Most drugs in use today bind to specific proteins and interfere with the activity of the protein. Some antibiotics do bind DNA by intercholating nonspecifically between base pairs and interfering with replication and transcription. An alternative new approach is to design drugs that bind to specific sequences of DNA and either promote or inhibit the transcription of key genes.
DNA Binding Drugs
β-hydroxybutyrate is an experimental drug now being used to treat sickle-cell anemia. Somehow it increases the transcription of fetal hemoglobin genes, which form proteins which do not aggregate as the sickle cell hemoglobin gene product does. These approach has been called chemical genetics. Stuart Schreiber at Harvard has been a leader in the this field.
Another way to inhibitor specific gene transcription is to bind a small single-stranded DNA to the dsDNA to form a triple helix. The single stranded DNA molecule can bind to exposed H bond donors and acceptors of bases not involved in Watson-Crick H-bonding interactions between complementary bases in the ds DNA.
Figure: Triple helix
The single strand binds to such donors that are accessible in the major grove of dsDNA.
Peptide-nucleic acid analogs, as shown below, are promising new drugs.They are less charged than nucleic acids (so can more easily cross a membrane) and are resistance to cleavage by proteases and nucleases. One could image them binding to specific nucleotide sequences and inhibiting processes like DNA replication and transcription.
Transcription factors that are generally activated by genetic events or upstream signaling pathways are key regulators of cell state. Due to the extensive protein-protein interfaces and general absence of hydrophobic pockets that might inhibit protein:protein interactions required for transcriptional regulation, it has been difficult to design drugs that bind to transcription factors and modulate their activity. Moellering et al. report a successful development of a direct-acting antagonist of an oncogenic transcription factor, NOTCH1. This antagonist consists of cell-permeable stabilized α-helical peptides, SAHMs that was "stapled" into a stable helix through addition of two unnatural alkenyl amino acids which through ring closure sterically restrained the peptide in an alpha helix. The helix mimicked one protein:protein interface region in the ternary complex of DNA:NOTCH1:MAML1 (which is a coactivator protein). The peptides antagonized on NOTCH signaling and cell proliferation in T-cell acute lymphoblastic leukemia cells (T-ALL).
DNA Binding and Genomic Analysis
Huge numbers (100,000 to 1 million) of different DNA molecules can be covalently attached to silicon or glass chips, as described above for the peptides. These sequences are located at specific x,y coordinates on the chip. DNA probes can then be made from cells (by adding reverse transcriptase to isolated mRNA. forming cDNA), which are then labeled with a fluorescent molecule. Easier yet, mRNA can be isolated from the cells and labeled with a fluorophore. When added to the chip, they will bind through complementary H-bond interactions to specific complementary DNA on the chip. Using this technique, an individual's entire genome could be analyzed in a short amount of time. For example, mutations in certain genes associated with cancer might be detected by fluorescent-DNA probes made from a possible mutant gene to specific DNA molecules in the chip that were designed to bind to mutant probes.
In an amazing variation, mRNA can be extracted from two different cells, a control and a tumor cell. The control mRNA can be labeled with a green fluorophore, while the tumor cell mRNA can be labeled with a red fluorphore. They can both be added to the chip containing a library of human genes. If the gene is expressed in both cell types, both types of labeled mRNA will bind and the spot on the chip will appear yellow.
If the gene is not expressed in either tissue, the spot will appear black. Genes that are only expressed in tumor cells will appear red and in control cells green. In a single experiment, the differential expression of genes in tumor cells can be determined. In this way, tumor-specific proteins can be identified, which could lead to the development of a vaccine against those tumor antigens. A typical microarray analysis for this type of experiment is shown below. (from Nature, 403, 699 (2000).
Array technologies have continued to evolve. Affymetrix has developed a array chip that contains over 38,000 genes, representing the entire human genome.
RNA Binding Drugs
One of the best new alternatives is to design a RNA molecule complementary to a given mRNA for a specific protein selected for inhibition. This antisense RNA forms a dsRNA when it binds to the mRNA and inhibits translation of the mRNA.
Figure: antisense RNA forms a dsRNA
Cells can be altered through genetic engineering to make an antisense RNA within the cells by inserting an inverted copy of the target cDNA for the gene of interest into the cell, and allowing the cell to manufacture its own drug!
Even more exciting is the use of RNAis (RNA inference) for disease therapy. In contrast to traditional drugs, RNAis are one of nature's proven methods to inhibit gene transcription or RNA translation (mainly from viruses). Even though it was only discovered five years ago, it may be only a few years before drug trials based on RNAi take place. Especially promising are trials for siRNAs that inhibit HIV. To show that it works in organisms, mice were infected with a fusion of hepatitis C gene segment and a gene for a fluorescent protein luciferase. Specific siRNAs-treated mice showed a dramatic decrease of fluorescence, but not those treated with a nonspecific RNAi. As with other drugs, getting them into target cells is proving difficult. Simple liposome-encapsulated siRNAs apparently are not as effective as hoped. Carrying the siRNA into the cells with virus is yet another idea. Some companies pursuing this technology are shown below.
- Alnylam Pharmaceuticals
- Antisense drugs: AVI BioPharma
- Calando: RONDEL (RNAi/Oligonucleotide Nanoparticle Delivery) in which siRNA is bound to a block polymer of a positive polymer interspersed among cyclodexdrins, protecting the. siRNA from degrations by nucleases. These self-associate to form nanopartaicles. Adamantine (hydophobic) linked to polyethylene glycol (polar) to which targeting proteins (like transferrin) are attached are added to the nanoparticles, leading to PEG-dependent stabilization of the nanoparticle (and si-RNA) and transferrin-dependent delivery to target cancer cells, which over express transferrin receptors.