HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications

Abstract

A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have Ki values of 0.2 μM, 50 pM and 700 pM. These findings demonstrate that the approach described here compares favorably to traditional MS based screening methods.

Rapid LC-MS Based High-Throughput Screening Method, Affording No False Positives or False Negatives, Identifies a New Inhibitor for Carbonic Anhydrase

“…While the data in Fig. 5 do not say which compound in Library B was the strong binder, we have previously demonstrated that this inhibitor (ethoxzolamide) can easily be identified as the strong binder in a batch of ~400 compounds, using the recently published HAMS screening method, where the highest-affinity ligand (of the 400 in the well) is identified in a complementary screen 26 .

Figure 5 Screening assay for CA binders with methoxzolamide as the reporter molecule.

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“…For many druggable targets, binding compounds are known, and we provide three relevant examples herein. For a protein with no known binding compounds, a complementary assay could be used to identify a weak-binding reporter molecule 26 .

Figure 1 Experimental workflow.

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“…The binder can be identified in a variety of ways. For example, we recommend using a complementary assay that we recently described, coined “HAMS” (high-affinity mass spectrometry screening) 26 . In that assay, the tightest binder in sets of 400 compounds is identified using a screening procedure that is similar to the workflow described here.…”

“…Immobilizations of carbonic anhydrase (CA) and pepsin were carried out by adjusting a previously published procedure 26 . Each protein was maintained at its optimal pH.…”

“…Recently, we reported a novel MS-based HTS method, High-Affinity Mass Spectrometry screening (HAMS), which uniquely evades the detection of false positive hits 26 . However, the method requires acquisition of two LC-MS datasets per set of 350 compounds, and this requirement limits throughput.…”