Intact Mass Screening for Covalent Compound Libraries: What and Why?

Recent publications highlight an “evolving toolbox” of techniques and technologies implemented in covalent inhibitor discovery and design. Though historically covalent compound discovery has been conducted via structure based drug design, direct covalent ligand screening of electrophilic compound libraries has been gaining popularity as a primary approach. Intact Mass Mass Spectrometry (Intact-MS) is a powerful tool for the identification of compounds which form an irreversible bond with a protein or nucleic acid target. A combination of high-throughput sample introduction platforms and automated data analysis programs allow for the screening of >10,000 covalent compounds in 2-3 days. The generation of “hit” lists has increased demands on secondary assays defining and rank ordering compound potency. Determination of the rate constant, Kinact/KI, is the potency metric of irreversible inhibitors akin to an IC50 value for reversible inhibitors. At Momentum, we offer primary and secondary Intact-MS screening to identify target modifying covalent compounds and characterize their kinetics.

Covalent Drugs: What and Why?

Covalent or irreversibly binding drugs are a powerful strategy to achieve prolonged target engagement or to bind proteins that lack easily accessible pockets for reversible binders — so called “undruggable” proteins. The oncology target KRAS eluded inhibition via traditional reversible compounds until 2021, when the covalent binder sotorasib was approved. Sotorasib occupies a previously unknown pocket on the KRAS G12C mutant, locking it in an inactive state and preventing activation of downstream growth and survival pathways

Covalent inhibitors have also been successfully employed in targeting the receptor tyrosine kinase EGFR, another popular oncology target. While reversible inhibitors (gefitinib and erlotinib) showed initial success in patients, disease eventually progressed, in part due to an acquired mutation that decreased the binding affinity. Second generation covalent inhibitors (afatinib) partially restored activity against the mutation but were non-specific to mutant EGFR T790M. A third generation of covalent inhibitors (osimertinib and rociletinib) was successfully developed to address the specificity issue, improving on-target potency and reducing off-target activity.

Ibrutinib (Imbruvica®) a first-in-class Bruton’s tyrosine kinase (Btk) inhibitor, is the biggest commercial success story so far of covalent inhibitors, reaching blockbuster status in its second year on the market. We used the ibrutinib-Btk model system to demonstrate our high-throughput kinetic characterization assay, determining potency Kinact/KI values for three approved and four Phase II-III candidates in a two-hour run. Check out our poster Validation of an Automated Process for the Determination of Covalent Modifier Potency: Kintact/Ki of the Btk/ Ibrutinib Model System which we presented at SLAS 2023 for more details.

Intact-MS: What and Why?

Renewed interest in targeted covalent modifying compounds, brought on in part by the commercial success of marketed drugs such as Imbruvica® and Tagrisso®, have fueled the advancement of covalent screening technologies. An assortment of MS-based based and non-MS-based systems have emerged for the determination and characterization of covalent interactions, including activity-based protein profiling (ABPP) via proteomic or gel based platforms and phenotypic screening techniques. Of the many screening approaches available, one of the highest throughput strategies is Intact-MS. Intact mass MS utilizes software tools to “deconvolute” m/z envelopes into a single “intact” mass of a large biomolecule, providing semi-quantitative information related to specific protein constructs and their post reaction “adducted” products.

Intact-MS is advantageous because of the high-throughput, automated workflow with quick assay set-up and robust detection. After incubating test compounds with the target, samples are analyzed using a RapidFire coupled to an Agilent Quadrupole Time-of-Flight MS (qToF) MS. Interrogation of the raw mass spectrum results in intact protein masses, revealing active covalent “hits”. By varying compound concentration and incubation time, the same process can be used to generate Kinact/KI data, ranking “hits” by their kinetic potency. Downstream of primary hit identification and secondary kinetic characterization, Intact-MS data can be paired with crystal structures to reveal mechanism of action and guide downstream optimization of therapeutic leads.

At Momentum, we have automated the end-to-end workflow from sample preparation to data analysis, enabling us to perform primary covalent modification screens of 12 x 384 well plates daily. Momentum can screen a client’s chemical library (if available) or provide access to the WuXi covalent compound collection (~12,000 compounds). The same process can be used to determine Kinact/KI values at a rate of ~15 minutes per compound, allowing for the rank ordering of “hits” by compound potency. When trying to unlock an undruggable target by accurately and efficiently screening covalent chemical libraries, Intact-MS is the go-to HTS method to accelerate your therapeutic program. Speak to one of our PhDs today to find your lead candidate using our Intact MS Screening for Covalent Compound Libraries.