Experimental screening of dihydrofolate reductase yields a "test set" of 50,000 small molecules for a computational data-mining and docking competition.

High-throughput screening (HTS) generates an abundance of data that are a valuable resource to be mined. Dockers and data miners can use "real-world" HTS data to test and further develop their tools. A screen of 50,000 diverse small molecules was carried out against Escherichia coli dihydrofolate reductase (DHFR) and compared with a previous screen of 50,000 compounds against the same target. Identical assays and conditions were maintained for both studies. Prior to the completion of the second screen, the original screening data were publicly released for use as a "training set", and computational chemists and data analysts were challenged to predict the activity of compounds in this second "test set". Upon completion, the primary screen of the test set generated no potent inhibitors of DHFR activity.

Multicopy suppressors for novel antibacterial compounds reveal targets and drug efflux susceptibility.

Gene dosage has frequently been exploited to select for genetic interactions between a particular mutant and clones from a random genomic library at high copy. We report here the first use of multicopy suppression as a forward genetic method to determine cellular targets and potential resistance mechanisms for novel antibacterial compounds identified through high-throughput screening. A screen of 8640 small molecules for growth inhibition of a hyperpermeable strain of Escherichia coli led to the identification of 49 leads for suppressor selection from clones harboring an E. coli genomic library. The majority of suppressors were found to encode the multidrug efflux pump AcrB, indicating that those compounds were substrates for efflux. Two leads, which produced clones containing the gene folA, encoding dihydrofolate reductase (DHFR), proved to target DHFR in vivo and were competitive inhibitors in vitro.

High-throughput screening identifies inhibitors of the SARS coronavirus main proteinase.

The causative agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus, SARS-CoV. The main proteinase of SARS-CoV, 3CLpro, is an attractive target for therapeutics against SARS owing to its fundamental role in viral replication. We sought to identify novel inhibitors of 3CLpro to advance the development of appropriate therapies in the treatment of SARS. 3CLpro was cloned, expressed, and purified from the Tor2 isolate. A quenched fluorescence resonance energy transfer assay was developed for 3CLpro to screen the proteinase against 50,000 drug-like small molecules on a fully automated system. The primary screen identified 572 hits; through a series of virtual and experimental filters, this number was reduced to five novel small molecules that show potent inhibitory activity (IC50 = 0.5-7 microM) toward SARS-CoV 3CLpro.

High throughput screening identifies novel inhibitors of Escherichia coli dihydrofolate reductase that are competitive with dihydrofolate.

This communication describes the high-throughput screen of a diverse library of 50,000 small molecules against Escherichia coli dihydrofolate reductase to detect inhibitors. Sixty-two compounds were identified as having significant inhibitory activity against the enzyme. Secondary screening of these revealed twelve molecules that were competitive with dihydrofolate, nine of which have not been previously characterized as inhibitors of dihydrofolate reductase. These novel molecules ranged in potency (K(i)) from 26 nM to 11 microM and may represent fresh starting points for new small molecule therapeutics directed against dihydrofolate reductase.