How to avoid metal artifacts

Back in 2017 we observed with characteristic subtlety that “heavy metals suck.” That post described a hit-finding campaign which foundered when the apparent activity of the fragments turned out to be due to contaminating zinc. A new paper in J. Med. Chem. by Thomas Gerstberger, Peter Ettmayer, and colleagues at Boehringer Ingelheim (BI) describes a similar story, along with suggestions of how to avoid being misled.

 

BI had a collaboration with FORMA Therapeutics that entailed screening roughly 1.7 million compounds against ten targets using biochemical and cell-based assays. The effort resulted in chemical probes against BCL6 and SOS1 and a clinical compound against the latter. Another target was the activated (GTP-loaded) form of KRAS^G12D. Of the 6917 hits from the primary AlphaScreen assay, 1535 gave dose-response curves and passed various counter screens. Of these, 87 representative compounds were tested in STD NMR and thermal shift assays. Only seven confirmed by STD NMR, but these did not confirm by SPR or crystallography.

 

In parallel, the researchers were successfully using FBLD to develop inhibitors of KRAS^G12D, which we wrote about here. Some of the fragment hits were structurally similar to those from the HTS screen, and further searching of the FORMA library led to fairly potent (high nanomolar or low micromolar) hits in the AlphaScreen assay. Two of these even yielded crystal structures, though despite their chemical similarity to one another they bound to the protein in completely different orientations.

 

Unfortunately, follow-up work “revealed erratic structure-activity relationships,” and upon resynthesis the compounds were much less active. At this point the researchers became suspicious, and analyses of the original samples showed they contained >20,000 ppm of palladium contamination. Furthermore, PdCl₂ itself turned out to be a low micromolar inhibitor in the assay.

 

Metals are frequently used as catalysts or reagents in organic synthesis and can be difficult to completely remove during purification. Worse, their presence is often not detectable using standard purity assessments such as HPLC and NMR. Particularly in the case of fragments, which are expected to have low affinities, a small amount of metal contaminant could give a reasonable-looking but misleading signal in an assay.

 

To avoid this problem in the future the researchers developed a Metal Ion Interference Set, or MIIS, consisting of a dozen different metal ions and other salts, all soluble in DMSO so as to be compatible with typical screens. The MIIS is now routinely screened before initiating HTS campaigns, and the results of 74 assays are summarized in the paper. Pd²⁺, Au³⁺, and Ag¹⁺ are particularly nasty, often giving IC₅₀ values < 1 µM, but every metal gave IC₅₀ values < 10 µM in at least two assays. Biochemical assays such as AlphaScreen or TR-FRET were more susceptible to artifacts, with 20.9% showing IC₅₀ < 10 µM, while biophysics assays such as mass spectrometry were better behaved, with only 2.3% showing IC₅₀ < 10 µM. Cellular assays were also surprisingly robust, with 6.3% showing IC₅₀ < 10 µM.

 

This is a nice paper showing that even a massive screen may produce no useful chemical matter. Soberingly, the fact that some of the fragments gave reasonable-looking crystal structures even though the functional activity came from metal contaminants is a salutary reminder that just because you have a crystal structure of a bound ligand doesn’t mean you have a viable starting point.

 

Forewarned is forearmed, and the MIIS appears to be a valuable tool for assessing assay sensitivity to metal ions, which are all too often lurking invisibly in compound samples.