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WEDNESDAY, MAY 14

8:30  Chairperson’s Remarks
Andrew Napper, Ph.D., Director, High-Throughput Screening, Penn Center for Molecular Discovery, University of Pennsylvania

8:40 Discovery of Protease Inhibitor Probes by Target Class Profiling
Andrew Napper, Ph.D.
As part of the Molecular Libraries Screening Center Network (MLSCN), the Penn Center for Molecular Discovery (PCMD) has a mandate to discover novel chemical probes to be made available to the wider drug discovery community. The PCMD has implemented a strategy for profiling multiple members of protease target classes against a library of 250,000 compounds. Rapid and reliable set up of assays has been achieved by pintool transfer of compounds formatted in 1536- and 384-well plates either individually or as mixtures. Screening of cysteine and serine proteases has led to the discovery of sub-nanomolar inhibitors as novel chemical genomic probes. At the PCMD these inhibitors have been shown to inhibit the propagation of the SARS and Ebola viruses and the malarial parasite Plasmodium falciparum in human host cells. Complete HTS results are available on PubChem, presenting a profile of activity that allows the flagging of non-selective compounds to facilitate the identification of selective inhibitors against newly discovered proteases.

9:10 HTS Assays For Lipid Kinases
Marshall Michener, Ph.D., Research Fellow, Primary Pharmacology, Inflammation Research, Pfizer Global Research and Development
HTS assay development for proteins kinases enjoys a wide variety of methodologies to choose from these days.  Lipid kinases, however, present unique challenges for HTS assay development due to the physical properties of the native substrates.  We will present assay development strategies and results for the comparison, development and validation of
HTS compatible assays for sphingosine kinase 1 and 2.  We will show the challenges in adapting protein kinase assay technologies, how they can be used for enzymatic characterization and our progress in identifying both medium and high throughput platforms.  Result metrics from targeted library screening and hit characterization will be summarized.

9:40 Optimization of Enzymatic Assays for HTS: Sampling the Biochemical Space Using a Parallel, Streamlined Approach
Daniele Carettoni, Ph.D., Head, Biochemistry, Axxam
High-throughput screening has radically changed drug discovery by applying automation, parallelization, and miniaturization along the entire process. Due to the growing size of the compound collections, however, protein supply has increasingly become a critical bottleneck in enabling screening campaigns on enzymatic targets.
Traditionally, optimization of the myriad parameters to increase protein yield and catalytic efficiency has been conducted by means of a low-throughput, labor-intensive and time-consuming process, often incompatible with the needs of drug discovery research. To comply with the requirements of HTS, we have developed a streamlined process for the optimization of the enzymatic assays, by applying high-throughput technologies. Each chimeric version of the target is expressed in insect cells cultured in miniaturized 24 deep-well format plates. The culture conditions, cell lines, time of expression, and media additives are each varied to create a matrix of 80 alternative conditions. In a similar manner, each target protein is purified by robot-assisted affinity chromatography in 96-well format using a matrix of 24 different conditions. The measurement of the expression and purification yield is accomplished by a high-throughput sensitive luminescence-based readout in 96-well format. The subsequent automated optimization of the enzymatic reaction in 384-well format on a matrix of 250 different conditions allows selection of the best conditions to further decrease the protein amount required for HTS.

10:10 Networking Coffee Break in the Exhibit Hall

10:55 Beyond Kinases: Interrogating the Purinome
Bob Lowery, Ph.D., President, CEO, Corporate, BellBrook Labs
The development of ATP-site ligands as protein kinase inhibitors raises questions about the scope of their off-target effects as well as possibilities for the therapeutic targeting of other ATP-utilizing enzymes. Adenine nucleotides are interconverted by diverse proteins  including, in addition to kinases, other types of transferases, phosphodiesterases, membrane transporters, DNA modifying enzymes, molecular chaperonins. Despite high diversity in the sequence motifs and folds that bind adenine nucleotides, there are commonalities in the ATP interaction networks across functionally diverse enzymes. To explore the ligand selectivity of ATP-binding sites, we screened a kinase-focused library across diverse ATP-utilizing enzymes using adenine nucleotide detection as a generic assay method. The assays rely on highly selective antibodies that distinguish between nucleotides on the basis of a single phosphate group. Homogenous fluorescent assays have been developed for both ADP and AMP, making it possible to interrogate a diverse panel of otherwise intractable ATP-utilizing enzymes in an HTS format. Of the eight ATP-utilizing enzymes used in the study, we found micromolar interactions of protein kinase inhibitors with one mammalian target and one bacterial target, indicating the potential for off-target effects. The approach used provides a framework for more systematic efforts to map the ligand selectivity of ATP-utilizing enzymes. It also raises the possibility of leveraging the large body of kinase inhibitor chemoselectivity data to address other target families in the purinome.

11:25 Multiparameter Biochemical uHTS for Small Molecules and Fragments
Dirk Ullmann, Senior Vice President, Discovery Biology, Evotec AG
Modern screening methods are characterized by minimized attrition in bringing primary hits into a hit-to-lead program. A key element is the application of multiparameter detection and data analysis methods. This talk will discuss advanced miniaturized fluorescent assays for uHTS by parallel acquisition of multiple readouts. Case studies on a number of enzyme and binding assays will be used to demonstrate the capabilities in eliminating falses for example due to fluorescent quenching and auto fluorescence. In addition it will be shown who these screening methods can be applied to both small molecule screening and fragment-based drug discovery in parallel (e.g. uHTS at very high compound concentrations).

12:55 Session Break

1:25 Chairperson’s Remarks

1:30  Applications of Biochemical Tests in HTS Hit Identification and Characterization
Ji-Hu Zhang, Hits Discovery Group, Novartis Institutes for BioMedical Research
Biochemical assays continue to be the predominant tool in HTS and SAR studies in early lead discovery.  The integration of a number of different measurement technologies in the winnowing process from HTS to lead discovery allows for the efficient separation of the true hits from the inactives and false positives. The application of a variety of biochemical tests in both hits identification and hit characterization, including LC/MS and biophysical and other readouts will be discussed along with the use of selectivity and orthogonal assays.

2:00  Drug Discovery in Academia: Two Examples of Novel Approaches to Biochemical Assays
Marcie Glicksman, Ph.D., Senior Director, Leads Discovery, Laboratory for Drug Discovery in Neurodegeneration, Brigham and Women’s Hospital
Neurodegenerative diseases are challenging from a drug discovery perspective with virtually no disease modifying agents available on the market.  The Laboratory for Drug Discovery at the Harvard NeuroDiscovery Center works with academic labs around the country in a collaborative model of drug discovery within academics.  We take projects from assay development, high-throughput screening, and exploratory medicinal chemistry with the goal to have a compound show efficacy in a relevant animal model.  I will highlight two programs currently at our center.

2:30  Ice Cream Refreshment Break in the Exhibit Hall (Last Chance for Viewing)

3:10  High-Throughput Screening for Inhibitors of p27Kip1 Proteolysis
Xuedong Liu, Ph.D., Associate Professor, Chemistry and Biochemistry, University of Colorado-Boulder
Decreased levels of cell cycle inhibitor p27Kip1 due to excessive degradation occur in a variety of aggressive human tumors. Since reduced p27 expression has been associated with a poor prognosis in many human cancers and resistance to certain anti-tumor therapies, it has been postulated that elevation of p27Kip1 expression could improve prognosis and perhaps even provide a cure for malignant cancers. However, this concept has not been proven or rigorously tested largely due to the absence of specific small molecule inhibitors that perturb abnormal reduction in p27 levels. The abundance of p27 is primarily controlled by the ATP-dependent ubiquitin-proteasome pathway. Ubiquitinaton of p27 is primarily catalyzed by SCFSkp2 , a multiprotein E3 ligase complex and requires specific interaction between Skp2 and Cks1. Mutations in Skp2 or Cks1 that disrupt the Cks1-Skp2 interaction perturbs p27 degradation. We have a non-radioactive, robust, high throughput screening assay for the Skp2-Cks1 interaction based on Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen^TM). Using this assay we screened small molecule compound libraries for inhibitors that disrupt Skp2-Cks1 binding. Several compounds were identified and shown to be able to inhibit p27 ubiquitination. Identifying specific small molecule inhibitors of p27 degradation is a first step towards evaluating whether inhibition of p27 degradation would be an effective anti-cancer therapy approach and translating what we have learned about the basic mechanisms of p27 degradation into potential new drug leads in cancer biology.        

3:50  Speaker to be Announced

4:20  The Discovery of Slowness: Enhancing Drug Efficiency by Developing NCEs with Long Kinase-Drug Residence Times
Doris Hafenbradl, Executive Vice President, Screening & Proteins, Proterus
Retrospective analysis has shown that among the successful kinase drugs a surprising large proportion shows slow drug-kinase binding kinetics. Broader analysis reveals that this phenomenon is not only true for kinase drugs but also for drugs throughout all target families. Despite this clear advantage of slow binding drugs no systematic development of drugs with this property is happening so far. This discrepancy is mainly due to the lack of time and cost efficient methods to measure kinase binding kinetics for large numbers of drug candidates. Here we present a novel technology that is overcoming this bottleneck and allows the rational design of drug candidates with slow kinetics. Moreover, we discuss how this new technology can be implemented into the current lead optimization process in order to produce better drugs.

4:50pm Close of Conference

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