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 rapid inactivation in water limits practical uses. This natural product covalently bonds to immunological protein MR1 in antigen presenting cells (APCs), enabling MR1 to traffic 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 develop several new series of water-stable compounds tailored for powerful and distinctive immunological functions. We report their water stability, capacity to bind MR1 and traffic it to the cell surface (EC50 17 nM), potent activation (EC50 56 pM) or inhibition (IC50 80 nM) of interacting MAIT cells, and develop compounds with diazirine-alkyne, biotin, or fluorophore labels for studying cellular MR1. Computer modelling casts new light on the molecular mechanism of activation, revealing that activators are first captured in MR1 via  pi-interactions and H-bonds, before tighter covalent bonding to Lys43 in MR1. This chemical study advances our molecular understanding of how bacterial metabolites are captured by MR1, influence cell surface expression of MR1, interact and modify human T cells; offering new clues for developing novel vaccine adjuvants, immunotherapeutics, and cancer drugs.

N-(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 N-(2-aminophenyl)-benzamide functionality as the zinc-binding group linked to various cap groups, including the amino acids pyroglutamic acid and proline. We have identified benzamides that inhibit HADC1 and HDAC2 at nanomolar concentrations, with antiproliferative activity at micromolar concentrations against A549 and SF268 cancer cell lines. Docking studies shed light on the mode of binding of benzamide inhibitors to HDAC1, whereas cellular analysis revealed downregulated expression of EGFR mRNA and protein. Two benzamides were investigated in a mouse model of bleomycin-induced pulmonary fibrosis, and both showed efficacy on a preventative dosing schedule. N-(2-Aminophenyl)-benzamide inhibitors of class I HDACs might lead to new approaches for treating fibrotic disorders.

Talarolides Revisited: Cyclic Heptapeptides from an Australian Marine Tunicate-Associated Fungus, Talaromyces 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 Talaromyces sp. CMB-TU011, facilitating access to the rare cycloheptapeptide talarolide A (1) along with three new analogues, B-D (2-4). Detailed spectroscopic analysis supported by Marfey's analysis methodology was refined to resolve N-Me-l-Ala from N-Me-d-Ala, l-allo-Ile from l-Ile and l-Leu, and partial and total syntheses of 2, and permitted unambiguous assignment of structures for 1 (revised) and 2-4. Consideration of diagnostic ROESY correlations for the hydroxamates 1 and 3-4, and a calculated solution structure for 1, revealed how cross-ring H-bonding to the hydroxamate moiety influences (defines/stabilizes) the cyclic peptide conformation. Such knowledge draws attention to the prospect that hydroxamates may be used as molecular bridges to access new cyclic peptide conformations, offering the prospect of new biological properties, including enhanced oral bioavailability.

Helical structure in cyclic peptides: effect of N-methyl amides versus 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, in silico) reveal why N-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, etc.) and the position of the cyclization linker (i to i + 4 or i to i + 7 bridges) in order to sculpt the macrocyclic size, stabilize its structure, and promote cell uptake. We find that rigidifying the macrocycle leads to higher alpha helicity, target affinity and proteolytic stability to different extents, whereas cell uptake of compounds shown here is mostly driven by hydrophobicity and aromaticity of the macrocycle.

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. A combination of fluorine and sulfur substituents on an aromatic ring induces microdipoles that enhance cell uptake of 12-residue peptide inhibitors of p53-HDM2 interaction and of cell-penetrating cyclic peptides. These aromatic motifs can be readily inserted into peptide sidechains to enhance their cell uptake.

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. Positional isomers, with the same molecular weight, hydrogen bond donors/acceptors, rotatable bonds, calculated LogP, topological polar surface area, and total hydrophobic surface area, can have different membrane permeabilities that correlate with the size of the largest continuous hydrophobic surface patch. These results illuminate a key local molecular determinant of membrane permeability.

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. CD and NMR spectra, NMR and X-ray structures, modelling, and MD simulations reveal that this first example of a synthetically incorporated long-range n to π* CO⋅⋅⋅Cγ =Ο interaction uniquely enforces an almost perfect and remarkably stable peptide α-helix in water but not in DMSO. This unusual interaction with a covalent amide bond outside the helical backbone suggests new approaches to synthetically stabilize peptide structures in water.

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

Wollamides are cyclic hexapeptides, recently isolated from an Australian soil Streptomyces isolate, that exhibit promising in vitro antimycobacterial activity against Mycobacterium bovis 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 in vitro growth-inhibitory activity against Mycobacterium tuberculosis (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). Substitution of other amino acid residues mainly reduced or ablated antimycobacterial activity. Significantly, whereas wollamide B2 was the most potent in restricting M. tuberculosisin vitro, wollamide B1 restricted M. tuberculosis intracellular burden in infected macrophages. Wollamide B1 synergized with pretomanid (PA-824) in inhibiting M. tuberculosisin vitro growth but did not antagonize prominent first- and second-line tuberculosis antibiotics. Furthermore, wollamide B1 exerted bactericidal activity against nonreplicating M. tuberculosis and impaired growth of multidrug- and extensively drug-resistant clinical isolates. In vivo pharmacokinetic profiles for wollamide B1 in rats and mice encourage further optimization of the wollamide pharmacophore for in vivo bioavailability. Collectively, these observations highlight the potential of the wollamide antimycobacterial pharmacophore.

Twists or turns: stabilising alpha vs. 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]-NH2, and stabilise 14-18 membered rings that mimic different kinds of non-regular secondary structures found in protein hotspots. 2D NMR spectra allowed determination of 3D structures for 14 cyclic tetrapeptides in water. Five formed two (i, i + 3) hydrogen bonds and a beta/gamma (6, 7) or beta (9, 19, 20) turn; eight formed one (i, i + 4) hydrogen bond and twisted into a non-helical (13, 18, 21, 22, 24) or helical (5, 17, 23) alpha turn; one was less structured (15). A beta or gamma turn was favoured for Z = Dab, Orn or Glu due to a χ1 gauche (+) rotamer, while an alpha turn was favoured for Z = Dap (but not X = Dap) due to a gauche (-) rotamer. Surprisingly, an unstructured peptide ARLARLARL could be twisted into a helix when either a helical or non-helical alpha turn (5, 13, 17, 18, 21-24) with Z = Dap was attached to the N-terminus. These structural models provide insights into stability for different turns and twists corresponding to non-regular folds in protein hotspots.

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 310-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. Cell uptake increases when hydrophobic residues and lactam linkers (i, i + 4) form a contiguous hydrophobic surface patch. Uptake increases further when both hydrophobic and positively charged (but not neutral or negative) residues are clustered into like surface patches. Amphipathicity alone is however insufficient for cell uptake of acyclic sequences. Changing the linker from lactam to hydrocarbon further increases uptake, but also promotes cell lysis. Helicity, positive charge and amphipathicity together promote cell permeability. Most known bioactive helical peptides do not optimally cluster residues for amphipathicity and so are likely unoptimised for cell uptake.

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 ß-turn between residues Glu9-Phe12 and a kinked alpha helix between Val16-Gly37. N-terminal turn constraints attenuated binding affinity and activity (compounds 1-8). Lys-Asp (i, i+4) crosslinks in the middle and at the C-terminus increased alpha helicity and cAMP stimulation without much effect on binding affinity or beta-arrestin 2 recruitment (compounds 9-18). Strategic positioning of helix-inducing constraints and amino acid substitutions (Tyr16, Ala22) increased peptide helicity and produced ten-fold higher cAMP potency (compounds 19-28) over GLP-1(7-37)-NH2. The most potent cAMP activator (compound 23) was also the most potent inducer of insulin secretion.

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. NMR structure analysis reveals a slight distortion from α-helicity at the C-terminal aspartate caused by torsional restraints imposed by the K(i)-D(i+4) lactam bridge. To investigate this effect on helix nucleation, the more water-soluble 2 was appended to N-, C-, or both termini of a palindromic peptide ARAARAARA (≤5 % helicity), resulting in 67, 92, or 100 % relative α-helicity, as calculated from CD spectra. From the C-terminus of peptides, 2 can nucleate at least six α-helical turns. From the N-terminus, imperfect alignment of the Asp5 backbone amide in 2 reduces helix nucleation, but is corrected by a second unit of 2 separated by 0-9 residues from the first. These cyclic peptides are extremely versatile helix nucleators that can be placed anywhere in 5-25 residue peptides, which correspond to most helix lengths in protein-protein interactions.

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 ß-turn and a C-terminal helix that differentially influenced affinity and agonist potency. These structures can inform development of small molecule agonists of the GLP-1 receptor to treat type 2 diabetes.

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. This likely anchors the receptor-activating N-terminus of nociception nearby for insertion in the membrane-spanning helices of ORL-1. In search of higher agonist potency, two lysine and two aspartate residues were strategically incorporated into the receptor-binding C-terminus of the nociceptin sequence and two Lys(i)→Asp(i+4) side chain-side chain condensations were used to generate lactam cross-links that constrained nociceptin into a highly stable α-helix in water. A cell-based assay was developed using natively expressed ORL-1 receptors on mouse neuroblastoma cells to measure phosphorylated ERK as a reporter of agonist-induced receptor activation and intracellular signaling. Agonist activity was increased up to 20-fold over native nociceptin using a combination of this helix-inducing strategy and other amino acid modifications. An NMR-derived three-dimensional solution structure is described for a potent ORL-1 agonist derived from nociceptin, along with structure-activity relationships leading to the most potent known α-helical ORL-1 agonist (EC50 40 pM, pERK, Neuro-2a cells) and antagonist (IC50 7 nM, pERK, Neuro-2a cells). These α-helix-constrained mimetics of nociceptin(1-17) had enhanced serum stability relative to unconstrained peptide analogues and nociceptin itself, were not cytotoxic, and displayed potent thermal analgesic and antianalgesic properties in rats (ED50 70 pmol, IC50 10 nmol, s.c.), suggesting promising uses in vivo for the treatment of pain and other ORL-1-mediated responses.

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.