When cancer slips past the “guardian of the genome” -
In healthy bodies, the number of new cells produced corresponds exactly to the body's needs. Sometimes, however, a cell veers off course from the rest—despite a number of protective mechanisms—and begins dividing uncontrollably. In other words: cancer develops. We now know that every malignant tumor is the result of underlying genetic mutations that affect several different classes of genes. Genes are sections of DNA, the molecule governing heredity that contains the “blueprint” for building proteins. But a damaged gene can produce a defective protein, too many or too few proteins or even no proteins at all—all with fatal consequences. The first step towards cancer occurs when what is known as an oncogene becomes hyperactive due to a mutation. These genes stimulate cellular growth, but the abnormal effect only becomes noticeable when a tumor suppressor gene, such as p53, becomes damaged. Often these two classes of genes are described using a car as an analogy: the oncogene is the gas pedal for triggering more cell growth while the tumor suppressor gene is the brake pedal.
“The cell is continually subjected to UV radiation and other stress factors that can damage the genetic code of the DNA molecule,” reports Boeckler. “The p53 molecule is seen as the response to all of these factors because, dependent on the cellular environment, it can activate a broad spectrum of genes whose proteins are responsible for apoptosis, which is the programmed cell death. Some other proteins are also responsible for DNA repair and anti-angiogenesis. That is the term for inhibition of the growth of new blood vessels. Tumors stimulate angiogenesis because they require their own blood vessels to ensure sufficient nourishment. The p53 protein therefore plays a key role in the body's own defense against cancerous cells. “But in about fifty percent of all tumors this gene is deactivated by mutations,” explains Boeckler. “At body temperature the protein is just at the level of stability, but if you have destabilizing mutations, it will be largely present in its unfolded state.”
Among the “top ten” oncogenic, or cancer-causing genetic mutations, is the mutation Y220C. It occurs in approximately 75,000 new cancer cases per annum and is the most frequent mutation, in which p53 is deactivated by thermodynamic destabilization — all because a single building block of the protein is replaced. This building block, the amino acid tyrosine, is replaced by the much smaller amino acid cysteine. “This has fatal results,” Boeckler explains, “because important protein–protein interactions are lost. Instead of two small binding sites on the surface of the p53 protein, a sort of large binding ‘pocket’ forms. Our goal was to use structure-based methods to find small stabilizing agents that selectively bind to this newly created pocket. After all, it is this pocket that is responsible for destabilization and therefore ultimately the deactivation of p53.”
With this goal in mind, scientists used what is known as Virtual High Throughput Screening (vHTS), the computer-based evaluation of enormous databases of structures, to analyze more than 2.7 million commercially available compounds for their ability to bind to the mutated p53 pocket. By this process, more than 80 promising substances were identified, each of which then underwent thorough testing, with success: one substance in particular proved especially effective, and was made even more effective by modifications to its structure. “The low molecular weight of the resulting molecule PhiKan083 makes it a very good candidate as a lead structure, which is a substance that should be developed into a drug through further chemical modifications,” explains Böckler. “What essentially happens is that, with increasing concentrations of this molecule, the melting point of the mutated p53 increases. This considerably slows down the unfolding of the protein at body temperature.”
The intention is now to develop this molecule so that it may actually be used as a treatment for cancer. “This follow-up project will be a cooperative effort between my team and Sir Alan Fersht's lab where the current research was done,” reports Boeckler. “The plan is for postgraduates to perform one part of the work in Munich and the other part in Cambridge. And we have already made a promising start, namely in determining the crystal structure of PhiKan083 and the affected domains of the mutated p53 protein. It is the first crystal structure that has ever been made from p53 or a p53 mutant with a drug-like stabilizer. These data provide us with an excellent starting point for further optimization of the lead structure by structure-based drug design. The hope is to develop a substance that binds to the mutated p53 with even greater affinity than our current hit, thus increasing stabilization even further.”
“Targeted rescue of a destabilized mutant of p53 by an in silico screened drug,”
Frank M. Boeckler et. al.
PNAS, 30 July, 2008.
Professor Dr. Frank Boeckler
Center for Drug Research
Department of Chemistry and Pharmacy at LMU Munich
Tel.: ++49 (0) 89 / 2180 – 77804
Fax: ++49 (0) 89 / 2180 – 77884