September 1, 2005 (Vol. 25, No. 15)

Building a Better Toolbox for Research by Increasing Availability of Molecular Probes

There have been significant advancements in drug discovery paradigms over the past decade with the emergence of high throughput screening (HTS) technologies at the center.

For a validated drug target, HTS is a powerful tool to explore chemical diversity space for developing a pharmacophore model and identifying a lead series of compounds for preclinical development. The sequencing of the human genome and development of microarray technologies that have enabled high throughput screening of gene and protein expression profiles have complemented these efforts and is allowing for an extensive exploration of new target diversity space.

Despite the advancements in screening technologies, the biological activity of many proteins is not fully understood, and functional assays are not available to translate genomic or proteomic data into useful biochemical or cell-based assays for HTS.

In what is becoming the post-genomics/proteomics era, considerably more attention is being placed on defining the biological function of proteins and understanding how they can be pharmacologically regulated to impact diseases. Among the estimated 25,000 proteins that can be produced by the human genome, as many as 10,000 have been projected to represent potential drug targets based on genetic analysis.

Currently, fewer than 500 different proteins are targeted by available FDA-approved drugs, with the majority representing enzyme or receptor targets (Science 287:1960).

The NIH recognizes that biological understanding drives drug discovery and and has recently established a network of HTS centers referred to as the Molecular Libraries Screening Center Network (MLSCN) to provide HTS resources and molecular probes to the scientific community.


The overall goal of the program is to create a national resource that will provide innovative HTS approaches for identifying small molecule probes to study the function of newly discovered proteins, cellular phenomena, physiological processes, and disease mechanisms.

Molecular probes are intended to provide investigators with tools that perturb protein or cellular function for dissection of molecular events that regulate complex biological processes. In special cases, the probes may have the necessary chemical and physical properties to become starting scaffolds, leading to new therapeutic development.

Additionally, some molecular probes may provide novel imaging tools, which can be useful for both basic and clinical research as markers of disease progression or drug efficacy. Molecular probes have been extensively used for such purposes in the past, although there has never been a unified, broad search for new probes prior to the establishment of the MLSCN.

A well-recognized example of using molecular probes are studies that have utilized various modulators of adenylate cyclase, phosphodiesterases, or protein kinase A as probes to investigate the involvement of cyclic AMP signaling in various cellular processes.

The MLSCN program is a cornerstone of the NIH Roadmap Initiative for medical research and is funded by all of the institutes of the NIH. The MLSCN program is being co-administered by the National Institute of Mental Health (NIMH) and the National Human Genome Research Institute (NHGRI).

This program is intended to establish HTS facilities in leading research institutes within the U.S. and to make screening data, chemical structures, and other physicochemical information relating to active molecular probes available to the scientific community via the publicly accessible database PubChem.

In addition to the newly established NIH Chemical Genomics Center, nine other centers have been awarded grants, and including the Southern Research Molecular Libraries Screening Center, Emory Chemistry-Biology Center in the MLSCN, MLSCN Center at Columbia University, New Mexico Molecular Libraries Screening Center, The Penn Center for Molecular Discovery, Pittsburgh Molecular Libraries Screening Center, San Diego Chemical Library Screening Center, Scripps Research Institute Molecular Screening Center, and the Vanderbilt Screening Center for PCRs, Ion Channels, and Transporters.

HTS Assays for the MLSCN

Each center within the network contains a multidisciplinary team of scientists and engineers with specialized expertise, equipment, methods and throughput capabilities. The centers will reproduce, optimize, and automate biochemical, cellular, and model organism-based HTS-ready assays submitted to the MLSCN by the biomedical research community.

Through a Program Announcement (PAR-05-147), the MLSCN is soliciting applications from investigators who have developed innovative assays, and who are interested in having their assay(s) screened in the MLSCN against a large number of compounds and expanding the utility of their assay(s) for producing useful in vitro and/or in vivo chemical probes. The MLSCN is especially interested in HTS-ready assays involving biological processes implicated in diseases where there are no or few known modulators.

As the NIH-NINDS High Throughput Screening Facility for Neurodegeneration, Southern Research has recently conducted a large-scale screening campaign of a 100,000 compound library to identify inducers of the promoter of the excitatory amino acid transporter, EAAT-2, which illustrates as example of a suitable assay for the MLSCN.

The loss of glutamate transporters has been implicated in the pathogenesis of certain neurodegenerative diseases such as Parkinson’s disease and amyotrophic lateral sclerosis. An HTS assay to screen compounds for ability to increase EAAT-2 expression is particularly appropriate for the MLSCN since there are few known inducers of EAAT-2.

In collaboration with Dr. Jeffrey Rothstein from the Johns Hopkins University Medical Center, we developed a HTS assay using COS-7 cells stably transfected with a construct containing the EAAT-2 promoter driving a luciferase reporter. HTS of the 100,000 compound NINDS library identified a series of compounds that were capable of increasing expression from the EAAT-2 promoter.

A representation of the results from the screening run is illustrated in Figure 3. As is likely to be the case for some MLSCN assays, the development of this assay proved challenging since initially there was no positive control compound available to automate the assay for HTS. To overcome this obstacle, we successfully identified a positive control by performing a pilot screen of a smaller library containing a collection of known bioactive compounds.

Following a detailed analysis of structure-activity relationships, “hits” from this assay are anticipated to provide valuable tools for further study of EAAT-2 function, assessing its utility as a drug target and providing insight to potential mechanisms of pharmacological intervention.

MLSCN Organizational Structure

The organizational structure of the MLSCN is illustrated in Figure 4. The overall program is managed by the NIH MLSCN Project Team made up of members representing most of the institutes at NIH. A Center Steering Committee, which is composed of NIH Program Directors and the Directors of each screening center, is responsible for network operations including oversight of assay assignments, compound distribution and development policies and procedures.

Each center is organized into teams of scientists and engineers with expertise in assay implementation, HTS implementation, chemistry and probe development, and informatics. Additional NIH-sponsored programs seek proposals that will promote HTS assay development, new chemical and natural product chemistry, new cheminformatic designs and instrumentation development to advance the screening capabilities and capacities of the MLSCN.

Compound libraries will be distributed to each center from an NIH central chemical repository that is managed by Discovery Partners International (San Francisco). The library will contain more than 500,000 chemically-diverse compounds including FDA-approved drugs, bioactive molecules, toxins, metabolites, and natural products.

The screening centers will analyze biological data by advanced cheminformatics methods to define structure-activity relationships. Certain probes may be chosen for chemical modification to optimize probe potency and selectivity. Each center will deposit screening data and chemical structures into the PubChem database that is managed by the National Library of Medicine at NIH.

PubChem will also contain comprehensive information regarding the activity and physicochemical properties of molecular probes that is intended to aid in vitro, as well as in vivo testing.

Impact of the MLSCN on Drug Discovery

In summary, the goal of the MLSCN is to build a better toolbox for medical researchers in both the public and private sectors by providing molecular probes that will perturb protein and cellular function for the study of complex biological processes relevant to physiology and disease.

The increased availability of molecular probes and resulting structure-activity relationships from the MLSCN is anticipated to provide tools and insight for the validation of novel drug targets and the development of pharmacophore models. Some molecular probes may also have suitable physicochemical and pharmacological properties to serve as an initial lead for preclinical efficacy testing.

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