Hydrocarbon-stapled peptides are a class of bioactive alpha-helical ligands developed to dissect and target protein interactions. While there is consensus that stapled peptides can be effective chemical tools for investigating protein regulation, their broader utility for therapeutic modulation of intracellular interactions remains an active area of study. In particular, the design principles for generating cell-permeable stapled peptides are empiric, yet consistent intracellular access is essential to in vivo application. Here, we used an unbiased statistical approach to determine which biophysical parameters dictate the uptake of stapled peptide libraries. We found that staple placement at the amphipathic boundary combined with optimal hydrophobic and helical content are the key drivers of cellular uptake, whereas excess hydrophobicity and positive charge at isolated amino acid positions can trigger membrane lysis at elevated peptide dosing. Our results provide a design roadmap for maximizing the potential to generate cell-permeable stapled peptides with on-mechanism cellular activity.
Hydrocarbon stapling has been applied to restore and stabilize the α-helical structure of bioactive peptides for biochemical, structural, cellular, and in vivo studies. The peptide sequence, in addition to the composition and location of the installed staple, can dramatically influence the properties of stapled peptides. As a result, constructs that appear similar can have distinct functions and utilities. Here, we perform a side-by-side comparison of stapled peptides modeled after the pro-apoptotic BIM BH3 helix to highlight these principles. We confirm that replacing a salt-bridge with an i, i + 4 hydrocarbon staple does not impair target binding affinity and instead can yield a biologically and pharmacologically enhanced α-helical peptide ligand. Importantly, we demonstrate by electron microscopy that the pro-apoptotic activity of a stapled BIM BH3 helix correlates with its capacity to achieve cellular uptake without membrane disruption and accumulate at the organellar site of mechanistic activity.
A previously healthy 19-year-old man presented with a 2-month history of intermittent suprapubic pressure-like abdominal pain, dysuria, and 1-week history of pink discoloration of urine. His vital signs, WBC count, lactate level, and creatinine level were within normal limits. Physical examination revealed a soft abdomen with suprapubic tenderness. Urinalysis result was significant for cloudy, pink urine with >300 mg/dL proteinuria, >100 WBCs per high-power field, large red blood cells with >20 RBCs per high-power field, >1 bacteriuria, and positive for the presence of leukocyte esterase.For the diagnosis and teaching points, see page e30. To view the entire collection of Images in Emergency Medicine, visit www.annemergmed.com
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