De novo Fc-based receptor dimerizers differentially modulate PlexinB1 function.

Signaling by single-pass transmembrane receptors often involves a formation of ligand-induced receptor dimers with particular conformation, and bivalent receptor binders can modulate receptor functions by inducing different receptor dimer conformations, although such agents are difficult to design. Here, we describe the generation of both antagonistic and agonistic receptor dimerizers toward PlexinB1 (PlxnB1), a receptor for semaphorin 4D (Sema4D), by grafting two different PlxnB1-binding peptides onto the human immunoglobulin G1 (IgG1) Fc protein. The function-modulating activity of a peptide Fc was strongly dependent on the type of the peptide as well as the grafting site, with the best variants showing activity at an nM concentration range. Structural analysis of each peptide-PlxnB1 complex revealed that the agonistic Fc dimerizes PlxnB1 in a face-to-face fashion similar to that induced by Sema4D, whereas antagonistic Fc would induce signaling-incompetent PlxnB1 dimer conformation, enforcing the idea that plexin activation is primarily controlled by the receptor orientation within the dimer.

Lasso-grafting of macrocyclic peptide pharmacophores yields multi-functional proteins.

Protein engineering has great potential for devising multifunctional recombinant proteins to serve as next-generation protein therapeutics, but it often requires drastic modifications of the parental protein scaffolds e.g., additional domains at the N/C-terminus or replacement of a domain by another. A discovery platform system, called RaPID (Random non-standard Peptides Integrated Discovery) system, has enabled rapid discovery of small de novo macrocyclic peptides that bind a target protein with high binding specificity and affinity. Capitalizing on the optimized binding properties of the RaPID-derived peptides, here we show that RaPID-derived pharmacophore sequences can be readily implanted into surface-exposed loops on recombinant proteins and maintain both the parental peptide binding function(s) and the host protein function. We refer to this protein engineering method as lasso-grafting and demonstrate that it can endow specific binding capacity toward various receptors into a diverse set of scaffolds that includes IgG, serum albumin, and even capsid proteins of adeno-associated virus, enabling us to rapidly formulate and produce bi-, tri-, and even tetra-specific binder molecules.

Development of cyclic peptides with potent in vivo osteogenic activity through RaPID-based affinity maturation.

Osteoporosis is caused by a disequilibrium between bone resorption and bone formation. Therapeutics for osteoporosis can be divided into antiresorptives that suppress bone resorption and anabolics which increase bone formation. Currently, the only anabolic treatment options are parathyroid hormone mimetics or an anti-sclerostin monoclonal antibody. With the current global increases in demographics at risk for osteoporosis, development of therapeutics that elicit anabolic activity through alternative mechanisms is imperative. Blockade of the PlexinB1 and Semaphorin4D interaction on osteoblasts has been shown to be a promising mechanism to increase bone formation. Here we report the discovery of cyclic peptides by a novel RaPID (Random nonstandard Peptides Integrated Discovery) system-based affinity maturation methodology that generated the peptide PB1m6A9 which binds with high affinity to both human and mouse PlexinB1. The chemically dimerized peptide, PB1d6A9, showed potent inhibition of PlexinB1 signaling in mouse primary osteoblast cultures, resulting in significant enhancement of bone formation even compared to non-Semaphorin4D-treated controls. This high anabolic activity was also observed in vivo when the lipidated PB1d6A9 (PB1d6A9-Pal) was intravenously administered once weekly to ovariectomized mice, leading to complete rescue of bone loss. The potent osteogenic properties of this peptide shows great promise as an addition to the current anabolic treatment options for bone diseases such as osteoporosis.

Facile Synthesis of Dimeric Thioether-Macrocyclic Peptides with Antibody-like Affinity against Plexin-B1.

Macrocyclic peptides have gained increasing attention due to their ease of discovery through various in vitro display platforms as well as their potential in possessing favorable properties of both small molecule and antibody drug classes. It is well-known that the avidity achieved through the bivalent binding mode of antibodies gives rise to their slow dissociation rates and thus high potency as drug molecules. Here, we report the synthesis of dimeric thioether-macrocyclic peptides through a branched synthesis approach allowing for synthesis of dimeric peptides in a comparable number of steps as monomers and tunability of linker lengths from 30 to 200 Å. Applying this method to synthesize dimers of a model PlexinB1-binding macrocyclic peptide showed close to 300-fold increases in their apparent binding affinity, bringing the KD down from 8 nM to 30 pM as well as affording improved biological activities when compared to their monomeric counterparts. These enhancements demonstrate that this is a simple synthetic strategy to harness the benefits of bivalence that antibodies naturally possess.

In vivo programming of endogenous antibodies via oral administration of adaptor ligands.

Vaccination is a reliable method of prophylaxis and a crucial measure for public health. However, the majority of vaccines cannot be administered orally due to their degradation in the harsh gut environment or inability to cross the GI tract. In this study, we report the first proof-of-concept study of orally producible chemically programmed antibodies via specific conjugation of adaptor ligands to endogenous antibodies, in vivo. Pre-immuniztion with 2,4-dinitrophenyl (DNP), or the reactive hapten, 1,3-diketone (DK), or a novel reactive hapten, vinyl sulfone (VS) in mice, followed by oral administration of adaptor ligands composed of the hapten and biotin to the pre-immunized mice resulted in successful in vivo formation of the biotin-hapten-antibody complexes within 2h. Pharmacokinetic evaluations revealed that apparent serum concentrations of programmed antibodies were up to 144nM and that the serum half-lives reached up to 34.4h. These findings show promise for the future development of orally bioavailable drug-hapten-antibody complexes asa strategy to quickly and easily modulate immune targets for aggressive pathogens as well as cancer.

Allosteric Inhibition of a Semaphorin 4D Receptor Plexin B1 by a High-Affinity Macrocyclic Peptide.

Semaphorin axonal guidance factors are multifunctional proteins that play important roles in immune response, cancer cell proliferation, and organogenesis, making semaphorins and their signaling receptor plexins important drug targets for various diseases. However, the large and flat binding surface of the semaphorin-plexin interaction interface is difficult to target by traditional small-molecule drugs. Here, we report the discovery of a high-affinity plexin B1 (PlxnB1)-binding macrocyclic peptide, PB1m6 (KD = 3.5 nM). PB1m6 specifically inhibited the binding of physiological ligand semaphorin 4D (Sema4D) in vitro and completely suppressed Sema4D-induced cell collapse. Structural studies revealed that PB1m6 binds at a groove between the fifth and sixth blades of the sema domain in PlxnB1 distant from the Sema4D-binding site, indicating the non-competitive and allosteric nature of the inhibitory activity. The discovery of this novel allosteric site can potentially be used to target plexin family proteins for the development of drugs that modulate semaphorin and plexin signaling.

Construction and screening of vast libraries of natural product-like macrocyclic peptides using in vitro display technologies.

Macrocyclic structure and backbone N-methylation represent characteristic features of peptidic natural products, which play critical roles in their biological activity. Although natural products have been the traditional source of such peptides, recent developments in synthesizing natural product-like macrocyclic peptides using reconstituted translation systems have enabled us to construct vast trillion-member libraries of non-standard macrocyclic peptides. In addition, a method for displaying such libraries on their corresponding mRNA templates allows us to rapidly screen them for potent ligands against various drug targets. This review describes methodologies for the ribosomal synthesis of novel natural product-like macrocyclic peptides and their recent applications in the discovery of bioactive molecules using in vitro display technologies.

Protein cocrystallization molecules originating from in vitro selected macrocyclic peptides.

Transmembrane proteins are intractable crystallization targets due to their low solubility and their substantial hydrophobic outer surfaces must be enclosed within a partial micelle composed of detergents to avoid aggregation. Unfortunately, encapsulation within a partial micelle diminishes specific protein-to-protein contacts needed for crystal lattice formation. In addition, the high conformational flexibility of certain transmembrane proteins reduces sample homogeneity causing difficulty in crystallization. Cocrystallization ligands, based on either antibody scaffolds or other proteinaceous non-antibody scaffolds, have greatly facilitated the crystallization of transmembrane proteins. Recently, in vitro selected macrocyclic peptide ligands have been shown to facilitate protein crystallization as well. In this review, we discuss selection strategies used for the discovery of macrocyclic peptide ligands and the three-dimensional crystal structure of the transporter PfMATE in complex with in vitro selected macrocyclic peptides.