RNA therapeutics offer great potential to transform the biomedical landscape, encompassing the treatment of hereditary conditions and the development of better vaccines. However, the delivery of RNAs into the cell is hampered, among others, by poor endosomal escape. This major hurdle is often tackled using special lipids, polymers, or protein-based delivery vectors. In this review, we will focus on the most prominent peptide- and protein-based endosomal escape strategies with focus on RNA drugs. We discuss cell penetrating peptides, which are still incorporated into novel transfection systems today to promote endosomal escape. However, direct evidence for enhanced endosomal escape by the action of such peptides is missing and their transfection efficiency, even in permissive cell culture conditions, is rather low. Endosomal escape by the help of pore forming proteins or phospholipases, on the other hand, allowed to generate more efficient transfection systems. These are, however, often hampered by considerable toxicity and immunogenicity. We conclude that the perfect enhancer of endosomal escape has yet to be devised. To increase the chances of success, any new transfection system should be tested under relevant conditions and guided by assays that allow direct quantification of endosomal escape.
Cell-Penetrating Peptides , Proteins , Humans , Proteins/metabolism , Endosomes/metabolism , Cell-Penetrating Peptides/metabolism , Transfection , RNA, Small Interfering/genetics
α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.
Crystallography, X-Ray , Receptors, Adrenergic, alpha-1/chemistry , Binding Sites , HEK293 Cells , Humans , Ligands , Lipids/chemistry , Models, Molecular , Quinazolines/chemistry , Quinazolines/metabolism , Quinoxalines/chemistry , Quinoxalines/metabolism , Receptors, Adrenergic, alpha-2/chemistry
Neurotensin receptor 1 (NTSR1) and related G protein-coupled receptors of the ghrelin family are clinically unexploited, and several mechanistic aspects of their activation and inactivation have remained unclear. Enabled by a new crystallization design, we present five new structures: apo-state NTSR1 as well as complexes with nonpeptide inverse agonists SR48692 and SR142948A, partial agonist RTI-3a, and the novel full agonist SRI-9829, providing structural rationales on how ligands modulate NTSR1. The inverse agonists favor a large extracellular opening of helices VI and VII, undescribed so far for NTSR1, causing a constriction of the intracellular portion. In contrast, the full and partial agonists induce a binding site contraction, and their efficacy correlates with the ability to mimic the binding mode of the endogenous agonist neurotensin. Providing evidence of helical and side-chain rearrangements modulating receptor activation, our structural and functional data expand the mechanistic understanding of NTSR1 and potentially other peptidergic receptors.
