We next tested substitutions at the 4-position of the indole to enhance labeling efficiency through ionic interactions with the p53 D228 carbonyl ( Fig. Indeed, a Cα methyl substitution on an acrylamide is known to substantially lower the reactivity toward thiols due to the steric hindrance of the nucleophile ( 18). However, the thermal stability was enhanced with a ΔTm of +3.563☌ ± 0.002☌ for KG6 compared with +1.4☌ ± 0.1☌ for KG3. We found that the indole methacrylamide KG6 did not react as efficiently as KG3, with 15% ± 1% labeling compared with 79% ± 1%, respectively ( Fig. To avoid this clash, the carbazole scaffold was replaced with an indole ( Fig. Insertion of a Cα methyl acrylamide onto the carbazole produced a labile amide bond to the carbazole presumably due to a steric clash with the phenyl ring of the carbazole. Interestingly, it was originally predicted through computational docking that PhiKan083 would adopt the same conformation that we observed for KG3 ( 8). 1E) or pyrrole in other reversible compounds ( 17). The covalent KG3–C220 bond orients the acrylamide Cα and Cβ outside of the Y220C hydrophobic cavity between C220 and L145, resulting in the loss of the van der Waals (vdW) interactions with p53 V147, L145, F109, L257, and V157, which were observed with the ethyl group in PhiKan083 ( Fig. The 3-positions of the two carbazoles face opposing sides of the p53 Y220C cavity, where the aldehyde on KG3 forms an H-bond with T150, whereas the 3-position methylamine on PhiKan083 forms an H-bond with the carbonyl backbone of D228 ( Fig. The overall fold of the p53 Y220C-CL is comparable with p53 Y220C–PhiKan083, with a root-mean-square deviation (RMSD) of 0.383 Å however, there are several differences in the small molecule's binding mode. To determine the binding mode of the covalent carbazole, we solved a cocrystal structure of p53 Y220C-CL bound to KG3 at 2.4 Å resolution ( Fig. The abundance of mutated p53 in tumors highlights the therapeutic potential of a small molecule capable of reverting mutant p53 to its WT form. Genetically engineered mouse models have shown that restoration of p53 WT activity in p53-deficient cancers promotes tumor regression and a cure ( 4, 5). The primary transcriptional targets of p53 WT are p21 ( CDKN1A), which functions as a potent cell-cycle inhibitor, MDM2 ( MDM2), the E3 ligase for p53, and proapoptotic Bcl-2 family proteins ( BBC3, BAX, and NOXA). Cells carrying a TP53 mutation accumulate high levels of mutant p53 protein, which drives a dominant-negative effect on the wild-type (WT) copy and the p53 homologs p63 and p73 ( 3). A germline mutation in one TP53 allele results in Li-Fraumeni syndrome, a disorder that increases the risk of cancer occurrence by 70% to 100% over an individual's lifetime ( 2). Unlike RB1, CDKN2A, and PTEN, which are lost in tumors through homozygous deletion, TP53 is most frequently found with a somatic missense mutation in either a heterozygous setting or with a 17p deletion and loss of the second allele ( 1). The most commonly mutated gene in cancer is the transcription factor and tumor suppressor TP53 (p53).
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