Galectins, a class of carbohydrate-binding proteins, play a crucial role in various physiological and disease processes. Therefore, the identification of ligands that efficiently bind these proteins could potentially lead to the development of new therapeutic compounds. In this study, we present a method that involves screening synthetic click glycopeptide libraries to identify lectin-binding ligands with low micromolar affinity. Our methodology, initially optimized using Concanavalin A, was subsequently applied to identify binders for the therapeutically relevant galectin 1. Binding affinities were assessed using various methods and showed that the selected glycopeptides exhibited enhanced binding potency to the target lectins compared to the starting sugar moieties. This approach offers an alternative means of discovering galectin-binding ligands as well as other carbohydrate-binding proteins, which are considered important therapeutic targets.

• Klíčová slova
• click chemistry, glycopeptide libraries, lectins, split-and-mix, sugars,
• MeSH
• galektin 1 chemie   metabolismus   MeSH
• galektiny chemie   metabolismus   MeSH
• glykopeptidy * chemie   metabolismus   MeSH
• konkanavalin A chemie   metabolismus   MeSH
• lidé MeSH
• ligandy MeSH
• peptidová knihovna MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• galektin 1 MeSH
• galektiny MeSH
• glykopeptidy * MeSH
• konkanavalin A MeSH
• ligandy MeSH
• peptidová knihovna MeSH

The binding process of insulin to its transmembrane receptor entails a sophisticated interplay between two proteins, each possessing two binding sites. Given the difficulties associated with the use of insulin in the treatment of diabetes, despite its remarkable efficacy, there is interest in smaller and more stable compounds than the native hormone that would effectively activate the receptor. Our study adopts a strategy focused on synthesizing extensive combinatorial libraries of bipodal compounds consisting of two distinct peptides linked to a molecular scaffold. These constructs, evaluated in a resin bead-bound format, were designed to assess their binding to the insulin receptor. Despite notable nonspecific binding, our approach successfully generated and tested millions of compounds. Rigorous evaluations via flow cytometry and specific antibodies revealed peptide sequences with specific interactions at either receptor binding Site 1 or 2. Notably, these sequences bear similarity to peptides discovered through phage display by other researchers. This convergence of chemical and biological methods underscores nature's beauty, revealing general principles in peptide binding to the insulin receptor. Overall, our study deepens the understanding of molecular interactions in ligand binding to the insulin receptor, highlighting the challenges of targeting large proteins with small synthetic peptides.

• Klíčová slova
• insulin, library, peptide, receptor, scaffold,
• MeSH
• inzulin metabolismus   chemie   MeSH
• knihovny malých molekul chemie   farmakologie   chemická syntéza   MeSH
• lidé MeSH
• ligandy MeSH
• molekulární struktura MeSH
• peptidová knihovna MeSH
• peptidy chemie   metabolismus   chemická syntéza   MeSH
• receptor inzulinu * metabolismus   chemie   MeSH
• techniky kombinatorické chemie * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• inzulin MeSH
• knihovny malých molekul MeSH
• ligandy MeSH
• peptidová knihovna MeSH
• peptidy MeSH
• receptor inzulinu * MeSH

The development of highly active and selective enzyme inhibitors is one of the priorities of medicinal chemistry. Typically, various high-throughput screening methods are used to find lead compounds from a large pool of synthetic compounds, and these are further elaborated and structurally refined to achieve the desired properties. In an effort to streamline this complex and laborious process, new selection strategies based on different principles have recently emerged as an alternative. Herein, we compare three such selection strategies with the aim of identifying potent and selective inhibitors of human carbonic anhydrase II. All three approaches, in situ click chemistry, phage-display libraries and synthetic peptide libraries, led to the identification of more potent inhibitors when compared to the parent compounds. In addition, one of the inhibitor-peptide conjugates identified from the phage libraries showed greater than 100-fold selectivity for the enzyme isoform used for the compound selection. In an effort to rationalize the binding properties of the conjugates, we performed detailed crystallographic and NMR structural analysis, which revealed the structural basis of the compound affinity towards the enzyme and led to the identification of a novel exosite that could be utilized in the development of isoform specific inhibitors.

• Publikační typ
• časopisecké články MeSH

Human aldo-keto reductase 1C3 (AKR1C3) stereospecifically reduces steroids and prostaglandins and is involved in the biotransformation of xenobiotics. Its role in various cancers makes it a potential therapeutic target for the development of inhibitors. Recombinant AKR1C3 with a thrombin-cleavable N-terminal His6 tag was expressed from a pET-28(+) vector for structural studies of enzyme-inhibitor complexes. A modified in situ proteolysis approach was applied to specifically remove the His tag by thrombin cleavage during crystallization screening trials. This improved the morphology and diffraction quality of the crystals and allowed the acquisition of high-resolution diffraction data and structure solution. This approach may be generally applicable to other proteins expressed using the pET-28(+) vector.

• Klíčová slova
• 17β-hydroxysteroid dehydrogenase 5, His tags, aldo-keto reductase 1C3, diffraction-quality improvement, in situ proteolysis, pET-28(+),
• MeSH
• difrakce rentgenového záření metody   MeSH
• histidin * genetika   MeSH
• krystalizace metody   MeSH
• krystalografie rentgenová metody   MeSH
• lidé MeSH
• protein AKR1C3 chemie   genetika   metabolismus   MeSH
• proteolýza MeSH
• sekvence aminokyselin MeSH
• trombin metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AKR1C3 protein, human MeSH Prohlížeč
• histidin * MeSH
• protein AKR1C3 MeSH
• trombin MeSH

Three novel antimicrobial peptides (AMPs), named panurgines (PNGs), were isolated from the venom of the wild bee Panurgus calcaratus. The dodecapeptide of the sequence LNWGAILKHIIK-NH₂ (PNG-1) belongs to the category of α-helical amphipathic AMPs. The other two cyclic peptides containing 25 amino acid residues and two intramolecular disulfide bridges of the pattern Cys8-Cys23 and Cys11-Cys19 have almost identical sequence established as LDVKKIICVACKIXPNPACKKICPK-OH (X=K, PNG-K and X=R, PNG-R). All three peptides exhibited antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, antifungal activity, and low hemolytic activity against human erythrocytes. We prepared a series of PNG-1 analogs to study the effects of cationicity, amphipathicity, and hydrophobicity on the biological activity. Several of them exhibited improved antimicrobial potency, particularly those with increased net positive charge. The linear analogs of PNG-K and PNG-R having all Cys residues substituted by α-amino butyric acid were inactive, thus indicating the importance of disulfide bridges for the antimicrobial activity. However, the linear PNG-K with all four cysteine residues unpaired, exhibited antimicrobial activity. PNG-1 and its analogs induced a significant leakage of fluorescent dye entrapped in bacterial membrane-mimicking large unilamellar vesicles as well as in vesicles mimicking eukaryotic cell membrane. On the other hand, PNG-K and PNG-R exhibited dye-leakage activity only from vesicles mimicking bacterial cell membrane.

• MeSH
• antibakteriální látky chemie   farmakologie   MeSH
• antifungální látky chemie   farmakologie   MeSH
• hydrofobní a hydrofilní interakce MeSH
• Hymenoptera metabolismus   MeSH
• kationické antimikrobiální peptidy chemie   metabolismus   farmakologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• povrchově aktivní látky MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza proteinů MeSH
• unilamelární lipozómy metabolismus   MeSH
• včelí jedy chemie   metabolismus   farmakologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• antifungální látky MeSH
• kationické antimikrobiální peptidy MeSH
• povrchově aktivní látky MeSH
• unilamelární lipozómy MeSH
• včelí jedy MeSH

In the venom of eusocial bee Lasioglossum laticeps, we identified a novel unique antimicrobial peptide named lasiocepsin consisting of 27 amino acid residues and two disulfide bridges. After identifying its primary structure, we synthesized lasiocepsin by solid-phase peptide synthesis using two different approaches for oxidative folding. The oxidative folding of fully deprotected linear peptide resulted in a mixture of three products differing in the pattern of disulfide bridges. Regioselective disulfide bond formation significantly improved the yield of desired product. The synthetic lasiocepsin possessed antimicrobial activity against both Gram-positive and -negative bacteria, antifungal activity against Candida albicans, and no hemolytic activity against human erythrocytes. We synthesized two lasiocepsin analogs cyclized through one native disulfide bridge in different positions and having the remaining two cysteines substituted by alanines. The analog cyclized through a Cys8-Cys25 disulfide bridge showed reduced antimicrobial activity compared to the native peptide while the second one (Cys17-Cys27) was almost inactive. Linear lasiocepsin having all four cysteine residues substituted by alanines or alkylated was also inactive. That was in contrast to the linear lasiocepsin with all four cysteine residues non-paired, which exhibited remarkable antimicrobial activity. The shortening of lasiocepsin by several amino acid residues either from the N- or C-terminal resulted in significant loss of antimicrobial activity. Study of Bacillus subtilis cells treated by lasiocepsin using transmission electron microscopy showed leakage of bacterial content mainly from the holes localized at the ends of the bacterial cells.

• MeSH
• antibakteriální látky chemická syntéza   chemie   farmakologie   MeSH
• antifungální látky chemická syntéza   chemie   farmakologie   MeSH
• Candida albicans účinky léků   MeSH
• cyklické peptidy chemická syntéza   chemie   farmakologie   MeSH
• cystin chemická syntéza   chemie   MeSH
• erytrocyty účinky léků   MeSH
• gramnegativní bakterie účinky léků   MeSH
• grampozitivní bakterie účinky léků   ultrastruktura   MeSH
• hemolýza MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• molekulární sekvence - údaje MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza proteinů MeSH
• včelí jedy chemická syntéza   chemie   farmakologie   MeSH
• včely chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• antifungální látky MeSH
• cyklické peptidy MeSH
• cystin MeSH
• lasiocepsin MeSH Prohlížeč
• včelí jedy MeSH

Two novel antimicrobial peptides, named halictines, were isolated from the venom of the eusocial bee Halictus sexcinctus. Their primary sequences were established by ESI-QTOF mass spectrometry, Edman degradation and enzymatic digestion as Gly-Met-Trp-Ser-Lys-Ile-Leu-Gly-His-Leu-Ile-Arg-NH2 (HAL-1), and Gly-Lys-Trp-Met-Ser-Leu-Leu-Lys-His-Ile-Leu-Lys-NH2 (HAL-2). Both peptides exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria but also noticeable hemolytic activity. The CD spectra of HAL-1 and HAL-2 measured in the presence of trifluoroethanol or SDS showed ability to form an amphipathic alpha-helical secondary structure in an anisotropic environment such as bacterial cell membrane. NMR spectra of HAL-1 and HAL-2 measured in trifluoroethanol/water confirmed formation of helical conformation in both peptides with a slightly higher helical propensity in HAL-1. Altogether, we prepared 51 of HAL-1 and HAL-2 analogs to study the effect of such structural parameters as cationicity, hydrophobicity, alpha-helicity, amphipathicity, and truncation on antimicrobial and hemolytic activities. The potentially most promising analogs in both series are those with increased net positive charge, in which the suitable amino acid residues were replaced by Lys. This improvement basically relates to the increase of antimicrobial activity against pathogenic Pseudomonas aeruginosa and to the mitigation of hemolytic activity.

• MeSH
• antibakteriální látky chemie   izolace a purifikace   farmakologie   MeSH
• Bacteria účinky léků   MeSH
• erytrocyty účinky léků   MeSH
• hemolýza účinky léků   MeSH
• hemolyziny chemie   izolace a purifikace   farmakologie   MeSH
• kationické antimikrobiální peptidy chemie   izolace a purifikace   farmakologie   MeSH
• krysa rodu Rattus MeSH
• molekulární sekvence - údaje MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• včelí jedy chemie   MeSH
• včely chemie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• hemolyziny MeSH
• kationické antimikrobiální peptidy MeSH
• včelí jedy MeSH

A novel homologue of insect defensin designated lucifensin (Lucilia defensin) was purified from the extracts of various tissues (gut, salivary glands, fat body, haemolymph) of green bottle fly (Lucilia sericata) larvae and from their excretions/secretions. The primary sequence of this peptide of 40 residues and three intramolecular disulfide bridges was determined by ESI-QTOF mass spectrometry and Edman degradation and is very similar to that of sapecin and other dipteran defensins. We assume that lucifensin is the key antimicrobial component that protects the maggots when they are exposed to the highly infectious environment of a wound during the medicinal process known as maggot therapy. We also believe that lucifensin is that long-sought larger molecular weight antimicrobial factor of the Lucilia sericata excretions/secretions believed to be effective against pathogenic elements of the wound microbial flora.

• MeSH
• antiinfekční látky chemie   izolace a purifikace   farmakologie   MeSH
• defensiny chemie   izolace a purifikace   farmakologie   MeSH
• Diptera metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• hojení ran účinky léků   MeSH
• larva metabolismus   MeSH
• mikrobiální testy citlivosti MeSH
• molekulární sekvence - údaje MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antiinfekční látky MeSH
• defensiny MeSH

Three novel structurally related pentadecapeptides, named lasioglossins, were isolated from the venom of the eusocial bee Lasioglossum laticeps. Their primary sequences were established as H-Val-Asn-Trp-Lys-Lys-Val-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-I), H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-II) and H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Val-Lys-NH(2) (LL-III). These lasioglossins exhibited potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, low haemolytic and mast cell degranulation activity, and a potency to kill various cancer cells in vitro. The lasioglossin CD spectra were measured in the presence of trifluoroethanol and sodium dodecyl sulfate solution and indicated a high degree of alpha-helical conformation. NMR spectroscopy, which was carried out in trifluoroethanol/water confirmed a curved alpha-helical conformation with a concave hydrophobic and convex hydrophilic side. To understand the role of this bend on biological activity, we studied lasioglossin analogues in which the Gly in the centre of the molecule was replaced by other amino acid residues (Ala, Lys, Pro). The importance of the N-terminal part of the molecule to the antimicrobial activity was revealed through truncation of five residues from both the N and C termini of the LL-III peptide. C-terminal deamidation of LL-III resulted in a drop in antimicrobial activity, but esterification of the C terminus had no effect. Molecular modelling of LL-III and the observed NOE contacts indicated the possible formation of a bifurcated H-bond between hydrogen from the Lys15 CONH peptide bond and one H of the C-terminal CONH(2) to the Ile11 oxygen atom. Such interactions cannot form with C-terminal esterification.

• MeSH
• antiinfekční látky chemická syntéza   chemie   farmakologie   MeSH
• gramnegativní bakterie účinky léků   MeSH
• grampozitivní bakterie účinky léků   MeSH
• hemolýza účinky léků   MeSH
• kationické antimikrobiální peptidy chemie   farmakologie   MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• mastocyty účinky léků   metabolismus   MeSH
• mikrobiální testy citlivosti MeSH
• nádorové buněčné linie MeSH
• peptidy chemická syntéza   chemie   farmakologie   MeSH
• proliferace buněk účinky léků   MeSH
• protinádorové látky chemická syntéza   chemie   farmakologie   MeSH
• screeningové testy protinádorových léčiv MeSH
• včelí jedy chemie   MeSH
• včely chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antiinfekční látky MeSH
• kationické antimikrobiální peptidy MeSH
• lasioglossin I MeSH Prohlížeč
• lasioglossin II MeSH Prohlížeč
• lasioglossin III MeSH Prohlížeč
• peptidy MeSH
• protinádorové látky MeSH
• včelí jedy MeSH

A novel antimicrobial peptide designated melectin was isolated from the venom of the cleptoparasitic bee Melecta albifrons. Its primary sequence was established as H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Ala-His-Met-Lys-NH(2) by Edman degradation and ESI-QTOF mass spectrometry. Synthetic melectin exhibited antimicrobial activity against both gram-positive and -negative bacteria and it degranulated rat peritoneal mast cells, but its hemolytic activity was low. The CD spectra of melectin measured in the presence of trifluoroethanol and sodium dodecyl sulfate showed a high content alpha-helices, which indicates that melectin can adopt an amphipathic alpha-helical secondary structure in an anisotropic environment such as the bacterial cell membrane. To envisage the role of the proline residue located in the middle of the peptide chain on biological activity and secondary structure, we prepared several melectin analogues in which the Pro11 residue was either replaced by other amino acid residues or was omitted. The results of biological testing suggest that a Pro kink in the alpha-helical structure of melectin plays an important role in selectivity for bacterial cells. In addition, a series of N- and C-terminal-shortened analogues was synthesized to examine which region of the peptide is related to antimicrobial activity.

• MeSH
• antibakteriální látky chemická syntéza   izolace a purifikace   farmakologie   MeSH
• cirkulární dichroismus MeSH
• degranulace buněk účinky léků   MeSH
• erytrocyty účinky léků   MeSH
• gramnegativní bakterie účinky léků   MeSH
• grampozitivní bakterie účinky léků   MeSH
• hemolýza účinky léků   MeSH
• hydrofobní a hydrofilní interakce MeSH
• inhibiční koncentrace 50 MeSH
• kationické antimikrobiální peptidy chemická syntéza   izolace a purifikace   farmakologie   MeSH
• krysa rodu Rattus MeSH
• mastocyty účinky léků   fyziologie   MeSH
• mikrobiální testy citlivosti MeSH
• molekulární sekvence - údaje MeSH
• potkani Wistar MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• včelí jedy chemie   MeSH
• včely * MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• kationické antimikrobiální peptidy MeSH
• melectin MeSH Prohlížeč
• včelí jedy MeSH