(1374-C) Screening for Success – Enhancing Hit Confidence from Affinity Selection Mass Spectrometry (ASMS) Screens
Tuesday, February 6, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: Affinity selection mass spectrometry (ASMS) screening promises a rapid, low-cost route to hit equity for challenging drug targets, including traditionally “undruggable” proteins, RNA and membrane proteins. Using automated variants of ASMS, such as ALIS (automated ligand identification system), large libraries >1M can be interrogated in a few days, with low reagent consumption (mg's), minimal assay development and little user intervention. ALIS is therefore a very attractive “fast first” approach to drug discovery, to assess ligandability, identify tool compounds or as a biophysical route to start points for development of PROTACs.
In ALIS, high throughput is achieved by screening targets against large compound pools, containing hundreds to thousands of compounds, and deploying high resolution LC-MS to identify putative hits. The ability to detect true binders depends strongly on the target and ligand concentrations, interaction affinity, depletion that occurs during the ALIS process and, importantly, the ionizability of the library molecule. Thus, binders with the same affinity and dissociation constants could vary in MS intensity by several orders of magnitude.
To sample the desired biophysical space across a suitable range of chemical diversity (and thus ionizability) highly sensitive mass spectrometers are routinely used. However, this makes identifying selective hits from background or promiscuous binders even more challenging. Initial hit rates can be >5-10% from a single primary screen without suitable hit triage. Further rounds of singleton hit confirmation or validation using orthogonal techniques may be required to reduce hit rate and remove false positives. This negates the primary advantages of ASMS for high throughput screening (HTS) – speed, simplicity and low reagent usage.
Here, we show that by combining compound pre-screening for ionizability, with novel primary screen design, multi-parameter hit calling, frequent hitter removal and chemical triage we can provide more confident hit finding at the primary screen stage. We show with model systems that false positives are removed along with promiscuous binders without affecting the true positive hit rate. This results in higher confidence hits with greater probability of orthogonal validation. Finally, we demonstrate that our enhanced approach workflow is capable of identifying even weak binders to challenging targets such as RNA.