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

Elizabeth Lipp

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

In a perfect high-throughput screening world, hit-to-lead discovery scientists would have many chemical series from which to choose. Unfortunately, as Tara Stauffer, research scientist at Pharmacopeia (www.pharmacopeia.com), noted, “determining the optimal series for further chemical development can be a daunting task.”

At GTCbio’s “Assay Development & Screening Technologies Conference,” held in San Francisco last month, the evolution of the search process for hit-to-lead compounds was highlighted. A few of the presentations that featured new technologies designed to streamline the discovery process are reviewed in this article.

“A key concern in early-stage drug discovery is making sure that we work on physiologically relevant targets and assays,” said Berta Strulovici, Ph.D., vp, basic research at Merck & Co. (www.merck.com). Dr. Strulovici is also head of the automated biotechnology unit, a center of excellence for lead and target identification at Merck Research Laboratories.

“My department spends about 50 percent of its time working toward identifying novel compounds that affect molecular targets of interest by screening a large library of compounds against molecular targets. The other 50 percent is spent on the identification of proprietary targets with genome-scale RNA interference technologies.”

Dr. Strulovici presented specific examples of the benefits of implementing complex, multiparametric cellular assays on a large, industrial scale along the drug discovery pipeline—from small molecule lead ID, to target ID, to identifying potential responder populations.

For target ID using RNAi technology in human cell lines, multiparametric assays generate biological activity profiles that provide valuable insight into complex disease models. “Our infrastructure is unique in the industry,” explained Dr. Strulovici. “We are working with highly miniaturized, fully automated systems, that enable us to assess each gene in the genome as a potential target for drug discovery.”

Dr. Strulovici presented three case studies to demonstrate how this infrastructure impacts overall effectiveness in providing solid leads. The first study detailed mouse adult neuronal stem cell research to identify compounds that induce either proliferation or differentiation toward various cell lineages. The other two case studies focused on genome-scale siRNA screening, one for the identification of novel targets for Alzheimer disease, the other for the identification of responders in the clinic for an oncology program.

Combined with miniaturization technologies and sophisticated data-analysis tools, “we have identified novel targets for several therapeutic areas, which are now either at target-validation stage or have progressed to lead ID,” Dr. Strulovici said. “In addition to this, we are continually developing and refining technology to be able to perform the high-throughput biology needed to further research and drug discovery.

“As much as we work with artificial methods,” added Dr. Strulovici, “physiological assay systems are key. The easiest way is not the best way, since the physiological relevance may not be there. For the clinicians, you need to bear in mind that you do not work in isolation, the work you do needs to be relevant in the field. The team you build to get the job done is also key. Hire the best people possible and give them the freedom they need to make discovery possible.”

Hit Parade

Highly engineered assays intended for high-throughput screening may not be ideal for selecting the best chemical series to pursue in a hit-to-lead campaign, according to Stauffer. “The bigger picture may include multiple assays to arrive at the best active series for chemical optimization,” she noted.

Pharmacopeia’s screening platform is based on its ECLiPS® (encoded combinatorial libraries on polymeric support) technology. Stauffer elaborated, “We basically go through three steps in the process: a primary HTS in which we screen the eluate from multiple beads; a follow up HTS, in which we screen single bead eluates from those sublibraries, which demonstrate activity in the assay; and finally, we submit beads for our unique decoding process, which is how we identify the structure of compounds in the active wells in step two.

“In the second step, we are searching for active compounds from a large random sample of beads out of a particular active sublibrary. Statistically, we will have multiple instances of the same bead (compound) in the screening plates. So in this process a truly active compound will produce multiple hits. A single compound might show up eight times, so the frequency of the hit demonstrates how strong the active signal is for that compound. This also reduces the incidence of false positives.”

The research she presented demonstrated how assays to explore differences in receptor binding, selectivity, and downstream functional activity were developed to reduce eight chemically distinct series, identified from a single HTS of six million compounds, to one pharmacologically relevant series that was suitable for further optimization. “Three were rejected out of hand for chemical properties, selectivity, and IP position,” noted Stauffer. “So the question remains, how do you choose the most viable option to pursue for development from the remaining five?”

The next round of compound vetting looked at binding and off-target testing; as a result, another compound series dropped out, she continued. “Looking at the functional pharmacology of the compounds was what eventually produced the best candidate series. Ultimately, evaluating compounds in the signaling pathway assay was what indicated which series would be the best to advance.”

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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