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Jul 1, 2008 (Vol. 28, No. 13)

Compound Discovery Tools Still Evolving

Finding the Best Chemical Series to Pursue in a Hit-to-Lead Campaign Need Not Be Daunting

  • Inhibitors for the Reversal of MDR

    Multidrug resistance (MDR) is a phenomenon by which tumor cells display or develop resistance to a number of structurally and functionally distinct anticancer drugs. “MDR is not just a problem for cancer, it is a major clinical obstacle that contributes to the marginal efficacy of other classes of drugs such as antibiotics, antifungals, and antiparasitic compounds,” explained Gary Piazza, program director, Southern Research Molecular Libraries Screening Center, Southern Research Institute (www.southernresearch.org).

    Dr. Piazza reported that a significant factor that contributes to MDR is the overexpression of certain ATP-dependent transporter proteins such as MRP1 in tumor cell membranes that cause the efflux of cytotoxic drugs, thereby reducing their intracellular concentration and limiting their effectiveness.

    Southern Research Molecular Libraries Screening Center, part of the NIH Roadmap Molecular Libraries Screening Initiative, developed a cell-based assay to identify compounds that reverse MRP1-mediated drug resistance. “In a nutshell, we identified a series of molecules that are potent inhibitors of MRP1,” said Dr. Piazza. “Our purpose was to run an HTS to identify novel inhibitors of MRP1 to probe the function of MRP1 and assess its utility as a drug target.”

    Dr. Piazza was quick to point out that this is only the beginning. “There is quite a bit of work ahead to develop compounds with desired pharmacological properties and selectivity and then focus on a particular type of cancer that overexpresses MRP. We have some interesting compounds that may have applications to fighting drug resistance.”

  • Screening via SPR Sensor

    A major driving force in early-phase drug discovery has been the need to increase throughput and decrease assay volumes. “These developments led FujiFilm (www.fujifilm.com) to develop the AP-3000 automated screening system based on SPR,” said Takayuki Yamada, application manager of FujiFilm’s life science group.

    SPR has been a valuable technology for the detailed study of biomolecular interactions. In spite of its many positive attributes (e.g., label-free, direct-binding measurements, and kinetic analysis), low throughput limited its use. “The AP-3000 maintains the principles of the SPR detection method, however, our Sensor Stick provides the sensitivity and throughput required for the modern drug discovery lab,” Don Janezic, business development manager for Fujifilm, noted.

    This fully automated workstation has a built-in 12 channel disposable tip pipette and robotics for moving microplates, tip boxes, reagents, and SPR sensors. Up to 3,840 discreet small-molecule/protein interactions can be measured in 24 hours with no user intervention, according to Janezic.

    Using a stop-flow sensor design, which produces low background and high sensitivity, the AP-3000 allows the screening of fragment molecules with molecular weights as low as 100 daltons. Janezic added. “The growing interest in label-free technologies and the increased throughput needed for hit confirmation, lead optimization, focused library screening, and the verification of in silico derived chemical structures makes the AP-3000 ideal for drug discovery.”.

  • Aptamer Discovery

    Aptamers are structured single-stranded oligonucleotides capable of highly specific target recognition. Shuhao Zhu, associate director, Archemix (www.archemix.com), noted that they are “an exciting new therapeutic modality.” His presentation focused on how Archemix is facilitating aptamer discovery.

    Dr. Zhu noted that there is currently only one approved aptamer therapeutic, Macugen. Several more aptamers are in various stages of clinical development. “The aptamer lead-discovery process is enabled by a series of platform assays that allow for efficient measurement of aptamer-target affinities,” Dr. Zhu continued. “Aptamers are easily synthesized chemically, which allows for rapid screening assay turn-around and SAR analysis.”

    Dr. Zhu described the use of SPR and 96-well flow cytometry-based assays to characterize, minimize, and optimize aptamers during the discovery stage and described how competitive ELISA and FACS assays have been used to prioritize aptamer hits.

    “One challenge we are facing is that we have many discovery programs, and we need to screen a huge amount of aptamers targeting different biological molecules. To simplify the early-stage screening process, we need screening toolboxes to address the needs of different discovery programs. The SPR-based binding assay provides real-time measurement of on- and off-rate without labeling, and our plate-based FACS-binding assay provides a measurement of aptamer affinity toward the cell-presented targets. These platform-based screening assays used at the discovery stage can be easily translated for use in PK/PD assessment during downstream development.”

    Recent technological advances have improved the speed and reliability of HTS, but the total number of compounds that can be assayed using these methods remains in the 106 range, said John W. Cuozzo, Ph.D., director, ELT lead discovery, GlaxoSmithKline (www.gsk.com)

    “Aptamer-, phage-, and antibody-screening libraries have significantly greater diversity and can be rapidly queried for hits by selection.”

    Encoded library technology (ELT) combines affinity-based enrichment with combinatorial chemistry to enable the rapid selection of hits from small molecule libraries containing up to several billion compounds. “Each ELT library molecule carries a unique DNA tag that encodes the chemical composition of the small molecule,” explained Dr. Cuozzo. “Libraries are selected against protein targets by affinity-based methods. Enriched binders are isolated, sequenced, and translated back into their corresponding chemical structures. The hit compounds are then synthesized without the DNA tag and assayed for activity against the target.”

    “We focus largely on soluble protein targets—approximately half of our targets are soluble proteins,” said Robert Hertzberg, Ph.D., vp, screening and compound profiling. “Not all targets are amenable to this approach, e.g., ion channels are currently not. However, we are working to extend this technology.”

    Drs. Cuozzo and Hertzberg agreed that key differentiators of ELT are the size of the library, currently some 10 billion compounds, against the key constants of economy and time. “Our primary goal is to increase the likelihood of quality hits,” noted Dr. Hertzberg. “We’re pushing to increase the number and diversity of compounds in the clinic.”


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