Current Drug Targets - Volume 4, Issue 8, 2003

Correct regulation of gene expression is essential both to normal development and to the correct functioning of the adult organism. Such regulation is usually achieved at the level of DNA transcription, a process that controls which genes are transcribed into RNA by the enzyme RNA polymerase, although post-transcriptional regulation is also important. The transcription of specific genes is controlled by regulatory proteins known as transcription factors. Transcription factors have been grouped in families on the basis of shared DNA-binding motifs. Other regions of the factors interact with RNA polymerase and its associated proteins to increase or decrease the rate of transcription. The vital role of these factors, together with the fact that a single factor can affect the expression of many genes, suggests that the inactivation of a transcription factor as a result of an inherited mutation is incompatible with survival. Therefore, numerous pharmacological drugs including steroids have been identified to affect the transcription factors. However, only few drugs have been developed initially to target the transcriptional regulation, since transcription factors work within nuclei. From early 1990, several researchers including us tried to modulate the transcription factors using molecular strategy. Initially, overexpression of TAR-containing sequences (TAR decoys) in a double copy murine retroviral vector was used to render cells resistant to HIV replication [1]. Currently, TAR decoys, short RNA oligonucleotides corresponding to the HIV TAR sequence, are used to inhibit HIV expression and replication by blocking the binding of the HIV regulatory protein Tat to the authentic TAR region. However, such RNA decoys are very difficult to use in vivo. In addition, the regulation of decoy expression is also problematic. To overcome these issues, synthetic ds DNA with high affinity for transcription factors has been developed as a “decoy” cis-element to bind the transcription factors and block the activation of genes mediating such diseases, resulting in an effective therapy for treating diseases, since transfection of ds ODN corresponding to the cis sequence will result in attenuation of the removal of the transfactors from the endogenous cis-element with subsequent modulation of gene expression [2]. This approach is particularly attractive for several reasons: 1) the potential drug targets (transcription factors) are plentiful and readily identifiable, 2) the synthesis of the sequence-specific decoy is relatively simple and can be targeted to specific tissues, 3) knowledge of the exact molecular structure of the target transcription factor is unnecessary, and 4) decoy ODN may be more effective than antisense ODN in blocking constitutively expressed factors as well as multiple transcription factors that bind to the same cis element. Although the mechanisms of actions of antisense ODN are still unclear, the principle of the transcription factor decoy approach is simply the reduction of promoter activity due to the inhibition of binding of a transcription factor to a specific sequence in the promoter region [ (Fig. 1). In 1996, clinical application of “decoy” against E2F was approved by FDA to treat neointimal “For figure please see the pdf file.” TF = transcription factor, decoy = decoy ODN, antisense = antisense ODN, siRNA = small interfering RNA.] hyperplasia in vein bypass grafts which results in failure in up to 50 % of grafts within a period of 10 years [3]. The present targets for decoy ODN are wide-spread from cell cycle regulatory transcription factors such as E2F to inflammatory regulatory transcription factors such as NFkB. Although there are still many unresolved issues in the clinical application of decoy strategy, its utility could be widespread as a useful tool for gene therapy in other diseases. Recent progress in the backborne of DNA in decoy including ab-anomeric, methylphosphonate- and phosphorothioate oligonucleotides have also given the opportunity to treat various diseases. In addition, circular dumbbell (ribbon) double-stranded ODN may also become a potent tool to develop decoy strategy. In this exciting hot topic issue, transcription factor decoy ODN have been discussed as the potential drugs to treat various diseases. REFERENCES [1] Bielinska. A., Shivdasani, R.A., Zhang, L., Nabel, G.J. (1990) Science 250, 997-1000. [2] Morishita, R., Higaki, J., Tomita, N., Ogihara, T. (1998) Circ. Res. 82, 1023-1028. [3] Mann, M.J., Whittemore, A.D., Donaldson, M.C., Belkin, M., Conte, M.S., Polak, J.F., Orav, E.J., Ehsan, A., Dell'Acqua, G., Dzau, V.J. (1999) Lancet 354, 1493-1498.

Efficient and minimally invasive drug delivery systems have been developed to treat intractable human diseases. One approach has been the development of chimeric vector systems combining at least two different vector systems. Based on this concept, chimeric drug delivery systems that combine viral and non-viral features have been developed. Fusigenic non-viral particles have been constructed by conferring viral fusion proteins onto non-viral vectors. HVJ (hemagglutinating virus of Japan; Sendai virus)-liposomes were constructed by the combination of DNA-loaded liposomes with a fusigenic envelope derived from HVJ (hemagglutinating virus of Japan, Sendai virus). Reconstituted HVJ-liposomes were also developed by the insertion of isolated fusion proteins of HVJ into DNA-loaded liposomes. Recently, the technology has been developed to incorporate macromolecules directly into inactivated HVJ particles without liposomes. The resulting HVJ envelope vector introduced plasmid DNA, efficiently and rapidly into both cultured cells in vitro and organs in vivo . Furthermore, proteins, synthetic oligonucleotides and drugs have also been effectively introduced into cells using the HVJ envelope vector. The HVJ envelope vector will be a promising tool for both ex vivo and in vivo gene therapy experiments.

Recent progress in cellular and molecular research has provided a new technique to inhibit target gene expression based on DNA technology such as antisense oligonucleotides (ODN) or decoy ODN. Especially, application of an antisense strategy to regulate the transcription of disease-related genes in vivo has important therapeutic potential to treat or cure a variety of diseases and abnormal physiological conditions. On the other hand, recently, a successful ODNbased approach termed decoy ODN has used synthetic ODN containing an enhancer element that can penetrate cells, to bind to sequence-specific DNA-binding proteins and interfere with transcription in vitro and in vivo. Transfection of ciselement double-stranded decoy ODN has been reported as a new powerful tool in a new class of anti-gene strategies to treat various diseases as gene therapy or as a research tool to examine the molecular mechanisms of expression of a specific gene . Transfection of double-stranded ODN corresponding to the cis-sequence will result in attenuation of the authentic cis-trans interaction, leading to removal of trans-factors from the endogenous cis-elements with subsequent modulation of gene expression. To date, we have chosen several target transcription factors such as NFkB (nuclear factor- kB) and E2F to prevent the progression of diseases, and negative regulatory element (NRE) for the renin gene and angiotensinogen gene-activating element (AGE) for the angiotensinogen gene to examine the molecular mechanisms of gene expression. In this section, we introduce the principles of the decoy strategy and how to design decoy ODN.

Recent progress in molecular biology has provided application of gene transfer methods in arthritis. Two clinical trails using ex vivo retrovirus mediated delivery of interleukin -1 receptor antagonist gene for rheumatoid arthritis has begun in USA and Germany. However, there are still many issues to be elucidated; one is the development of gene delivery system, and the other is the selection of therapeutic gene. Arthritis is nonlethal disease, and safety is one of the important issues. Currently viral mediated vectors are major even in clinical trials however, non viral efficient gene transfer system should be developed in future. Recently the application of DNA technologies, such as antisense oligonucleotide (ODN) strategies to regulate the transcription of disease-related genes in vivo, has significant therapeutic potential. Transfection of cis-element double-stranded oligonucleotides (decoy ODN) for nuclear factor kB binding site has been reported as a new powerful tool in arthritis. The concept of regulation the disease related gene expression at the level of transcriptional factor may be more therapeutic effects compared with monotherapy in arthritis.

Molecular therapy is emerging as a potential strategy for the treatment of cardiovascular disease such as restenosis after angioplasty, vascular bypass graft occlusion, transplant coronary vasculopathy, homozygous familial hypercholesterolemia and cystic fibrosis, for which no known effective therapy exists. Molecular biology and pathophysiology of the cardiovascular system have started to emerge, and the time is ripe for the introduction of gene therapy to the management of cardiovascular disorders. In this review, we have focused on the future potential of oligonucleotide-based gene therapy for restenosis after angioplasty, which still remains an issue in the field of cardiovascular disease.

Decoy oligodeoxynucleotides (ODN) that can reduce the trans-activity of transcription factors may be highly useful in gene therapy and the study of transcriptional regulation. Several different types of these double-stranded DNA decoys have been developed, including unmodified oligonucleotide duplexes, αβ-anomeric oligonucleotides, and oligonucleotide duplexes with methylphosphonate- and phosphorothioate-modified linkages. The latter ODNs have been particularly extensively studied but suffer from a number of limitations, including their insensitivity to polymerases, their lack of sequence specificity, and their tendency to activate immune responses. To resolve these problems, circular dumbbell (CD) double-stranded ODNs were developed. These CD ODNs are constructed by the circularization of the 3' and 5' ends of the oligonucleotides and enzymatic ligation. They exhibit high resistance to nucleases, are easily taken up by cells, and have a nontoxic unmodified backbone that resembles natural DNA. In this article, we review the method of constructing CD ODNs and their advantages compared to other modified ODNs for use as transcription decoys.

Recanalization therapy remains the most effective way for treatment of evolving myocardial infarction and thereby salvaging jeopardized tissue. However, the efficacy of reperfusion in limiting infarction and improving recovery of contractile function depends on the amount of irreversible damage occurring prior to initiating reperfusion and is related to failure of energy production to meet the basal needs of the injured myocardium. In recent years, a variety of metabolic therapies that enhance myocardial metabolism and attenuate changes in sodium and calcium homeostasis during ischemia have been proposed. They focus on (a) increasing myocardial glucose metabolism during ischemia or (b) inhibiting fatty acid metabolism to increase glucose use, and (c) inhibiting sodium and calcium influx pathways that deplete high energy phosphates. Recent studies from our laboratory showed that inhibition of aldose reductase, a key regulatory enzyme in the substrate flux via polyol pathway, reduces ischemic injury and improves functional and metabolic recovery after ischemia-reperfusion in hearts. These and subsequent studies have generated considerable interest in the use of aldose reductase inhibitors as potential therapeutic adjuncts in treating evolving myocardial infarction in patients. This review will discuss the mechanisms by which aldose reductase inhibitors protect ischemic myocardium and provide rationale for their use as cardioprotective drugs.

Synthetic peptide sequences constitute a useful tool to understand protein related diseases. A preliminary study consists of the analysis of peptide interaction with model membranes. The simplest one is based on monomolecular films of lipids spread at the air-water interface that imitate the interfacial environment in which some proteins function. Monolayer methodology provides a reliable screen of the extent to which hydrophobic interactions, charges, dipole potentials and subphase composition drive protein-lipid interaction. One step forward is based on the use of liposomes (lipid-based vesicles) that were originally introduced in 1965 as models of lipid bilayer membranes. Later, they have been widely studied as drug delivery systems mainly due to their safety, structural versatility, composition, fluidity and also because of their ability to incorporate almost any molecule regardless of its structure. In this sense, liposomes have been used as carriers of proteins and peptide antigens. Antigenic materials can be attached to the outer surface, encapsulated within the internal aqueous spaces or reconstituted within the lipid bilayers of the liposomes. In the present review we describe the steps going from the selection of peptides related to viral hepatitis proteins to its diagnostic and therapeutic application, with special emphasis on the use of model membranes to predict peptide mode of interaction with the target cell.

Increasing bacterial resistance to virtually all available antibiotics causes an urgent need for new antimicrobial drugs, drug targets and therapeutic concepts. This review focuses on strategies to render bacteria highly susceptible to the antimicrobial arsenal of the immune system by targeting bacterial immune escape mechanisms that are conserved in a major number of pathogens. Virtually all innate molecules that inactivate bacteria, ranging from antimicrobial peptides such as defensins and cathelicidins to bacteriolytic enzymes such as lysozyme and group IIA phospholipase A2, are highly cationic in order to facilitate binding to the anionic bacterial cell envelopes. Bacteria have found ways to modulate their anionic cell wall polymers such as peptidoglycan, lipopolysaccharide, teichoic acid or phospholipids by introducing positively charged groups. Two of these mechanisms involving the transfer of D-alanine into teichoic acids and of L-lysine into phospholipids, respectively, have been identified and characterized in Staphylococcus aureus, a major human pathogen in community- and hospital-acquired infections. Inactivation of the responsible genes, dltABCD for alanylation of teichoic acids and mprF for lysinylation of phosphatidylglycerol, renders S. aureus highly susceptible to many human animicrobial molecules and leads to profoundly attenuated virulence in several animal models. dltABCD- and mprFrelated genes are found in the genomes of many bacterial pathogens indicating that the escape from human host defenses by modulation of the cell envelope is a general trait in pathogenic bacteria. This review suggests that inhibitors of DltABCD or MprF should have great potential in complementing or replacing the conventional antibiotic therapies.

Peripheral arterial disease (PAD) is an important manifestation of systemic atherosclerosis that is characterized by obstruction of the arteries in the lower limbs. Experimental and epidemiological studies suggested a key role for oxidative stress in initiation and progression of the atherosclerotic process. The results of these studies provided a good basis for interventional trials with antioxidants, particularly with vitamin E, but the findings were conflicting. In this paper we review the observational and interventional studies with antioxidants, and ask whether vitamins supplementation should or should be not be recommended for PAD patients.

The ubiquitin-proteasome pathway plays a role in the degradation of the bulk of proteins in the cytoplasmic and nuclear compartments. In this pathway proteins are targeted for degradation by covalent ligation with ubiquitin, a reaction that requires ATP. Following the binding of the first ubiquitin molecule with the e-amino group of a lysine residue of the substrate protein, a polyubiquitin chain is usually formed, in which the C-terminus of each ubiquitin unit is linked to a specific Lys residue of the previous ubiquitin. Central to this pathway is the 26S proteasome, a high molecular mass multifunctional protease which requires ATP for its catalytic activity. Substrates of the 26S proteasome are not only old or damaged proteins, but also short lived proteins functioning as regulatory factors in a large array of cellular processes, such as cell cycle progression, cell growth and gene expression, inflammatory response and immune surveillance. A number of inhibitors of the catalytic activity of proteasomes have been developed and successfully employed in the study of their functional and structural properties, as well as of their involvement in different cellular processes. Some of these molecules due to their toxicity are used only as experimental research tools; others instead are now in clinical trials for treatment of a variety of hematologic malignancies and solid tumors and of reperfusion injury occurring after cerebral ischemia and myocardial infarction. Furthermore, proteasome inhibitors are described to interfere with HIV maturation, budding and aggressiveness, and cytostatic drugs, as well as antiretroviral agents used in HAART, have been shown to behave in vitro and in cultured cell lines as inhibitors of proteasome proteolytic activity at therapeutic dosages.