Compound CSH18 binds to p38γ. (A) CSH18 is predicted to bind to the LBS of p38γ, with a Glide XP docking score of −10.9 kcal/mol. (B) Chemical structure of compound CSH18. (C) Mass spectrometry data: 1-dimensional spectra of the ¹H NMR aromatic region of compound CSH18 at 25°C; (i) molar ratio between CSH18 and p38γ is 50:1 and (ii) free CSH18 with a concentration of 50 μM. (D) Substitution in CSH18: CSH18 has a Br group, which might act as an alkylator, potentially causing nonspecific binding. To reduce this risk, we replaced Br with CN in CSH18 to create CSH18CN. This substitution maintains a similar conformation in the lipid-binding domain of p38γ without functioning as an alkylator. (E) Top chemical shift residues of p38γ in complex with CSH18/CN by 2-dimensional NMR (¹H-¹⁵N). Many residues around the ATP binding sites exhibit chemical shifts, indicating that CSH18CN likely binds at the ATP site of p38γ. (F) 3-Dimensional (3D) structure of the ATP pocket of p38γ. The 3D structure of p38γ with its inhibitor SU005 is calculated based on the docking pose of 6 inhibitors to the p38γ and p38α (PMID: 27431267). The differences of ATP pockets’ residues in p38γ and p38α listed at the top corner of the figure. SU005 demonstrates the highest specificity for p38γ in the ATP pocket among the compounds tested (SU001-SU006). This inhibitor preferentially binds to the p38γ isoform because of its unique structural interactions with residues in the ATP binding pocket, specifically M109, F111, T114, and K118. CSP, chemical shift perturbation; β-OG, n-octyl-β-D-glucoside.