Synthetic Cross-linking of Peptides: Molecular Linchpins for Peptide Cyclization.

Peptide-derived drugs constitute a significant fraction of therapeutic agents. In 2013, The global market of peptide therapeutics was ca. $19 billion; this value does not include revenue from insulin derivatives of $28 million. The combined sales of insulin and non-insulin peptide drugs is estimated to exceed $70 billion by 2019. A significant fraction of peptide-derived drugs is composed of an amino acid sequence and additional chemical functionalities that improve biological and pharmacological properties of the drug. In this review, we focus on synthetic cross-linkers that we refer to as "linchpins", which are commonly used to constrain the secondary structure of peptides and equip them with added benefits such as resistance to proteolytic degradation and conformational stability. The latter property leads to an increase in binding potency and increased bioavailability due to increased permeation through biological membranes. Some linchpins can even introduce properties not found in natural peptides such as light-responsiveness. Peptides cyclized by linchpins can be viewed as a sub-class of a larger family of peptide-derived drugs with desired pharmacological performance in vivo. To understand how chemical modifications by linchpins improve drug discovery, this review also briefly summarizes canonical examples of chemical modification used in modern peptide therapeutics.

Light-responsive bicyclic peptides.

In this paper, we describe a method for the synthesis of light-responsive (LR) bicyclic macrocycles from linear peptides composed of 20 natural amino acids. Small molecules, peptide macrocycles, and protein conjugates that reversibly turn their function on and off in response to visible light enabled the fields of photopharmacology and optochemical genetics. Bioactive LR molecules could be produced by grafting azobenzene or other LR-structures onto molecules with known biological functions (e.g., alpha-helical peptides). It is also possible to discover such LR ligands de novo by selecting compounds with a desired function-such as binding to a target-from a library of LR-compounds or a genetically-encoded (GE) library of LR-macrocycles. The bicyclic topology of ligands offers added value such as improved binding and stability when compared to monocyclic peptides, but approaches for the design of bicyclic light-responsive architectures are limited. To address this need, we developed a tridentate C2-symmetric hydroxyl amine and di-chlorobenzene containing azobenzene (HADCAz) LR-linker with two orthogonally reactive functionalities (chlorobenzyl and hydroxylamine) to convert a linear unprotected peptide into a bicyclic peptide in a one-pot, two-step reaction. This linker reversibly isomerizes from the trans to cis form upon irradiation with blue light (365 nm). The resulting bicyclic peptide contains two loops of amino acids, one of which is constrained with an azobenzene moiety that can change the conformation in response to visible light. A scalable synthetic route to the HADCAz linker allowed us to demonstrate its application in multiple synthetic bicyclic peptides with loops that contain 2-5 amino acids.

Allene Functionalized Azobenzene Linker Enables Rapid and Light-Responsive Peptide Macrocyclization.

In this manuscript, we describe the efficient synthesis of a bis(allenamide) functionalized water-soluble azobenzene reagent (BSBDA) and its application as a new tool for the rapid generation of visible light-responsive macrocyclic peptides and peptide libraries displayed on the surface of bacteriophage. The allenamide functionality promotes cysteine ligation in model peptides and those displayed on phage with rates 2-3 orders of magnitude faster than the established alkyl halide containing azobenzenes.

Influence of muscarinic receptor modulators on interacerebroventricular injection of arachydonylcyclopropylamide induced antinociception in mice.

The interaction between antinociception induced by CB1 agonist and muscarinic receptor modulators has not been studied yet. In the present study, the effect of pilocarpine (a muscarinic agonist) and atropine (a muscarinic antagonist) on arachidonylcyclopropylamide (ACPA, a CB1 agonist) induced antinociception was studied in mice. In this study the antinociceptive effect of intracerebroventricular administration of ACPA (0.001-2 µg/mice) or intraperitoneal injection of pilocarpine (2.5-20mg/kg) or atropine (1 and 5mg/kg) were studied individually. Then the effect of co-administration of pilocarine (2.5mg/kg) or atropine (5mg/kg) and ACPA (0.001-2 µg/mice) were studied as well. ACPA and pilocarpine induced antinociception in mice but atropine did not. Pilocarpine potentiated but atropine antagonized the antinociceptive effect of ACPA. It is concluded that ACPA induced antinociception is influenced by muscarinic receptor modulators in mice.

Discovery of light-responsive ligands through screening of a light-responsive genetically encoded library.

Light-responsive ligands are useful tools in biochemistry and cell biology because the function of these ligands can be spatially and temporally controlled. Conventional design of such ligands relies on previously available data about the structure of both the ligand and the receptor. In this paper, we describe de novo discovery of light-responsive ligands through screening of a genetically encoded light-responsive library. We ligated a photoresponsive azobenzene core to a random CX7C peptide library displayed on the coat protein of M13 phage. A one-pot alkylation/reduction of the cysteines yielded a photoresponsive library of random heptapeptide macrocycles with over 2 × 10(8) members. We characterized the reaction on-phage and optimized the yield of the modifications in phage libraries. Screening of the library against streptavidin yielded three macrocycles that bind to streptavidin in the dark and cease binding upon irradiation with 370 nm light. All ligands restored their binding properties upon thermal relaxation and could be turned ON and OFF for several cycles. We measured dissociation constants, Kd, by electrospray ionization mass spectrometry (ESI-MS) binding assay. For ligand ACGFERERTCG, the Kd of cis and trans isomers differed by 22-fold; an incomplete isomerization (85%), however, resulted in the apparent difference of 4.5-fold between the dark and the irradiated state. We anticipate that the selection strategy described in this report can be used to find light-responsive ligands for many targets that do not have known natural ligands.

Uniform amplification of phage display libraries in monodisperse emulsions.

In this paper, we describe a complete experimental setup for the uniform amplification of libraries of phage. Uniform amplification, which multiplies every phage clone by the same amount irrespective of the growth rate of the clone is essential for phage-display screening. Amplification of phage libraries in a common solution is often non-uniform: it favors fast-growing clones and eliminates those that grow slower. This competition leads to elimination of many useful binding clones, and it is a major barrier to identification of ligands for targets with multiple binding sites such as cells, tissues, or mixtures of proteins. Uniform amplification is achieved by encapsulating individual phage clones into isolated compartments (droplets) of identical volume. Each droplet contains culture medium and an excess of host (Escherichia coli). Here, we describe microfluidics devices that generate mono-disperse droplet-based compartments, and optimal conditions for amplification of libraries of different size. We also describe the detailed synthesis of a perfluoro surfactant, which gives droplets exceptional stability. Droplets stabilized by this compound do not coalesce after many hours in shaking culture. We identified a commercially available compound (Krytox), which destabilizes these droplets to recover the amplified libraries. Overall, uniform amplification is a sequence of three simple steps: (1) encapsulation of mixture of phage and bacteria in droplets using microfluidics; (2) incubation of droplets in a shaking culture; (3) destabilization of droplets to harvest the amplified phage. We anticipate that this procedure can be easily adapted in any academic or industrial laboratory that uses phage display.

Quantitative synthesis of genetically encoded glycopeptide libraries displayed on M13 phage.

Phage display is a powerful technology that enables the discovery of peptide ligands for many targets. Chemical modification of phage libraries have allowed the identification of ligands with properties not encountered in natural polypeptides. In this report, we demonstrated the synthesis of 2 × 10(8) genetically encoded glycopeptides from a commercially available phage-displayed peptide library (Ph.D.-7) in a two-step, one-pot reaction in <1.5 h. Unlike previous reports, we bypassed genetic engineering of phage. The glycan moiety was introduced via an oxime ligation following oxidation of an N-terminal Ser/Thr; these residues are present in the peptide libraries at 20-30% abundance. The construction of libraries was facilitated by simple characterization, which directly assessed the yield and regioselectivity of chemical reactions performed on phage. This quantification method also allowed facile yield determination of reactions in 10(9) distinct molecules. We envision that the methodology described herein will find broad application in the synthesis of custom chemically modified phage libraries.

Bacteriophages and viruses as a support for organic synthesis and combinatorial chemistry.

Display of polypeptide on the coat proteins of bacteriophages and viruses is a powerful tool for selection and amplification of libraries of great diversity. Chemical diversity of these libraries, however, is limited to libraries made of natural amino acid side chains. Bacteriophages and viruses can be modified chemically; peptide libraries presented on phage thus can be functionalized to yield moieties that cannot be encoded genetically. In this review, we summarize the possibilities for using bacteriophage and viral particles as support for the synthesis of diverse chemically modified peptide libraries. This review critically summarizes the key chemical considerations for on-phage syntheses such as selection of reactions compatible with protein of phage, modification of phage "support" that renders it more suitable for reactions, and characterization of reaction efficiency.

Diversity of phage-displayed libraries of peptides during panning and amplification.

The amplification of phage-displayed libraries is an essential step in the selection of ligands from these libraries. The amplification of libraries, however, decreases their diversity and limits the number of binding clones that a screen can identify. While this decrease might not be a problem for screens against targets with a single binding site (e.g., proteins), it can severely hinder the identification of useful ligands for targets with multiple binding sites (e.g., cells). This review aims to characterize the loss in the diversity of libraries during amplification. Analysis of the peptide sequences obtained in several hundred screens of peptide libraries shows explicitly that there is a significant decrease in library diversity that occurs during the amplification of phage in bacteria. This loss during amplification is not unique to specific libraries: it is observed in many of the phage display systems we have surveyed. The loss in library diversity originates from competition among phage clones in a common pool of bacteria. Based on growth data from the literature and models of phage growth, we show that this competition originates from growth rate differences of only a few percent for different phage clones. We summarize the findings using a simple two-dimensional "phage phase diagram", which describes how the collapse of libraries, due to panning and amplification, leads to the identification of only a subset of the available ligands. This review also highlights techniques that allow elimination of amplification-induced losses of diversity, and how these techniques can be used to improve phage-display selection and enable the identification of novel ligands.

Influence of nicotinic receptor modulators on CB2 cannabinoid receptor agonist (JWH133)-induced antinociception in mice.

Delta9-tetrahydrocannabinol is the active component in cannabis and has long been associated with pain relief. This effect is believed to be mediated through central and peripheral CB1 and peripheral CB2 receptors. We have explored the possible antinociceptive effect of a CB2 receptor agonist, JWH133, using the formalin test in mice. The drug was administered by the intracerebroventricular and intraperitoneal routes. Although no antinociceptive effect was observed after intracerebroventricular administration of JWH133, when the drug was administered by the intraperitoneal route, it produced an analgesic effect. The influence of nicotinic cholinergic receptor modulators, nicotine and mecamylamine, on antinociceptive effect of JWH133 was also studied. Nicotine increased and mecamylamine decreased the antinociceptive effect of JWH133. It is concluded that JWH133-induced analgesia is influenced by nicotinic cholinergic receptor activity.

Influence of cholinergic system modulators on morphine state-dependent memory of passive avoidance in mice.

Memories are shown to be impaired in mice during step-down passive avoidance tasks with substantial residual effects lasting as long as 24 h after the pre-training administration of morphine. Administration of the same dose of morphine as a pre-test treatment restored memory. Since the cholinergic system has been reported to be involved in several actions of morphine, e.g.: modulation of memory and analgesia, we have investigated the part played by cholinergic modulator drugs, on the memory recall in mice. The locomotor activity of animals was studied as well. Administration of either atropine, a peripheral-central muscarinic antagonist, or mecamylamine, a peripheral-central nicotinic antagonist, failed to alter memory themselves, but significantly prevented morphine-induced memory recall following co-administration with morphine. Neither hexamethonium, a peripheral nicotinic antagonist, nor neostigmine, a peripheral anticholinesterase, showed intrinsic activity or a significant change in morphine-induced memory recall. Finally, physostigmine, a peripheral-central anticholinesterase, not only induced memory recall itself, but also increased morphine-induced retrieval. Memory recall of the step-down passive avoidance task following drug combinations was not related to locomotor activity changes. Thus, morphine-induced memory recall appears to be influenced by central cholinergic activity.

Influence of central administration ATP-dependent K+ channel on morphine state-dependent memory of passive avoidance.

Pre-training injection of a moderate dose of morphine (5-10 mg/kg) in a step-down passive avoidance task induced state-dependent learning with impaired memory retrieval on the test day. The impairment of memory was restored after the pre-test administration of the same dose of the drug. We have studied the effect of intracerebroventricular administration of naloxone and K(ATP) channel modulators (glibenclamide and diazoxide) on the test day on restoration of memory by morphine in mice. The effect of scopolamine on restoration of memory on the test-day by glibenclamide was studied as well. Naloxone pretreatment (0.006, 0.025 and 0.1 microg/mouse) reversed the effect of pre-test morphine administration. The K(ATP) channel blocker, glibenclamide (0.1, 0.5 and 1 microg/mouse), showed effects similar to those of pre-test administration of morphine. Glibenclamide tended to potentiate the morphine response. Scopolamine (0.15 and 0.30 microg/mouse) prevented the effect of glibenclamide on the restoration of memory. The pre-test administration of different doses of diazoxide (1.7, 5 and 15 microg/mouse), a K(ATP) channel opener, showed no effect on restoration of memory when used alone but decreased morphine state-dependence. Diazoxide blocked the effects of glibenclamide on memory restoration. It is concluded that K(ATP) channel modulators may be involved, at least in part, in morphine state dependence through a cholinergic system mechanism.