Kallikrein 5 (KLK5) is a serine protease expressed in the outer skin layers, where it regulates the barrier function by cleaving desmosomal proteins. Elevated KLK5 causes excessive proteolysis, leading to corneocyte overdesquamation and barrier compromise. Increased KLK5 activity has been linked to atopic dermatitis (AD), a chronic inflammatory disease affecting up to 20% of children, highlighting the need for therapies that restore barrier integrity. While many serine protease inhibitors have been developed, most lack KLK5 selectivity. To address this, novel analogues of the sunflower trypsin inhibitor were designed and evaluated. Lead 7 emerged as a potent (IC50 = 14 ± 4 nM; Ki = 11 nM) selective KLK5 inhibitor. In keratinocytes from a Netherton syndrome patient, lead 7 significantly reduced the KLK5 activity and improved epithelial barrier integrity, as shown by transepithelial electrical resistance. These findings suggest lead 7 as a potential therapy for AD and other conditions with elevated KLK5 activity.

Epidermal proteases, such as human tissue kallikrein-related peptidase, regulate skin barrier function through keratinocyte desquamation, antimicrobial defence, inflammatory response and barrier maintenance. It has been reported that the activity of kallikreins (KLKs) is upregulated in inflammatory skin disorders, including atopic dermatitis (AD; eczema). AD is a very common, chronic and relapsing inflammatory skin disease affecting 15–20% of children in the UK. Studies have shown that enhanced KLK5 results in AD-like skin architecture with an impaired skin barrier function. In this study, we aimed to develop a potent and selective inhibitor that targets KLK5 and restores epidermal barrier dysfunction in AD. Nature-based sunflower trypsin inhibitor 1 (SFTI), a small, circular peptide exhibiting wide-ranging serine protease inhibition can be engineered and modified to become a potent inhibitor specific for tissue kallikreins; our previous study also indicated that it was able to permeate through the outermost layer to exert its inhibitory action on serine proteases. In this study, we wanted to develop a more potent and specific inhibitor of KLK5 based on a lead SFTI analogue (‘analogue 6’) that was previously developed by our research group. Computer modelling was employed to assess the structure and binding affinity between KLK5 and SFTI-1. Designed SFTI analogues were then synthesized using solid phase peptide synthesis. The inhibitory effect was evaluated by its half maximal inhibitory concentration (IC50) against KLK5 using a 7-amido-4-methylcoumarin assay and further tested by protease-activated receptor 2-dependent intracellular calcium influx assay in keratinocytes. Computer modelling then indicated that an endogenous inhibitor was comparable with the analogue 6 we developed previously. Analogue 6 was further modified, based on the endogenous inhibitor structure, and synthesized. The new analogue achieved an IC50 of 14 nmol L–1 for KLK5, which was a fourfold increase in potency compared to analogue 6 (IC50 56 nmol L–1) and a 16-fold increase vs. SFTI-1 wild type (IC50 230 nmol L–1). Further, the new analogue could inhibit proteinase-activated receptor 2 (PAR2)-dependent intracellular calcium influx by modulating the activation of PAR2 receptor, a KLK5 downstream molecule in keratinocytes at a minimum inhibitor-to-enzyme molar ratio of 1:1. The novel potent inhibitor for KLK5 could be a potential therapeutic intervention for AD and other inflammatory skin conditions with enhanced KLK5.

Electricity is applied to native chemical ligation and related products. A 1 V potential applied to platinum electrodes in 0.15 M TCEP solution converts Cys to Ala in cyclic peptides, and cleaves the 2-mercapto-2-phenethyl acyl transfer auxiliary.

Homogeneous glycoprotein syntheses have become possible in the last decade due to advances in chemical ligation strategies, particularly Native Chemical Ligation (NCL). For native glycoproteins this still requires laborious and technically challenging syntheses of glycopeptide components, combined with multi-segment ligation reactions. Here we explore new reactions between sugar-linked acyl transfer auxiliaries and peptide thioesters. We show that native glycoproteins are difficult to produce using this approach but various related analogues are accessible. The results show that site-specific neoglycoconjugation is a viable route to simply glycosylated proteins, which may be extended using well-documented enzymatic processes.

Head-to-sidechain macrocylic peptides, and neoglycopeptides, were readily prepared by site-specific amidation of aspartic and glutamic acid sidechain hydrazides. Hydrazides, serving as latent thioesters, were introduced through regioselective opening of the corresponding Nα-Fmoc protected anhydride precursors.

Herein we report the first synthesis of protected boronocysteine. The target compound was prepared via copper-catalysed diaste-reoselective nucleophilic borylation of a sulfinimine. After deprotection to give the amine as the hydrochloride salt, four boronocysteine amide derivatives were prepared through reaction with a variety of different active acylating agents.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Microwave irradiation of 1,6-diynes, RC≡C(CH2)4C≡CR, with Fe(CO)5 in dimethylether leads to the facile and clean formation of cyclopentadienone complexes [{η4-C4R2C(O)C4H8}Fe(CO)3] in good yields resulting from a [2 + 2 + 1] cycloaddition. The molecular structures of three examples (R = Ph, 2,4-F2C6H3, 4-MeOC6H4) have been obtained. The addition of HBF4 leads to the clean and reversible formation of cationic hydroxycyclopentadienyl complexes [{η5-C4R2C(OH)C4H8}Fe(CO)3][BF4]. Sequential addition of hydroxide and acid has also been carried out in an attempt to prepare hydroxycyclopentadienyl–hydride complexes. These were largely unsuccessful but in one case a Shvo-type complex with a bridging hydride was detected by 1H NMR spectroscopy. Reasons for the differing behaviour of [{η4-C4(SiMe3)2C(O)C4H8}Fe(CO)3] and the related aryl-functionalised derivatives are considered.

BACKGROUND:Upregulation of kallikreins (KLKs) including KLK5 has been reported in atopic dermatitis (AD). KLK5 has biological functions that include degrading desmosomal proteins and inducing proinflammatory cytokine secretion through protease-activated receptor 2 (PAR2). However, due to the complex interactions between various cells in AD inflamed skin, it is difficult to dissect the precise and multiple roles of upregulated KLK5 in AD skin. OBJECTIVE:We investigated the effect of upregulated KLK5 on the expression of epidermal-related proteins and cytokines in keratinocytes and on skin architecture. METHODS:Lesional and nonlesional AD skin biopsies were collected for analysis of morphology and protein expression. The relationship between KLK5 and barrier-related molecules was investigated using an ex vivo dermatitis skin model with transient KLK5 expression and a cell model with persistent KLK5 expression. The influence of upregulated KLK5 on epidermal morphology was investigated using an in vivo skin graft model. RESULTS:Upregulation of KLK5 and abnormal expression of desmoglein 1 (DSG1) and filaggrin, but not PAR2 were identified in AD skin. PAR2 was increased in response to transient upregulation of KLK5, whereas persistently upregulated KLK5 did not show this effect. Persistently upregulated KLK5 degraded DSG1 and stimulated secretion of IL-8, IL-10, and thymic stromal lymphopoietin independent of PAR2 activity. With control of higher KLK5 activity by the inhibitor sunflower trypsin inhibitor G, restoration of DSG1 expression and a reduction in AD-related cytokine IL-8, thymic stromal lymphopoietin, and IL-10 secretion were observed. Furthermore, persistently elevated KLK5 could induce AD-like skin architecture in an in vivo skin graft model. CONCLUSIONS:Persistently upregulated KLK5 resulted in AD-like skin architecture and secretion of AD-related cytokines from keratinocytes in a PAR2 independent manner. Inhibition of KLK5-mediated effects may offer potential as a therapeutic approach in AD.

N→S Acyl transfer is a popular method for the postsynthesis production of peptide C α-thioesters for use in native chemical ligation and for the synthesis of head-to-tail cyclic peptides. Meanwhile thioester formation at the side chain of aspartic or glutamic acids, leading to tail-to-side-chain-cyclized species, is less common. Herein we explore the potential for cysteine to function as a latent thioester when appended to the side chain of glutamic acid. Initial insights gained through study of C-terminal β-alanine as a model for the increased chain length were ultimately applied to peptide macrocyclization. Our results emphasize the increased barrier to acyl transfer at the glutamic acid side chain and indicate how a slow reaction, facilitated by cysteine itself, may be accelerated by fine-tuning of the stereoelectronic environment.

It is not highly sophisticated, yet the N→S acyl transfer reaction of a native peptide sequence potentially fills an important technology gap. While several routes to synthetic peptide thioesters exist, only one is routinely applicable for biologically derived samples. Using the naturally occurring amino acid cysteine as the sole activator for N→S acyl transfer we have demonstrated transformation of synthetic and biologically derived precursors into thioesters for use in Native Chemical Ligation, providing a viable alternative for biological samples. Further refinement will be key to realising the full potential of this intriguing process, and increase the number of applications in peptide engineering and therapeutics.1 Introduction2 N→S acyl transfer in ‘normal’ peptide sequences3 Reduced reactivity of internal Xaa-Cys motifs as an advantage in head-to-tail peptide cyclisation4 Reduced reactivity of internal Xaa-Cys motifs as an advantage in modification and cyclisation of biologically produced precursors5 Hydrazinolysis of Xaa-Cys motifs and the acyl hydrazide as a stable thioester equivalent6 Rapid thioester formation via an N→Se acyl shift7 Outlook and conclusions

Tissue kallikreins (KLKs), in particular KLK5, 7 and 14 are the major serine proteases in the skin responsible for skin shedding and activation of inflammatory cell signaling. In the normal skin, their activities are controlled by an endogenous protein protease inhibitor encoded by the SPINK5 gene. Loss-of-function mutations in SPINK5 leads to enhanced skin kallikrein activities and cause the skin disease Netherton Syndrome (NS). We have been developing inhibitors based on the Sunflower Trypsin Inhibitor 1 (SFTI-1) scaffold, a 14 amino acids head-to-tail bicyclic peptide with a disulfide bond. To optimize a previously reported SFTI-1 analogue (I10H), we made five analogues with additional substitutions, two of which showed improved inhibition. We then combined those substitutions and discovered a variant (Analogue 6) that displayed dual inhibition of KLK5 (tryptic) and KLK7 (chymotryptic). Analogue 6 attained a tenfold increase in KLK5 inhibition potency with an Isothermal Titration Calorimetry (ITC) Kd of 20nM. Furthermore, it selectively inhibits KLK5 and KLK14 over seven other serine proteases. Its biological function was ascertained by full suppression of KLK5-induced Protease-Activated Receptor 2 (PAR-2) dependent intracellular calcium mobilization and postponement of Interleukin-8 (IL-8) secretion in cell model. Moreover, Analogue 6 permeates through the cornified layer of in vitro organotypic skin equivalent culture and inhibits protease activities therein, providing a potential drug lead for the treatment of NS.

Understanding the factors that influence N → S acyl transfer in native peptide sequences, and discovery of new reagents that facilitate it, will be key to expanding its scope and applicability. Here, through a study of short model peptides in thioester formation and cyclisation reactions, we demonstrate that a wider variety of Xaa-Cys motifs than originally envisaged are capable of undergoing efficient N → S acyl transfer. We present data for the relative rates of thioester formation and cyclisation for a representative set of amino acids, and show how this expanded scope can be applied to the production of the natural protease inhibitor Sunflower Trypsin Inhibitor-1 (SFTI-1).

3/4-Mercaptobenzyl sulfonates were investigated as aryl thiol catalysts for native chemical ligation (NCL). Whilst catalysing NCL processes at a similar rate to 4-mercaptophenyl acetic acid (MPAA), the increased polarity and solubility of 3-mercaptobenzyl sulfonate in particular may favour its selection as NCL catalyst in many instances.

Sunflower Trypsin Inhibitor (SFTI-1) analogues have been prepared from simple linear precursors produced either by chemical synthesis or following purification from Escherichia coli. We have shown, for the first time that these linear SFTI-1 derived peptide sequences can be converted to circular peptides via selective consecutive acyl transfer reactions, and that the products derived from synthetic and bacterial origin are identical. Preliminary analysis of the semi-synthetic SFTI-1 analogues confirmed SFTI-I10H as an inhibitor of Kallikrein-5 (KLK5) protease that could also mediate its action on human keratinocytes. The preliminary results obtained serve as a useful starting point for the biological production of SFTI-1 based, selective KLK5 inhibitors for the treatment of atopic dermatitis. (C) 2014 The Authors. Published by Elsevier Ltd.

Respiratory syncytial virus is a leading cause of lower respiratory tract illness among infants, the elderly and immunocompromised individuals. Currently, there is no effective vaccine or disease modifying treatment available and novel interventions are urgently required. Cathelicidins are cationic host defence peptides expressed in the inflamed lung, with key roles in innate host defence against infection. We demonstrate that the human cathelicidin LL-37 has effective antiviral activity against RSV in vitro, retained by a truncated central peptide fragment. LL-37 prevented virus-induced cell death in epithelial cultures, significantly inhibited the production of new infectious particles and diminished the spread of infection, with antiviral effects directed both against the viral particles and the epithelial cells. LL-37 may represent an important targetable component of innate host defence against RSV infection. Prophylactic modulation of LL-37 expression and/or use of synthetic analogues post-infection may represent future novel strategies against RSV infection.