Multiparameter Kinetic Analysis for Covalent Fragment Optimization by Using Quantitative Irreversible Tethering (qIT)

Abstract Chemical probes that covalently modify cysteine residues in a protein‐specific manner are valuable tools for biological investigations. Covalent fragments are increasingly implemented as probe starting points, but the complex relationship between fragment structure and binding kinetics makes covalent fragment optimization uniquely challenging. We describe a new technique in covalent probe discovery that enables data‐driven optimization of covalent fragment potency and selectivity. This platform extends beyond the existing repertoire of methods for identifying covalent fragment hits by facilitating rapid multiparameter kinetic analysis of covalent structure–activity relationships through the simultaneous determination of K i, k inact and intrinsic reactivity. By applying this approach to develop novel probes against electrophile‐sensitive kinases, we showcase the utility of the platform in hit identification and highlight how multiparameter kinetic analysis enabled a successful fragment‐merging strategy.


Supplementary Figures
: Crystal structure of Cdk2(ES) Figure S2: Hit identification and validation for acrylamide S1 Figure S3: Mass spectrometric analysis of tryptic digests of labelled Cdk2(ES) Figure S4: In vitro inhibition of Cdk2(ES) kinase activity Figure S5: Crystal structure of S1-Cdk2(ES) Figure S6: Crystal structure of 6-Cdk2(ES) Figure S7: Synthesis of merged acrylamide fragments 9, 10 and 11 Figure S8: Crystal structure of 9-Cdk2(ES) Figure S9: Crystal structure of 11-Cdk2(ES)  Supplementary Figure S4 | In vitro inhibition of Cdk2(ES) kinase activity. WT, ES or modified pCdk2 was incubated with cyclin A2 to form active holoenzymes. The holoenzymes were incubated with peptide substrate, ATP, NADH, PEP, LD and PK at 37 ˚C and the absorbance measured over time in clear 384-well plates. Kinase activity is determined from the gradient of the absorbance over time (between 1000-3000 s) and normalised relative to pCdk2(WT) = 100% and cyclin A2 only = 0% (n = 4; error bars denote s.e.m).

A. Chemical synthesis
All non-aqueous reactions were carried out under an inert atmosphere (argon) with flame-dried glassware, using standard techniques. Anhydrous solvents were obtained by filtration through drying columns (DMF, CH2Cl2, THF).
Flash column chromatography was performed using 230-400 mesh silica, with the indicated solvent system according to standard techniques. Analytical thin-layer chromatography (TLC) was performed on precoated aluminium-backed silica gel plates. Visualisation of the developed chromatogram was performed by UV absorbance (254 nm) and/or stained with aqueous potassium permanganate solution, aqueous ceric ammonium molybdate, or a ninhydrin solution in ethanol.
Nuclear magnetic resonance spectra were recorded on 400 MHz or 500 MHz spectrometers. Chemical shifts for 1 H NMR spectra are recorded in parts per million from tetramethylsilane with the residual protic solvent resonance as the internal standard (chloroform: δ 7.27 ppm, methanol: δ 3.31 ppm). Data are reported as follows: chemical shift (multiplicity [s = singlet, d = doublet, t = triplet, m = multiplet and br = broad], coupling constant (in Hz), integration). 13 C NMR spectra are recorded with complete proton decoupling. Chemical shifts are reported in parts per million from tetramethylsilane with the solvent resonance as the internal standard ( 13 CDCl3: δ 77.0 ppm, 13 CD3OD: δ 49.0 ppm). Assignments of 1 H and 13 C spectra were based upon the analysis of δ and J values, as well as DEPT, COSY, HMBC, HSQC and nOe experiments where appropriate.
Commercial reagents were used as supplied or purified by standard techniques where necessary. Compounds 1 and 2 were used as commercially supplied.