family members, and VDAC has been also proposed to control the release of cytochrome c without mPTP opening , , .

[97 100 101 ] Taken together, these data make VDAC a putative target in ischemia-reperfusion. Interestingly, a strategy developed to inhibit VDAC was shown to reduce myocardial ischemia-reperfusion injury. Perfusion of rat hearts with a cell permeable peptide corresponding to the BH4 domain of Bcl-X , which had been reported to close VDAC and to prevent the VDAC-mediated release of cytochrome c , was

L [102 ]
shown to inhibit creatine kinase release and to reduce myocardial cell death . In the same way, the Ca -dependent actin-regulatory

[84 ] 2 +
protein gelsolin was suggested to inhibit apoptosis by blocking mitochondrial VDAC activity . This is in disagreement with recent [103 ] results that indicate that closure of VDAC, not opening, leads to mitochondrial outer membrane permeabilization and apoptosis . It is [104 ] clear that a better knowledge of the role of VDAC, if any, in ischemic injury, will take time, given the lack of proven pharmacological compounds specifically targeting VDAC. blockers inhibit . In the same way, recent data show that the myocardial protection conferred by ischemic post-conditioning is

Mitochondrial potassium (mitoK

[111 -113 ]
blocked by the mitoK channel blocker 5-hydroxydecanoate and thus achieved at least in part by opening of mitoK channels .

ATP ATP

[114 ]

The mechanism for mitoK channels opening during pre-and post-conditioning is currently the subject of intense investigation.

ATP

Several recent studies have linked the protein kinase C epsilon (PKC ), which translocation to mitochondria seems important for ε pre-conditioning , to mitoK channel opening. Other kinases have also been involved .

[ induced by elevated Ca concentrations in isolated mitochondria. However, this hypothesis has been disputed, because mitochondrial 2 + membrane depolarization generated by K flux does not appear to be sufficient to affect Ca transport and has not been observed in

+ 2 + [122 ]
intact cardiac myocytes . Moreover, a higher depolarization may lead to deleterious phenomena more frequently associated with cell

[123 ]
death, than t protection (such as decreased ATP synthesis and even mPTP opening).

The second hypothesis proposes that perturbed ROS production is responsible for mitoK channel effect. opening-mediated ROS release during the preconditioning phase. In this scheme, mPTP opening may act as a protective mechanism in response to different stimuli during the pre-conditioning period to inhibit its deleterious opening during the reperfusion phase. This is an attractive mechanism but is still highly controversial However, the full demonstration of the involvement of mitoK channels in cardioprotection is hampered by the absence of selective ATP agents for the mitoK channel; the mitoK channel openers and inhibitors which have been used to demonstrate the cardioprotective ATP ATP effects of these channels (most notably diazoxide and 5-hydroxydecanoate), are claimed to be specific for this channel, but numerous studies argue against this specificity (see the review of Halestrap et al.

).

[134 ]

A better understanding of the cellular mechanisms and of the role of mitoK channels will require the development of more specific ATP and more selective pharmacological entities; however, they still appear to be a promising target for the development of new cardioprotective agents.

The mitochondrial translocator protein (TSPO)

TSPO (formerly known as Peripheral Benzodiazepine Receptor

) is a ubiquitous high affinity cholesterol binding 18 kDa protein [135 ] primarily located on the outer mitochondrial membrane, where it is associated with VDAC . It has been implicated in numerous

[136 ]
biological functions , including regulation of mPTP opening , but the exact physiological role of TSPO remains unclear

[135 ]

[136 137 ] (except in steroid-producing tissues where TSPO mediates the transport of cholesterol from the outer to the inner mitochondrial membrane and promotes pregnenolone synthesis

). TSPO is also present in non steroidogenic tissues, especially in the heart, where its function

[138 ]
is unknown. Several reports suggest that TSPO ligands might modulate apoptotic responses and play an anti-apoptotic role in [139 ] oxidative stress conditions such as ischemia-reperfusion . In this context, TSPO expression correlated with the quality of kidney

[140 ]
preservation, indicating that it might serve as an index of kidney and mitochondrial viability during storage . Thus, one means to

[141 ]
improve resistance of the cells to oxidative stress generated during ischemia would be to increase TSPO expression levels using pharmacological tools to increase TSPO transcription. Leducq showed that the specific TSPO ligand 4 -chlorodiazepam ( ) reduced infarct size in both global and regional models of myocardial ] ′

Fig. (4)

ischemia-reperfusion in rats. The protective mechanism involved limiting mitochondrial membrane permeabilization, notably a resistance of mitochondria to mPTP opening, associated with a reorganization of the balance between pro-and anti-apoptotic proteins of the Bcl-2

family proteins at the level of mitochondrial membranes . Taken together, these data demonstrate a role for TSPO as a modulator of

[143 ]
necrotic and apoptotic cell death induced by ischemia-reperfusion, suggesting that TSPO could constitute a possible target for cardioprotection.

Pharmacological strategies acting indirectly on mitochondrial membrane permeability Ischemic pre-and post-conditioning

Ischemic pre-conditioning (IPC) is one of the more powerful processes to protect the heart from lethal ischemia-reperfusion injury.

IPC was originally described by Murry et al.

, who demonstrated that a sequence of successive short periods of ischemia reduced [144 ] infarct size produced by a subsequent long ischemic period. Studies revealed that IPC caused the release of G protein-coupled receptors agonists such as adenosine, bradykinin or opioids that activated a cascade of cardioprotective kinases referred to as Reperfusion Injury " Salvage Kinase (RISK) pathway . This cascade includes PKC, the PI3kinase-AKT pathway, the ERK1/2 pathway, and downstream

" [145 ]
proteins such as glycogen synthase kinase-3 (GSK-3 ), PKG, Bad or endothelial nitric oxide synthase (eNOS). PKC is a primary demonstrated that brief repetitive ischemic periods of ischemia followed by reperfusion after a lethal ischemic period results in a robust reduction in infarct size, termed ischemic post-conditioning (IPOC). Subsequent studies have shown that IPC and IPOC use a similar signalling pathway, i.e. the RISK pathway, and that classical ligands able to trigger IPC, such as adenosine or opioids, were also able to induce IPOC . For a detailed characterization of the signalling pathways involved in IPC and IPOC see . From a clinical point

β β ε cardioprotective PKC isoform,
[148 ] [115 ]
of view the discovery of IPOC is fundamental as it can be applied in patients .

[149 ]

The cellular mechanisms governing IPC and IPOC protection are not fully understood, but increasing lines of evidence suggest that indirect inhibition of mitochondrial membrane permeability, through inhibition of mPTP opening during reperfusion, may be the target of the RISK pathway . The mechanism by which the RISK pathway inhibits mPTP is unclear, but several candidates have phosphorylation and inhibition of GSK-3 , a downstream target of AKT, which have been demonstrated to inhibit mPTP , . II activation .

[160 ]

However, whatever the mechanism(s) connecting the RISK pathway to mPTP inhibition, the activation of this cascade of kinases offers the opportunity to promote mPTP inhibition and thus cardioprotection at reperfusion. This property can be exploited therapeutically as described in the following studies.

Targeting of the RISK pathway by ischemic post-conditioning

IPOC was recently described, but small clinical studies have already been initiated . In these studies, IPOC was induced in [161 -163 ] patients by a series of successive episodes of inflation-deflation of the angioplasty balloon during percutaneous coronary intervention.

IPOC significantly protected the heart against ischemia-reperfusion-induced injury and, importantly, the protective effect is of significant duration .

[163 ]

Targeting the RISK pathway via pharmacological agents

The administration of high doses of adenosine at reperfusion in acute myocardial infarction resulted in myocardial protection in two small clinical studies , . A reduction in infarct size was also observed in larger randomized controlled studies in which adenosine [164 165 ] was administrated as an adjunct to thrombolytic therapy after the beginning of reperfusion , . However, this pharmacological [166 167 ] strategy did not improve clinical outcomes .

[167 ]

Morphine is another G protein-coupled receptor agonist which is known to activate the RISK pathway and to cause potent against myocardial infarction through inhibition of mPTP opening. Thus, the use of morphine, a well-known drug which can be administrated acutely in safe conditions, as an adjunct of myocardial reperfusion, may be a useful clinical strategy in acute myocardial infarction.

cardioprotection
It is also possible to act upstream in the RISK pathway, as several groups have shown that inhibitors of GSK-3 reduced infarct size in β mice and rats, and that this effect was associated with the inhibition of mPTP opening , , .

Mitochondria-targeted antioxidants in limiting mitochondrial membrane permeability

In physiological conditions, ROS are formed within mitochondria and are eliminated by powerful antioxidant systems . Under

[174 ] pathological conditions, especially during post-ischemic reperfusion, the sudden influx of oxygen to the mitochondrial respiratory chain leads to a burst of ROS (as a superoxide anion; ) which can overwhelm endogenous antioxidant systems and deplete the reducing

[175 ]
compounds protecting mitochondria against oxidative insults. It should be noted that the respiratory chain is not the only source of ROS in mitochondria . For instance, MAO-A has been shown to represent an important source of hydrogen peroxide in rat heart . ROS

[174 ] [176 ]
cause non-specific damage to lipids, proteins and mitochondrial DNA. When associated with mitochondrial Ca overload and adenine 2 + nucleotide depletion, conditions that pertain during myocardial reperfusion, high levels of ROS can induce opening of the mPTP .

[44 ]

ROS generated during early reperfusion have been found to be primary activators of mPTP and cardiomyocyte death , and Clarke et

[177 ]
al.

have reported that IPC-induced inhibition of mPTP is probably mediated by a reduction in oxidative stress. This property might

[178 ] also contribute to the cardioprotective effect of some therapeutic drugs such as pyruvate , the -adrenoceptor blocking agent

[179 ] β carvedilol or the anaesthetic propofol, frequently used for cardiac surgery .

[180 ]

[181 ]

These data highlight the major role of ROS in the increase in mitochondrial membrane permeability. Thus, any antioxidant treatment is likely to protect mitochondria and ultimately the myocardium at reperfusion. dismutase mimetic drug which was able to eliminate ROS in the microenvironment of the mPTP and to inhibit its opening, leading to the protection of the myocardium against myocardial ischemia-reperfusion ( ). Fig. [START_REF] Yan | Autophagy in chronically ischemic myocardium[END_REF] An interesting result was also obtained with the free radical scavenger edavarone which was shown to inhibit mPTP opening and to prevent cardiac reperfusion injury . In a recent clinical study, edavarone delivered via intravenous injection during percutaneous

[184 ]
coronary intervention reduced infarct size and reperfusion arrhythmias, and improved short term clinical outcomes in acute myocardial infarction . However, most of the clinical studies with conventional antioxidants have yielded disappointing results .

[185 ]

[186 ]

Several reasons can be evoked to explain this failure; among them, the main reason may be the difficulty of delivering the drug to mitochondria . To overcome these limitations, investigators have synthetized amphiphilic cations from well-established antioxidant in situ compounds, which are sufficiently lipophilic to cross lipid bilayer membranes and which can be selectively taken up into the mitochondria by the large, negative inside, inner membrane potential , . These drugs include alpha tocopherol (MitoVit E) and ubiquinone [187 188 ] (MitoQ) (

). A recent study demonstrated that feeding rats with MitoQ significantly limited cardiac reperfusion-injury ; this Fig. [START_REF] Yan | Autophagy in chronically ischemic myocardium[END_REF] [189 ] approach might provide a promising pharmacological strategy.

Using a similar idea, a novel class of small cell-permeable antioxidant peptides ( ) that target mitochondria have been reported Fig. [START_REF] Yan | Autophagy in chronically ischemic myocardium[END_REF] [ . Their antioxidant effect is due to the presence of basic amino acids, tyrosine and dimethyltyrosine, which are effective in scavenging 190 ] ROS. These peptides enter the mitochondria in a potential-independent manner (contrary to MitoQ and MitoVitE) because they include a sequence motif targeted to mitochondria. These peptides reduce mitochondrial ROS production, inhibit mPTP opening, prevent the release of cytochrome c from mitochondria and protect against myocardial ischemia-reperfusion ex vivo and in vivo .

[190 ] [191 ] Another alternative antioxidant approach would be to prevent mitochondrial ROS production to protect mitochondrial membrane. A first possibility would be the development of specific MAO-A inhibitors, since MAO-A inhibition has been shown to reduce myocardial damage induced by ischemia-reperfusion . This can also be achieved by mild uncoupling , decreasing ROS generation by [192 ] [193 ] dissipating the electrochemical potential of H , allowing an increase in electron flux at the expense of ubiquinol. Mild uncoupling might + represent an antioxygen defense mechanism during the reperfusion period, preventing the sudden increase in membrane potential, the overproduction of ROS, and the increase in mitochondrial membrane permeability.

Concluding remarks

This manuscript highlights that mitochondria represent a pertinent target to develop cardioprotective pharmacological therapies and the crucial role of the preservation of mitochondrial membrane integrity to protect cardiac cells during myocardial ischemia-reperfusion.

Beside their essential roles in energy production, Ca homeostasis and ROS production, mitochondria control necrotic and apoptotic 2 + myocardial cell death by regulating the permeability of its membranes. Indeed, mPTP may be considered as a central event in necrosis (although a controversy persists concerning the molecular composition of mPTP), whereas the permeabilization of mitochondrial outer membranes by proteins of the Bcl-2 family leads to apoptosis. On the whole, the studies presented above reveal that both anti-necrotic and anti-apoptotic strategies targeting mitochondrial permeability are able to protect cardiomyocytes from death during ischemia-reperfusion.

However, the signaling mechanisms mediating mitochondrial membrane permeabilization in ischemia-reperfusion are diverse, so it seems likely that a combination of pharmacological therapies, i.e. compounds combining different pharmacological effects or co-administration of several drugs acting simultaneously on distinct targets, should afford a better protection against myocardial ischemia-reperfusion injury.

Ackowledgements:

R. Assaly was supported by a doctoral grant from region Ile-de France and S. Paradis was supported by the Minist re de la Recherche et de la that other mechanisms than Bax and BH-3 only proteins have been involved in outer membrane permeability. However, they were not described during myocardial ischemia-reperfusion.

Fig. (3)

The inhibition of mitochondrial permeabilization as a pharmacological approach to limit myocardial ischemia-reperfusion injury Schematic representation of the pharmacological strategies which have been or might be used to limit mitochondrial membrane

  -Val CsA, Debio 025 or NIM811, , , , ) confirming the relevance of this approach. Sanglifehrin A also prevents 4 ] [21 40 46 47 ] myocardial ischemia-reperfusion injury , . This drug is unrelated to CsA but potently inhibits mPTP by inhibiting the [48 49 ] peptidyl-prolyl cis-trans isomerase activity of CyP-D ( and ( )). Fig. (3) 4

[150 - 153 ]

 153 emerged which may act in concert to mediate mPTP inhibition (). These include: Fig.[START_REF] Takagi | The role of autophagy in mediating cell survival and death during ischemia and reperfusion in the heart[END_REF] eNOS generation of NO, which alone , or through the PKG-PKC -mitoK channel can limit mPTP opening , , phosphorylation and/or complex formation with cytosolic proteins like PKC can interact with mPTP , , whereas the activity ε [155 156 ] of PKC and its translocation to mitochondria at the onset of reperfusion mediates apoptosis ,

β[

  150 153 ] Nishihara et al. suggested that the binding of GSK-3 to ANT might be responsible for mPTP inhibition and the myocardial [158 ] β protection afforded by IPC, inhibition of downstream signals of the RISK pathway, such as Bax translocation from cytoplasm to mitochondria or hexokinase [159 ]

  [153 168 169 ] Similarly, beside their lipid-lowering actions, statines (3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors) have been shown to exhibit cardioprotective properties , . The mechanism seems to involve stimulation of several kinases in the [170 171 ] RISK pathway but also activation of the mitoK channel through NO generation and inhibition of mPTP opening . ATP [172 ]Another possible strategy is to directly target PKC via PKC activators or PKC inhibitors. A recent small clinical study of acute ε δ myocardial infarction using the PKC inhibitory peptide, KAI-9803, which was delivered via intracoronary injection during percutaneous δ coronary intervention, revealed signs of cardioprotection. Unfortunately, the observed improvements were not significant, probably because of the small size of the sample[173 ]

è

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Fig. ( 1 ) 2 +Fig. ( 2 )

 122 Fig. (1) Scheme illustrating the mitochondrial sequence of events triggered by myocardial ischemia-reperfusion The scheme summarizes the processes described in the text, showing the relationships between oxygen consumption, ATP production, Ca 2 + accumulation, reactive oxygen species (ROS) generation and the initiation of cell death through activation of the mitochondrial permeability transition pore (mPTP). During ischemia, cellular acidosis along with a high concentration of ADP prevents mPTP opening although favourable conditions prevail ( decrease, high Pi, slight increase in ROS). During reperfusion, high matrix concentrations of Ca and Pi ΔΨ m 2 +associated with a burst of ROS favour mPTP opening despite the antagonizing effect of recovery.ΔΨ m

Fig. ( 4 )

 4 Fig. (4)Chemical structures of some pharmacological agents capable of limiting mitochondrial membrane permeabilization.

Fig. ( 5 )

 5 Fig. (5) Hypothetical scheme showing the cardioprotective pathways activated at the time of reperfusion by both ischemic pre-and post-conditioning. Ligands of receptors coupled to G-proteins or growth factors initiate cardioprotection by activating a cascade of kinase leading to mPTP inhibition and thus protect the cell against apoptotic and necrotic death. mPTP indicates permeability transition pore; K : mitochondrial + ATP potassium channel. ERK1/2, extracellular regulated kinase; MEK1/2 mitogen-activated protein kinase/ERK1/2 kinase; PI3K, , Phosphoinositide 3-kinase; GSK-3 , glycogen synthase kinase 3 ; eNOS, endothelial NOS; P70S6K, p70 ribosomal S6 protein kinase β β

  Jennings et al. established that 35 of ATP utilization

	[13 ]	1 0	[14 ]	%

observed during the first 90 minutes of complete ischemia in the canine heart is due to F F -ATPase activity. Mitochondria are believed 1 0 to hydrolyse ATP under conditions of oxygen deficit to maintain the mitochondrial membrane potential. However, this effect destroys any available ATP and favours Ca accumulation, which may contribute to the reduced performance of the heart (stunning) during 2 +

  The mechanism by which activation of mitoK results in cardioprotection is not well-understood, but implies a limitation in ATP mitochondrial membrane permeability. Several hypotheses have been proposed. The first one considers that the beneficial effect of mitoK activation could be the consequence of a partial dissipation of the membrane potential caused by the net influx of K .

	115 ]	ATP				[116 -117 ]
	ATP					+ [118 ]	This would
	reduce Ca entry or release an excess of Ca , preventing Ca overload 2 + 2 + 2 +	[119 ]	and thus mPTP opening during reperfusion. This is
	consistent with the results of Korge et al.	[120 ]	and Facundo et al.	[121 ]	, who reported that diazoxide prevents opening of the mPTP

  the cellular damages resulting from oxidative stress and cardiac injuries induced by ischemia-reperfusion in rodents, andObame et al. [143

	et al.	[142 ]	found that the irreversible TSPO ligand SSR180575 prevented

[START_REF] Zini | The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation[END_REF]

[START_REF] Zini | The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation[END_REF]

[START_REF] Zini | The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation[END_REF]

[START_REF] Zini | The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation[END_REF]

[START_REF] Zini | The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation[END_REF]

[START_REF] Zini | The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation[END_REF]

[START_REF] Zini | The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation[END_REF]

[START_REF] Zini | The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation[END_REF]

[START_REF] Zini | The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation[END_REF]

[START_REF] Zini | The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation[END_REF]

[START_REF] Zini | The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation[END_REF]

[START_REF] Zini | The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation[END_REF]

Author Keywords mitochondria ; heart ; ischemia-reperfusion ; necrosis ; apoptosis ; mitochondrial membrane permeability.


Lazaros Mavridis

David W Ritchie

3D-BLAST: 3D PROTEIN STRUCTURE ALIGNMENT, COMPARISON, AND CLASSIFICATION USING SPHERICAL POLAR FOURIER CORRELATIONS

This paper presents a novel sequence-independent method of aligning protein structures using three-dimensional spherical polar Fourier (SPF) representations of protein shape. The approach is demonstrated by clustering subsets of the CATH database for each of the four main CATH fold types, and by searching the entire CATH database of some 12,000 structures using several protein structures as queries. Overall, the automatic SPF clustering approach agrees very well with the expert-curated CATH classification, and ROCplot analyses of the database searches show that the approach has very high precision and recall. Database query times can be reduced considerably by using a simple rotationally-invariant pre-filter in tandem with a more sensitive rotational search with little or no reduction in accuracy. Hence it should soon be possible to perform on-line 3D structural searches in interactive time-scales.

Introduction

The BLAST and FASTA sequence alignment programs are probably considered by most biologists as the standard tools for searching genomic nucleotide or amino acid sequence databases. However, it is known that in nature protein structure is more conserved than protein sequence. Hence, structural alignments can provide significant insights about protein function and can help classify protein families into functional super-families [1]. Considering the large and rapidly growing number of three-dimensional (3D) protein structures available in the Protein Databank (PDB [2]), it is important to develop new methods to align and compare protein structures [3]. Current protein structure alignment methods such as SSM [START_REF] Krissinel | Proceedings of the Fifth international Conference on Molecular Structural Biology[END_REF], CE [START_REF] Shindyalov | [END_REF], VAST [6], SSAP [10], and DALI [1] typically use graphmatching and dynamic programming techniques to identify and align cliques of backbone Cα atoms or secondary structural features. However, these approaches are slow compared to conventional sequence alignment methods. Furthermore, finding the best way to perform 3D structural alignments remains an open question [7]. The most widely used protein structure classifications are the CATH [8] and SCOP [9] databases, both of which are curated by human experts. In CATH, the classification is initially performed using SSAP, whereas SCOP relies more on visual inspection by the curators. In both cases, it would be desirable to be able to assemble and update structural classifications in a more automated way.

In a significant step towards performing protein structure comparisons more efficiently, Mak et al. [11] and Sael et al. [12] showed that the 3D shapes of large protein molecules could be compared and classified very rapidly using special 3D pose-invariant descriptors derived from spherical harmonic (SH) and Zernike polynomials [13]. Hence this kind of approach offers the possibility of being able to search a 3D database of protein structures very rapidly. Similarly, it has been shown previously that the related spherical polar Fourier (SPF) representation provides a fast way to perform protein-protein docking correlations [14,15]. However, until now, the SPF approach has not been used to superpose and compare protein shapes. Conceptually, the use of Zernike descriptors has close parallels with the SPF representation, although Mak et al. and Sael et al. did not exploit the special rotational properties of the SH basis functions. Hence their approaches can detect similar protein shapes, but cannot superpose them. There is growing interest in the use of SH-based shape representation techniques [16][17][18][19][20][21][22][23][24][25]. The novelty of our SPF representation compared to other SH-based approaches is its use of orthonormal Laguerre-Gaussian (GL) radial functions to give a 3D density-based representation of molecular shape. This removes the requirement that molecular surfaces should be star-like with respect to the chosen coordinate origin [26], and it allows both rotational an translational correlation expressions to be calculated analytically [27]. In this paper, we demonstrate that SPF correlations may be used to superpose and compare protein structures in an efficient and completely sequenceindependent manner. We present results obtained by clustering multiple protein structures selected from the CATH database, and we demonstrate the utility of our approach by performing queries of single protein structures against the entire CATH database of some 12,000 protein structures.

Methods

Spherical Polar Fourier Shape Density Functions

In the SPF approach, protein shapes are represented as 3D density functions expressed as expansions of orthonormal basis functions:

ܴሺܚሻ ൌ ܽ ܴ ሺݎሻݕ ሺߴ, ߮ሻ ሺ1ሻ ୀି ିଵ ୀ ே ୀ1
where N is the order of the expansion, ܴ ሺݎሻ are Laguerre-Gaussian radial functions, ݕ ሺԂ, φሻ are real spherical harmonics, and ܽ are the expansion coefficients which are calculated numerically as described previously [14].

Figure 1 shows the SPF representations of a pair of similar nitrogenase domains at several expansion orders. In order to superpose a pair of protein structures we calculate a rotation-dependent Carbo-like similarity score S ROT using:

2 1 2 2 1 2             = ∑ ∑ ∑ N nlm nlm N nlm nlm nlm N nlm nlm ROT b a b a S ሺ2ሻ
Conceptually, one protein is held fixed and a six-dimensional (6D) rotational/translational search over positions of the second protein is performed. However, in practice it is more efficient to implement the search using one translational and five Euler angle rotational coordinates [14].

Rotationally Invariant Fingerprints

Protein superpositions can be calculated relatively quickly by using FFT techniques to accelerate the 6D search [14]. However, it is necessary to develop even faster comparison techniques in order to search very large 3D structural databases. Noting that expansion coefficients with the same values of m transform amongst themselves under rotation, it is natural to use the vector interpretation of SH coefficients to construct rotationally invariant (RI) fingerprints (RIFs) as:

ܣ ൌ ඩ ܽ 2 ୀି ሺ3ሻ
If the coefficients a nlm define the shape density of a protein, then the rotation-invariant descriptors A n together encode the protein's radial mass distribution. By analogy to Eq 2, the RIF similarity score is written as:

2 1 1 2 1 1 1             = ∑ ∑ ∑ = = = N n n N n n n N n n RIF B A B A S ሺ4ሻ

Implementation

Our approach has been implemented in a C program called 3D-Blast. We have used 3D-Blast to calculate and store SPF coefficients for all the proteins of the CATH database. However, the approach could equally be used to store SPF descriptions of other protein structure databases such as SCOP, for example. Given a protein query structure in PDB format, 3D-Blast will calculate its SPF representation and then use only the resulting SPF expansion coefficients to search its database.

Data Preparation

In order to evaluate the SPF approach, clustering experiments were performed on selected proteins from the CATH database [8,10], and the entire CATH database was searched using SPF density functions as queries. In CATH, proteins are assigned to a super-family according to their fold class, architecture, topology, and homology. This classification scheme is essentially hierarchical, with the top-level class consisting of four possible fold types: All-α, All-β, α+β, and irregular. Each of the four levels in the CATH hierarchy is identified by a numeric code. Additionally, CATH names each protein according to its four-letter PDB code, its chain letter, and the number of domains (e.g. 1IOMA02). These naming conventions are followed here. For each clustering experiment, five or six super-families with the same architecture were selected in such a way as to give around 30 protein structures for each CATH fold class. Hence the aim of these experiments is to assess how well our approach can identity proteins with the same topology and homology within a given fold architecture. The details of the super-families used here are shown in Table 1.

For the initial database search experiment, asparagine synthetase (PDB code 12AS, CATH super-family 3.30.930.10) was selected as the query structure. The 3.30.930.10 super-family has 27 members, and these were treated as "positives" while the remaining proteins in the database were treated as "negatives" with respect to the query. If a scoring function were to reproduce exactly the CATH classification, the 27 positives would appear at the top of the ranked list. However, such an ideal outcome is seldom observed in practice. Hence Receiver-Operator-Characteristic (ROC) curves [28] are used to analyse objectively the precision/recall characteristics of the scoring functions. In a ROC analysis, each element of the ranked list is considered in turn, and the number of positives and negatives in the sublists to each side of the current element are counted. Here, we call the high similarity sublist the "hit list". A true positive (TP) is assigned when an element in the hit list contains an original positive, and a false positive (FP) is assigned if that element contains a negative. Conversely, a true negative (TN) is assigned when an original negative falls outside the hit list, and a false negative (FN) is assigned if that position is occupied by a positive member. ROC plots are produced by plotting the true positive rate (TPR) on the y-axis against the false positive rate (FPR) on the x-axis, where TPR and FPR are given by:

ܴܶܲ ൌ ܶܲ ܶܲ ܰܨ ሺ5ሻ ܴܲܨ ൌ ܲܨ ܲܨ ܶܰ ሺ6ሻ
The quality of a scoring function can quickly be assessed from the shape of a ROC plot. For example, a random scoring function would give a diagonal line (TPR=FPR), whereas a perfect scoring function would give a horizontal line (TPR=1) with a maximum value for the area under the curve (AUC=1).

Expansion Resolution and Protein Superposition

It was shown previously that SPF expansions of order N ≥ 25 are required for protein-protein docking correlations [14]. However, from our previous results on small-molecule virtual screening [29], we expected that considerably lower order expansions would be sufficient to calculate satisfactory protein shape-density superpositions. In order to test this hypothesis, we selected four example protein pairs with sequence identities ranging from 28% to 43% previously identified by Levitt and Gerstein [30], and we superposed them using different expansion orders. The root mean square deviation (RMSD) for each superposed pair was calculated between corresponding Cα atoms identified by ProFit [31]. Figure 2 shows the RMSD as a function of the expansion order N. Figure 2 shows that using an expansion order of N ≥3 is generally sufficient to obtain good superpositions (with RMSDs ranging from 2Å to 5Å), requiring about 0.25 seconds per superposition. However, we choose to use expansions to order N=6 in order to disambiguate cases where flipping about an axis might give similar scores [29]. Figure 2 shows that calculating N=6 correlations is only marginally more expensive than using N=3. Figure 3 shows the N=6 superposition of the nitrogenase proteins shown in Figure 1.

Clustering Protein Structures

For the All-α clustering experiment, five super-families were selected, as listed in Table 1. For each pair of proteins in this set, a correlation search was performed to find the orientation that gives the maximum Carbo similarity score (Eq 2). Ward's agglomerative clustering [32] was then applied to the resulting table of pair-wise similarity scores. The clustering results in Figure 4 show that the 1.10.230.10 and 1.10.167.10 super-families are correctly assigned to two separate groups. Although there exist some differences in the clusters produced for the remaining superfamilies, there is still a very good agreement between the calculated SPF clusters and the CATH hierarchy. The most notable exception is that suposin (PDB code 1N69) is grouped with the 1.10.30.10 super-family. From visual inspection of Figure 4 it can be seen that the overall fold of suposin is much closer to that of the 1.10.30.10 super- For the All-β class, six super-families were selected. As can be seen in Figure 5, SPF clustering correctly distinguishes all six groups, but two proteins are misplaced according to the CATH classification. These are the carboxy-terminal LIM domain (PDB code 1CTL) and the influenza virus hemagglutinin (PDB code 2VIR) which are grouped with the singleton heparin-binding domain (PDB code 1KMX). This seems to occur because 1CTL and 2VIR are calculated to be less similar to the other members of their respective CATH super-families, and they are grouped with 1KMX largely because all three proteins have similar steric bulk.

For the α + β class, five super-families were selected. From these five super-families the 3.10.130.10 and 3.10.120.10 super-families are correctly assigned into two groups, as shown in Figure 6. The other three superfamilies (3.10.110.10, 3.10.150.10, and 3.10.170.10), present a similar case to the All-α results, whereby one superfamily group (3.10.110.10) is split into two sub-groups and two super-family groups (3.10.170.10 and 3.10.150.10) are merged into one. Nonetheless, despite these differences, the overall consistency of the SPF clustering with the CATH hierarchy is clearly very good.

For the irregular class, six super-families were selected. As in the All-β example, SPF clustering is completely consistent with the CATH hierarchy. However, two proteins are misplaced with respect to the CATH classification, namely bikunin from the inter-alpha-inhibitor complex (PDB code 1BIK) and the tick anticoagulant peptide (PDB code 1D0D), which are both grouped with the 4.10.490.10 super-family. This seems to be due to the difference in size between those proteins and the rest of their super-family of factor XA inhibitors. For example, Figure 7 shows that bikunin has a repeat of the same motif as the other factor XA inhibitors. Hence, it is sterically too large to be clustered with the other XA inhibitors, and is instead placed with the larger proteins of the 4.10.490.10 super-family.

Database Searching

Because it is relatively time-consuming to calculate high order comparisons, different rotational and rotationinvariant parameters were tested to explore the extent to which the speed of queries on a large database can be improved while still performing accurate searches. In each case, the asparagine synthetase structure (PDB code 12AS, CATH super-family 3.30.930.10) was superposed onto each protein in the database using N=6 SPF correlation searches, and the database structures were ranked in order to similarity to the query. Figure 8 shows the resulting ROC curves obtained for a range of expansion orders when querying the CATH database, from which it can be seen that our approach gives very good precision and recall. Figure 9 shows the 27 members of this superfamily, which were treated as true positives with respect to the query. To analyse the results further, the TPs were clustered into 5 groups. The query belongs to group 1 and when using an expansion order of N ≥6, all members of this group were found in the top 10 hits. groups 2 and 3 have similar β-sheet structures to group 1, but different arrangements of α-helices. All proteins in groups 2 and 3 are ranked in the top 20% of the database. All proteins in group 4 are ranked in the top 30%. The singleton group 5 is an obvious outlier due to its extra α-helical domain.

Figure 8 also shows results for the RIF scoring function. Compared to the rotation-dependent scoring function, the RIF function generally performs remarkably well. However, the two functions behave rather differently on the first percentages of the database. For example, the rotational searches give a TPR of around 40% on the first 0.1% of the database, whereas the RIF searches give a TPR of only around 10%. Hence, the RIF function is not sufficiently sensitive to be used on its own but it could usefully be used as a fast pre-filter on the database so that the more expensive rotation-dependent function is only applied to the most promising candidates. In order to test the notion, the CATH database was searched using the RIF and rotational scoring functions in tandem using several protein structures as queries: asparagine synthetase, ALF4-activated Giα1 protein (PDB code 1AGR), chicken cysteine-rich protein (PDB code 1B8T), dihydrolipoyllysine-residue acetyl transferase (PDB code 1W4E), and UbcH7 (PDB code 1C4Z). Using a RIF pre-filter similarity threshold of 0.99, which selects from 2% to 15% of the database for rotational re-scoring, each tandem search takes less than 10 minutes compared to 75 minutes for full rotational searches on a 2.3GHz Pentium Xeon processor. Figure 10 shows the resulting ROC plots for the rotational, RIF, and the tandem searches. This figure shows that tandem searches achieve the same high level of performance as the rotational searches. The very high precision/recall values further support the utility of the SPF scoring functions.

Discussion and Conclusions

It has been shown that low resolution SPF expansions provide a reliable and fast sequence-independent way to superpose and compare protein structures. The clustering results of the four CATH super-families show that SPF clustering generally agree with the CATH classification. The database search results show that a large protein structure database can be queried accurately using SPF expansions to order N=6. In order to accelerate further the scoring calculation, the use of a simple rotationally invariant scoring function was investigated. Our RIF scoring function is significantly faster to calculate, but it is less precise than a rotational search. Nonetheless, the RIF function may still be used effectively as an initial filter when searching large databases. Using the RIF function in tandem with the rotational function to re-score only the top matches of the database gives very promising results.

Thus tandem searches are much faster than full rotational searches, yet the recall/precision is almost equivalent. Currently a limitation of our approach is that proteins larger than a typical domain of around 100-150 residues are not represented well due to the exponential decay of the GL functions at large radial distances. However, we are working to extend the resolution range of our approach to be able to describe proteins of any size, and we are enhancing the approach to be able to detect the presence of symmetrical domains and repeated motifs, for example. We also aim to develop a web-based interface for general use by the biological community. As well as being able to calculate rapidly sequence-independent protein structure superpositions, we believe that the SPF approach could provide an automatic and objective way to enhance the quality of protein structure classifications.

Figure 1 .

 1 Figure 1. Two similar nitrogenase proteins, represented as SPF expansions at expansion orders N=5, 10, 15, and 20. The protein in the top row is from azotobacter vinlandii (PDB code 2MIN). The protein in the bottom row is from clostridium pasteurianum (PDB code 1MIO).

Figure 2 .

 2 Figure 2. On the left, the RMSD values of four protein pairs are plotted against the expansion order N. On the right, the time per pair-wise search is plotted against the expansion order N.

Figure 3 .

 3 Figure 3. The superposition of a pair of nitrogenase proteins calculated using N=6 SPF correlations, shown as ribbon cartoons (left), backbone traces (middle), and as 3D SPF density expansions to order N=25 (right). The protein in the top row is from azotobacter (PDB code 2MIN). Top row: PDB code 2MIN; middle row PDB code 1MIO; bottom row their superposed orientation. The two proteins have a sequence identity of 43%.

  family than the CATH assignment of 1.10.225.10. This suggests that the automatic SPF classification could potentially help the CATH curators resolve unusual or ambiguous cases.

Figure 4 .

 4 Figure 4. SPF clustering results of the All-α class. Left: the dendrogram obtained using N=6 with five clusters; Right: the corresponding groups and the representative proteins for each group.

Figure 5 .

 5 Figure 5. SPF clustering of the All-β class. Left: the dendrogram obtained using N=6 with six clusters; Right: the corresponding groups and the representative proteins for each group.

Figure 6 .

 6 Figure 6. SPF clustering of α + β class. Left: the dendrogram obtained using N=6 with five clusters; Right: the corresponding groups and the representative proteins for each group.

Figure 7 .

 7 Figure 7. SPF clustering of the irregular class. Left: the dendrogram obtained using N=6 with six clusters; Right: the corresponding groups and the representative proteins of each group.

Figure 8 .Figure 9 .Figure 10 .

 8910 Figure 8. ROC plot analyses obtained when querying the entire CATH database function (Eq 2); B: close-up view of (A); C: RIF scoring function (Eq

Table 1 .

 1 The 23 CATH super-families used in the four clustering experiments, grouped according to class and architecture. For each super-family the name of each representative protein is provided according to the CATH naming convention.

	Class +	Topology +	Protein Name	Representative
	Architecture	Homology	and Function	member
		230.10	Cytochrome p450-Terp; Domain 2	1iomA02
	All-α	120.10	Bifunctional Trypsin/Alpha-Amylase Inhibitor	1beaA00
	Orthogonal Bundle	225.10	NK -Lysin	1l9lA00
	(1.10)	167.10	Regulator of G-Protein Signalling 4; Domain 2	1dk8A02
		30.10	DNA Binding (I) , subunit A	1qrvA00
		109.10	Umud Fragment, subunit A	1jhfB00
		150.10	Urease, subunit B	1ejxB00
	All-β	110.10	Cysteine Knot Cytokines, subunit B	1g47A00
	Ribbon	77.10	Hemagglutinin; Chain A, domain 2	1jsdA02
	(2.10)	160.10	Vascular Endothelial Growth Factor -165, Heparin -binding Domain	1kmxA00
		10.10	Seminal Fluid Protein PDC -109 (Domain B)	1h8pA02
		130.10	P-30 Protein	1dy5A00
	α+β	170.10	Elastate; Domain 1	1u4gA01
	Roll	150.10	DNA Polymerase III; Chain A, Domain 2	1ok7A01
	(3.10)	110.10	Ubiquitin Conjugating Enzyme

Acknowledgements

This work is funded by the Agence Nationale la Recherche, grant reference: ANR-08-CEXC-017-01.