Glyco‐Scan: Varying Glycosylation in the Sequence of the Peptide Hormone PYY3‐36 and Its Effect on Receptor Selectivity

The increasing prevalence of obesity worldwide calls for safe and highly efficacious satiety drugs. PYY3‐36 has been implicated in food intake regulation, and novel peptide analogues with high Y2 receptor‐subtype selectivity and potency have potential as drugs for the treatment of obesity. It has been hypothesized that PYY3‐36 associates with the plasma membrane prior to receptor activation such that the amphipathic α‐helix of PYY3‐36 possibly guides the C‐terminal pentapeptide into the correct conformation for receptor activation. Ala‐scans are used routinely to study the effect of individual amino acids in a given peptide sequence. Here we report the glyco‐scan of the peptide hormone PYY3‐36, in which hydroxyl side‐chain functionalities were glycosylated; in addition new glycosylation sites were introduced. An array of novel PYY3‐36 analogues with a glycan positioned in the water–membrane interface or in the N terminal were screened for Y‐receptor affinity and selectivity as well as metabolic stability. Interestingly, in contrast to the Y1 and Y4 receptors, the Y2 receptor readily accommodated glycosylations. Especially glycosylations in the α‐helical region of PYY3‐36 were favorable both in terms of Y‐receptor selectivity and endopeptidase resistance. We thus report several PYY3‐36 analogues with enhanced Y‐receptor selectivity. Our results can be used in the design of novel PYY analogues for the treatment of obesity. The glyco‐scan concept, as systematically demonstrated here, has the potential for a wider applicability.     

The Impact of Adrenomedullin Thr22 on Selectivity within the Calcitonin Receptor‐like Receptor/Receptor Activity‐Modifying Protein System

Adrenomedullin (ADM) is a peptide hormone of the calcitonin gene‐related peptide (CGRP) family. It is involved in the regulation of cardiovascular processes such as angiogenesis, vasodilation, and the reduction of oxidative stress. ADM mediates its effects by activation of the ADM‐1 and ‐2 receptors (AM₁R/AM₂R), but also activates the CGRP receptor (CGRPR) with reduced potency. It binds to the extracellular domains of the receptors with its C‐terminal binding motif (residues 41–52). The activation motif, consisting of a disulfide‐bonded ring structure (residues 16–21) and an adjacent helix (residues 22–30), binds to the transmembrane region and stabilizes the receptor conformation in the active state. While it was shown that the binding motif of ADM guides AM₁R selectivity, there is little information on the activation motif itself. Here, we demonstrate that Thr22 of ADM contributes to the selectivity. By using solid‐phase peptide synthesis and cAMP‐based signal transduction, we studied the effects of analogues in the activation motif of ADM on AM₁R and CGRPR activity. Our results indicate that Thr22 terminates the α‐helix and orients the ring segment by hydrogen bonding. Using olefin stapling, we showed that the α‐helical arrangement of the ring segment leads to decreased AM₁R activity, but does not affect CGRPR activation. These results demonstrate that the conformation of the ring segment of ADM has a strong impact on the selectivity within the receptor system.     

Iterative Nonproteinogenic 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 nonproteinogenic residues while retaining the target‐binding epitope by using iterative unnatural 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.     

A Parallel Semisynthetic Approach for Structure–Activity Relationship Studies of Peptide YY

The gut hormone peptide YY (PYY) is postprandially secreted from enteroendocrine L cells and is involved in the regulation of energy homeostasis. The N‐terminal truncated version PYY(3–36) decreases food intake and has potential as an anti‐obesity agent. The anorectic effect of PYY(3–36) is mediated through Y₂ receptors in the hypothalamus, vagus, and brainstem regions, and it is well known that the C‐terminal tetrapeptide sequence of PYY(3–36) is crucial for Y₂ receptor activation. The aim of this work was to develop a semisynthetic methodology for the generation of a library of C‐terminally modified PYY(3–36) analogues. By using an intein‐based expression system, PYY(3–29) was generated as a C‐terminal peptide α‐thioester. Heptapeptides bearing an N‐terminal cysteine and modifications at one of the four C‐terminal positions were synthesized in a 96‐well plate by parallel solid‐phase synthesis. In the plate format, an array of [Ala30]PYY(3–36) analogues were generated by ligation, desulfurization, and subsequent solid‐phase extraction. The generated analogues, in which either Arg33, Gln34, Arg35, or Tyr36 had been substituted with proteinogenic or non‐proteinogenic amino acids, were tested in a functional Y₂ receptor assay. Generally, substitutions of Tyr36 were better tolerated than modifications of Arg33, Gln34, and Arg35. Two analogues showed significantly improved Y₂ receptor selectivity; therefore, these results could be used to design new drug candidates for the treatment of obesity.     

Design of Lanthanide Fingers: Compact Lanthanide‐Binding Metalloproteins

Lanthanides have interesting chemical properties; these include luminescent, magnetic, and catalytic functions. Toward the development of proteins incorporating novel functions, we have designed a new lanthanide‐binding motif, lanthanide fingers. These were designed based on the Zif268 zinc finger, which exhibits a ββα structural motif. Lanthanide fingers utilize an Asp₂Glu₂ metal‐coordination environment to bind lanthanides through a tetracarboxylate peptide ligand. The iterative design of a general lanthanide‐binding peptide incorporated the following key elements: 1) residues with high α‐helix and β‐sheet propensities in the respective secondary structures; 2) an optimized big box α‐helix N‐cap; 3) a Schellman α‐helix C‐cap motif; and 4) an optional D ‐Pro‐Ser type II’ β‐turn in the β‐hairpin. The peptides were characterized for lanthanide binding by circular dichroism (CD), NMR, and fluorescence spectroscopy. In all instances, stabilization of the peptide secondary structures resulted in an increase in metal affinity. The optimized protein design was a 25‐residue peptide that was a general lanthanide‐binding motif; this binds all lanthanides examined in a competitive aqueous environment, with a dissociation constant of 9.3 μM for binding Er³⁺. CD spectra of the peptide‐lanthanide complexes are similar to those of zinc fingers and other ββα proteins. Metal binding involves residues from the N‐terminal β‐hairpin and the C terminal α‐helical segments of the peptide. NMR data indicated that metal binding induced a global change in the peptide structure. The D ‐Pro‐Ser type II’ β‐turn motif could be replaced by Thr–Ile to generate genetically encodable lanthanide fingers. Replacement of the central Phe with Trp generated genetically encodable lanthanide fingers that exhibited terbium luminescence greater than that of an EF‐hand peptide.     

Studies of the structure of the N-terminal domain from the Y4 receptor - a G protein-coupled receptor - and its interaction with hormones from the NPY family

Binding of peptide hormones to G protein-coupled receptors is believed to be mediated through formation of contacts of the ligands with residues of the extracellular loops of family 1 GPCRs. Here we have investigated whether additional binding sites exist within the N-terminal domain, as studied in the form of binding of peptides from the neuropeptide Y (NPY) family to the N terminus of the Y4 receptor (N-Y4). The N-terminal domain of the Y4 receptor has been expressed in isotopically enriched form and studied by solution NMR spectroscopy. The peptide is unstructured in solution, whereas a micelle-associated helical segment is formed in the presence of dodecylphosphocholine (DPC) or sodium dodecylsulfate (SDS). As measured by surface plasmon resonance (SPR) spectroscopy, N-Y4 binds with approximately 50 microM affinity to the pancreatic polypeptide (PP), a high-affinity ligand to the Y4 receptor, whereas binding to neuropeptide Y (NPY) and peptide YY (PYY) is much weaker. Residues critical for binding in PP and in N-Y4 have been identified by site-directed mutagenesis. The data indicate that electrostatic interactions dominate and that this interaction is mediated by acidic ligand and basic receptor residues. Residues of N-Y4 are likely to contribute to the binding of PP, and in addition might possibly also help to transfer the hormone from the membrane-bound state into the receptor binding pocket.     

Design of a Short Thermally Stable α‐Helix Embedded in a Macrocycle

Although helices play key roles in peptide–protein and protein–protein interactions, the helical conformation is generally unstable for short peptides (10–15 residues) in aqueous solution in the absence of their binding partners. Thus, stabilizing the helical conformation of peptides can lead to increases in binding potency, specificity, and stability towards proteolytic degradation. Helices have been successfully stabilized by introducing side chain‐to‐side chain crosslinks within the central portion of the helix. However, this approach leaves the ends of the helix free, thus leading to fraying and exposure of the non‐hydrogen‐bonded amide groups to solvent. Here, we develop a “capped‐strapped” peptide strategy to stabilize helices by embedding the entire length of the helix within a macrocycle, which also includes a semirigid organic template as well as end‐capping interactions. We have designed a ten‐residue capped‐strapped helical peptide that behaves like a miniprotein, with a cooperative thermal unfolding transition and T_m≈70 °C, unprecedented for helical peptides of this length. The NMR structure determination confirmed the design, and X‐ray crystallography revealed a novel quaternary structure with implications for foldamer design.     

Solution Structure of the Leader Sequence of the Patellamide Precursor Peptide,PatE1–34

The solution structure of the leader sequence of the patellamide precursor peptide was analysed by using CD and determined with NOE‐restrained molecular dynamics calculations. This leader sequence is highly conserved in the precursor peptides of some other cyanobactins harbouring heterocycles, and is assumed to play a role in targeting the precursor peptide to the post‐translational machinery. The sequence was observed to form an α‐helix spanning residues 13–28 with a hydrophobic surface on one side of the helix. This hydrophobic surface is proposed to be the site of the initial binding with modifying enzymes.     

Synthesis,Characterization, and Initial Biological Evaluation of [99mTc]Tc‐Tricarbonyl‐labeled DPA‐α‐MSH Peptide Derivatives for Potential Melanoma Imaging

α‐Melanocyte stimulating hormone (α‐MSH) derivatives target the melanocortin‐1 receptor (MC1R) specifically and selectively. In this study, the α‐MSH‐derived peptide NAP‐NS1 (Nle‐Asp‐His‐d ‐Phe‐Arg‐Trp‐Gly‐NH₂) with and without linkers was conjugated with 5‐(bis(pyridin‐2‐ylmethyl)amino)pentanoic acid (DPA‐COOH) and labeled with [^99mTc]Tc‐tricarbonyl by two methods. With the one‐pot method the labeling was faster than with the two‐pot method, while obtaining similarly high yields. Negligible trans‐chelation and high stability in physiological solutions was determined for the [^99mTc]Tc‐tricarbonyl–peptide conjugates. Coupling an ethylene glycol (EG)‐based linker increased the hydrophilicity. The peptide derivatives displayed high binding affinity in murine B16F10 melanoma cells as well as in human MeWo and TXM13 melanoma cell homogenates. Preliminary in vivo studies with one of the [^99mTc]Tc‐tricarbonyl–peptide conjugates showed good stability in blood and both renal and hepatobiliary excretion. Biodistribution was performed on healthy rats to gain initial insight into the potential relevance of the ^99mTc‐labeled peptides for in vivo imaging.     

α‐Keto Phenylamides as P1′‐Extended Proteasome Inhibitors

The major challenge for proteasome inhibitor design lies in achieving high selectivity for, and activity against, the target, which requires specific interactions with the active site. Novel ligands aim to overcome off‐target‐related side effects such as peripheral neuropathy, which is frequently observed in cancer patients treated with the FDA‐approved proteasome inhibitors bortezomib ( 1 ) or carfilzomib ( 2 ). A systematic comparison of electrophilic headgroups recently identified the class of α‐keto amides as promising for next generation drug development. On the basis of crystallographic knowledge, we were able to develop a structure–activity relationship (SAR)‐based approach for rational ligand design using an electronic parameter (Hammett’s σ) and in silico molecular modeling. This resulted in the tripeptidic α‐keto phenylamide BSc4999 [(S)‐3‐(benzyloxycarbonyl‐(S)‐leucyl‐(S)‐leucylamino)‐5‐methyl‐2‐oxo‐N‐(2,4‐dimethylphenyl)hexanamide, 6 a ], a highly potent (IC₅₀=38 nM ), cell‐permeable, and slowly reversible covalent inhibitor which targets both the primed and non‐primed sites of the proteasome’s substrate binding channel as a special criterion for selectivity. The improved inhibition potency and selectivity of this new α‐keto phenylamide makes it a promising candidate for targeting a wider range of tumor subtypes than commercially available proteasome inhibitors and presents a new candidate for future studies.     

Acetylcholine Promotes Binding of α‐Conotoxin MII at α3β2 Nicotinic Acetylcholine Receptors

α‐Conotoxin MII (α‐CTxMII) is a 16‐residue peptide with the sequence GCCSNPVCHLEHSNLC, containing Cys2–Cys8 and Cys3–Cys16 disulfide bonds. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel–ligand interactions on ligand‐binding affinity, homology models of the heteropentameric α₃β₂‐nAChR were constructed. The models were created in MODELLER with the aid of experimentally characterized structures of the Torpedo marmorata‐nAChR (Tm‐nAChR, PDB ID: 2BG9) and the Aplysia californica‐acetylcholine binding protein (Ac‐AChBP, PDB ID: 2BR8) as templates for the α₃‐ and β₂‐subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α‐CTxMII. The nAChR homology models described here bind ACh with binding energies commensurate with those of previously reported systems, and identify critical interactions that facilitate both ACh and α‐CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α₃β₂‐nAChR for α‐CTxMII with ACh bound to the receptor, and this was confirmed through two‐electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α‐CTxMIIs on nAChRs.     

Selective DNA‐Binding by Designed Bisbenzamidine‐Homeodomain Chimeras

We report the construction of conjugates between three variants of the helix 3 region of a Q50K engrailed homeodomain and bisbenzamidine minor‐groove DNA binders. The hybrid featuring the sequence of the native protein failed to bind to DNA; however, modifications that increased the α‐helical folding propensity of the peptide allowed specific DNA binding by a bipartite (major/minor groove) interaction.     

Galectin‐1–Asialofetuin Interaction Is Inhibited by Peptides Containing the Tyr‐Xxx‐Tyr Motif Acting on the Glycoprotein

Galectin‐1 (Gal‐1), a ubiquitous β‐galactoside‐binding protein expressed by various normal and pathological tissues, has been implicated in cancer and autoimmune/inflammatory diseases in consequence of its regulatory role in adhesion, cell viability, proliferation, and angiogenesis. The functions of Gal‐1 depend on its affinity for β‐galactoside‐containing glycoconjugates; accordingly, the inhibition of sugar binding blocks its functions, hence promising potential therapeutic tools. The Tyr‐Xxx‐Tyr peptide motifs have been reported to be glycomimetic sequences, mainly on the basis of their inhibitory effect on the Gal‐1–asialofetuin (ASF) interaction. However, the results regarding the efficacy of the Tyr‐Xxx‐Tyr motif as a glycomimetic inhibitor are still controversial. The present STD and trNOE NMR experiments reveal that the Tyr‐Xxx‐Tyr peptides studied do not bind to Gal‐1, whereas their binding to ASF is clearly detected. ¹⁵N,¹H HSQC titrations with ¹⁵N‐labeled Gal‐1 confirm the absence of any peptide–Gal‐1 interaction. These data indicate that the Tyr‐Xxx‐Tyr peptides tested in this work are not glycomimetics as they interact with ASF via an unrevealed molecular linkage.     

Discovery of α‐Substituted Imidazole‐4‐acetic Acid Analogues as a Novel Class of ρ1 γ‐Aminobutyric Acid Type A Receptor Antagonists with Effect on Retinal Vascular Tone

The ρ‐containing γ‐aminobutyric acid type A receptors (GABA_ARs) play an important role in controlling visual signaling. Therefore, ligands that selectively target these GABA_ARs are of interest. In this study, we demonstrate that the partial GABA_AR agonist imidazole‐4‐acetic acid (IAA) is able to penetrate the blood–brain barrier in vivo; we prepared a series of α‐ and N‐alkylated, as well as bicyclic analogues of IAA to explore the structure–activity relationship of this scaffold focusing on the acetic acid side chain of IAA. The compounds were prepared via IAA from l ‐histidine by an efficient minimal‐step synthesis, and their pharmacological properties were characterized at native rat GABA_ARs in a [³H]muscimol binding assay and at recombinant human α₁β₂γ_2S and ρ₁ GABA_ARs using the FLIPR? membrane potential assay. The (+)‐α‐methyl‐ and α‐cyclopropyl‐substituted IAA analogues ((+)‐ 6 a and 6 c , respectively) were identified as fairly potent antagonists of the ρ₁ GABA_AR that also displayed significant selectivity for this receptor over the α₁β₂γ_2S GABA_AR. Both 6 a and 6 c were shown to inhibit GABA‐induced relaxation of retinal arterioles from porcine eyes.     

Two‐Face,Two‐Turn α‐Helix Mimetics Based on a Cross‐Acridine Scaffold: Analogues of the Bim BH3 Domain

The design of a cross‐acridine scaffold mimicking the i, i+3, i+5, and i+7 residues distributed over a two‐face, two‐turn α‐helix is described. Docking studies and 2D ¹H,¹⁵N HSQC NMR spectroscopy provide compelling evidence that compound 3 d accurately reproduces the arrangement of four hotspots in the Bim BH3 peptide to permit binding to the Mcl‐1 and Bcl‐2 proteins (K_i 0.079 and 0.056 μM , respectively). Furthermore, the hotspot mutation could also be mimicked by individual or multiple deletions of side chains on the scaffold.     

Small‐Molecule Proteomimetic Inhibitors of the HIF‐1α–p300 Protein–Protein Interaction

The therapeutically relevant hypoxia inducible factor HIF‐1α–p300 protein–protein interaction can be orthosterically inhibited with α‐helix mimetics based on an oligoamide scaffold that recapitulates essential features of the C‐terminal helix of the HIF‐1α C‐TAD (C‐terminal transactivation domain). Preliminary SAR studies demonstrated the important role of side‐chain size and hydrophobicity/hydrophilicity in determining potency. These small molecules represent the first biophysically characterised HIF‐1α–p300 PPI inhibitors and the first examples of small‐molecule aromatic oligoamide helix mimetics to be shown to have a selective binding profile. Although the compounds were less potent than HIF‐1α, the result is still remarkable in that the mimetic reproduces only three residues from the 42‐residue HIF‐1α C‐TAD from which it is derived.     

Thermoresponsive poly(γ‐propyl‐l‐glutamate)‐graft‐(oligo ethylene glycol)s: Synthesis,characterization, and properties

A series of thermoresponsive poly(γ‐propyl‐_L‐glutamate)‐graft‐(oligo ethylene glycol)s (PPLG‐g‐OEGs) with different main‐chain and side‐chain lengths have been synthesized via copper‐mediated alkyne‐azide 1,3‐dipolar cycloaddition between poly(γ‐azidopropyl‐_L‐glutamate)s (PAPLG) and propargyl terminated oligo ethylene glycols (Pr‐OEGs). Fourier transform infrared spectrometer analysis revealed that PAPLG₁₀ adopted 39.4% β‐sheet, 47.4% α‐helix, and 13.2% random coil while PAPLG with longer main‐chain length (DP = 37 and 88) and PPLG‐g‐OEGs adopted exclusive α‐helix in the solid state. Circular dichroism analysis revealed that PPLG‐g‐OEGs adopted α‐helical conformations with helicities in the range of 50～100%. The thermoresponsive behaviors of PPLG‐g‐OEGs in water have been studied by dynamic light scattering. The polymer concentration, main‐/side‐chain length, and helicity collectively affected their cloud point temperatures. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 41022.     

Designing Anticancer Peptides by Constructive Machine Learning

Constructive (generative) machine learning enables the automated generation of novel chemical structures without the need for explicit molecular design rules. This study presents the experimental application of such a deep machine learning model to design membranolytic anticancer peptides (ACPs) de novo. A recurrent neural network with long short‐term memory cells was trained on α‐helical cationic amphipathic peptide sequences and then fine‐tuned with 26 known ACPs by transfer learning. This optimized model was used to generate unique and novel amino acid sequences. Twelve of the peptides were synthesized and tested for their activity on MCF7 human breast adenocarcinoma cells and selectivity against human erythrocytes. Ten of these peptides were active against cancer cells. Six of the active peptides killed MCF7 cancer cells without affecting human erythrocytes with at least threefold selectivity. These results advocate constructive machine learning for the automated design of peptides with desired biological activities.