Core Technologies

High-throughput Screening (HTS)

Our high-throughput screening (HTS) facility occupies 10,000 square feet of laboratory space and is located in Farmingdale (NY). The facility contains a variety of automated workstations, plate readers and equipment necessary to support a broad range of cell-based and biochemical high-throughput screens in 384-well plates. We have also compiled a screening library containing diversity sets and focused collections targeting kinases, GPCRs, proteases methyltransferases and protein-protein interaction interfaces.

We also use affinity-based screening for discovering hit compounds against poorly characterized targets that are less amenable to HTS. The method involves incubating the target protein with mixtures of up to 500 compounds at a time. Unbound compounds are separated from hits that bind to the target by size exclusion chromatography (SEC) and the binders are identified through liquid chromatography coupled with mass spectrometry (LC-MS). This screening approach facilitates drug discovery for more challenging targets and is a powerful complement to HTS.

The screening operation is so effective that an entire screening library can be tested against a biological target in as little as two weeks. An industrial-scale robotic cherry-picker retrieves orders of screening “hits” for confirmation testing within 24 hours. All the data generated throughout the discovery cascade is stored in a company-wide data management system. Our scientists use querying tools to analyze the data and identify the compounds with the preferred properties. The most desirable HTS hits are developed into drug leads through medicinal chemistry.

your alt text

High-throughput screening of small molecules in 384-well assay plates (top); Selection of screening hits using the robotic cherry picker (bottom).

Metabolic and Physicochemical Profiling

We apply a number of laboratory screens to profile compounds for drug-likeness, which allows us to predict how compounds may behave when administered to patients. Compounds are tested for solubility and permeability, factors that are critical in determining how well a drug is absorbed when taken by mouth. Another assay measures how much a drug is metabolized by subcellular liver microsomes which allows us to predict how stable a drug is to metabolic inactivation by the liver. Compounds are also tested for their inhibitory effect on cytochrome P450 enzymes, which we use to predict possible drug-drug interactions. Computational methods are also used for determining physicochemical properties of compounds. Together, this information is used throughout the drug discovery cycle to optimize the properties of lead compounds and to identify those compounds that have the greatest chance of succeeding as drugs.

High-Speed Analoging

Modern drug discovery requires the efficient production of drug-like molecules to provide an understanding of Structure-Activity relationships (SAR) and systematically guide the optimization of screening hits to potential drugs.   At OSI, the application of systematic High-Speed Analoging (HSA) approaches, involving cutting-edge parallel synthesis and high-throughput purification techniques coupled with computational chemistry input, enables the rapid generation of  intelligently designed iterations of novel compound libraries to expedite our “HTS hit-to-lead” and “lead optimization” drug discovery processes.

24 Compounds are synthesized simultaneously during a High Speed Analoging (HSA) synthesis (left); HSA products are purified using parallel and high-throughput methodologies (right).

Structure-Based Drug Design & Virtual Screening

Our approach at OSI is to combine theoretical and experimental insights to accelerate an informed decision-making process critical to success in drug discovery.   The Computational Chemistry team at OSI applies their significant technical expertise, strong hardware platform, state-of-the-art software suites, robust databases and top line virtual screening (VS) technologies to enhance OSI’s drug discovery team efforts in numerous respects. The application of VS approaches to computationally “dock” small molecules into the active sites of our molecular targets internally has been proven to successfully capture potential “false negatives” from high-throughput screens against individual therapeutic targets that would otherwise have been overlooked. In cases where a full high-throughput screen may not be feasible, combining virtual screening technologies with OSI’s rapid cherry-picking capacity permits the selection of chemical matter with the highest probability of inhibiting our targets from among our extensive collections.   When applied to the millions of compounds accessible to us in external collections, VS allows the rational target-driven selection of compounds for acquisition as potential drug leads.

In OSI’s drug discovery programs, computational chemists collaborate with medicinal and high-speed analoging chemists to predict compound properties and design potential drug molecules. In this process we utilize information from x-ray crystal structures and/or homology models in which the initial small molecule “lead” is bound to the protein target to improve the “fit” and efficiently guide the rational optimization into potent and selective drugs for treating cancer, diabetes, obesity and other diseases. Drug discovery teams routinely visualize and probe molecular interactions in 3D using advanced computational graphics systems to understand the forces that contribute to enhanced binding. This insight drives our iterative cycle of hypothesis, design, synthesis, testing, and refinement, often with co-crystallization of the improved agents with the target protein, to produce medicines with superior potency, selectivity, and efficacy. As an example, we show the binding of one of our lead compounds in the active site of the target protein.

Molecular Target Identification and Protein Profiling Using Affinity Chromatography and Mass Spectrometry

Identifying protein complexes by affinity chromatography and mass spectrometry is becoming a mainstay method of defining signal transduction pathways. This technique is most frequently used to compare disease and normal tissue states. At OSI we use the technique to define signaling cascades that mediate drug actions. Such studies may identify the functional genomics of new drug compounds, both upstream and downstream, with current protein targets involved in human disease. The advantage of affinity chromatography is that it can measure changes in protein function rather than simple changes in protein expression.

RNA Interference (RNAi): An Important Tool in Rapid Target Validation

From the studies of plant genetics and the roundworm C. elegans , the process of RNA interference (RNAi) reveals a natural cellular process that can be utilized to knock out the function of a particular gene. The ability to individually “knock out” the function of a gene via RNAi allows the systematic study of the function of different cancer-causing genes and their cell signaling pathways. We have used this revolutionary technology to discover gene targets important for cancer cells to proliferate and survive. The identification of these important cancer targets paves the road to develop molecular targeted therapies.

Molecular Imaging

Molecular imaging techniques allow for the non-invasive assessment of biological and biochemical processes in living animals. These techniques are used to enhance our understanding of disease progression and drug treatment response at both the pre-clinical and clinical levels. Our laboratory uses both luminescence and fluorescence imaging to visualize and quantitate non-invasively specific molecular targets, biochemical pathways and physiological effects of novel drug candidates in vivo. Luminescence imaging utilizes light produced from a chemical reaction without an external excitation source, whereas in fluorescence imaging, energy from an external source (excitation) is absorbed and re-emitted virtually immediately at a longer, lower energy wavelength (emission). Careful design of pre-clinical experiments can provide a rapid understanding of an observed biological process, thereby accelerating drug discovery and potentially better predicting clinical outcome of a given agent. With our research focus on EMT (Epithelial-to Mesenchymal Transition), we are currently developing and utilizing molecular probes in the near-infrared (NIR) range that target specific facets of this biochemical cascade to detect early changes in cancer progression, efficacy and drug sensitivity. Our goal is to successfully integrate molecular imaging into our drug discovery process to increase our efficiency in selecting and developing the most suitable drug candidates by providing early and more predictive preclinical data.

Molecular imaging at OSI. A 3-D rendition of an orthotopic tumor (left; deep red mass) using bioluminescence imaging. Fluorescence image of a tumor bearing mouse (right) with tumor mass observed in lower right flank.

References and Suggested Reading

  1. Boisclair MD et al. In: Teicher, BA & Andrews, PA (eds) Cancer drug discovery and development: Anticancer drug development guide: Preclinical screening, clinical trials and approval 2004; 2nd Ed. Humana Press Inc. Totowa, NJ.
  2. Jinhua W et al. Structural basis for activation-loop trans-phosphorylation and small-molecule inhibition of the insulin-like growth factor-1 receptor kinase. EMBO Journal 2008; 27(14): 1985-1994.
  3. Buck, E et al. Feedback mechanisms promote cooperativity for small molecule inhibitors of EGFR and IGF-1R. Cancer Research 2008; 68 (20): 8322-8332.
  4. Ji Q-S et al. A novel, potent and selective Insulin-like growth factor-I receptor (IGF-IR) small molecule inhibitor blocks activation of IGF-IR signaling in vitro and inhibits IGF-IR dependent tumor growth in vivo. Molecular Cancer Therapeutics, 2007; 6(8): 2158-2167.
  5. Li AH et al. A Highly Effective One-Pot Synthesis of Quinolines from o-Nitroarylcarbaldehydes. Organic & Biomolecular Chemistry 2007; 5(1): 61-64.
  6. Mulvihill, M et al. Regio- and stereospecific syntheses of syn- and anti-1,2-imidazolylpropylamines from the reaction of 1,1'-carbonyldiimidazole with syn- and anti-1,2-aminoalcohols. Journal of Organic Chemistry 2004; 69(15): 5124-5127.
  7. Thomson S et al. Kinase switching in mesenchymal-like non-small cell lung cancer lines contributes to EGFR inhibitor resistance through pathway redundancy. Clin Exp Metastasis. 2008; 25(8):843-54.
  8. Petti F et al. Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930. Mol Cancer Ther. 2005; 4(8):1186-97.
  9. Thelemann A et al. Phosphotyrosine signaling networks in epidermal growth factor receptor overexpressing squamous carcinoma cells. Mol Cell Proteomics. 2005; 4(4):356-76.
  10. Weissleder R, Ntziachristos V. Shedding Light onto Live Molecular Targets, Nature Medicine 2003; 9(1): 123-128
  11. Hoffman R. The Multiple Uses of Fluorescent Proteins to Visualize Cancer In Vivo. Nature Reviews 2005; 5: 796-806
  12. Willmann J et al. Molecular Imaging in Drug development, Nature Reviews 2008; 591-607.
  13. Urano Y et al. Selective Molecular Imaging of Viable Cancer Cells with pH-Activatable Fluorescence Probes, Nature Medicine 2008; (On-Line Publication), 1-6.