Potent Immunomodulators Developed from an Unstable Bacterial Metabolite of Vitamin B2 Biosynthesis.

Bacterial synthesis of vitamin B2 generates a by-product, 5-(2-oxopropylideneamino)-d-ribityl-aminouracil (5-OP-RU), with potent immunological properties in mammals, but it is rapidly degraded in water. This natural product covalently bonds to the key immunological protein MR1 in the endoplasmic reticulum of antigen presenting cells (APCs), enabling MR1 refolding and trafficking to the cell surface, where it interacts with T cell receptors (TCRs) on mucosal associated invariant T lymphocytes (MAIT cells), activating their immunological and antimicrobial properties. Here, we strategically modify this natural product to understand the molecular basis of its recognition by MR1.

-(2-Aminophenyl)-benzamide Inhibitors of Class I HDAC Enzymes with Antiproliferative and Antifibrotic Activity.

Inhibitors of histone deacetylases (HDACs) have received special attention as novel anticancer agents. Among various types of synthetic inhibitors, benzamides constitute an important class, and one is an approved drug (chidamide). Here, we present a novel class of HDAC inhibitors containing the -(2-aminophenyl)-benzamide functionality as the zinc-binding group linked to various cap groups, including the amino acids pyroglutamic acid and proline.

Talarolides Revisited: Cyclic Heptapeptides from an Australian Marine Tunicate-Associated Fungus, sp. CMB-TU011.

Application of a miniaturized 24-well plate system for cultivation profiling (MATRIX) permitted optimization of the cultivation conditions for the marine-derived fungus sp. CMB-TU011, facilitating access to the rare cycloheptapeptide talarolide A () along with three new analogues, B-D (-). Detailed spectroscopic analysis supported by Marfey's analysis methodology was refined to resolve -Me-l-Ala from -Me-d-Ala, l--Ile from l-Ile and l-Leu, and partial and total syntheses of , and permitted unambiguous assignment of structures for (revised) and -.

Helical structure in cyclic peptides: effect of -methyl amides esters.

An alpha helical turn can be reproduced in a cyclic pentapeptide if the first and fifth amino acid sidechains are correctly joined. Here structural studies (CD, NMR, ) reveal why -methylation at positions not involved in hydrogen bonds disrupts helicity whereas ester bonds can maintain helicity and promote greater cell uptake.

Landscaping macrocyclic peptides: stapling hDM2-binding peptides for helicity, protein affinity, proteolytic stability and cell uptake.

Cyclic peptides that modulate protein-protein interactions can be valuable therapeutic candidates if they can be delivered intact to their target proteins in cells. Here we systematically compare the effects of different helix-inducing cyclization constraints on the capacity of a macrocyclic peptide component to confer α-helicity, protein-binding affinity, resistance to degradative proteases and cell uptake to a 12-residue peptide fragment of tumor suppressor protein p53. We varied the helix-inducing constraint (hydrocarbon, lactam, aliphatic or aromatic thioether, ) and the position of the cyclization linker ( to + 4 or to + 7 bridges) in order to sculpt the macrocyclic size, stabilize its structure, and promote cell uptake.

Tuning Electrostatic and Hydrophobic Surfaces of Aromatic Rings to Enhance Membrane Association and Cell Uptake of Peptides.

Aromatic groups are key mediators of protein-membrane association at cell surfaces, contributing to hydrophobic effects and π-membrane interactions. Here we show electrostatic and hydrophobic influences of aromatic ring substituents on membrane affinity and cell uptake of helical, cyclic and cell penetrating peptides. Hydrophobicity is important, but subtle changes in electrostatic surface potential, dipoles and polarizability also enhance association with phospholipid membranes and cell uptake.

Shear-induced mixing of granular materials featuring broad granule size distributions.

Granular flows during a shear-induced mixing process are studied using discrete element methods. The aim is to understand the underlying elementary mechanisms of transition from unmixed to mixed phases for a granular material featuring a broad distribution of particles, which we investigate systematically by varying the strain rate and system size. Here the strain rate varies over four orders of magnitude and the system size varies from ten thousand to more than a million granules.

Connecting Hydrophobic Surfaces in Cyclic Peptides Increases Membrane Permeability.

N- or C-methylation in natural and synthetic cyclic peptides can increase membrane permeability, but it remains unclear why this happens in some cases but not others. Here we compare three-dimensional structures for cyclic peptides from six families, including isomers differing only in the location of an N- or Cα-methyl substituent. We show that a single methyl group only increases membrane permeability when it connects or expands hydrophobic surface patches.

Twists or turns: stabilising alpha beta turns in tetrapeptides.

Protein-protein interactions involve hotspots as small as 4 sequential amino acids. Corresponding tetrapeptides have no structure in water. Here we report linking side chains of amino acids X and Z to form 24 cyclic tetrapeptides, cyclo-[XAAZ]-NH, and stabilise 14-18 membered rings that mimic different kinds of non-regular secondary structures found in protein hotspots.

Mirror image pairs of cyclic hexapeptides have different oral bioavailabilities and metabolic stabilities.

Rule-of-five parameters and membrane permeabilities have been routinely used to guide development of orally bioavailabile drugs. Here we compare enantiomeric pairs of cyclic hexapeptides with identical rule-of-five parameters and membrane permeabilities. For each enantiomeric pair, the isomer with more l- than d-amino acids is much more orally bioavailable in rats, more metabolically stable to rat liver microsomes, and cleared more slowly in vivo.

A Novel Long-Range n to π* Interaction Secures the Smallest known α-Helix in Water.

The introduction of an amide bond linking side chains of the first and fifth amino acids forms a cyclic pentapeptide that optimally stabilizes the smallest known α-helix in water. The origin of the stabilization is unclear. The observed dependence of α-helicity on the solvent and cyclization linker led us to discover a novel long-range n to π* interaction between a main-chain amide oxygen and a uniquely positioned carbonyl group in the linker of cyclic pentapeptides.

Structure-Activity Relationships of Wollamide Cyclic Hexapeptides with Activity against Drug-Resistant and Intracellular .

Wollamides are cyclic hexapeptides, recently isolated from an Australian soil isolate, that exhibit promising antimycobacterial activity against Bacille Calmette Guérin without displaying cytotoxicity against a panel of mammalian cells. Here, we report the synthesis and antimycobacterial activity of 36 new synthetic wollamides, collated with all known synthetic and natural wollamides, to reveal structure characteristics responsible for growth-inhibitory activity against (H37Rv, H37Ra, CDC1551, HN878, and HN353). The most potent antimycobacterial wollamides were those where residue VI d-Orn (wollamide B) was replaced by d-Arg (wollamide B1) or d-Lys (wollamide B2), with all activity being lost when residue VI was replaced by Gly, l-Arg, or l-Lys (wollamide B3).

Glucuronic acid as a helix-inducing linker in short peptides.

A new strategy is demonstrated for making peptides helical, using a carbohydrate to bridge between sidechains at each end of a pentapeptide. CD and NMR spectra establish that both an α-helix and a 3-helix structure can form depending upon the bridge.

Contiguous hydrophobic and charged surface patches in short helix-constrained peptides drive cell permeability.

Most protein-protein interactions occur inside cells. Peptides can inhibit protein-protein interactions but tend not to enter cells. We systematically compare cell permeability for 8-12 residue model peptides with helix-inducing lactam/hydrocarbon linkers between amino acid sidechains.

Helixconstraints and amino acid substitution in GLP-1 increase cAMP and insulin secretion but not beta-arrestin 2 signaling.

Glucagon-like peptide (GLP-1) is an endogenous hormone that induces insulin secretion from pancreatic islets and modified forms are used to treat diabetes mellitus type 2. Understanding how GLP-1 interacts with its receptor (GLP-1R) can potentially lead to more effective drugs. Modeling and NMR studies of the N-terminus of GLP-1 suggest a β-turn between residues Glu9-Phe12 and a kinked alpha helix between Val16-Gly37.

Helix Nucleation by the Smallest Known α-Helix in Water.

Cyclic pentapeptides (e.g. Ac-(cyclo-1,5)-[KAXAD]-NH2 ; X=Ala, 1; Arg, 2) in water adopt one α-helical turn defined by three hydrogen bonds.

Flexibility versus Rigidity for Orally Bioavailable Cyclic Hexapeptides.

Cyclic peptides and macrocycles have the potential to be membrane permeable and orally bioavailable, despite often not complying with the "rule of five" used in medicinal chemistry to guide the discovery of oral drugs. Here we compare solvent-dependent three-dimensional structures of three cyclic hexapeptides containing d-amino acids, prolines, and intramolecular hydrogen bonds. Conformational rigidity rather than flexibility resulted in higher membrane permeability, metabolic stability and oral bioavailability, consistent with less polar surface exposure to solvent and a reduced entropy penalty for transition between polar and nonpolar environments.

Short Hydrophobic Peptides with Cyclic Constraints Are Potent Glucagon-like Peptide-1 Receptor (GLP-1R) Agonists.

Cyclic constraints are incorporated into an 11-residue analogue of the N-terminus of glucagon-like peptide-1 (GLP-1) to investigate effects of structure on agonist activity. Cyclization through linking side chains of residues 2 and 5 or 5 and 9 produced agonists at nM concentrations in a cAMP assay. 2D NMR and CD spectra revealed an N-terminal β-turn and a C-terminal helix that differentially influenced affinity and agonist potency.

Facile synthesis of mono- and bis-methylated Fmoc-Dap, -Dab and -Orn amino acids.

A new methodology for the synthesis of side chain mono- or bis-methylated Fmoc-Dap, -Dab and -Orn amino acids was developed by probing the reactivity of commercially available Fmoc amino acids.

Helix-constrained nociceptin peptides are potent agonists and antagonists of ORL-1 and nociception.

Nociceptin (orphanin FQ) is a 17-residue neuropeptide hormone with roles in both nociception and analgesia. It is an opioid-like peptide that binds to and activates the G-protein-coupled receptor opioid receptor-like-1 (ORL-1, NOP, orphanin FQ receptor, kappa-type 3 opioid receptor) on central and peripheral nervous tissue, without activating classic delta-, kappa-, or mu-opioid receptors or being inhibited by the classic opioid antagonist naloxone. The three-dimensional structure of ORL-1 was recently published, and the activation mechanism is believed to involve capture by ORL-1 of the high-affinity binding, prohelical C-terminus.

Cyclic Penta- and Hexaleucine Peptides without N-Methylation Are Orally Absorbed.

Development of peptide-based drugs has been severely limited by lack of oral bioavailability with less than a handful of peptides being truly orally bioavailable, mainly cyclic peptides with N-methyl amino acids and few hydrogen bond donors. Here we report that cyclic penta- and hexa-leucine peptides, with no N-methylation and five or six amide NH protons, exhibit some degree of oral bioavailability (4-17%) approaching that of the heavily N-methylated drug cyclosporine (22%) under the same conditions. These simple cyclic peptides demonstrate that oral bioavailability is achievable for peptides that fall outside of rule-of-five guidelines without the need for N-methylation or modified amino acids.

Improving on nature: making a cyclic heptapeptide orally bioavailable.

The use of peptides in medicine is limited by low membrane permeability, metabolic instability, high clearance, and negligible oral bioavailability. The prediction of oral bioavailability of drugs relies on physicochemical properties that favor passive permeability and oxidative metabolic stability, but these may not be useful for peptides. Here we investigate effects of heterocyclic constraints, intramolecular hydrogen bonds, and side chains on the oral bioavailability of cyclic heptapeptides.

Comparative α-helicity of cyclic pentapeptides in water.

Helix-constrained polypeptides have attracted great interest for modulating protein-protein interactions (PPI). It is not known which are the most effective helix-inducing strategies for designing PPI agonists/antagonists. Cyclization linkers (X1-X5) were compared here, using circular dichroism and 2D NMR spectroscopy, for α-helix induction in simple model pentapeptides, Ac-cyclo(1,5)-[X1-Ala-Ala-Ala-X5]-NH2, in water.

Truncated and helix-constrained peptides with high affinity and specificity for the cFos coiled-coil of AP-1.

Protein-based therapeutics feature large interacting surfaces. Protein folding endows structural stability to localised surface epitopes, imparting high affinity and target specificity upon interactions with binding partners. However, short synthetic peptides with sequences corresponding to such protein epitopes are unstructured in water and promiscuously bind to proteins with low affinity and specificity.

Total synthesis, structure, and oral absorption of a thiazole cyclic peptide, sanguinamide A.

The first total synthesis and three-dimensional solution structure are reported for sanguinamide A, a thiazole-containing cyclic peptide from the sea slug H. sanguineus. Solution phase fragment synthesis, solid phase fragment assembly, and solution macrocyclization were combined to give (1) in 10% yield.