Current Pharmaceutical Design - Volume 11, Issue 22, 2005

DNA (antisense and other oligonucleotides) drug design represents a direct genetic approach for cancer treatment. Such an approach takes advantage of mechanisms that activate genes known to confer a growth advantage to neoplastic cells. The ability to block the expression of these genes allows exploration of normal growth regulation. Progress in DNA drug technology has been rapid, and the traditional antisense inhibition of gene expression is now viewed on a genomic scale. This global view has led to a new vision in antisense technology, the elimination of nonspecific and undesirable side effects, and ultimately the generation of more effective and less toxic nucleic acid medicines. Several antisense oligonucleotides are in clinical trials, are well tolerated, and are potentially active therapeutically. DNA drugs are promising molecular medicines for treating human cancer in the near future.

Prevention, improved screening, and better treatment regimens have improved cancer incidence and mortality in the last decade. Chemoradiation continues to cause high morbidity in patients undergoing treatment. DNA therapeutics have the potential to modify the genes that cause tumor progression in order to produce a response that is tumor-specific, efficacious and systemic without toxicity to normal cells. The most widely used and most experimentally advanced DNA therapeutic is the antisense oligonucleotide. These oligomers are predominantly used to inhibit mRNA expression. For cancer chemotherapy, the Bcl-2 antisense oligonucleotide is currently in phase III clinical trials. Transcription factor decoys form DNA:protein heteroduplexes and produce cellular responses at the genomic rather than transcriptional level. The use of other transcription factor decoys as oncologic reagents is now being developed. The phenomenon of RNA interference has only recently been discovered to occur in plants as a response to viral infection. Small interfering RNAs cause mRNA inhibition. siRNAs also inhibit expression of mRNA, however the intracellular cascade is quite different. siRNA could prove to be more powerful and longer lasting than antisense. Several DNA therapeutics are currently being studied. This review will focus on antisense oligonucleotides, transcription factor decoys and siRNA with an emphasis on how they can be employed as anticancer agents. Mechanism of action and design strategies will be summarized, as well as therapeutic targets and demonstrated clinical efficacy for each reagent.

The ability of certain DNA sequences to form G-quartet structures has been exploited recently to develop novel anti-cancer agents including small molecules that promote G-quartet formation within the c-MYC promoter thereby repressing c-MYC transcription and introducing G-quartet-forming oligodeoxynucleotides (GQ-ODN) into cancer cells resulting in p53-dependent cell cycle arrest and inhibition of DNA replication. GQ-ODNs also have been developed as potent inhibitors of signal transducer and activator of transcription (STAT) 3, a critical mediator of oncogenic signaling in many cancers. This review summarizes the rational design of G-quartet forming DNA drugs as Stat3 inhibitors. Topics that are reviewed include the strategy of structure-based drug design, establishment of a structure-activity relationship, development of a novel intracellular delivery system for G-quartet-forming DNA agents and in vivo drug testing to assess the anti-cancer effects of DNA drugs in tumor xenografts. Results to date with GQ-ODN targeting Stat3 are encouraging, and it is hoped that continued pursuit of the methodology outlined here may lead to development of an effective agent for treatment of metastatic cancers, such as prostate and breast, in which Stat3 is constitutively activated.

Most anticancer drugs presently used clinically target genomic DNA. The selectivity of these anticancer drugs for tumor tissues is probably due to tumor-specific defects suppressing cell cycle checkpoints and DNA repair, and enhancing apoptotic response in the tumor. We will review the molecular interactions within the ATM-Chk2 pathway implicating the DNA damage sensor kinases (ATM, ATR and DNA-PK), the adaptor BRCT proteins (Nbs1, Brca1, 53BP1, MDC1) and the effector kinases (Chk2, Chk1, Plk3, JNK, p38). The molecular interaction map convention (MIM) will be used for presenting this molecular network (http://discover.nci.nih.gov/mim/). A characteristic of the ATM-Chk2 pathway is its redundancy. First, ATM and Chk2 phosphorylate common substrates including p53, E2F1, BRCA1, and Chk2 itself, which suggests that Chk2 (also known as CHECK2, Cds1 in fission yeast, and Dmchk2 or Dmnk or Loki in the fruit fly) acts as a relay for ATM and/or as a salvage pathway when ATM is inactivated. Secondly, redundancy is apparent for the substrates, which can be phosphorylated/activated at similar residues by Chk2, Chk1, and the polo kinases (Plk's). Functionally, Chk2 can activate both apoptosis (via p53, E2F1 and PML) and cell cycle checkpoint (via Cdc25A and Cdc25C, p53, and BRCA1). We will review the short list of published Chk2 inhibitors. We will also propose a novel paradigm for screening interfacial inhibitors of Chk2. Chk2 inhibitors might be used to enhance the tumor selectivity of DNA targeted agents in p53-deficient tumors, and for the treatment of tumors whose growth depends on enhanced Chk2 activity.

Advances in the molecular biology of oncogenesis have established a key role for transcription factors in malignant transformation. In some cases the activity of the transcription factor itself is altered by mutation. In many other cases, the activity of the transcription factor is affected by mutations in upstream signaling or regulatory proteins. This review highlights four transcription factors - Stat3, Stat5, NF-kB, and HIF-1 - which are associated with cancer development. The evidence for the involvement of these factors in oncogenesis is reviewed. Further, we examine the efforts to specifically target these transcription factors for therapeutic intervention. Such strategies include using peptidomimetics, antisense oligonucleotides, small molecule inhibitors, and G-quartet oligonucleotides. Inhibition of transcription factor activity may occur at the level of activation, translocation, or DNA binding. Application of these approaches to in vitro and in vivo models of tumorigenesis is discussed.

Oligodeoxynucleotides containing deoxycytidyl-deoxyguanosine dinucleotides (CpG ODNs) activate the host immune system, leading to innate and acquired immune responses. The immune stimulatory effects of CpG ODNs are being exploited as a novel therapeutic approach to treatment of human diseases, and some CpG ODNs are being evaluated in clinical trials. The cellular recognition of CpG motifs requires the presence of the Toll-like receptor (TLR) 9, which triggers cell signaling and immune responses. There are three main types of first-generation CpG ODNs, which mimic the immunostimulatory activity of bacterial DNA and are recognized by TLR9, A-, B- and C-Class ODNs. Although all three CpG ODN classes stimulate TLR9-dependent signaling, there are striking differences in the cell types they activate and their dose-dependent immunostimulatory efficacy. Second-generation CpG ODNs, with advanced nucleic acid chemistry and unique modifications to their sequences and structures are being developed. Medicinal chemistry studies suggest that the immunomodulatory activity of CpG ODNs can be altered by site-specific incorporation of modifications in order to develop disease-specific drugs. Both first- and second-generation CpG ODNs have potential for treatment of various human diseases, such as infections, immunodeficiencies, and cancers. This article will focus on the recent advances in developing CpG ODNs as novel anti-cancer therapeutic agents.

Natural toxins are the product of a long-term evolution, and have captured crucial events in the most essential and vital processes of living organisms. They can attack components of the protein synthesis machinery (as in the case of Diphteria and Shiga toxins, and Ribosome inactivating proteins), actin polymerization (Clostridium botulinum type C, C2, toxins and Enterotoxin A), signal transduction pathways (Cholera toxin, Heat-labile enterotoxins, Pertussis and Adenylate cyclase toxins), intracellular trafficking of vesicules (for Tetanus and Botulinum neurotoxin type C) as well as immune and/or inflammatory responses (Pyrogenic exotoxins, Cholera and Pertussis toxins). Of interest is the fact that several bacterial and vegetal toxins can either kill selectively cells infected with the human immunodeficiency virus (HIV) or exert inhibitory effects on its life cycle. In particular both pertussis toxin (PTX) and its nontoxic B-oligomeric component (PTX-B) can block the infectious process in vitro at multiple levels, by preventing the entry of CCR5-dependent (R5) HIV strains and by inhibiting both R5 and CXCR4-dependent HIVs at post-entry level(s). In addition, some toxins possess immunostimulating properties that have been exploited in terms of adjuvancy and induction of specific cytotoxic T lymphocytes responses to different vaccine preparations, including some experimental vaccine against HIV infection. Thus, toxins may represent a relatively unexplored exhibition of powerful biological agents that could either prevent infection or attack HIV-infected cells.

Human pancreatic islet transplantation has recently been shown to be successful in replacing pancreatic endocrine function into type 1 diabetic recipients. A major drawback, however, is the high amount of pancreatic ß cells required to render one single patient insulin-independent. Given the shortage of human ß cell donors, the majority of type 1 diabetic patients remain excluded from this therapeutic option. High number of islets are requested since substantial islet cell death and dysfunction occur within the first few hours and days after islet transplantation. Impaired vascularization of the engraft, the non-specific inflammatory reaction at the site of transplantation, together with the presence of active or memory autoimmune responses to islet autoantigens and allogeneic recognition contribute to apoptosis of ß cells and subsequent early graft function loss. This review will focus on ex vivo engineering of the islet graft by gene transfer to improve islet engraftment. An overview of currently used gene transfer techniques will be given and their potential will be discussed.