October 15, 2011 (Vol. 31, No. 18)

Tool Combinations Lay Waste to Obstacles in Small Molecule Discovery and Development

Caliper Life Sciences recently hosted a meeting where scientists described use of the company’s technologies to support high-quality drug candidate discovery and optimization. The application of Caliper’s imaging technology, automation/liquid-handling robotics, and chip-based microfluidics technologies to meet and overcome technical challenges in both drug discovery and in developing in vitro and in vivo assays were among the topics discussed.


Michelle Palmer, Ph.D., director of discovery and preclinical research, chemical biology platform, Broad Institute of Harvard and MIT, described the institute’s approach to integrating novel technologies for identifying small molecules that drive translation research and therapeutics.

The chemical biology and novel therapeutics platforms were established at the Broad to discover small molecules that impact biology and medicine and to innovate the process through which probes and drugs are both discovered and developed.

The Broad enterprise, she explained, “is part of a large network, the MLSMR, funded by NIH. Through that network, we collaborate on 25 new projects a year. Projects start with assay development, then progress to hit selection, and prioritization. The hits are then put through a battery of secondary assays to determine which are the most promising for chemical optimization. We work in close collaboration with our chemistry team. We then provide the data and the chemical tool back to the research community through NIH’s probe report.”

Dr. Palmer said that the Broad applies standard HTS assays as well as novel technologies to prioritize hits coming out of screens and then to discover and understand the mechanism of action of a particular compound. “This knowledge informs the medicinal chemistry process and ability to detect, quantify, and understand the effects of the compound in various cell and animal models.”

The Broad applies a variety of integrated technologies to achieve this level of detail. These include next-generation chemistry (diversity-oriented synthesis), small molecule microarrays (SMMs), microfluidic-based chromatin modifying enzyme assays, multiplexed gene-expression measurements, and SILAC proteomics for target ID.

Dr. Palmer believes that automation has had a big impact on the small molecule characterization process, because, “the more you can do to automate a process and improve data consistency, quality, and reproducibility, the easier it is to think about the data. The data-visualization tools that are evolving to allow us to look at large quantities and types of data permit a much more unbiased approach to identifying the mechanism.”

Assay Optimization

Edward Esposito, Ph.D., senior research scientist at Blue Sky Biotech, talked about optimization of tyrosine kinase assays using the company’s template-directed receptor assembly (TDA2.0) platform.

Blue Sky designed its TDA2.0 platform to replicate the biological context of receptor domains in vivo and enable the development of in vitro assays with more relevant biochemical activity enabling the identification of novel small molecule drugs.

TDA2.0 technology consists of full-length cytoplasmic domains of receptor tyrosine kinases (RTKs) coupled to lipid vesicles. “We developed a lipid-based nanosphere that has nickel NTA groups present on its surface. This fluid matrix allows anything that’s bound, like a histidine-tagged protein or peptide, to move around on its surface and interact with co-localized binding partners. In the case of a receptor kinase, it mimics the inside of the cell surface,” Dr. Esposito said.

By putting the catalytic portion of a membrane-associated kinase in a membrane environment, the enzyme’s conformation is dictated by the close proximity of its binding partners, other catalytic subunits that form homodimers or homoligomers and are regulated by their interactions with one another.

“For years, people have made recombinant proteins, like the insulin receptor cytoplasmic domain for in vitro screening, adding substrate and kinase, to determine whether phosphorylation occurs. But you don’t get good activity because the insulin receptor in a physiological setting interacts with other insulin receptors. This doesn’t happen in the absence of a membrane-docking domain. These things in solution often have very low affinity for each other, so no native biomimetic assembly occurs.”

Dr. Esposito described a multicomponent kinase assay that previously “would have been impossible” without the TDA2.0 system and Caliper’s LabChip EZ Reader, a microfluidic, integratable drug-screening platform that enables a range of enzymatic assays.

The LabChip EZ Reader II is a benchtop instrument that analyzes enzyme activity by “sipping” reactions from 96- or 384-well microtiter plates into a Caliper LabChip. The data signature is generated by the shift in mobility of nonphosphorylated peptide substrates and phosphorylated products by electrophoresis in the chip and detected via LED-induced fluorescence.

The assay, developed by Blue Sky in collaboration with Pfizer, measured PDK1 and mTor activity via a fluorescently labeled peptide substrate of AKT.

“Combining the TDA2.0 system and the LabChip EZ Reader enabled us to determine, in real time, the kinetics of AKT activity in the presence and absence of TDA2.0, PDK1, and mTor. We expect that these multicomponent assays and detection systems will allow us to look at kinase pathways and their responses to small molecule drug candidates in a truly biological context.”

Many scientists believe small molecule drug discovery operations can be enhanced by using an approach based on chemical biology and new therapeutics platforms. [Olivier-Fotolia.com]

Safety Liabilities

Karen Leach, Ph.D., associate research fellow in the compound safety prediction group in worldwide medicinal chemistry at Pfizer Global Research & Development, discussed methods and technologies for predicting safety liabilities early in the drug development process.

Since drug toxicity accounts for about 60% of the novel drug candidate attrition rate, she said, a “more holistic approach” is needed that builds predictive frameworks incorporating structure activity relationships (SAR) data, physical chemistry, ADME, pharmacology, and safety data for selecting potential drug series and molecules.

Researchers at Pfizer use its Compound Safety Evaluator v2.0, analytical software that provides a way to rank compounds by their potential for inducing toxicity and combines multiple data sources to derive a single value. The lower the score, Dr. Leach said, the greater the potential safety liability.

Project teams use this tool to assess the potential safety liabilities of compounds and series early in the drug discovery process, well before any in vivo studies are conducted. In this way, teams can concentrate resources on advancing compounds with lower safety risks.

Dr. Leach explained that Pfizer also has been working with Caliper in developing a number of cell-based kinase assays, using Luminex technology. Pfizer utilizes in vitro kinase assays to characterize selectivity of kinase inhibitors. These assays primarily use recombinant enzymes and Km levels of ATP. However, the challenge is knowing whether that activity translates to inhibition of functional kinase activity.

Luminex technology is an antibody-based approach that utilizes a capture antibody and a phospho-specific antibody to directly measure substrate phosphorylation in cells for each of the kinases. Using these assays, the Pfizer groups hope to identify the activity profiles of kinase inhibitors in a more physiological, cellular context. This information will be used by project teams to understand compound selectivity and drive SAR.

Small Molecule Inhibitors

Victoria Korboukh, Ph.D., post-doctoral scientist at the University of North Carolina’s Center for Integrative Chemical Biology & Drug Discovery, described her team’s research on finding small molecule inhibitors for histone methyltransferase G9a, an enzyme of the protein lysine methyltransferases (PKMTs) group.

These enzymes mediate methylation of histone proteins in nucleosomes, the structures that comprise the basic units of chromosomes. Nucleosomes consist of double-stranded DNA wrapped around a protein octamer containing two copies each of the histone proteins H2A, H2B, H3, and H4.

Histone proteins undergo a variety of post-translational modifications including methylation, occurring within the histone core region as well as on the N-terminal tails that protrude from the core region. Methylation, among other modifications, affects DNA regulatory processes including replication, repair, and transcription.

In particular, the PKMTs and PRMTs that modify lysine and arginine residues within histone proteins have been correlated with a variety of human disease states including rheumatoid arthritis, cancer, heart disease, diabetes, as well as neurodegenerative disorders such as Parkinson and Alzheimer diseases.

As part of understanding the catalytic mechanisms of these enzymes, Dr. Korboukh and her colleagues are looking for small molecule chemical probes that could be used to validate targets either in cell-based or in vivo experiments. In light of the importance of these enzymes in a large variety of human disease states, it is critical to elucidate their catalytic mechanisms, in particular G9a and GLP, both of which modulate transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of nonhistone targets.

To facilitate chemical exploration of these proteins, Dr. Korboukh and her colleagues, working with Caliper technology, developed a highly quantitative microfluidic capillary electrophoresis assay to enable full mechanistic studies of these enzymes, as well as the kinetics of their inhibition. This technology separates small biomolecules, in this case peptides, based on their charge-to-mass ratio.

But since methylation doesn’t change the charge of peptide substrates, the investigators used a methylation-sensitive endoproteinase strategy to separate methylated from unmethylated peptides. The assay was validated on a lysine methyltransferase (G9a) and a lysine demethylase (LSD1) and was employed to investigate the inhibition of G9a by small molecules.

Because methylation produces only a minor change in the substrate’s mass and itself bears no charge, separation of the substrate and the product has been impossible, Dr. Korboukh noted.

Typically, she said, researchers use radiolabeled co-factor, s-adenosylmethionine, to study this class of enzymes, as well as coupled fluorescent assay. In the fluorescent assay, SAHH (s-adenosylhomocysteine hydrolase) and adenosine deaminase convert the methyltransferase reaction by-product (s-adenosyl homocysteine) to homocysteine and inosine. Homocysteine is then quantified using ThioGlo.

Dr. Korboukh explained that her group has developed a high-throughput assay employing Endo LysC proteinase, which cleaves proteins on the C-terminal side of the lysine residues. Activity of the Endo LysC is inhibited if the lysine is methylated.

“In our assays we use short fluorescently labeled peptides as substrates that are subjected to Endo LysC proteolysis after methylation reaction. If the PKMT is active—the methyl mark on the lysine will protect the peptide from cleavage, if the activity is inhibited—unmethylated peptide will be cleaved with Endo LysC.

“Now we have a significant change in mass-to-charge ratio of the product vs. substrate, and the two can be separated and quantified using the Caliper EZ Reader platform. Although coupling methylation reaction with Endo LysC makes it an endpoint assay, this platform still allows detailed kinetic studies of the enzyme of interest.”

Use of this assay to study enzyme activity and kinetics allowed the development of a probe, UNC638, which Dr. Korboukh and her colleagues said provides high potency, excellent selectivity, low cell toxicity, and robust on-target activities in cells, making it the preferred chemical probe of G9a/GLP34.

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