Histidine-Covalent Stapled Alpha-Helical Peptides Targeting hMcl-1

Several novel and effective cysteine targeting (Cys) covalent drugs are in clinical use. However, the target area containing a druggable Cys residue is limited. Therefore, methods for creating covalent drugs that target different residues are being looked for; examples of such ligands include those that target the residues lysine (Lys) and tyrosine (Tyr). Though the histidine (His) side chain is more frequently found in protein binding locations and has higher desirable nucleophilicity, surprisingly limited research has been done to specifically target this residue, and there are not many examples of His-targeting ligands that have been rationally designed. In the current work, we created novel stapled peptides that are intended to target hMcl-1 His 252 covalently. We describe the in vitro (biochemical, NMR, and X-ray) and cellular design and characterization of such agents. Our findings further suggest that the use of electrophiles to specifically target His residues is warranted.


Table S2
page S6 Mass-spectrometry data of stapled peptides.

Figure S1
page S7 Schematic representation of the synthesis of stapled compound 1.

Figure S2
page S8 Schematic representation of the synthesis of stapled compound 9.

Figure S3
page S9 CD curves of linear peptide 138E12.

S4
Table S1.Summary of structural parameters for the crystal structure of hMcl-1(172-323) in complex with peptide 155H1.Data collection and refinement statistics.

Figure
Figure S26 page S29Superposition of the X-ray structures of hMcl-1(172-323) covalently bound to 138E12, represented in pink (PDB ID 6VBX) and to compound 155H1, represented in purple (PDB ID 8VJP).A close-up view of the covalent sulfonamide bond resulting from the reaction of the for the highest-resolution shell are shown in parentheses.

Figure S1 .
Figure S1.Schematic representation of the synthesis of stapled Compound 1 and mechanism of

Figure S2 .
Figure S2.Schematic representation of the synthesis of stapled Compound 9 and mechanism of

Figure S3 .
Figure S3.CD curves of linear peptide 138E12 in pure water (blue) and in 15 mM Phosphate

Figure S5 .
Figure S5.CD curve of stapled Compound 2 in MQ water.

Figure S6 .
Figure S6.CD curve of stapled peptide Compound 3 in MQ water.

Figure S7 .
Figure S7.CD curve of stapled peptide Compound 4 in MQ water.

Figure S8 .
Figure S8.CD curve of stapled peptide Compound 5 in MQ water.

Figure S9 .
Figure S9.CD curve of stapled peptide Compound 6 in MQ water.

Figure S10 .
Figure S10.CD curve of stapled peptide Compound 7 in MQ water.

Figure S11 .
Figure S11.CD curve of stapled peptide Compound 8 in MQ water.

Figure S12 .
Figure S12.CD curve of stapled peptide Compound 9 in MQ water.

Figure S13 .
Figure S13.CD curve of stapled peptide Compound 10 in MQ water.

Figure S15 .
Figure S15.Dose−response DELFIA displacement assay curves comparing the ability of non-

Figure S19 .
Figure S19.Detection of ligand binding via time-dependent 1 H and 13 C  -Met NMR measurement.

Figure S21 .
Figure S21.His side chain long range so-fast HMQC [ 15 N, 1 H] correlations spectra of 50M wt

Figure S22 .
Figure S22.His side chain long range so-fast HMQC [ 15 N, 1 H] correlations spectra of 50M wt

Figure S23 .
Figure S23.His side chain long range so-fast HMQC [ 15 N, 1 H] correlations spectra of 50M wt

Figure S24 .
Figure S24.His side chain long range so-fast HMQC [ 15 N, 1 H] correlations spectra of 50M wt

Table S2 .
Mass-spectrometry data of stapled peptides.All the compounds were analyzed using an Agilent 6545 QTOF LC/MS instrument.