αβ-PEPTIDE MIMICS OF Z-DOMAIN PEPTIDES

Described are αβ-peptide mimics of Z-domain scaffold pep tides, methods of making them, and methods of using them. The αβ-peptide mimics include B-amino acid residues and, optionally, disulfide bonds to stabilize the conformation of the mimics. The compounds may be truncated as compared to conventional Z-domain scaffold peptides and are resistant to proteolytic degradation due to the presence off-amino acid residues. The mimics can be made so as to bind selectively to a desired target

Iterative non-proteinogenic residue incorporation yields α/β-peptides with a helix-loop-helix tertiary structure and high affinity for VEGF

Inhibition of specific protein-protein interactions is attractive for a range of therapeutic applications, but the large and irregularly shaped contact surfaces involved in many such interactions make it challenging to design synthetic antagonists. Here, we describe the development of backbone-modified peptides containing both α- and β-amino acid residues (“α/β-peptides”) that target the receptor-binding surface of vascular endothelial growth factor (VEGF). Our approach is based on the Z-domain, which adopts a three-helix bundle tertiary structure. We show how a two-helix “mini-Z-domain” can be modified to contain β and other non-proteinogenic residues while retaining the target-binding epitope using iterative non-natural residue incorporation. The resulting α/β-peptides are less susceptible to proteolysis than is their parent α-peptide, and some of these α/β-peptides match the full-length Z-domain in terms of affinity for receptor-recognition surfaces on the VEGF homodimer

Two interdependent mechanisms of antimicrobial activity allow for efficient killing in nylon-3-based polymeric mimics of innate immunity peptides

AbstractNovel synthetic mimics of antimicrobial peptides have been developed to exhibit structural properties and antimicrobial activity similar to those of natural antimicrobial peptides (AMPs) of the innate immune system. These molecules have a number of potential advantages over conventional antibiotics, including reduced bacterial resistance, cost-effective preparation, and customizable designs. In this study, we investigate a family of nylon-3 polymer-based antimicrobials. By combining vesicle dye leakage, bacterial permeation, and bactericidal assays with small-angle X-ray scattering (SAXS), we find that these polymers are capable of two interdependent mechanisms of action: permeation of bacterial membranes and binding to intracellular targets such as DNA, with the latter necessarily dependent on the former. We systemically examine polymer-induced membrane deformation modes across a range of lipid compositions that mimic both bacteria and mammalian cell membranes. The results show that the polymers' ability to generate negative Gaussian curvature (NGC), a topological requirement for membrane permeation and cellular entry, in model Escherichia coli membranes correlates with their ability to permeate membranes without complete membrane disruption and kill E. coli cells. Our findings suggest that these polymers operate with a concentration-dependent mechanism of action: at low concentrations permeation and DNA binding occur without membrane disruption, while at high concentrations complete disruption of the membrane occurs. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova

Toward a soluble model system for the amyloid state

The formation and deposition of amyloids is associated with many diseases. β-Sheet secondary structure is a common feature of amyloids, but the packing of sheets against one another is distinctive relative to soluble proteins. Standard methods that rely on perturbing a polypeptide’s sequence and evaluating impact on folding can be problematic for amyloid aggregates because a single sequence can adopt multiple conformations and diverse packing arrangements. We describe initial steps toward a minimum-sized, soluble model system for the amyloid state that supports comparisons among sequence variants. Critical to this goal is development of a new linking strategy to enable intersheet association mediated by side chain interactions, which is characteristic of the amyloid state. The linker design we identified should ultimately support exploration of relationships between sequence and amyloid state stability for specific strand-association modes

Backbone modification of a polypeptide drug alters duration of action in vivo

because of protease-catalyzed degradation. We used PTHR1 signaling to evaluate a strategy for creating active and biostable backbone-modified analogs of the well-known agonist PTH(1-34). PTH is an 84-residue protein that controls key physiological processes, including the maintenance of extracellular levels of calcium and phosphate and bone homeostasis 1 . PTH(1-34) matches full-length PTH in potency and efficacy at PTHR1 and is the active ingredient in the osteoporosis drug teriparatide (Forteo). As with many other peptide-based therapeutics, PTH(1-34) has a short half-life in the bloodstream (<30 min) 2 . Therapeutic effects for osteoporosis treatment appear to be maximized by pulsatile rather than continuous exposure to PTH(1-34), but the optimal exposure cycle is unclear 3 . We generated new analogs of PTH(1-34) by replacing selected α-amino acid residues with homologous β-amino acid residues, an unconventional approach that alters the backbone but can maintain the natural side chain complement. The results show that this technically straightforward strategy can provide hormone analogs that display native-like receptor activation potencies and prolonged residency in the bloodstream. The C-terminal portion of PTH(1-34) forms an α-helix upon binding to the receptor, but the bioactive conformation of the N-terminal segment is unknown. The backbone-modification strategy described here is based on previous studies showing that α-helical segments Systematic modification of the backbone of bioactive polypeptides through b-amino acid residue incorporation could provide a strategy for generating molecules with improved drug properties, but such alterations can result in lower receptor affinity and potency. Using an agonist of parathyroid hormone receptor-1 (PTHR1), a G protein-coupled receptor in the B-family, we present an approach for a→b residue replacement that enables both high activity and improved pharmacokinetic properties in vivo. involved in protein-recognition processes can be mimicked by oligomers containing mixtures of α and β residues 4,5 . Other types of unnatural oligomers, such as peptides composed of D-α-amino acid residues 6 , peptoids 7 and β-peptides 8 , have been explored for functional mimicry of bioactive α-helices; however, none of these alternative molecular scaffolds allows faithful mimicry of a long α-helix 5 , as required for potent analogs of PTH. In previous studies, PTH analogs containing one to three β-residue replacements were used to probe local conformational requirements, and many of these replacements caused profound declines in agonist activity We prepared all four PTH(1-34) analogs containing five α→β 3 replacements in an αααβ pattern 13 within the C-terminal region (A5-D5 in PTHR1 has two distinct functional states: RG, which forms when the intracellular portion contacts G αS (a heterotrimeric G-protein responsible for activating adenylate cyclase upon receptor activation); and R 0 , which forms independent of G αS 15,16 . An agonist's affinity for the RG state correlates with PTHR1 activation potency, whereas R 0 affinity correlates with the duration of some in vivo response

Differential membrane binding of α/β-peptide foldamers: implications for cellular delivery and mitochondrial targeting

The intrinsic pathway of apoptosis is regulated by the Bcl-2 family of proteins. Inhibition of the anti-apoptotic members represents a strategy to induce apoptotic cell death in cancer cells. We have measured the membrane binding properties of a series of peptides, including modified α/β-peptides, designed to exhibit enhanced membrane permeability to allow cell entry and improved access for engagement of Bcl-2 family members. The peptide cargo is based on the pro-apoptotic protein Bim, which interacts with all anti-apoptotic proteins to initiate apoptosis. The α/β-peptides contained cyclic β-amino acid residues designed to increase their stability and membrane-permeability. Dual polarisation interferometry was used to study the binding of each peptide to two different model membrane systems designed to mimic either the plasma membrane or the outer mitochondrial membrane. The impact of each peptide on the model membrane structure was also investigated, and the results demonstrated that the modified peptides had increased affinity for the mitochondrial membrane and significantly altered the structure of the bilayer. The results also showed that the presence of an RRR motif significantly enhanced the ability of the peptides to bind to and insert into the mitochondrial membrane mimic, and provide insights into the role of selective membrane targeting of peptides

Structural and functional diversity among agonist-bound states of the GLP-1 receptor

Recent advances in G-protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multidimensional signal propagation mediated by these receptors depends, in part, on their conformational mobility; however, the relationship between receptor function and static structures is inherently uncertain. Here, we examine the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide 1 receptor (GLP-1R), an important clinical target. We use variants of the peptides GLP-1 and exendin-4 (Ex4) to explore the interplay between helical propensity near the agonist N terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an Ex4 analog, the GLP-1R and Gs heterotrimer revealed two receptor conformers with distinct modes of peptide-receptor engagement. Our functional and structural data, along with molecular dynamics (MD) simulations, suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy.</p