Affinity Selection Mass Spectrometry (ASMS) is a high throughput screening (HTS) technique for identifying small molecule test compounds based on their binding affinity to a target. Unlike traditional biochemical assays that test a compound’s functional activity, ASMS is a biophysical assay which directly measures the binding affinity between a test compound and the target. Confirmed binders can then be further interrogated to determine their binding affinity by running test compound titration curves. Insights into the binding site of the target can also be gained through competitive binding experiments, such as orthosteric (primary, functional) or allosteric (secondary, regulatory) binding. At Momentum Biotechnologies, we offer a solution-based version of ASMS known as Affinity Ligand Identification System (ALIS).

Typically, ASMS is used for high throughput screening of rule-of-five compliant small molecule chemical libraries. Recently we have demonstrated the ability to screen macrocyclic peptides and related macrocycles which are of great interest for certain classes of targets, such as enzymes and ion channels.

An ASMS high-throughput screen begins with incubation of a target with a pooled mixture of test compounds, usually ~250 compounds per well. Each test compound is at 1 uM while the target is at a higher concentration (typically 5 uM) to ensure sufficient binding capacity should there be multiple binders in a given pool. The pools are then subjected to size exclusion chromatography (SEC), fractionating the large molecule target(s) from the much smaller test compounds. The SEC fraction containing the target elutes early and is further interrogated by reversed-phase chromatography coupled to a high-resolution mass spectrometer. A test compound which elutes from the SEC column in the target fraction is a putative binder which can be confirmed and characterized in downstream experiments.

Targets that can be analyzed by ASMS include proteins and oligonucleotides (RNA and DNA), as well as protein-protein and protein-oligonucleotide complexes. The required features for targets are that they are both soluble and large enough to be fractionated from the test compounds (generally 10KDa or larger).

ASMS offers many distinct advantages as a HTS lead identification technique, such as:

• ASMS is binding site agnostic, identifying potential leads acting through a variety of mechanisms, including targeted degradation and disruption of protein-protein or protein-oligonucletide interactions. ASMS screens are especially useful on targets where there is uncertainty around the mechanism of action. ASMS does not require synthetic modification of compounds or targets, which can impact their activity and subsequent target binding in addition to synthesis being slow and costly.
• ASMS is compatible with a range of targets. ASMS has found hits for protein, RNA, DNA, protein-protein, and protein-nucleic acid targets without the need for tagging or surface immobilization, ensuring the results are physiologically relevant. ASMS can even be used without selecting a specific target to screen macromolecular complexes.
• ASMS uses minimal target, typically requiring only 50 picomoles of target per experiment. This traslates to about 1 mg of a 50 KDa protein for a screen of 100k test compounds.
• ASMS assay development is typically limited to buffer optimization and can therefore be set up in a fraction of the time of traditional biochemical HTS methods like FRET and enzymatic assays that require synthesis of radioisotopes, chromophores, or fluorophores. Often the storage buffer used for a specific target can be used as the assay buffer.
• ASMS is generally 2-3 orders of magnitude faster to run than other biophysical assays such as SPR, interferometry, DSF, Mesoscale, ITC, and NMR. Furthermore, because SPR immobilizes the target on the surface, it is not ideal for screening flexible RNA.
• ASMS allows compounds to be screened in pools of 250 saving clients time and money. At Momentum, 100,000 test compounds can be screened in a less than 48 hours. Compound identification and quantification is based on the isotope cluster corresponding to the molecular formula of each ligand, enabling ASMS to be run on pooled mixtures of non-encoded, unmodified libraries. This avoids time consuming chemical tagging that can alter the structure and behavior of modified products. For example, DNA encoded library (DEL) screening approaches require synthesis of DNA tagged libraries, which are limited in structural diversity by the constraints of DNA compatible chemistry and often lack drug like properties as defined by Lipinski’s Rule of Five. DEL is also incompatible with RNA targets due to unwanted crosslinking between the DNA tags and the nucleic acid target. While the initial DEL screen may be faster and cheaper than ASMS, after hit identification compounds need to be resynthesized without the DNA tag, negating the time and cost savings.
• ASMS is immune to false positives resulting from impurities or the breakdown of test compounds (compound identification is entirely based on molecular formula). ASMS is very commonly used as an orthogonal hit confirmation tool for biochemical and functional assays where biologically active impurities or breakdown products can create significant problems during data interpretation.
• ASMS can rank the relative affinities of compounds if the target is not provided in molar excess. ASMS detects compounds with affinity ranging from tens of micromolar to tighter. Beyond identifying candidates, ASMS can also help elucidate the binding site through competitive binding experiments.

Finally, ASMS covers broad chemical space — positive mode electrospray ionization is compatible with 95% of drug-like test compounds. This enables the screening of most test compound libraries in their entirety. Momentum can screen a client’s chemical library (if available) or provide our own compound libraries (as needed). Whether trying to unlock an undruggable target, or accurately and efficiently screen chemical libraries, ASMS is the go-to HTS method to accelerate a therapeutic program from target discovery to lead optimization. Speak to one of our PhDs today to find your lead candidate using ASMS.