C-terminal Amide Research Articles

Signaling Modulation via Minimal C-Terminal Modifications of Apelin-13.

Apelin is an endogenous peptide that is involved in many diseases such as cardiovascular diseases, obesity, and cancer, which has made it an attractive target for drug discovery. Herein, we explore the penultimate and final sequence positions of [Pyr1]-apelin-13 (Ape13) via C-terminal N α-alkylated amide bonds and the introduction of positive charges, potentially targeting the allosteric sodium pocket, by assessing the binding affinity and signaling profiles at the apelin receptor (APJ). Synthetic analogues modified within this segment of Ape13 showed high affinity (K i 0.12-0.17 nM vs Ape13 K i 0.7 nM), potent Gαi1 activation (EC50 Gαi1 0.4-0.9 nM vs Ape13 EC50 1.1 nM), partial agonist behavior disfavoring β-arrestin 2 recruitment for positively charged ligands (e.g., 49 (SBL-AP-058), EC50 β-arr2 275 nM, E max 54%) and high plasma stability for N-alkyl ligands (t 1/2 > 7 h vs Ape13 t 1/2 0.5 h). Combining the benefits of the N α-alkylated amide bond with the guanidino substitution in a constrained ligand led to 63 (SBL-AP-049), which displayed increased plasma stability (t 1/2 5.3 h) and strong reduction of β-arrestin 2 signaling with partial maximal efficacy (EC50 β-arr 864 nM, E max 48%), significantly reducing the hypotensive effect in vivo.

Elucidating the molecular determinants in the process of gastrin C-terminal pentapeptide amide end activating cholecystokinin 2 receptor by Gaussian accelerated molecular dynamics simulations.

Gastrin plays important role in stimulating the initiation and development of many gastrointestinal diseases through interacting with the cholecystokinin 2 receptor (CCK2R). The smallest bioactive unit of gastrin activating CCK2R is the C-terminal tetrapeptide capped with an indispensable amide end. Understanding the mechanism of this smallest bioactive unit interacting with CCK2R on a molecular basis could provide significant insights for designing CCK2R antagonists, which can be used to treat gastrin-related diseases. To this end, we performed extensive Gaussian accelerated molecular dynamics simulations to investigate the interaction between gastrin C-terminal pentapeptide capped with/without amide end and CCK2R. The amide cap influences the binding modes of the pentapeptide with CCK2R by weakening the electrostatic attractions between the C-terminus of the pentapeptide and basic residues near the extracellular domain in CCK2R. The C-terminus with the amide cap penetrates into the transmembrane domain of CCK2R while floating at the extracellular domain without the amide cap. Different binding modes induced different conformational dynamics of CCK2R. Residue pairs in CCK2R had stronger correlated motions when binding with the amidated pentapeptide. Key residues and interactions important for CCK2R binding with the amidated pentagastrin were also identified. Our results provide molecular insights into the determinants of the bioactive unit of gastrin activating CCK2R, which would be of great help for the design of CCK2R antagonists.

PS-BPB09-2: DEVELOPMENT OF LONG-ACTING HUMAN ADRENOMEDULLIN FC FUSION PROTEINS

Background: Human adrenomedullin (hAM) is a hypotensive peptide hormone with strong anti-inflammatory effects. AM consists of 52 amino acid residues with an amidated C-terminus and a ring structure formed by intramolecular disulfide bonds. Both of these structures are necessary for binding to receptors and to exert their biological functions. AM has also shown therapeutic effects in various animal disease models, including inflammatory bowel disease, ischemic heart disease, sepsis and stroke. However, the short half-life of native AM in the blood required continuous administration for treatment. To solve this, we have developed four human IgG1 and IgG4 Fc fusion proteins containing full-length human AM or AM(6–52). Methods: Recombinant Fc-AM derivatives were generated using mammalian cells. After that, we tested the biological activities of Fc-fusion AMs (Fc-AMs) to stimulate the intracellular accumulation of cAMP in HEK-293 cells stably expressing the AM1 receptor. Next, we compared IgG1-AM (6–52) and IgG4-AM (6–52) concentrations in the peripheral blood and tissues of rats after subcutaneous injection. In addition, the antihypertensive effect of Fc-AM was examined on spontaneously hypertensive rats (SHRs) which were given high salt diets. Results: We have developed four Fc-AMs, which are long-acting AM derivatives in mammalian cells. Fc-AMs produced in mammalian cells do not require refolding or amidation. Indeed, Fc-AMs could be measured by mAM assay, which recognizes the amidated form, indicating that Fc-AMs produced in mammalian cells were amidated. Fc-AMs, generated in a mammalian cell production system can therefore easily be produced in large amounts with few purification steps. The Fc-AMs stimulated intracellular cAMP production in cultured cells stably expressing the AM type I receptor in vitro. Fc-AM (6–52) induced higher cAMP levels for the receptor than Fc-AM. The pharmacokinetic study was performed using N-terminal deficient AM derivatives, which have strong receptor binding ability. Sufficient concentrations of IgG1-AM (6–52) and IgG4-AM (6–52) were observed in blood 14 days after a single subcutaneous administration. Tissue transfer to the kidney and small intestine was observed after administration of IgG1-AM (6–52) or IgG4-AM (6–52) to rats. In addition, the single subcutaneous administration of IgG4-AM (6–52) suppressed the increase in blood pressure in SHRs which were loaded with high salt diets. Conclusion: Fc-AM produced from mammalian cells is easily prepared and seems to be an effective new therapeutic agent for hypertension.

A Program for Predicting the Retention Time of Peptides with Post-Translational Modifications

This paper describes the Retention Time Predictor (RTP) program and web service for predicting the retention time of peptides on a chromatographic column in mass spectrometry experiments. Taking into account post-translational modifications of peptides the program represents a modification of the well-known SSRCalc version 3 (Krokhin, Anal. Chem. 2006, 78(22), 7785-7795). The values of retention coefficients for modified amino acid residues and the algorithm for calculating the isoelectric point value were from the pIPredict program (Skvortsov et al., Biomed. Chem. Res. Meth. 2021, 4(4), e00161). Modifications described in the program include (i) Tandem Mass Tag (TMT) and Isobaric Tags for Relative and Absolute Quantification (iTRAQ) labels; (ii) acetylation, formylation, and methylation of the N-terminal residue and/or lysine side chain; (iii) carbamidomethylation of cysteine, asparagine, and glutamic acid residues; (iv) oxidation and double oxidation of methionine and proline residues; (v) phosphorylation of serine, threonine, and tyrosine residues; (vi) C-terminal amidation of lysine and arginine residues; (vii) formation of propionamide with a cysteine residue. Retention coefficient estimation was based on data from 25 mass spectrometry experiments for which identification was performed from the raw data deposited in the ProteomeXchange database. The RTP program and web service are freely available at http://lpcit.ibmc.msk.ru/RTP.

One-pot and sustainable liquid-phase peptide extension for synthesis of C-terminal amidation peptides aided by small molecular tags

A series of phosphonate or aliphatic-based tags were developed to achieve sustainable peptide synthesis, resulting in over a 10-fold reduction in solvent usage compared to SPPS.

Quantitating α-amidated peptide degradation by separative technologies and ultra-high resolution mass spectrometry

α-amidation of peptides is a C-terminal modification allowing to improve half-life of therapeutic peptides. However, due to storage conditions, this C-terminal amide function can be partially hydrolyzed into a carboxylic acid moiety, resulting in a decrease of the molecule bioactivity. It is therefore necessary to be able to detect and quantify any trace of this degradation product when dealing with such class of peptides. For this purpose, LC-UV, CE-UV, LC/MS and LC/MS/MS are conventional analytical technologies used in pharmaceutical laboratories to monitor peptide integrity. Since the production of the carboxylated peptide issued from the amidated starting compound is scrutinized, ultra-high-resolution mass spectrometry was investigated without the recourse to any separative method (direct infusion mode) to achieve rapid straightforward relative quantitative analyses from the detected isotopic clusters of both peptides. Indeed, low or high-resolution MS instruments present in pharmaceutical laboratories (fitted with Q, IT or ToF mass analyzers) allow to distinguish both molecular ions that differ from 0.984 Da (NH2 formally substituted by OH). However, the analytical difficulty comes from the fact that the A+1 ion (13C contribution) of the amidated peptide and the A ion (12C contribution) of any produced carboxylated sequence are overlapping hampering the detection of minute amount of the hydrolyzed product. Actually, to differentiate these two isobaric ions, mass analyzers should possess at least a resolution of 90 000 for 7 residues peptides (molecular ions around 700–800 Da) which is presumably attainable with any FT-MS instrument. With an ultra-high-resolution mass spectrometer (FT-ICR MS) the observed fine isotopic structure displaying all expected isotopologues permitted in a single fast analysis to unambiguously detect both compounds (non-hydrolyzed and hydrolyzed one) and subsequently define the relative amount of the carboxylated peptide contaminant. Finally, these results were confronted with conventional measurements recorded with orthogonal methodologies such as LC-UV and CE-UV. The pro and cons of all quantitative strategies are discussed.

AI protein structure prediction-based modeling and mutagenesis of a protostome receptor and peptide ligands reveal key residues for their interaction

The protostome leucokinin (LK) signaling system, including LK peptides and their G protein-coupled receptors, has been characterized in several species. Despite the progress, molecular mechanisms governing LK peptide–receptor interactions remain to be elucidated. Previously, we identified a precursor protein for Aplysia leucokinin-like peptides (ALKs) that contains the greatest number of amidated peptides among LK precursors in all species identified so far. Here, we identified the first ALK receptor from Aplysia, ALKR. We used cell-based IP1 activation assays to demonstrate that two ALK peptides with the most copies, ALK1 and ALK2, activated ALKR with high potencies. Other endogenous ALK-derived peptides bearing the FXXWX-amide motif also activated ALKR to various degrees. Our examination of cross-species activity of ALKs with the Anopheles LK receptor was consistent with a critical role for the FXXWX-amide motif in receptor activity. Furthermore, we showed, through alanine substitution of ALK1, the highly conserved phenylalanine (F), tryptophan (W), and C-terminal amidation were each essential for receptor activation. Finally, we used an artificial intelligence–based protein structure prediction server (Robetta) and Autodock Vina to predict the ligand-bound conformation of ALKR. Our model predicted several interactions (i.e., hydrophobic interactions, hydrogen bonds, and amide-pi stacking) between ALK peptides and ALKR, and several of our substitution and mutagenesis experiments were consistent with the predicted model. In conclusion, our results provide important information defining possible interactions between ALK peptides and their receptors. The workflow utilized here may be useful for studying other ligand–receptor interactions for a neuropeptide signaling system, particularly in protostomes.

The neuropeptide galanin adopts an irregular secondary structure

Human galanin is a 30-residue neuropeptide targeted for development of analgesics, antidepressants, and anticonvulsants. While previous work from our group and others has already produced significant insights into galanin's N-terminal region, no extant structures of galanin in databases include its full-length sequence and the function of its C-terminus remains ambiguous. We report the NMR solution structure of full-length human galanin C-terminal amide, determined from 2D 1H–1H COSY, TOCSY, and ROESY NMR data. Galanin adopts an irregular helical structure across its N-terminus, likely the average of several coiling states. We present the NMR structure of a peptide encompassing the C-terminus of galanin as a stand-alone fragment. The C-terminus of full-length galanin appears to indirectly assist the intramolecular association of hydrophobic sidechains within its N-terminus, remotely rigidifying their position when compared to previously studied N-terminal galanin fragments. By contrast, there is flexibility in the C-terminus of galanin, characterized by two i to i + 2 hydrogen-bonded turns within an otherwise dynamic backbone. The C-terminal portion of the peptide renders it soluble, and plays a hitherto undescribed biophysical role in pre-organizing the galanin receptor binding epitope. We speculate that hydrophilic microdomains of signaling peptides, hormones, and perhaps intrinsically disordered proteins may also function similarly.

Revealing C-terminal peptide amidation by the use of the survival yield technique

α-amidation of peptide sequences is a common post-translational modification in the living world. Since the majority of these C-terminal amidated peptides are bioactive, there is hence a great interest to identify and characterize them from biological matrices and natural extracts. Regarding conventional separative methods dedicated to peptides (such as HPLC or CE), elution protocols must be carefully optimized hampering straightforward LC-MS analysis of complex samples. From a mass spectrometry point of view, they are difficult to pinpoint owing to the only 1 Da mass difference between the post-translational amidated and the corresponding native carboxylated forms producing overlapping isotopic contributions of both molecular ions. To circumvent this analytical difficulty, usage of energy-resolved tandem mass spectrometry experiments and of the survival yield technique was investigated. Pair of peptides were thus dissociated in positive and negative mode according to the survival yield technique, in MS2 and MS3 experiments, in order to separate them giving a reliable MS/MS methodology to detect such post-translationally modified sequence.

Observation of Heavy-Chain C-Terminal Amidation in Human Endogenous IgG

Therapeutic IgG mAbs expressed from Chinese hamster ovary (CHO) cells are known to contain three C-terminal variants in their heavy chains, namely, the unprocessed C-terminal lysine, the processed C-terminal lysine, and C-terminal amidation. Although the presence of C-terminal amidation in CHO-expressed IgGs is well studied, the biological impact of the variant on the safety and efficacy of biotherapeutics has not been well understood. To further our biological understanding of C-terminal amidation, we analyzed a series of IgG samples, including both endogenous human IgGs as well as recombinant IgGs of different subclasses expressed from both CHO and murine cell lines, for their heavy-chain C-terminal variants by LC-MS/MS based peptide mapping. The results demonstrate that heavy-chain C-terminal amidation is a common variant occurring in IgG of all four subclasses (IgG1, IgG2, IgG3 and IgG4). The variant is generally present in recombinant IgG mAbs expressed from CHO cell lines but not in IgG mAbs expressed from murine cell lines, whereas the IgGs expressed from murine cell lines contain a much larger amount of unprocessed C-terminal lysine. Additionally, a significant amount of heavy-chain C-terminal amidation is observed in endogenous human IgGs, indicating that small amount of the variant present in therapeutic IgGs does not pose a safety concern.

Structural variation between neuropeptide isoforms affects function in the lobster cardiac system

Neuronal responses to peptide signaling are determined by the specific binding of a peptide to its receptor(s). For example, isoforms of the same peptide family can drive distinct responses in the same circuit by having different affinities for the same receptor, by having each isoform bind to a different receptor, or by a combination of these scenarios. Small changes in peptide composition can alter the binding kinetics and overall physiological response to a given peptide. In the American lobster (Homarus americanus), native isoforms of C-type allatostatins (AST-Cs) usually decrease heartbeat frequency and alter contraction force. However, one of the three AST-C isoforms, AST-C II, drives a cardiac response distinct from the response elicited by the other two. To investigate the aspects of the peptide that might be responsible for these differential responses, we altered various features of each peptide sequence. Although the presence of an amide group at the end of a peptide sequence (amidation) is often essential for determining physiological function, we demonstrate that C-terminal amidation does not dictate the AST-C response in the lobster cardiac system. However, single amino acid substitution within the consensus sequence did account for many of the differences in specific response characteristics (e.g. contraction frequency or force).

Engineering and Structural Insights of a Novel BBI-like Protease Inhibitor Livisin from the Frog Skin Secretion.

The Bowman–Birk protease inhibitor (BBI) family is a prototype group found mainly in plants, particularly grasses and legumes, which have been subjected to decades of study. Recently, the discovery of attenuated peptides containing the canonical Bowman–Birk protease inhibitory motif has been detected in the skin secretions of amphibians, mainly from Ranidae family members. The roles of these peptides in amphibian defense have been proposed to work cooperatively with antimicrobial peptides and reduce peptide degradation. A novel trypsin inhibitory peptide, named livisin, was found in the skin secretion of the green cascade frog, Odorrana livida. The cDNA encoding the precursor of livisin was cloned, and the predicted mature peptide was characterized. The mature peptide was found to act as a potent inhibitor against several serine proteases. A comparative activity study among the native peptide and its engineered analogs was performed, and the influence of the P1 and P2′ positions, as well as the C-terminal amidation on the structure–activity relationship for livisin, was illustrated. The findings demonstrated that livisin might serve as a potential drug discovery/development tool.

Reductive site-selective atypical C,Z-type/N2-C2 cleavage allows C-terminal protein amidation.

Biomolecule environments can enhance chemistries with the potential to mediate and modulate self-modification (e.g., self-cleavage). While these enhanced modes are found in certain biomolecules (e.g., RNA ribozymes), it is more rare in proteins. Targeted proteolytic cleavage is vital to physiology, biotechnology, and even emerging therapy. Yet, purely chemically induced methods for the site-selective cleavage of proteins remain scarce. Here, as a proof of principle, we designed and tested a system intended to combine protein-enhanced chemistry with tag modification to enable synthetic reductive protein chemistries promoted by diboron. This reductively driven, single-electron chemistry now enables an operationally simple, site-selective cleavage protocol for proteins directed to readily accessible dehydroalanine (Dha) residues as tags under aqueous conditions and in cell lysates. In this way, a mild, efficient, enzyme-free method now allows not only precise chemical proteolysis but also simultaneous use in the removal of affinity tags and/or protein-terminus editing to create altered N- and C-termini such as protein amidation (─CONH2).

Determination of the Relationships between the Chemical Structure and Antimicrobial Activity of a GAPDH-Related Fish Antimicrobial Peptide and Analogs Thereof.

The structure–activity relationships and mode of action of synthesized glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-related antimicrobial peptides were investigated. Including the native skipjack tuna GAPDH-related peptide (SJGAP) of 32 amino acid residues (model for the study), 8 different peptide analogs were designed and synthesized to study the impact of net charge, hydrophobicity, amphipathicity, and secondary structure on both antibacterial and antifungal activities. A net positive charge increase, by the substitution of anionic residues or C-terminal amidation, improved the antimicrobial activity of the SJGAP analogs (minimal inhibitory concentrations of 16–64 μg/mL), whereas the alpha helix content, as determined by circular dichroism, did not have a very definite impact. The hydrophobicity of the peptides was also found to be important, especially for the improvement of antifungal activity. Membrane permeabilization assays showed that the active peptides induced significant cytoplasmic membrane permeabilization in the bacteria and yeast tested, but that this permeabilization did not cause leakage of 260 nm-absorbing intracellular material. This points to a mixed mode of action involving both membrane pore formation and targeting of intracellular components. This study is the first to highlight the links between the physicochemical properties, secondary structure, antimicrobial activity, and mechanism of action of antimicrobial peptides from scombrids or homologous to GAPDH.

基于精氨酸酶切的蛋白质C端肽段富集方法的优化及评估

基于聚合物的蛋白质C端反向富集策略是用于研究蛋白质C端最为广泛的策略之一。目前,基于胰蛋白酶(trypsin)切割精氨酸残基C端(ArgC型酶切)的蛋白C端组学方法对蛋白质C端的鉴定深度仍有待提高。为解决这一问题,该研究对此方法进行了优化和评估:建立了基于“V型”过滤装置的“一锅法”富集流程,避免了副反应的干扰,缩短了样本的制备时间;优化了蛋白水平乙酰化反应条件,最大限度地降低了丝氨酸、苏氨酸、酪氨酸残基上的副反应,提高了肽段鉴定的可信性;优化了基于固相萃取枪头膜片过滤柱(StageTip柱)的样品分离过程,使C端肽段的鉴定深度增加至原来的4倍。通过以上优化,按照肽段水平错误发现率(FDR)<0.01、离子分数(ion score)≥20,且C端带有乙醇胺修饰的数据筛选标准,从人HEK 293T细胞中共鉴定出696个蛋白质C端。若仅要求肽段水平FDR<0.01,鉴定数目进一步增加到933个,这是基于聚合物富集策略的蛋白质C端组学方法所得的最大数据集之一。探索了胰蛋白酶镜像酶(LysargiNase)切割精氨酸残基N端(ArgN型酶切)与不同肽段N端衍生化修饰组合对蛋白质C端鉴定数目和种类的影响,“LysargiNase酶切+肽段N端乙酰化”新策略在原有“胰蛋白酶酶切+肽段N端二甲基化”策略的基础上将鉴定蛋白质C端的种类提升了47%。综上,该研究通过对基于Arg型酶切的蛋白C端组学方法的优化,提升了C端肽段的鉴定深度,扩大了C端肽段鉴定的覆盖范围。该方法将有望成为系统性表征蛋白质C端的有力工具。

Synthetic Analogues of Huwentoxin-IV Spider Peptide With Altered Human NaV1.7/NaV1.6 Selectivity Ratios.

Huwentoxin-IV (HwTx-IV), a peptide discovered in the venom of the Chinese bird spider Cyriopagopus schmidti, has been reported to be a potent antinociceptive compound due to its action on the genetically-validated NaV1.7 pain target. Using this peptide for antinociceptive applications in vivo suffers from one major drawback, namely its negative impact on the neuromuscular system. Although studied only recently, this effect appears to be due to an interaction between the peptide and the NaV1.6 channel subtype located at the presynaptic level. The aim of this work was to investigate how HwTx-IV could be modified in order to alter the original human (h) NaV1.7/NaV1.6 selectivity ratio of 23. Nineteen HwTx-IV analogues were chemically synthesized and tested for their blocking effects on the Na+ currents flowing through these two channel subtypes stably expressed in cell lines. Dose-response curves for these analogues were generated, thanks to the use of an automated patch-clamp system. Several key amino acid positions were targeted owing to the information provided by earlier structure-activity relationship (SAR) studies. Among the analogues tested, the potency of HwTx-IV E4K was significantly improved for hNaV1.6, leading to a decreased hNaV1.7/hNaV1.6 selectivity ratio (close to 1). Similar decreased selectivity ratios, but with increased potency for both subtypes, were observed for HwTx-IV analogues that combine a substitution at position 4 with a modification of amino acid 1 or 26 (HwTx-IV E1G/E4G and HwTx-IV E4K/R26Q). In contrast, increased selectivity ratios (>46) were obtained if the E4K mutation was combined to an additional double substitution (R 26A/Y33W) or simply by further substituting the C-terminal amidation of the peptide by a carboxylated motif, linked to a marked loss of potency on hNaV1.6 in this latter case. These results demonstrate that it is possible to significantly modulate the selectivity ratio for these two channel subtypes in order to improve the potency of a given analogue for hNaV1.6 and/or hNaV1.7 subtypes. In addition, selective analogues for hNaV1.7, possessing better safety profiles, were produced to limit neuromuscular impairments.

New Intracellular Peptide Derived from Hemoglobin Alpha Chain Induces Glucose Uptake and Reduces Blood Glycemia.

Intracellular peptides were shown to derive from proteasomal degradation of proteins from mammalian and yeast cells, being suggested to play distinctive roles both inside and outside these cells. Here, the role of intracellular peptides previously identified from skeletal muscle and adipose tissues of C57BL6/N wild type (WT) and neurolysin knockout mice were investigated. In differentiated C2C12 mouse skeletal muscle cells, some of these intracellular peptides like insulin activated the expression of several genes related to muscle contraction and gluconeogenesis. One of these peptides, LASVSTVLTSKYR (Ric4; 600 µg/kg), administrated either intraperitoneally or orally in WT mice, decreased glycemia. Neither insulin (10 nM) nor Ric4 (100 µM) induced glucose uptake in adipose tissue explants obtained from conditional knockout mice depleted of insulin receptor. Ric4 (100 µM) similarly to insulin (100 nM) induced Glut4 translocation to the plasma membrane of C2C12 differentiated cells, and increased GLUT4 mRNA levels in epididymal adipose tissue of WT mice. Ric4 (100 µM) increased both Erk and Akt phosphorylation in C2C12, as well as in epididymal adipose tissue from WT mice; Erk, but not Akt phosphorylation was activated by Ric4 in tibial skeletal muscle from WT mice. Ric4 is rapidly degraded in vitro by WT liver and kidney crude extracts, such a response that is largely reduced by structural modifications such as N-terminal acetylation, C-terminal amidation, and substitution of Leu8 for DLeu8 (Ac-LASVSTV[DLeu]TSKYR-NH2; Ric4-16). Ric4-16, among several Ric4 derivatives, efficiently induced glucose uptake in differentiated C2C12 cells. Among six Ric4-derivatives evaluated in vivo, Ac-LASVSTVLTSKYR-NH2 (Ric4-2; 600 µg/kg) and Ac-LASVSTV[DLeu]TSKYR (Ric4-15; 600 µg/kg) administrated orally efficiently reduced glycemia in a glucose tolerance test in WT mice. The potential clinical application of Ric4 and Ric4-derivatives deserves further attention.

The eptinezumab:CGRP complex structure – the role of conformational changes in binding stabilization

ABSTRACT To further elucidate the mechanism of action and binding properties of eptinezumab to calcitonin gene-related peptide (CGRP), X-ray crystallography, computational alanine scanning, and molecular dynamics were used. X-ray diffraction data were collected to determine the three-dimensional structures of the unbound eptinezumab antigen-binding fragment (Fab) and the Fab:CGRP complex. Molecular dynamics simulations were performed to analyze the transition between uncomplexed and complex states. The amidated C-terminus of CGRP was shown to bind in a pocket formed by the Fab heavy and light chains. There was extensive contact between all six complementarity-determining regions (CDRs; composed of light-chain [L1, L2, and L3] and heavy-chain [H1, H2, H3]) of eptinezumab and CGRP. The complex demonstrated a high ligand-binding surface area dominated by aromatic residues. CDR L3 contains a disulfide bond that stabilizes the loop, contributes surface area to the binding pocket, and provides van der Waals contacts. Comparison of the uncomplexed and complex structures revealed motion near the binding cleft. The CDR loops H2 and H3 were displaced ~1.4–2.0 Å and residue H-Tyr33 changed conformation, creating a ‘latch-and-lock’ mechanism for binding CGRP and preventing dissociation. Computational alanine scanning of CGRP identified energetic ‘hot spots’ that contribute to binding energy; mutating these positions to residues in homologous neuropeptides resulted in unfavorable binding energies. The attributes of the Fab region and the conformational changes that occur in eptinezumab during binding to CGRP contribute to the specificity, durability, and strength of the interaction, and likely underlie the rapid and sustained migraine preventive effect observed in clinical studies.

On-Resin Ugi Reaction for C-Terminally Modified and Head-to-Tail Cyclized Antibacterial Peptides.

Here we report a method to synthesize C-terminally modified peptides on resin. A four-component Ugi reaction of isocyanide resin, an Fmoc-protected amino acid, an amine, and a 6-nitroveratrylaldehyde gives C-terminal photocaged peptide amides, which can be photolyzed to generate C-terminal peptide amides. Changing the amine component in the Ugi reaction gives peptides with different C-terminal modifications including substituted anilides, alkyne, and azide. By installing an N-terminal azide and C-terminal alkyne, we synthesized a head-to-tail cyclized antibacterial peptide through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The cyclized peptide exhibited higher proteolytic stability and antibacterial activity than the linear peptide.

MECHANISMS IN ENDOCRINOLOGY: The physiology of neuronostatin.

In 2008, the first evidence of a new hormone called neuronostatin was published. The hormone was discovered using a bioinformatic method and found to originate from the same preprohormone as somatostatin. This small peptide hormone of 13 amino acids and a C-terminal amidation was soon found to exert pleiotropic physiological effects. In animal studies, neuronostatin has been shown to reduce food intake and delay gastric emptying and gastrointestinal transit. Furthermore, neuronostatin has been shown to affect glucose metabolism by increasing glucagon secretion during situations when glucose concentrations are low. Additionally, neuronostatin has been shown to affect neural tissue and cardiomyocytes by suppressing cardiac contractility. The effects of neuronostatin have not yet been delineated in humans, but if the effects found in animal studies translate to humans it could position neuronostatin as a promising target in the treatment of obesity, hypertension and diabetes. In this review, we describe the discovery of neuronostatin and the current understanding of its physiological role and potential therapeutic applicability.