High-Throughput Kinetic Characterization of Irreversible Covalent Inhibitors of KRAS^G12C by Intact Protein MS and Targeted MRM

Abstract

With recent advances and success in several drugs designed to treat acute and chronic diseases, targeted covalent inhibitors show a resurgence in drug discovery. As covalent inhibition is time-dependent, the preferred quantitative potency metric of irreversible inhibitors is the second-order rate constant k inact/K i, rather than IC50. Here, we present the development of a mass spectrometry-based platform for rapid kinetic analysis of irreversible covalent inhibitors. Using a simple liquid handling robot for automated sample preparation and a solid-phase extraction-based RapidFire–MS system for rapid MS analysis, kinetic characterization of covalent inhibitors was performed in high throughput both by intact protein analysis and targeted multiple reaction monitoring (MRM). In addition, a bimolecular reaction model was applied to extract k inact/K i in data fitting, providing tremendous flexibility in the experimental design to characterize covalent inhibitors with various properties. Using KRASG12C inhibitors as a test case, the platform was demonstrated to be effective for studying covalent inhibitors with a wide range of k inact/K i values from single digit to 3 × 105 M–1 s–1.

Fragment Optimization of Reversible Binding to the Switch II Pocket on KRAS Leads to a Potent, In Vivo Active KRASG12C Inhibitor

“… a k inact / K I , k inact , K I , IC 50 , and EC 50 values are shown as geometric mean ± SD ( n ≥ 3) and were determined, as described in the Experimental Section. b 4895 and 9900 M –1 s –1 previously reported. , c 6 nM previously reported …”

“…The mixture was concentrated, two drops of triethylamine were added, and the product was isolated via preparative RP HPLC (Agilent, Waters XBridge C18 50 × 150 mm 5 μm, 10−70% MeCN in basic water), yielding 75 mg (67%) of 46 as an off-white solid. (23). Acryloyl chloride (1 M in acetone, 419 μL, 0.419 mmol) was added to a mixture of potassium carbonate (57.8 mg, 0.419 mmol), acetone (3.7 mL), and water (0.74 mL).…”

Technologies for Direct Detection of Covalent Protein–Drug Adducts

“…Time-dependent covalent occupancy was calculated from the total ion count (TIC) of the deconvoluted mass of covalent HOIP-fragment adduct relative to the unbound HOIP. LC-MS approaches were also employed to assess the potency of covalent KRAS G12C inhibitors [37,96,97]. Differences in ionization efficiency of the unbound protein and adduct are only a minor concern as the protein size is significantly larger than the covalent ligand.…”

“…Quantification of covalent occupancy. Covalent target engagement is often quantified indirectly from the depletion of the unmodified proteolytic peptide in the treated sample relative to the untreated sample [96,97,101] as unbiased quantification of the (modified) proteolytic peptide can be challenging: ionization efficiency differences can occur following modification with a covalent ligand. The (LC-)MS/MS methods to overcome this bias will be discussed in the next section [100].…”

Covalent Library Screening by Targeted Mass Spectrometry for Rapid Binding Site Identification

“…With the goal of streamlining the typical covalent screening and hit follow-up assay cascade, we developed CoMPAS ( Co- valent M apping by P eptide A ttenuation S creening), a high-throughput assay that provides binding site information in the primary screen. The CoMPAS method relies on the quantitation of peptides from digested protein using targeted MS detection techniques, such as multiple reaction monitoring (MRM, also known as SRM) − or parallel reaction monitoring (PRM, also known as targeted MS 2 or MRM-HR). ,, The CoMPAS method circumvents the pitfalls associated with conjugated peptide detection by employing an alternate strategy: accurate quantitation of unmodified peptides that remain after the covalent reaction. Any attenuation of unmodified peptide abundance is inferred to be a result of covalent modification of that peptide.…”