With the discovery of suitable molecular targets — cellular molecules along pathways crucial for sustaining the life of cancer cells — comes the perplexing dilemma of where to find the next therapeutics that will bind to and disable those targets. While the possibilities for drug designs are near-limitless, the methods to screen drug databases and repositories are often problematic or ill-suited for the particular needs of researchers.
At the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, researchers reported new means of delving into vast stores of data in search of potential therapies, whether to find the next natural cancer fighter or to discover new classes of therapeutics.
Targeting neuroblastoma tumor-initiating cells
While research has yielded exceptional advances in treatment and therapeutics for most adult cancers, there has been little improvement in survival rates for patients with the deadly childhood cancer neuroblastoma for the past 30 years. Armed with advances in stem cell knowledge, researchers at The Hospital for Sick Children in Toronto, Canada, are screening currently approved drugs for new neuroblastoma therapies that kill cancer while sparing children exposure to excessive amounts of toxic therapeutics.
Using their screening process, the researchers searched more than 5,000 drugs and uncovered 47 candidates that show good potential against neuroblastoma, including rapamycin, on which the researchers are currently focusing.
“Neuroblastoma is particularly difficult to treat without aggressive chemotherapy and, even when treated successfully, the chemotherapies currently in use frequently have side effects that can have devastating repercussions later in life,” said Kristen Smith, Ph.D., a postdoctoral fellow at The Hospital for Sick Children. “We have developed an efficient screening process based on stem cells present in the growing bodies of children, cells that might be susceptible to harm from the necessary blunt force use of therapeutics.”
Smith and her colleagues used a cell-based assay program that pits chemotherapeutics against neuroblastoma tumor-initiating cells (TICs) and skin-derived precursors (SKPs). As their full-name suggests, TICs are cancer stem cells developed from tumor samples removed from children. SKPs, however, are normal non-cancerous stem cells found in the skin. Both varieties of stem cells originate from the neural crest, the portion of a developing embryo that eventually comprises the peripheral nervous system.
“The idea is to find a drug that can kill a neuroblastoma TIC without harming an SKP,” Smith said. “We reasoned that if the drug is potent enough to kill a tumor stem cell — without damaging a normal stem cell — then we may lessen the risk of SKPs or other stem cells becoming cancerous later in life.”
According to Smith, 40 of the 47 drugs that were recognized in the screening have never been used to treat neuroblastoma. The researchers are currently studying the highlighted drugs in TICs from multiple neuroblastoma patients. One drug in particular, rapamycin, has already been studied in an animal model of neuroblastoma, with promising results and is in clinical studies, Smith says.
The work was performed in collaboration with clinicians at The Hospital for Sick Children, Alessandro Datti, Ph.D., at the Mt. Sinai Robotics Facility, and Herman Yeger, Ph.D. and Sylvain Baruchel, M.D. at the Hospital for Sick Children.
The research was funded by the National Cancer Institute of Canada, Canadian Stem Cell Network, McLaughlin Centre for Molecular Medicine, The James Birrell and Lilah Funds for Neuroblastoma Research, and the Sick Kids Foundation.
Identification of inhibitors for MDM2 ubiquitin ligase activity from natural products by a novel high throughput electrochemiluminescent screen
Scientists laboring intensively to develop new therapeutics often turn to naturally produced molecules used by plants or microrganisms to ward off predators. The effectiveness of natural products such as Taxol (derived from tree bark) or rapamycin (derived from soil bacteria) prompted the National Cancer Institute (NCI)’s Natural Products Repository to collect and store over 220,000 biodiverse samples, derived from marine organisms, microbes, and plant life gathered from locations across the globe.
Researchers at NCI’s Center for Cancer Research (CCR) report their successful use of a new technology capable of mass-screening extracts from natural products for new potential therapies. In an initial run of the high-throughput screen, the Repository team uncovered a plant compound that blocks the breakdown of the well known tumor suppressor protein called p53.
“The samples in the Repository exist as extracts from specimens that have been collected in the oceans and forests of the world and shipped here — each containing thousands of compounds,” said Barry O’Keefe, Ph.D., a researcher at NCI’s campus in Frederick, Maryland.
“Somewhere among these samples are natural molecules that have been honed by nature that could have great therapeutic value, but finding them amid the clutter of other natural compounds is difficult.”
Their latest natural products screen uses an “electrochemiluminescent” assay, developed by CCR researcher Allan Weissman, M.D., which tags the target proteins and causes them to emit photons, or “light up” when an electrical current is passed through them. If the activity of the target protein is blocked (a sign that some molecule is “inhibiting” the target), the reaction goes dark.
To verify that the electrochemiluminescent assay worked properly, the Repository team searched for a molecule that inhibits the known ability of MDM2 to signal for the destruction of the pro-apoptosis (cell suicide) protein p53. In normal cells, MDM2 and p53 exist in a state of benign equilibrium — balanced to assure that cell suicide does not occur.
The researchers screened over 144,000 samples and uncovered almost 2,000 potential “hits” against MDM2. These hits were further refined, yielding 372 extracts from which chemists are now isolating active compounds. Among the active compounds recovered, one plant chemical called sempervirine was found to induce apoptosis in cancer cell lines.
“Searching through the literature we discovered that sempervirine had been previously considered by French cancer researchers in the 1980s, but since the roles of p53 and MDM2 were poorly understood at the time, sempervirine research took a different direction,” O’Keefe said. “Now we will take another look at this compound while we continue to analyze the other extracts.”
Identification of equal MDMX/MDM2-p53 interaction small molecule inhibitors
Half of all cancers occur because of a mutation in the tumor suppressor gene p53, while in numerous other cancers its protein is deregulated, taken out of service before it can do its job as a potent anti-cancer regulator. Now, researchers at St. Jude Children’s Research Hospital in Memphis, Tennessee have developed a strategy for stopping two key regulators of p53 that can contribute to cancer progression: proteins called MDMX and MDM2. Using biochemical assays developed at St. Jude, the researchers report the discovery of two small inhibitor molecules that can keep both MDMX and MDM2 from deregulating p53.
“We now have an understanding of how MDMX and MDM2 target functional p53, but the real challenge has been to find a means of controlling both MDMX and MDM2,” said Damon Reed, M.D., a researcher at St. Jude Children’s Research Hospital. “We are looking for a single therapeutic that will knock out both proteins, thereby allowing p53 to do its job, that is, to kill cancerous cells.”
While Reed and his colleagues have developed their process to look for new therapeutics for retinoblastoma, a rare childhood cancer of the eye, they believe small molecule inhibitors they have developed will have a much broader impact. “There are a number of cancers in which there is nothing wrong with p53, but the genes for MDM2 and MDMX are over-expressed, such as instances of retinoblastoma, leukemia, breast, lung, prostate, and colon cancers,” Reed said.
Through funding from the National Cancer Institute, Reed adapted two biochemical assays, fine tuning them to test over 6,000 biologically active compounds for those that could, ideally, bind to both MDM2 and MDMX. In the first test, fluorescence polarization, the researchers linked fluorescent tracers — molecules with the property to rotate light — to a p53-like molecule. If a candidate molecule binds to the MDMX protein it prevents the p53 binding, and, therefore, changes the signal of the fluorescent light.
The second assay, an AlphaScreen test, involves attaching small beads to both the p53-like molecule and either MDM2 or MDMX. If the tested compound binds to MDMX or MDM2, it blocks a chain between the two beads, which decreases the amount of light emitted by the beads.
The St. Jude researchers ran the 6,000 compounds through both the AlphaScreen and the fluorescence polarization assay and discovered two small molecules which bound MDMX and MDM2. According to Reed, the St. Jude team is continuing testing on the two identified molecules in cell culture, and is preparing the molecules for further testing in animal models.
In addition, the St. Jude team has expanded its search for novel, high affinity MDMX/MDM2 inhibitors using a 350,000 compound chemical library.
Targeted approach towards inhibition of telomere-hnRNP A1 interaction
Immortality is a term often used to describe the sustained longevity of cancer cells, which allows them to grow out of control and spread. The lifespan of a cell is determined by portions of DNA called telomeres, which stabilize the cap-ends of the chromosome structures of cellular DNA. Researchers at Gemin X Pharmaceuticals, Inc. in Montreal, Canada, report the development of a combined computer/laboratory system to address the labor-intensive task of screening millions of molecular compounds for the ability to disrupt telomere maintenance. Through their screening process, the researchers have identified two molecules that serve as potent inhibitors of A1 and A2, proteins that sustain telomeres and thus the immortality of cancer cells.
Like the plastic aglets at the ends of shoe strings, telomeres are regions of the chromosome that keep the DNA from fraying at the ends. The telomere consists of a short repeated segment of six DNA nucleotide subunits –thymine, guanine and adenine — in the order of TTAGGG. Gradually, telomeres erode, a trait that has evolved to enforce a cell’s mortality: a cell can only grow and divide so many times before its DNA becomes too unstable. This instability occurs when telomeres shorten below a critical threshold. In cancers, the telomere structures are maintained but they require capping proteins such as A1 and A2 in order to permit cell immortality.
In many cancers, the genes that encode A1 and A2 are over-expressed, leading to an overabundance of the proteins and, therefore, longer-lasting telomeres.
“We are seeking to halt tumor growth by taking the immortality out of cancer cells. Moreover, by targeting A1 and A2, the immediate response of the cancer cell is cell death,” said Richard C. Marcellus, Ph.D., a researcher at Gemin X.
“Since the A1 and A2 proteins bind directly to DNA, we were looking to find a molecule that could block this specific protein/DNA interaction,” Marcellus said. “However, the chemistry involved in building small molecules that are able to inhibit protein/DNA binding is daunting, so most drug developers have looked elsewhere for easier targets.”
To find these previously unidentified small molecules, the researchers at Gemin X began with the active area found in the A1 and A2 proteins. While they are slightly different molecules, both proteins bind to the same portion of the six nucleotides found in repeated telomere sequences — the central TAG component of TTAGGG — and the researchers used previously published structural data to create a molecular “footprint,” the shape needed to bind DNA.
They then created a computer model of this footprint, which they could use to screen through commercially available databases of small molecules without an exhaustive laboratory assay.
“We acquired as many molecular libraries as we could acquire, totaling some two million potential candidates,” Marcellus said. “It was an initial, brute force approach that we could use to quickly discard candidates that wouldn’t work.”
The initial screen winnowed the field of potential A1 and A2 inhibitors down to two thousand candidates, enabling researchers to move from the in silico approach to the more traditional “wet lab.” The researchers then ran the remaining candidates through a gamut of six separate assays, each designed to further weed out inappropriate molecules. The testing included determining whether the molecule actually bound to A1, a solubility assay to discard molecules that stuck to other molecules without specificity, binding studies to determine if the molecule stuck to DNA, binding studies to determine if the molecule stuck to unrelated proteins and an assessment of the molecule’s ability to bind to TAG.
Any molecules that made it through those assays were met with one final test: cytotoxicity — could the candidate, in fact, kill cancer cells? The researchers uncovered five classes of compounds that could halt growth and induce death in skin and lymphoma cancer cells. From those five, Marcellus said, they have identified two classes that would be suitable candidates for further refinement, a necessary step before testing in animal models.
