Cytosolic delivery of peptidic STAT3 SH2 domain inhibitors

Parallel Screening Using the Chloroalkane Penetration Assay Reveals Structure-Penetration Relationships

“…One possible reason for the apparent lesser cytosolic penetration of heterochiral peptides is increased susceptibility to degradation for homochiral l -peptides, which could lead to a false positive CAPA signal. While we cannot rule this out completely, previous work has shown little evidence of degradation effects on CAPA data, as measured with diverse linear and stapled peptides and canonical CPPs with similar lengths and overall sequences. ,,,, The poorer cytosolic penetration by heterochiral peptides is consistent with prior work showing that display of arginine side chains in a structured array, such as on one face of an α-helix, contributes to efficient cytosolic penetration and/or endosomal escape. ,, While these peptides are too short to maintain secondary structures at RT, optimal binding to membrane components could be promoted by a structured conformation such as an α-helix. , If this is the case, the heterochiral library would be generally expected to have poorer helical propensity and thus poorer membrane binding and/or endosomal escape. Still, within Library 2, a subset of peptides appeared more penetrant than others with the same number of d -arginines, such as het43 (Figure B).…”

“…The chloroalkane penetration assay (CAPA) has been used by our group and others to test the cytosolic penetration of a large variety of peptides, peptidomimetics, and small molecules. − CAPA uses a simple pulse-chase format with HeLa cells that express HaloTag protein in the cytosol. , When a chloroalkane-tagged molecule is applied in the pulse step, it can penetrate into the cytosol and covalently bind HaloTag. After wash steps, the cells are chased with a cell-permeable, chloroalkane-tagged dye.…”

Stapled β-Hairpins Featuring 4-Mercaptoproline

“…One of the potential advantages of incorporating non-proteinogenic amino acids and covalent staples is resistance to proteolytic degradation. To test whether the designed β-hairpins were stable to degradation, we adapted a cell lysate stability assay recently applied for development of olefin-stapled α-helices. − Because HeLa cell lysate contains all cellular proteases including lysosomal proteases, this represents a relatively rigorous test for metabolic stability of peptide therapeutics. Control peptides HP7 and ΔHP7 were degraded rapidly, with roughly 0% and 19%, respectively, remaining after only 2 h (Figure a).…”

Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting Its Protein–Protein Interactions

“…With regard to the second issue, several studies have demonstrated that efficient intracellular delivery of phosphopeptide mimics is possible, for instance, by conjugation to cell-penetrating sequences. 40 , 82 − 85 …”

“…Here we successfully overcame the first hurdle, thanks to the introduction of non-natural amino acids. With regard to the second issue, several studies have demonstrated that efficient intracellular delivery of phosphopeptide mimics is possible, for instance, by conjugation to cell-penetrating sequences. ,− …”