Drug Mechanism Studies
The comprehensive study of metabolic reactions is producing a large amount of information, as global biochemical profiling is becoming the hot technology.
“We are employing metabolomics investigations to clarify the mechanism of action of several new compounds,” said John Ryals, Ph.D., CEO at Metabolon. The company is using its metabolomics platform technology in a range of studies, including following the global effects of disease processes on metabolism in order to uncover new biomarkers.
At the meeting, Dr. Ryals discussed studies with the putative anticancer compound GMX1777/1778, a member of a class of molecules known as cyanoguanidinopyrimidines that are thought to be involved in the inhibition of NF-κB.
The approach followed by Dr. Ryals and his collaborators is essentially similar to that of the Hill group, in which cells are exposed to the agent and the changes in their metabolome followed over time. The most dramatic change in the global profile of the cells was an increase in the levels of glutamine and α-glutamylglutamine and a decrease in the levels of nicotinamide and NAD+.
Cell rescue experiments demonstrated that nicotinic acid and nicotinamide will save the cells from the killing effects of the anticancer agent. These observations are reminiscent of studies on cancer cells of almost a century, in which Otto Warburg demonstrated the heavy reliance of tumor cells on glycolysis for ATP generation, driven by NAD+.
These straightforward investigations point the way toward the understanding of the mechanism of action of GMX1777/1778, demonstrating that the metabolic intermediate NamPRT is the primary target and not NF-κB.
In an unrelated project, Metabolon looked for a biomarker that would complement PSA, a protein long used for detection of prostate cancer. Screening a large number of metabolites in normal, benign, and malignant cells, they found sarcosine to be a marker for aggressive prostate cancer.
Glycine is converted to sarcosine by S-adenosyl-methionine transfer as a result of methylation scavenging, and hypermethylation appears to be correlated with tumor aggressivity. Preliminary investigations indicate that sarcosine may be a useful marker of prostate cancer invasion and aggressivity. If so, sarcosine may have potential both as a biomarker and as a therapeutic target.
A third project aims to identify biomarkers for insulin resistance, aiming at validation of a compound that can predict the level of insulin resistance in nondiabetic at-risk patients. One marker, alpha hydroxybutyrate is better than adiponectin at correlating to insulin resistance, Dr. Ryals said. To validate this marker, the team followed individuals who underwent dramatic weight loss following bariatric surgery. “Alpha hydroxylbutyrate was the most significantly altered metabolite at one year post-bariatric surgery.”
“We have developed a high-throughput screening assay that allows us to detect small molecules with tumor-suppressing activity,” explained Gerhard Wagner, Ph.D., professor at Harvard Medical School. Dr. Wagner and his associates have studied the protein complex elF4E/elF4G, which is required for elongation of protein molecules on the ribosome. Misregulation of this complex is responsible for a variety of ills, including tumorigenesis and other forms of aberrant cell growth. For this reason the complex is a tantalizing target for small molecules that act as inhibitors.
Dr. Wagner and his co-workers devised a high-throughput fluorescence polarization assay that was validated with peptides designed for the task. This tool was applied to the massive screening of 16,000 compounds in the Cambridge DiverSet E Library, from which a high-binding candidate, christened 4EGl-1, was retrieved. This material disrupts the ElF4F complex, bringing translation to a grinding halt.
“Our results show that we can inhibit the interaction between the cap binding proteins with small molecules identified through our library screening,” Dr. Wagner stated. “It is likely that inhibition of cap-dependent translation will thus have therapeutic value against a wide range of cancer types.”