SignificanceUsing D-amino acids as the building blocks for bioactive peptides can dramatically increase their potency. However, simply swapping regular levorotary amino acids for dextrorotary (D)-amino acids alters the peptide surface topology and function is lost. Current methods to overcome this are not generally applicable and exclude the majority of therapeutic targets. By creating a mirror image of all 111,867 protein structures in the Protein Data Bank (PDB), we convert this repository into a D-peptide database with 2.8 million D-peptide structures. This D-PDB can be searched to find therapeutically active topologies, demonstrated here by the discovery of D-peptide GLP1R and PTH1R agonists. Evaluation of D-PDB coverage suggests that it holds candidates for most therapeutic targets and, thus, potentially contains hundreds of potent drug leads.

Small multidrug resistance (SMR) pumps represent a minimal paradigm of proton-coupled membrane transport in bacteria, yet no high-resolution structure of an SMR protein is available. Here, atomic-resolution structures of the efflux-multidrug resistance E () multidrug transporter in ligand-bound form are refined using microsecond molecular dynamics simulations biased using low-resolution data from X-ray crystallography. The structures are compatible with existing mutagenesis data as well as NMR and biochemical experiments, including pKas of the catalytic glutamate residues and the dissociation constant ([Formula: see text]) of the tetraphenylphosphonium cation. The refined structures show the arrangement of residue side chains in the active site occupied by two different ligands and in the absence of a ligand, illustrating how can adopt structurally diverse active site configurations. The structures also show a stable, well-packed binding interface between the helices H4 of the two monomers, which is believed to be crucial for dimerization. Guided by the atomic details of this interface, we design proteolysis-resistant stapled peptides that bind to helix H4 of an monomer. The peptides are expected to interfere with the dimerization and thereby inhibit drug transport. Optimal positions of the peptide staple were determined using free-energy simulations of peptide binding to monomeric Three of the four top-scoring peptides selected for experimental testing resulted in significant inhibition of proton-driven ethidium efflux in live cells without nonspecific toxicity. The approach described here is expected to be of general use for the design of peptide therapeutics.

Membrane proteins play the central roles in a variety of cellular processes, ranging from nutrient uptake and signalling, to cell-cell communication. Their biological functions are directly related to how they fold and assemble; defects often lead to disease. Protein-protein interactions (PPIs) within the membrane are therefore of great interest as therapeutic targets. Here we review the progress in the application of membrane-insertable peptides for the disruption or stabilization of membrane-based PPIs. We describe the design and preparation of transmembrane peptide mimics; and of several categories of peptidomimetics used for study, including d-enantiomers, non-natural amino acids, peptoids, and β-peptides. Further aspects of the review describe modifications to membrane-insertable peptides, including lipidation and cyclization via hydrocarbon stapling. These approaches provide a pathway toward the development of metabolically stable, non-toxic, and efficacious peptide modulators of membrane-based PPIs. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.