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Scientific Report 2007
Scripps Florida
Translational Research Institute
Probe and Drug Discovery: The Lead Identification Department
P.
Hodder, P. Baillargeon, P. Chase, L. DeLuca, F. Madoux, B. Mercer, D. Minond, S. Saldanha, L. Scampavia, T. Spicer, P. Subramaniam, L. Sullivan
The
Lead Identification Department is responsible for developing and executing high-throughput
screening (HTS) assays and for supporting downstream medicinal chemistry and probe
development efforts. The anchors of the department are 2 fully automated robotic
platforms (Fig. 1). One supports screening of 384- and 1536-well microtiter plates
in a variety of biochemical and cell-based assay formats. The other is used to manage
and characterize the screening library of more than 600,000 compounds used for drug
discovery at Scripps Research. The facility also contains an assay development laboratory
with equipment for tissue culture and semiautomated liquid handling and detection.
Supporting this operation is an integrated laboratory information management system,
which is used to track HTS data and compound usage and quality. Additionally, we
are involved in developing metallo-β-lactamase
chemical probes.
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| Fig. 1. The Scripps Research
uHTS platform. A, An industrial anthropomorphic robotic arm moves assay and compound
microtiter plates. B, A pin tool is used to transfer test compounds rapidly from
compound plates to assay plates. C, Liquid handlers capable of dispensing up to
32 different reagents and of washing plates are integrated into the platform. D,
Incubators, each capable of holding approximately 700,000 samples in 1536-well format,
are used to store microtiter plates at a variety of temperatures and gas concentrations.
E, A multimode plate reader measures absorbance, luminescence, fluorescence, or
fluorescence resonance energy transfer (with time resolution) from microtiter plates.
F, A kinetic imaging plate reader allows measurement of second-messenger or ion
channel activity in live cells. Not shown is a compound management robot capable
of storing and retrieving desirable compounds from the Scripps Research screening
file.
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The Scripps Research Institute Molecular Screening Center
Established
in 2005, the Scripps Research Institute Molecular Screening Center is a national
resource for small-molecule screening and the development of chemical probes. It
is 1 of 10 members in the Molecular Libraries Screening Centers Network, a translational
research initiative sponsored by the National Institutes of Health (NIH) and part
of the NIH Roadmap Initiative. The mission of the Scripps center is to screen the
NIH library of more than 150,000 individual compounds against peer-reviewed targets;
the goal is to discover proof-of-concept probes. The results are available to the
scientific community through the PubChem Web site of the National Center for Biotechnology
Information: http://pubchem.ncbi.nlm.nih.gov. Currently, the Lead Identification
Department serves as the HTS core within the Scripps screening center; our responsibilities
are to develop biological and biochemical assays, perform HTS campaigns, manage
the resulting data, steward the NIH screening library, and provide assay support
for the development of probes.
Other Screening Activities
Since the inauguration
of the ultra-HTS (uHTS) operation in November of 2005, we have also been actively
screening the Scripps collection of compounds against drug discovery targets not
only from the MLSCN but also from scientists at Scripps Research and from outside
partners. So far, members of the department have initiated and successfully completed
more than 30 uHTS-related collaborations (Table 1).
Discovery and Development of Class B Metallo-β-Lactamase Inhibitors
The diversity
of bacterial β-lactamases
continues to outpace the development of useful β-lactam–based
antibiotics. Although the development of class B β-lactamase
inhibitors has been an active area of research, an array of potent, class-specific
small-molecule inhibitors has yet to be fully characterized in the clinically relevant
VIM-2 metallo-β-lactamase
system. Additionally, VIM-2 inhibitors that are effective inhibitors of other class
B β-lactamases
will be of great interest. Such compounds will be useful as tools for characterizing
gram-negative pathogens or as adjuvants in antibiotic therapy.
One of our
goals is to develop HTS-ready assays suitable for rapid identification of compounds
that modulate the activity of Ambler molecular class B (Bush-Jakoby-Medeiros group
3) metallo-β-lactamases,
specifically the VIM-2 and IMP-1 enzymes. In preliminary research efforts, we have
developed HTS-ready fluorescence- and absorbance-based VIM-2 and IMP-1 inhibition
assays. In collaboration with K.B. Sharpless, Department of Chemistry, we have screened
a diverse click-chemistry library of compounds designed specifically to inhibit
metallo-β-lactamases.
Currently, we are developing several novel scaffolds that appear to be specific
inhibitors.
Publications
Lauer-Fields,
J.L., Spicer, T.P., Cudic, M., Burstein, G.D., Nagase, H., Hodder, P., Fields, G.B.
Screening of potential ADAMTS-4 inhibitors utilizing a collagen-model FRET substrate.
Anal. Biochem., in press.
T., Minond, D., Weiser, A., Dao, C., Habel, J., Spicer, T., Chase, P., Baillargeon,
P., Scampavia, L., Schürer, S., Chung, C., Mader, C., Southern, M., Tsinoremas,
N., LoGrasso, P., Hodder, P. Comparison
of miniaturized time-resolved fluorescence energy transfer and enzyme-coupled luciferase
high-throughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II).
J. Biomol. Screen., in press.
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