March 15, 2007 (Vol. 27, No. 6)
Refined Strategies Shed Light on Biological Processes and Improve Drug Selectivity and Safety Profiling
Although not everyone agrees on the definition of chemogenomics, most believe the field holds great promise for the pharmaceutical industry. In its broadest sense, chemogenomics describes how a chemical or family of compounds affects the genome. At the end of the day, this systematic approach combined with genomic targets represents a new technology coming of age.
Scientists are using chemical genomics to reveal biological synergies that impact multiple cellular networks and to enhance drug safety by improving selectivity in the discovery phase.
Synergistic Drug Combinations
The traditional drug discovery paradigm of “one-drug, one-target” may soon be replaced by a new approach that targets synergy in biological systems, according to scientists at CombinatoRx (www.combinatorx.com). “Researchers are increasingly recognizing the complexity of biological networks, and the need for coordinated action at multiple molecular targets,” says Glenn Short, Ph.D., senior scientist, technology and platform development. “In order to boost both efficacy and specificity of drugs, agents that target multiple pathways are needed.”
Grant Zimmerman, Ph.D., director of discovery sciences and technology, agrees. “Identifying multitarget therapeutics requires a different discovery approach. Our company has developed a combination high-throughput screening technology, cHTS™, that allows rapid and effective surveys of millions of combinations of drugs to identify synergistic combinations of compounds acting at multiple points within cellular networks.
“This is particularly important and unique to CombinatoRx because these combinations can reveal unexpected interactions between signaling pathways or genes that traditional discovery methods would miss. Another advantage is that synergistic combination pharmaceuticals often can be used at lower doses than needed for single use, thus reducing potential side effects.
“This cHTS technology rapidly screens thousands of compounds using disease-relevant phenotypic assays to detect synergistic multitarget mechanisms. To make this possible, we have developed custom analytical solutions, specific numerical models, databases, and novel hardware and software,” notes Dr. Zimmerman.
Initially, the company looked at combinations of many different known drugs. According to Curtis Keith, Ph.D., senior vp of research, “This first-tier approach was taken because known drugs have recognized pharmacology, toxicity, and safety profiles as well as proven manufacturing processes. Now, we are beginning to expand our approaches to discover combinations of different types of agents such as chemical probes, siRNAs, and antibodies.”
Systems Biology Approach
Scientists are increasingly focusing on understanding the complex interactions of a drug within the cell. Such integrative approaches assess effects on many cellular networks. Avalon Pharmaceuticals (www.avalonrx.com) is following such a systems biology approach.
“We have developed a unique, fully integrated systems biology engine that allows understanding the complex interactions of drugs and drug candidates based on their effects over multiple signaling pathways,” Kenneth Carter, Ph.D., president and CEO reports. “We don’t just monitor binary endpoints such as with enzyme assays or in clinical settings with the up- or down-regulation of biomarkers. We look across the entire genome in real time to assess the mechanistic effects.”
The Avalon Rx® Drug Discovery Engine aims to meet those challenges. It has two major components, according to Dr. Carter. “First, we prepare cellular material from tissue and monitor activity of many different genes based upon our novel transcriptional assay technology. This identifies a genetic fingerprint correlated with a desired biological outcome. Secondly, we perform high-throughput screening of libraries of compounds against such signature sets of expressed genes. This allows us to rapidly identify desirable response profiles. So, this approach permits evaluation of compounds against many different targets, both known and unknown, in a single assay.”
The company is conducting clinical trials of its lead candidate, AVN944 (an anti-cancer compound). Studies will examine generated biomarkers to assist in patient stratification, dosing, and combination therapy choices, among other parameters.
Avalon is also utilizing its approach to target pathways traditionally considered undruggable. According to Dr. Carter, “A primary example is beta-catenin, a protein known to be misregulated in cancers, especially colorectal. Conventional screens can’t work because this is not an enzyme. But we have used an RNAi technology to knock out function in order to obtain a gene signature of what needs to be shut down by drug compounds. This has helped us find corresponding candidates to further pursue. We now have a secondary drive to optimize these compounds in animal studies.”
Drug selectivity is a serious safety issue that must be addressed, especially if kinase inhibitors are to be applied as chemogenomics tools, indicates Patrick Zarrinkar, Ph.D., senior director, technology development and alliance management at Ambit Biosciences (www.ambitbio.com).
“The use of kinase inhibitors as therapeutics is in its early stages,” says Dr. Zarrinkar. “Because the relationship between selectivity, safety, and efficacy is still poorly understood, we are applying a novel competition binding assay to systematically explore these issues. In our assay, we evaluate a wide variety of known kinase inhibitors against a panel of more than 300 protein kinases. We can profile entire compound libraries using this approach.”
The technology, called KinomeScan, is an ATP site-dependent competition-binding assay employing Ambit’s amplifiable fusion protein (AFP™) system. This approach fuses protein kinases of interest to bacteriophage to facilitate production, isolation, and quantification.
Dr. Zarrinkar suggests that selectivity profiling has another important benefit: It is much more cost efficient. “Based upon the profile of a library against multiple targets of interest, we can more intelligently decide what compounds to pursue from that library based on knowledge of both potency and selectivity. This can streamline the process and allow better choices as to which programs to pursue because we know a starting point that is easier to optimize.
Serenex (www.serenex.com) is also focusing on enhancing selectivity using chemogenomics approaches. The company has developed a series of affinity media that reversibly bind proteins from multiple gene families, enabling the screening of compounds across thousands of targets in parallel.
“Our media are designed to be promiscuous in order to bind multiple protein classes,” Steven E. Hall, Ph.D., senior vp R&D, says. Initially, we are targeting the super-family of proteins that have binding sites for purines, e.g., ATP or NADH. This group, referred to as purine-binding proteins, includes in excess of 2,000 targets. The discovery process begins by incubating cell and tissue lysates with the media. Next, the protein-bound media is challenged with a library of small compounds that compete for the purine site on the protein. This results in compound-specific eluents that represent the binding partners for each compound. Separation of the proteins and analysis completes the process. The efficiency of the approach means that thousands of compounds can be profiled in just a few days.”
In one of its screens, the company identified Hsp90, a chaperone protein that is key to the proper folding of a number of proteins, including oncogenic kinases. “If Hsp90 is inhibited, kinases won’t fold properly and will be degraded,” Dr. Hall explains. “Thus, we have used this chemoproteomics approach to identify novel, orally active Hsp90 inhibitors. Previous compounds, like geldanamycin, were too toxic for development. By enhancing selectivity for Hsp90, the compounds we identified have decreased side effects.”
Dr. Hall suggests that inhibitors of Hsp90 not only have uses as oncology therapeutics, but also for antiviral, antifungal, and neuroprotective applications.
Inhibiting Ion Channels
Off-target effects have plagued therapeutics targeting ion channels. Improving selectivity in this arena is a long sought-after goal. Hydra Biosciences (www.hydrabiosciences.com) is focusing on the transient receptor potential (TRP) family of ion channels. “Ion channels are critically important for the flow of information into and out of the cell,” says Magdalene Moran, Ph.D., senior scientist and director of the channel group’s target discovery efforts.
“The TRP family comprises a group of nonselective cation channels distinct from others. TRPs act as multimodal integrators of signals and represent about 20% of all ion channels. The TRP members have low homology to each other, which allows more specific modulation and limits the potential for off-target effects.”
Dr. Moran reports that they have developed a high-throughput screen for identifying compounds to probe the in vivo role of a TRP channel (TRPV3). “This high-throughput screen identifies selective antagonists. We look at calcium flux in mammalian cells that express TRP. We then verify the results using the patch-clamp technique as a means for measuring ion flux in individual mammalian cells. This helps to confirm hits by providing a direct measurement of ion flow in and out of the cells.”
According to Dr. Moran, to confirm the in vitro effects, they quickly advance active compounds to a rat model to better understand what the channel does with regard to pain modulation.
“We believe these studies represent the first example of a selective antagonist to the TRPV3 channel and also validate it as a target for the treatment of pain. We are now in the process of optimizing candidates.”