Getting across the cell membrane: an overview for small molecules, peptides, and proteins

Abstract

The ability to efficiently access cytosolic proteins is desired in both biological research and medicine. However, targeting intracellular proteins is often challenging, because to reach the cytosol, exogenous molecules must first traverse the cell membrane. This review provides a broad overview of how certain molecules are thought to cross this barrier, and what kinds of approaches are being made to enhance the intracellular delivery of those that are impermeable. We first discuss rules that govern the passive permeability of small molecules across the lipid membrane, and mechanisms of membrane transport that have evolved in nature for certain metabolites, peptides, and proteins. Then, we introduce design strategies that have emerged in the development of small molecules and peptides with improved permeability. Finally, intracellular delivery systems that have been engineered for protein payloads are surveyed. Viewpoints from varying disciplines have been brought together to provide a cohesive overview of how the membrane barrier is being overcome.

Fig. 1

Possible routes of cytosolic entry. Molecules may passively diffuse across the cell membrane, or be shuttled in via natural or artificial delivery mechanisms. Membrane transporters allow the passage of various ions and metabolites. Protein toxins and viruses have evolved complex translocation mechanisms, hijacking the host’s ER transporters in some instances. Engineering approaches to improve a payload’s permeability may involve physically disrupting the membrane, chemically modifying the payload, or attaching the pay-load—covalently or non-covalently—to an intracellular delivery system that can disrupt cell membranes. In any case, the translocation event can occur across the plasma membrane, or across internal cellular membranes following endocytosis (termed “endosomal escape”). Images were adapted from Servier Medical Art

Fig. 2

(a) Cyclosporin A (CsA) in its closed conformation in nonpolar solvent [186]. The four intramolecular hydrogen bonds (dotted lines in blue) are thought to shield the polarity of the molecule. (b) The TAT peptide segment excerpted from the NMR structure of HIV-1 TAT protein (adapted from PDB 1TIV) [187]. The guanidinium nitrogens (blue) are thought to enhance the interaction between TAT and the cell membrane. (c) A slanted top- down view of the pre-pore formed by anthrax toxin protective antigens (PAs) (blue) in complex with lethal factors (LFs) (gray), which are translocated across the full pore. Shown in the figure are eight molecules of PA bound to four molecules of LF (PA ₈(LF_N)₄) (PDB 3KWV) [188]. (d) The neuraminidase inhibitor Zanamivir (top) and Zanamivir-L-Val (bottom) [189]. The conjugated valine (blue) has been proposed to render Zanamivir into a substrate for amino acid transporters