ALARM NMR:  A Rapid and Robust Experimental Method To Detect Reactive False Positives in Biochemical Screens

Abstract

High-throughput screening (HTS) of large compound collections typically results in numerous small molecule hits that must be carefully evaluated to identify valid drug leads. Although several filtering mechanisms and other tools exist that can assist the chemist in this process, it is often the case that costly synthetic resources are expended in pursuing false positives. We report here a rapid and reliable NMR-based method for identifying reactive false positives including those that oxidize or alkylate a protein target. Importantly, the reactive species need not be the parent compound, as both reactive impurities and breakdown products can be detected. The assay is called ALARM NMR (a La assay to detect reactive molecules by nuclear magnetic resonance) and is based on monitoring DTT-dependent (13)C chemical shift changes of the human La antigen in the presence of a test compound or mixture. Extensive validation has been performed to demonstrate the reliability and utility of using ALARM NMR to assess thiol reactivity. This included comparing ALARM NMR to a glutathione-based fluorescence assay, as well as testing a collection of more than 3500 compounds containing HTS hits from 23 drug targets. The data show that current in silico filtering tools fail to identify more than half of the compounds that can act via reactive mechanisms. Significantly, we show how ALARM NMR data has been critical in identifying reactive compounds that would otherwise have been prioritized for lead optimization. In addition, a new filtering tool has been developed on the basis of the ALARM NMR data that can augment current in silico programs for identifying nuisance compounds and improving the process of hit triage.

Iniparib Nonselectively Modifies Cysteine-Containing Proteins in Tumor Cells and Is Not a Bona Fide PARP Inhibitor

“…The DNA sequence encoding amino acids 223 to 324 of Human La antigen was cloned into pAG5 vector with a Cterminal his-tag and expressed in BL21(DE3)/plays (25). The protein was purified with a Ni-NTA column.…”

“…The structure of iniparib-met indicates that this molecule is highly reactive and likely to interact nonspecifically with many proteins containing cysteine residues. To test this, we incubated iniparib-met with several proteins containing cysteine residues, including La antigen (25), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), PARP-1 zinc finger-1, or ubiquitin, a protein that does not contain cysteine. We used DMSO as a negative control and tested the ability of cmpd-B and iniparib-met to react with these proteins.…”

A Simple Assay for Detection of Small-Molecule Redox Activity

“…In an ALARM NMR experiment, a test compound is mixed with a 224-amino acid portion of the human La protein, and resulting shift changes and line broadening in the 13 C NMR spectrum of La are observed upon modification of specific residues. 3 In a typical jump dilution experiment, an inhibitor is preincubated at > 10× IC 50 with the protein of interest and then rapidly diluted > 100-fold into a reaction containing substrate. Reversible compounds upon dilution below IC 50 display a return of enzymatic activity, whereas reactive compounds do not.…”

Shaping a Screening File for Maximal Lead Discovery Efficiency and Effectiveness: Elimination of Molecular Redundancy

“…Applying both filters showed a surprising number of compounds picked up by only a single set of the filters. The merging of the Pfizer and Wyeth screening file gave us the opportunity to revisit the existing filter sets, to combine the experiences from both companies, and incorporate also new findings of structural filters from the literature, [40][41][42][43][44][45][46][47][48] in addition to the creation of a small number of new proprietary filters to remove for example, compounds with very low feature content (no heteroatoms and low structural complexity) and compounds with multiple unattractive features, such as several nitro groups. 48 This knowledge integration resulted in the generation of a combined set of around 540 molecular filters, 55 which were applied to eliminate compounds with structural flaws ahead of the redundancy reduction.…”

“…In parallel, surveying the recent literature describing structural filters allowed us to include additional substructure filters. [40][41][42][43][44][45][46][47][48] Overall, around 140 filters were newly created and combined with the existing filter set, resulting in 540 unique substructure queries.…”