Current Medicinal Chemistry - Volume 10, Issue 16, 2003

The voltage dependent anion channel and the adenine nucleotide translocase are the principal proteins found in the mitochondrial outer and inner membranes, respectively. The two proteins can associate to form a junctional complex that establishes contact sites between the two membranes. This complex in turn recruits a range of proteins depending on the function to be executed. Among these, the junctional complexes can bind Bax and other proapoptotic proteins. Bax regulates the involvement of mitochondria in the apoptotic signalling pathway by controlling the the release of apoptogenic proteins from the mitochondrial intermembrane space to the cytosol. Another protein recruited to ANT is cyclophilin-D. Cyclophilin-D is a peptidylprolyl cis-trans-isomerase located in the intramitochondrial (matrix) compartment which stabilizes a “deformed” conformation of ANT in which its native gating properties are lost. Although the deformed state, when extensive, is lethal, cells can tolerate this conformational change when it occurs transiently. There is now a large body of data that implicates the voltage dependent anion channel, the adenine nucleotide translocase and cyclophilin-D, both separately and together, in the mitochondrial reactions of apoptosis. But there is no consensus over how, or indeed if, they are involved. This article examines the data relevant to this question and considers why a complex of these three proteins may be essential for the action of Bax and other proapoptotic proteins in permeabilizing the outer membrane to intermembrane space proteins.

The mitochondrial permeability transition (MPT) plays an important role in damage-induced cell death, and agents inhibiting the MPT may have a therapeutic potential for treating human conditions such as ischemia / reperfusion injury, trauma, and neurodegenerative diseases. The mitochondrial matrix protein, cyclophilin D (CYP D), a member of a family of highly homologous peptidylprolyl cis-trans isomerases (PPIases), plays a decisive role in MPT, being an integral constituent of the MPT pore. Other putative MPT pore proteins include the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC). In an alternative model, the MPT pore is formed by clusters of misfolded membrane proteins outlining aqueous channels that are regulated by CYP D and other chaperone-like proteins. Like cyclophilin A (CYP A) and other cyclophilin family members, CYP D is targeted by the immunosuppressant cyclosporin A (CsA). CsA is cytoprotective in many cellular and animal models, but protection may result from either inhibition of the MPT through an interaction with CYP D or inhibition of calcineurin-mediated dephosphorylation of BAD through an interaction with CYP A. The relevance of MPT inhibition by CsA for its cytoprotective effects is well documented in many cellular models. Mechanisms of action in vivo are more difficult to define, and accordingly the evidence is as yet less compelling in in vivo animal models of ischemia / reperfusion injury, trauma and neurodegenerative diseases. Notwithstanding, CYP D is a drug target of high interest. Structural considerations suggest feasibility of designing CYP D ligands without immunosuppressant properties. This is highly desirable, since they have the potential of being useful therapeutic agents in a variety of disease states. It might be a tougher challenge to obtain compounds specific for CYP D vs. other cyclophilins, and / or of small molecular weight, allowing brain penetration to make them suitable for treating neurodegenerative diseases.

In addition to its normal function, the adenine nucleotide translocase (ANT) forms the inner membrane channel of the mitochondrial permeability transition pore (MPTP). Binding of cyclophilin-D (CyPD) to its matrix surface (probably on Pro61 on loop 1) facilitates a calcium-triggered conformational change converting it from a specific transporter to a non-specific pore. The voltage dependent anion channel (VDAC) binds to the outer face of the ANT, at contact sites between the inner and outer membranes, and together VDAC, ANT and CyP-D probably represent the minimum MPTP configuration. The evidence for this is critically reviewed as is the structure and molecular mechanism of the carrier in its normal physiological mode. This provides helpful insights into MPTP regulation by adenine nucleotides, membrane potential and ANT ligands such as carboxyatractyloside and bongkrekic acid. Oxidative stress activates the MPTP by glutathionemediated cross-linking of Cys159 and Cys256 on matrix-facing loops of the ANT that inhibits ADP binding and enhances CyP-D binding. Molecular modeling of the loop containing the ADP binding site suggests an arrangement of aspartate and glutamate residues that may provide a calcium binding site. There are other proteins that may bind to the ANT, modulating MPTP opening and hence cell death. These included members of the Bax / Bcl-2 family (both oncoproteins and tumor suppressors) and viral proteins. Vpr from HIV-1 can bind to ANT and convert it into a pro-apoptotic pore, whereas vMIA from cytomegalovirus interacts to inhibit opening. Thus the ANT may provide a molecular link between physiopathological mechanisms of infection and the regulation of MPTP function and so represents a potential therapeutic target.

Voltage-dependent anion channels (VDAC) are major constituents of the outer mitochondrial membrane. Over the past few years, several hypotheses and mechanisms have been forwarded for and against a role for VDAC in outer mitochondrial membrane permeability and the subsequent release of apoptosis promoting factors. The current review outlines current knowledge related to the role of VDAC in the regulation of cell death and speculates on possible therapeutic applications through VDAC modulation.

Hexokinase II is often highly expressed in poorly differentiated and rapidly growing tumors that exhibit a high rate of aerobic glycolysis. Hexokinase II binds to the mitochondrial membrane through its interaction with the outer membrane voltage-dependent anion channel (VDAC), preferentially at contact sites between the outer and inner mitochondrial membrane. This location is thought to be important for the integration of glycolysis with mitochondrial energy metabolism. VDAC is a critical component of the mitochondrial phase of apoptosis and its interaction with Bcl-2 family proteins controls the rate of release of mitochondrial intermembrane space proteins that activate the execution phase of apoptosis. The proteins involved in the contact sites also constitute the mitochondrial permeability transition, one of the mechanisms by which mitochondrial protein release can be mediated. Hexokinase II binding to VDAC suppresses the release of intermembrane space proteins and inhibits apoptosis, thereby contributing to the survival advantage of tumor cells. This interaction places hexokinase II in a position to integrate glycolytic metabolism of the tumor cell with the control of apoptosis at the mitochondrial level. Mitochondrial binding of hexokinase II may constitute an attractive target for therapeutic intervention to suppress tumor growth.

The Bcl-2 family of proteins provide the most unambiguous link between mitochondrial functions and apoptosis, as their only (or principal) functions appear to be as regulators of this cell death pathway. Rational drug design to manipulate the functions of these proteins has been hampered by the lack of a clear understanding of a biochemical or molecular function, with disruption of intra-family protein-protein interactions as the only known, but daunting, objective. There has been substantial progress in this task using molecular modeling and drug leads. The prospects are also good for development of chemical tools for functional analysis of the Bcl-2 proteins.

The peripheral benzodiazepine receptor (PBR) is a critical component of the mitochondrial permeability transition pore (MPTP), a multiprotein complex located at the contact site between inner and outer mitochondrial membranes, which is intimately involved in the initiation and regulation of apoptosis. PBR is a small evolutionary conserved protein, located at the surface of the mitochondria where it is physically associated with the voltage-dependent anion channel (VDAC) and adenosine nucleotide translocase (ANT) that form the backbone of MPTP. PBR is widely distributed throughout the body and has been associated with numerous biological functions. Consistent with its localization in the MPTP, PBR is involved in the regulation of apoptosis, but also in the regulation of cell proliferation, stimulation of steroidogenesis, immunomodulation, porphyrin transport, heme biosynthesis, anion transport and regulation of mitochondrial functions. The recent literature on PBR is reviewed here. Specifically, we highlight numerous results suggesting that the use of specific PBR ligands to modulate PBR activity may have potential therapeutic applications and might be of significant clinical benefit in the management of a large spectrum of different indications including cancer, auto-immune, infectious and neurodegenerative diseases. In addition, we present the proposed mechanisms by which the molecules exerted these effects, particularly oriented on the modulation of the MPTP activities.

Mitochondria are key players in fundamental processes such as energy production and adaptive responses to cellular stress, including apoptosis. Mitochondrial membranes may undergo permeabilization when perturbed by a number of intracellular stress mediators, and consequently may allow the release of intramitochondrial proteins. This event triggers and amplifies the cellular apoptotic program, provided sufficient energy is available. Mitochondrial membranes are therefore both targets and regulators of intracellular pathways controlling cell fate. Evidence is emerging that the integration and biological outcome of these pathways might be critically dependent on the unique lipid composition of mitochondrial membranes.

Tryptophan is an essential amino acid and the least abundant constituent of proteins. In parallel it represents a source for two important biochemical pathways: the generation of neurotransmitter 5- hydroxytryptamine (serotonin) by the tetrahydrobiopterin-dependent tryptophan 5-hydroxylase, and the formation of kynurenine derivatives and nicotinamide adenine dinucleotides initiated by the enzymes tryptophan pyrrolase (tryptophan 2,3-dioxygenase, TDO) and indoleamine 2,3-dioxygenase (IDO). Whereas TDO is located in the liver cells, IDO is expressed in a large variety of cells and is inducible by the cytokine interferon-γ. Therefore, accelerated tryptophan degradation is observed in diseases and disorders concomitant with cellular immune activation, e. g. infectious, autoimmune, and malignant diseases, as well as during pregnancy. According to the cytostatic and antiproliferative properties of tryptophan-depletion on T lymphocytes, activated T-helper type 1 (Th-1) cells may down-regulate immune response via degradation of tryptophan. Especially in states of persistent immune activation availability of free serum tryptophan is diminished and as a consequence of reduced serotonin production, serotonergic functions may as well be affected. Accumulation of neuroactive kynurenine metabolites such as quinolinic acid may contribute to the development of neurologic / psychiatric disorders. Thus, IDO seems to represent a link between the immunological network and neuroendocrine functions with far reaching consequences in regard to the psychological status of patients. These observations provide a basis for the better understanding of mood disorder and related symptoms in chronic diseases.

Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), the rate-limiting enzyme of cholesterol synthesis. In recent years, statins have become the major choice of treatment for hypercholesterolemia. Emerging evidence from both animal and human studies indicates that mechanisms independent of cholesterol lowering effects contribute to the observed clinical benefits of statins. The anti-hypertrophy effect of statins on the cardiac tissue represents one of such mechanisms. The beneficial effects of statins on cardiac hypertrophy and cardioprotection may be attributed to their functional influences on small G proteins such as Ras and Rho, resulting in an increase of endogenous nitric oxide (NO), reduction of oxidative stress, inhibition of inflammatory reaction, and decrease of the renin-angiotensin system activity as well as C-reactive protein (CRP) levels in cardiac tissues. Recent findings from in vitro and in vivo studies of statins on cardioprotective effects are summarized in this review. The unveiled novel mechanisms support the use of statins as the new mainstay therapeutic agents for various cardiovascular diseases and complications.

The Nuclear Factor Kappa B (NF-κB) is a lymphoid-specific transcription factor, which is sequestered in the cytoplasm by the protein IκB. NF-κB plays a major role in the regulation of HIV-1 gene expression. Upon activation, NF-κB is released from IκB, moves to the nucleus, and binds to its sites on the HIV long terminal repeat to start transcription of integrated HIV genome. The present review focuses on the NF-κB as a potential target for the development of chemotherapy against HIV-1. Beginning from the viral-binding to reverse transcription, integration, and gene expression, to the virion maturation, the life cycle of HIV presents drug-targets at all the stages. As a result, many drugs have been developed and have entered clinical trials. Some of the most important of these are reverse transcriptase and protease inhibitors, which have been used mostly in clinical studies in the form of combined therapy. But, this combined therapy has presented the problem of resistance, due to mutations in the virus. However, targeting NF-κB for the suppression of virus does not present the problem of resistance, as NF-κB is a normal part of the human T-4 cell, and is not subject to mutations, as is the virus. An overview of the NF-κB system and its role in HIV-1 is presented, followed by a critical review of its current and potential synthetic inhibitors. The drugs studied against NF-κB fall mainly into three categories: (1) Antioxidants, against oxidative stress conditions, which aid in NF-κB activation, (2) IκB phosphorylation and degradation inhibitors (the phosphorylation and degradation of IκB is necessary to make NF-κB free and move to the nucleus), and (3) NF-κB-DNA binding inhibitors. The antioxidants include N-Acetyl-L-cysteine (NAC), α-Lipoic acid, glutathione monoester, pyrrolidine dithiocarbamate, and tepoxalin, of which NAC is the best studied. The IκB phosphorylation and degradation inhibitors, which have been studied in the context of HIV-1 include the salicylates (sodium salicylate, and acetylsalicylic acid (aspirin)). Finally, the NF-κB-DNA binding inhibitors, which have received attention only recently, are reviewed. These include the most potential, aurine tricarboxylic acid (ATA), a chelating agent, which has been found to inhibit NF-κB-DNA binding at a low concentration of 30 μM. The probable mechanism of action of these drugs is discussed alongwith relevant suggestions and conclusions.