Evidence of specificity of 18CN to p38γ. (A) Six residues were identified using the ligand virtual screening strategy and the druggable site prediction method based on the hydrophobic interaction ranking of β-octyl glucoside with p38γ within a distance of <5 Å. They are Phe249, 3.56 Å; Leu294, 3.75 Å; Leu198, 3.80 Å; Pro246, 4.12 Å; and Val293, 4.91 Å. The 2 p38γ mutants were designed with the following amino acid substitutions: pLVXTP-3FLAG-p38γ mut_3M (3M-p38γ): contains 3-point mutations, Pro246Gly, Val 293Gly, and Leu 294Gly; pLVXTP-3FLAG-p38γ mut_3M-3m (3M-3m-p38γ): contains 6-point mutations: Pro246Gly, Val 293Gly, Leu 294Gly, Leu198Ala, Pro245Ala, and Phe249Ala. The mutant constructs were validated by Sanger sequencing (the supplemental Methods). (B) The WB analysis of 2 mutants targeting the lipid-binding domain of p38γ, which were designed based on the 6 amino acids depicted in panel A. The first mutant, 3M-p38γ, carries 3-point mutations (P246G-V293G-L294G) and functions as a dominant-negative p38γ in Hut 78 cells, referred to as Hut-3M. The second mutant, 3M-3m-p38γ, includes an additional 3 mutations (P246G-V293G-L294G) on top of the Hut-3M construct, creating a 6-site mutant expressed in Hut 78 cells. As a control, Hut-WT cells were derived from an empty lentiviral vector (pLVXTP) expressing WT p38γ – 3× Flag tags in Hut 78 cells. Flag indicates plasmid p38γ expression. Red brackets indicate reduced protein level upon treatment. (C) The 3 most druggable sites on p38γ were identified using the “all docking pose” method.1 Site 1: the LBS, in which CSH18/CN exhibits the highest binding affinity (MaxGScore of −10.9 kcal/mol). Site 2: the ATP-binding site, in which CSH18/CN also binds with a MaxGScore of −10.1 kcal/mol), as the second-preferentially site. Site 3: the N-terminal region above the ATP-binding site, with a MaxGScore of −9.2 kcal/mol. (D) Expression of LBS mutants of p38γ (3M-p38γ and 3M-3m-p38γ) affects cell proliferation. The p38γ-3M mutant is lethal upon transduction. However, a low-expression clone of 3M-p38γ, exhibiting reduced levels of both FLAG-tagged and endogenous p38γ, was successfully isolated. Cell proliferation was assessed using cell counting and trypan blue exclusion assays with an automated cell counter. The data indicate that disruption of the LBS on p38γ redirects CSH18CN to preferentially bind the ATP site, resulting in cell death. WT, wild-type.
Figure 7.

Evidence of specificity of 18CN to p38γ. (A) Six residues were identified using the ligand virtual screening strategy and the druggable site prediction method based on the hydrophobic interaction ranking of β-octyl glucoside with p38γ within a distance of <5 Å. They are Phe249, 3.56 Å; Leu294, 3.75 Å; Leu198, 3.80 Å; Pro246, 4.12 Å; and Val293, 4.91 Å. The 2 p38γ mutants were designed with the following amino acid substitutions: pLVXTP-3FLAG-p38γ mut_3M (3M-p38γ): contains 3-point mutations, Pro246Gly, Val 293Gly, and Leu 294Gly; pLVXTP-3FLAG-p38γ mut_3M-3m (3M-3m-p38γ): contains 6-point mutations: Pro246Gly, Val 293Gly, Leu 294Gly, Leu198Ala, Pro245Ala, and Phe249Ala. The mutant constructs were validated by Sanger sequencing (the supplemental Methods). (B) The WB analysis of 2 mutants targeting the lipid-binding domain of p38γ, which were designed based on the 6 amino acids depicted in panel A. The first mutant, 3M-p38γ, carries 3-point mutations (P246G-V293G-L294G) and functions as a dominant-negative p38γ in Hut 78 cells, referred to as Hut-3M. The second mutant, 3M-3m-p38γ, includes an additional 3 mutations (P246G-V293G-L294G) on top of the Hut-3M construct, creating a 6-site mutant expressed in Hut 78 cells. As a control, Hut-WT cells were derived from an empty lentiviral vector (pLVXTP) expressing WT p38γ – 3× Flag tags in Hut 78 cells. Flag indicates plasmid p38γ expression. Red brackets indicate reduced protein level upon treatment. (C) The 3 most druggable sites on p38γ were identified using the “all docking pose” method.1 Site 1: the LBS, in which CSH18/CN exhibits the highest binding affinity (MaxGScore of −10.9 kcal/mol). Site 2: the ATP-binding site, in which CSH18/CN also binds with a MaxGScore of −10.1 kcal/mol), as the second-preferentially site. Site 3: the N-terminal region above the ATP-binding site, with a MaxGScore of −9.2 kcal/mol. (D) Expression of LBS mutants of p38γ (3M-p38γ and 3M-3m-p38γ) affects cell proliferation. The p38γ-3M mutant is lethal upon transduction. However, a low-expression clone of 3M-p38γ, exhibiting reduced levels of both FLAG-tagged and endogenous p38γ, was successfully isolated. Cell proliferation was assessed using cell counting and trypan blue exclusion assays with an automated cell counter. The data indicate that disruption of the LBS on p38γ redirects CSH18CN to preferentially bind the ATP site, resulting in cell death. WT, wild-type.

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