Acid Residue Peptide Research Articles

Reinforcement learning-driven exploration of peptide space: accelerating generation of drug-like peptides.

Using amino acid residues in peptide generation has solved several key problems, including precise control of amino acid sequence order, customized peptides for property modification, and large-scale peptide synthesis. Proteins contain unknown amino acid residues. Extracting them for the synthesis of drug-like peptides can create novel structures with unique properties, driving drug development. Computer-aided design of novel peptide drug molecules can solve the high-cost and low-efficiency problems in the traditional drug discovery process. Previous studies faced limitations in enhancing the bioactivity and drug-likeness of polypeptide drugs due to less emphasis on the connection relationships in amino acid structures. Thus, we proposed a reinforcement learning-driven generation model based on graph attention mechanisms for peptide generation. By harnessing the advantages of graph attention mechanisms, this model effectively captured the connectivity structures between amino acid residues in peptides. Simultaneously, leveraging reinforcement learning's strength in guiding optimal sequence searches provided a novel approach to peptide design and optimization. This model introduces an actor-critic framework with real-time feedback loops to achieve dynamic balance between attributes, which can customize the generation of multiple peptides for specific targets and enhance the affinity between peptides and targets. Experimental results demonstrate that the generated drug-like peptides meet specified absorption, distribution, metabolism, excretion, and toxicity properties and bioactivity with a success rate of over 90$\%$, thereby significantly accelerating the process of drug-like peptide generation.

Discrimination of Aspartic and Isoaspartic Acid Residues in Peptides by Tandem Mass Spectrometry with Hydrogen Attachment Dissociation.

Long-lived proteins undergo chemical modifications that can cause age-related diseases. Among these chemical modifications, isomerization is the most difficult to identify. Isomerization often occurs at the aspartic acid (Asp) residues. In this study, we used tandem mass spectrometry equipped with a newly developed ion activation method, hydrogen attachment dissociation (HAD), to analyze peptides containing Asp isomers. Although HAD preferentially produces [cn + 2H]+ and [zm + 2H]+ via N-Cα bond cleavage, [cn + 58 + 2H]+ and [zm - 58 + 2H]+ originate from the fragmentation of the isoAsp residue. Notably, [cn + 58 + 2H]+ and [zm - 58 + 2H]+ could be used as diagnostic fragment ions for the isoAsp residue because these fragment ions did not originate from the Asp residue. The detailed fragmentation mechanism was investigated by computational analysis using density functional theory. According to the results, hydrogen attachment to the carbonyl oxygen in the isoAsp residue results in the Cα-Cβ bond cleavage. The experimental and theoretical joint study indicates that the present method allows us to discriminate Asp and isoAsp residues, including site identification of the isoAsp residue. Moreover, we demonstrated that the molar ratio of peptide isomers in the mixture could be estimated from their fragment ion abundance. Therefore, tandem mass spectrometry with HAD is a useful method for the rapid discrimination and semiquantitative analysis of peptides containing isoAsp residues.

The synthetic peptide, PnPP-15, derived from the PnTx2-6 toxin of the spider Phoneutria nigriventer, induces peripheral antinociception involving neprilysin, opioid, and cannabinoid systems

BackgroundThe PnTx2–6 toxin is isolated from the Phoneutria nigriventer spider venom. The synthetic peptide PnPP-19 is derived from this toxin and induces antinociception. When topically applied, it is cleaved by proteases generating a 15 amino acid residues peptide, PnPP-15. PnPP-15 showed an antinociceptive effect in neuropathic pain developed by streptozotocin-induced diabetic mice. The present work aimed to investigate if PnPP-15 is also effective against the inflammatory pain induced by PGE2. We also evaluated its cytotoxicity in vitro and studied its possible action mechanism in the pain pathway. MethodsThe nociceptive threshold was assessed using the mechanical paw pressure test. Paw tissue was collected for protein extraction and western blot analyses. The FRET enzymatic assay measured neprilysin activity. The two-electrode voltage clamp technique measured evoked electrical currents in Xenopus laevis oocytes. Cytotoxicity was measured using multiple cell lines. ResultsPnPP-15 has peripheral antinociceptive activity via µ-opioid and CB1 cannabinoid receptors and inhibits neprilysin. In addition, it induced direct activation of mu-opioid receptors expressed in Xenopus laevis oocytes. PnPP-15 was not cytotoxic against mammalian kidney, liver, heart, nerve, or lung cells. ConclusionThis work demonstrated the antinociceptive effect of PnPP-15 in an inflammatory pain model, suggesting its use as a possible tool for developing new analgesic drugs.

Introduction of a fatty acid chain modification to prolong circulatory half-life of a radioligand towards glucose-dependent insulinotropic polypeptide receptor

BackgroundThe beneficial role of glucose-dependent insulinotropic polypeptide receptor (GIPR) in weight control and maintaining glucose levels has led to the development of several multi-agonistic peptide drug candidates, targeting GIPR and glucagon like peptide 1 receptor (GLP1R) and/or the glucagon receptor (GCGR). The in vivo quantification of target occupancy by these drugs would accelerate the development of new drug candidates. The aim of this study was to evaluate a novel peptide (GIP1234), based on previously reported ligand DOTA-GIP-C803, modified with a fatty acid moiety to prolong its blood circulation. It would allow higher target tissue exposure and consequently improved peptide uptake as well as in vivo PET imaging and quantification of GIPR occupancy by novel drugs of interest. MethodA 40 amino acid residue peptide (GIP1234) was synthesized based on DOTA-GIP-C803, in turn based on the sequences of endogenous GIP and Exendin-4 with specific amino acid modifications to obtain GIPR selectivity. A palmitoyl fatty acid chain was furthermore added at Lys14 via a glutamic acid linker to prolong its blood circulation time by the interaction with albumin. GIP1234 was conjugated with a DOTA chelator at the C-terminal cysteine residue to achieve 68Ga radiolabeling. The resulting PET probe, [68Ga]Ga-DOTA-GIP1234 was evaluated for receptor binding specificity and selectivity using HEK293 cells transfected with human GIPR, GLP1R, or GCGR. Blocking experiments with tirzepatide (2 μM) were conducted using huGIPR HEK293 cells to investigate binding specificity. Ex vivo and in vivo organ distribution of [68Ga]Ga-DOTA-GIP1234 was studied in rats and a pig in comparison to [68Ga]Ga-DOTA-C803-GIP. Binding of [68Ga]Ga-DOTA-GIP1234 to albumin was assessed in situ using polyacrylamide gel electrophoresis (PAGE). The stability was tested in formulation buffer and rat blood plasma. Results[68Ga]Ga-DOTA-GIP1234 was synthesized with non-decay corrected radiochemical yield of 88 ± 3.7 % and radiochemical purity of 97.8 ± 0.8 %. The molar activity for the radiotracer was 8.1 ± 1.1 MBq/nmol. [68Ga]Ga-DOTA-GIP1234 was stable and maintained affinity to huGIPR HEK293 cells (dissociation constant (Kd) = 40 ± 12.5 nM). The binding of [68Ga]Ga-DOTA-GIP1234 to huGCGR and huGLP1R cells was insignificant. Pre-incubation of huGIPR HEK293 cell sections with tirzepatide resulted in the decrease of [68Ga]Ga-DOTA-GIP1234 binding by close to 90 %. [68Ga]Ga-DOTA-GIP1234 displayed slow blood clearance in pigs with SUV = 3.5 after 60 min. Blood retention of the tracer in rat was 2-fold higher than that of [68Ga]Ga-DOTA-C803-GIP. [68Ga]Ga-DOTA-GIP1234 also demonstrated strong liver uptake in both pig and rat combined with decreased renal excretion. The concentration dependent binding of [68Ga]Ga-DOTA-GIP1234 to albumin was confirmed in situ by PAGE. Conclusion[68Ga]Ga-DOTA-GIP1234 demonstrated nanomolar affinity and selectivity for huGIPR in vitro. Addition of a fatty acid moiety prolonged blood circulation time and tissue exposure in both rat and pig in vivo. However, the liver uptake was also increased which may make PET imaging of abdominal tissues such as pancreas challenging. The investigation of the influence of fatty acid moiety on the biological performance of the peptide ligand paved the way for further rational design of GIPR ligand analogues with improved characteristics.

Antibacterial and anticancer activity of two NK-lysin-derived peptides from the Antarctic teleost Trematomus bernacchii

The NK-lysin antimicrobial peptide, first identified in mammals, possesses both antibacterial and cytotoxic activity against cancer cell lines. Homologue peptides isolated from different fish species have been examined for their functional characteristics in the last few years. In this study, a NK-lysin transcript was identified in silico from the head kidney transcriptome of the Antarctic teleost Trematomus bernacchii. The corresponding amino acid sequence, slightly longer than NK-lysins of other fish species, contains six cysteine residues that in mammalian counterparts form three disulphide bridges. Real time-PCR analysis indicated its predominant expression in T. bernacchii immune-related organs and tissues, with greatest mRNA abundance detected in gills and spleen. Instead of focusing on the full T. bernacchii derived NK-lysin mature molecule, we selected a 27 amino acid residue peptide (named NKL-WT), corresponding to the potent antibiotic NK-2 sequence found in human NK-lysin. Moreover, we designed a mutant peptide (named NKL-MUT) in which two alanine residues substitute the two cysteines found in the NKL-WT. The two peptides were obtained by solid phase organic synthesis to investigate their functional features. NKL-WT and NKL-MUT displayed antibacterial activity against the human pathogenic bacterium Enterococcus faecalis and the ESKAPE pathogen Acinetobacter baumannii, respectively. Moreover, at the determined Minimum Inhibitory Concentration and Minimum Bactericidal Concentration values against these pathogens, both peptides showed high selectivity as they did not exhibit any haemolytic activity on erythrocytes or cytotoxic activity against mammalian primary cell lines. Finally, the NKL-MUT selectively triggers the killing of the melanoma cell line B16F10 by means of a pro-apoptotic pathway at a concentration range in which no effects were found in normal mammalian cell lines. In conclusion, the two peptides could be considered as promising candidates in the fight against antibiotic resistance and tumour proliferative action, and also be used as innovative adjuvants, either to decrease chemotherapy side effects or to enhance anticancer drug activity.

Peptide Diversification through Addition Reaction of Free Carboxylic Acids to Ynamides

Peptide modification has emerged as an important topic in the academic community and pharmaceutical industry. However, they are primarily focused on the diversification of amines, thiols, and alcohols. Direct and chemoselective modification of acid residues in peptides is relatively underdeveloped. In this context, we report a novel and efficient method for the direct functionalization of acid residues in peptides. By using ynamides as reaction partners, the adducts are rapidly obtained in moderate to excellent yields at room temperature in water. This approach shows excellent chemoselectivity and a broad scope including dipeptides bearing unprotected Trp or Tyr residue and free Ser or Gln residue.

Computational Investigation of Conformational Properties of Short Azapeptides: Insights from DFT Study and NBO Analysis.

Azapeptides have gained much attention due to their ability to enhance the stability and bioavailability of peptide drugs. Their structural preferences, essential to understanding their function and potential application in the peptide drug design, remain largely unknown. In this work, we systematically investigated the conformational preferences of three azaamino acid residues in tripeptide models, Ac-azaXaa-Pro-NHMe [Xaa = Asn (4), Asp (5), Ala (6)], using the popular DFT functionals, B3LYP and B3LYP-D3. A solvation model density (SMD) was used to mimic the solvation effect on the conformational behaviors of azapeptides in water. During the calculation, we considered the impact of the amide bond in the azapeptide models on the conformational preferences of models 4-6. We analyzed the effect of the HB between the side-chain main chain and main-chain main-chain on the conformational behaviors of azapeptides 4-6. We found that the predicted lowest energy conformation for the three models differs depending on the calculation methods. In the gas phase, B3LYP functional indicates that the conformers tttANP-1 and tttADP-1 of azapeptides 4 and 5 correspond to the type I of β-turn, the lowest energy conformation with all-trans amide bonds. Considering the dispersion correction, B3LYP-D3 functional predicts the conformers tctANP-2 and tctADP-3 of azapeptide 4 and 5, which contain the cis amide bond preceding the Pro residue, as the lowest energy conformation in the gas phase. The results imply that azaAsx and Pro residues may involve cis-trans isomerization in the gas phase. In water, the predicted lowest energy conformer of azapeptides 4 and 5 differs from the gas phase results and depends on the calculational method. For azapeptide 6, regardless of calculation methods and phases, tttAAP-1 (β-I turn) is predicted as the lowest energy conformer. The results imply that the effect of the side chain that can form HBs on the conformational preferences of azapeptides 4 and 5 may not be negligible. We compared the theoretical results of azaXaa-Pro models with those of Pro-azaXaa models, showing that incorporating azaamino acid residue in peptides at different positions can significantly impact the folding patterns and stability of azapeptides.

A class of peptides designed to replicate and enhance the Receptor for Hyaluronic Acid Mediated Motility binding domain

The extra-cellular matrix (ECM) is a complex and rich microenvironment that is exposed and over-expressed across several injury or disease pathologies. Biomaterial therapeutics are often enriched with peptide binders to target the ECM with greater specificity. Hyaluronic acid (HA) is a major component of the ECM, yet to date, few HA adherent peptides have been discovered. A class of HA binding peptides was designed using B(X7)B hyaluronic acid binding domains inspired from the helical face of the Receptor for Hyaluronic Acid Mediated Motility (RHAMM). These peptides were bioengineered using a custom alpha helical net method, allowing for the enrichment of multiple B(X7)B domains and the optimization of contiguous and non-contiguous domain orientations. Unexpectedly, the molecules also exhibited the behaviour of nanofiber forming self-assembling peptides and were investigated for this characteristic. Ten 23-27 amino acid residue peptides were assessed. Simple molecular modelling was used to depict helical secondary structures. Binding assays were performed with varying concentrations (1-10 mg/mL) and extra-cellular matrices (HA, collagens I-IV, elastin, and Geltrex). Concentration mediated secondary structures were assessed using circular dichroism (CD), and higher order nanostructures were visualized using transmission electron microscopy (TEM). All peptides formed the initial apparent 310/alpha-helices, yet peptides 17x-3, 4, BHP3 and BHP4 were HA specific and potent (i.e., a significant effect) binders at increasing concentrations. These peptides shifted from apparent 310/alpha-helical structures at low concentration to beta-sheets at increasing concentration and also formed nanofibers which are noted as self-assembling structures. Several of the HA binding peptides outperformed our positive control (mPEP35) at 3-4 times higher concentrations, and were enhanced by self-assembly as each of these groups had observable nanofibers. Statement of significanceSpecific biomolecules or peptides have played a crucial role in developing materials or systems to deliver key drugs and therapeutics to a broad spectrum of diseases and disorders. In these diseased tissues, cells build protein/sugar networks, which are uniquely exposed and great targets to deliver drugs to. Hyaluronic acid (HA) is involved in every stage of injury and is abundant in cancer. To date, only two HA specific peptides have been discovered. In our work, we have designed a way to model and trace binding regions as they appear on the face of a helical peptide. Using this method we have created a family of peptides enriched with HA binding domains that stick with 3-4 higher affinity than those previously discovered.

Proteomic Identification of Plasma Components in Tachypleus tridentatus and Their Effects on the Longitudinal Bone Growth Rate in Rats.

Tachypleus tridentatus (T. tridentatus) is a marine animal and traditional Chinese medicine. T. tridentatus plasma is a valuable resource for important medical and health-based functions. In this experiment, in order to evaluate the effect and mechanism of T. tridentatus plasma with respect to the promotion of bone tissue growth in rats, the processes of ultrafiltration and mass spectrometry were first used to separate and identify the components of T. tridentatus plasma. Then, a comparison of the effects of the T. tridentatus plasma samples, which each possessed different molecular weights, regarding the growth of the long bones of rats was conducted. Finally, transcriptomics, proteomics, and bioinformatics were all used to analyze the biological functions and related signaling pathways of the T. tridentatus plasma in order to promote rat bone growth. The results showed that the contents of amino acid residues in peptides are related to the growth promotion that was contained in the 10-30 kDa plasma group. Moreover, the T. tridentatus plasma samples were found to be higher in this respect than those in the whole plasma group. In addition, the 10-30 kDa plasma group could significantly promote bone growth activity in rats. The proteomic analysis showed that the proteins that were differentially expressed in the 10-30 kDa plasma group were mainly enriched in the PI3K-AKT signal pathway. Our study suggested that the T. tridentatus plasma possesses promising potential for the purposes of clinical use, whereby it can serve the role of a growth-promoting agent.

A New Regioselective Synthesis of the Cysteine-Rich Peptide Linaclotide.

Linaclotide is a 14-amino acid residue peptide approved by the FDA for the treatment of irritable bowel syndrome with constipation (IBS-C), which activates guanylate cyclase C to accelerate intestinal transit. Here we show a new method for the synthesis of linaclotide through the completely selective formation of three disulfide bonds in satisfactory overall yields via mild oxidation reactions of the solid phase and liquid phase, using 4-methoxytrityl (Mmt), diphenylmethyl (Dpm) and 2-nitrobenzyl (O-NBn) protecting groups of cysteine as substrate, respectively.

Surface-enhanced Raman spectroscopy for drug discovery: peptide-RNA binding.

The ever-growing demand for new drugs highlights the need to develop novel cost- and time-effective techniques for drug discovery. Surface-enhanced Raman spectroscopy (SERS) is an emerging ultrasensitive and label-free technique that allows for the efficient detection and characterization of molecular interactions. We have recently developed a SERS platform for detecting a single protein molecule linked to a gold substrate (Almehmadi et al. Scientific Reports 2019). In this study, we extended the approach to probe the binding of potential drugs to RNA targets. To demonstrate the proof of concept, two 16-amino acid residue peptides with close primary structures and different binding affinities to the RNA CUG repeat related to myotonic dystrophy were tested. Three-microliter solutions of the RNA repeat with these peptides at nanomolar concentrations were probed using the developed approach, and the binding of only one peptide was demonstrated. The SER spectra exhibited significant fluctuations along with a sudden strong enhancement as spectra were collected consecutively from individual spots. Principal component analysis (PCA) of the SER spectral datasets indicated that free RNA repeats could be differentiated from those complexed with a peptide with 100% accuracy. The developed SERS platform provides a novel opportunity for label-free screening of RNA-binding peptides for drug discovery. Schematic representation of the SERS platform for drug discovery developed in this study.

Hit-to-Lead Short Peptides against Dengue Type 2 Envelope Protein: Computational and Experimental Investigations.

Data from the World Health Organisation show that the global incidence of dengue infection has risen drastically, with an estimated 400 million cases of dengue infection occurring annually. Despite this worrying trend, there is still no therapeutic treatment available. Herein, we investigated short peptide fragments with a varying total number of amino acid residues (peptide fragments) from previously reported dengue virus type 2 (DENV2) peptide-based inhibitors, DN58wt (GDSYIIIGVEPGQLKENWFKKGSSIGQMF), DN58opt (TWWCFYFCRRHHPFWFFYRHN), DS36wt (LITVNPIVTEKDSPVNIEAE), and DS36opt (RHWEQFYFRRRERKFWLFFW), aided by in silico approaches: peptide–protein molecular docking and 100 ns of molecular dynamics (MD) simulation via molecular mechanics using Poisson–Boltzmann surface area (MMPBSA) and molecular mechanics generalised Born surface area (MMGBSA) methods. A library of 11,699 peptide fragments was generated, subjected to in silico calculation, and the candidates with the excellent binding affinity and shown to be stable in the DI-DIII binding pocket of DENV2 envelope (E) protein were determined. Selected peptides were synthesised using conventional Fmoc solid-phase peptide chemistry, purified by RP-HPLC, and characterised using LCMS. In vitro studies followed, to test for the peptides’ toxicity and efficacy in inhibiting the DENV2 growth cycle. Our studies identified the electrostatic interaction (from free energy calculation) to be the driving stabilising force for the E protein–peptide interactions. Five key E protein residues were also identified that had the most interactions with the peptides: (polar) LYS36, ASN37, and ARG350, and (nonpolar) LEU351 and VAL354; these residues might play crucial roles in the effective binding interactions. One of the peptide fragments, DN58opt_8-13 (PFWFFYRH), showed the best inhibitory activity, at about 63% DENV2 plague reduction, compared with no treatment. This correlates well with the in silico studies in which the peptide possessed the lowest binding energy (−9.0 kcal/mol) and was maintained steadily within the binding pocket of DENV2 E protein during the MD simulations. This study demonstrates the use of computational studies to expand research on lead optimisation of antiviral peptides, thus explaining the inhibitory potential of the designed peptides.

Interaction of Aβ1-42 and Aβ1-40 with lipid membranes studied by circular dichroism and fluorescence spectroscopy

Aggregates of the amyloid β (Aβ) peptide exert neurotoxic effects that contribute to Alzheimer's disease. One of the mechanisms of Aβ toxicity is disruption of cellular ionic homeostasis either through modulation of existing ion channels or by de-novo formation of ion-conducting membrane pores. Aβ is present in human brain in various forms; the most abundant and toxic species are the 40- and 42-amino acid residue peptides, Aβ1-40 and Aβ1-42. Permeabilization of cellular membranes by Aβ1-42 has been established, but contradictory data have been reported for such activity of Aβ1-40.

Peptides as Therapeutic Agents for Atherosclerosis.

More than three decades ago, as a test for the amphipathic helix theory, an 18 amino acid residue peptide and its analogs were designed with no sequence homology to any of the exchangeable apolipoproteins. Based on the apolipoprotein A-I (the major protein component of high density lipoproteins, HDL) mimicking properties, they were termed as ApoA-I mimicking peptides. Several laboratories around the world started studying such de novo-designed peptides for their antiatherogenic properties. The present chapter describes the efforts in bringing these peptides as therapeutic agents for atherosclerosis and several lipid-mediated disorders.

Novel disintegrin-like peptides derived from an amphibian skin cDNA sequence of Hypsiboas punctatus.

Disintegrins comprise a family of small proteins that bind to and alter the physiological function of integrins, especially integrins that mediate platelet aggregation in blood. Here, we report a lysine-glycine-aspartic acid (KGD) disintegrin-like motif present in a 15-amino acid residue peptide identified in a cDNA library of the amphibian Hypsiboas punctatus skin. The original peptide sequence was used as a template from which five new analogs were designed, chemically synthesized by solid phase, and tested for disintegrin activity and tridimensional structural studies using NMR spectroscopy. The original amphibian peptide had no effect on integrin-mediated responses. Nevertheless, derived peptide analogs inhibited integrin-mediated platelet function, including platelet spreading on fibrinogen.

Oxidative Damage of S‐Containing Amino Acids by the Environmental Radical NO3.: A Kinetic, Product and Computational Study

AbstractMethionine, cysteine and cystine, which were acetylated at the N‐terminus and methylated at the C‐terminus, react rapidly with the environmental nitrate radical (NO3.) with rate coefficients of 7.7×109, 3.4×109 and 2.0×109 M−1 s−1, respectively, in acetonitrile. Methionine (Ac−Met−OMe) is successively oxidized via the sulfoxide (Ac−MetO−Me) to the sulfone (Ac−MetO2−OMe), with the latter step being also extremely fast with a rate coefficient of 1.2×109 M−1 s−1. Computations predict formation of an initial charge transfer complex between NO3. and the S or SO moiety in Ac‐Met‐OMe, Ac‐MetO‐OMe and cystine (Ac−Cys(S−S)Cys‐OMe), respectively, which is followed by simultaneous S−O bond formation and NO2. expulsion. Calculations for the reaction of cysteine (Ac‐Cys‐OMe) with NO3. revealed side‐chain oxidation to give a thiyl radical or a sulfenic acid as likely pathways. These findings highlight the potential harmful impact of NO2./O3 pollution on S‐containing amino acid residues in peptides.

Ischemia-induced retinal injury is attenuated by Neurovespina, a peptide from the venom of the social wasp Polybia occidentalis

Neurovespina is a synthetic peptide modified from Occidentalin-1202, a nine amino acid residue peptide isolated from the venom of the social wasp Polybia occidentalis. Previous studies showed that this peptide has a neuroprotective effect on the central nervous system, but its action on the eye has not been explored. So, the objective of this work was to investigate the neuroprotective effect of Neurovespina on the retina and its angiogenic potential in the chicken chorioallantoic membrane (CAM). Retinal ischemia was induced in rats by acute elevation of intraocular pressure (IOP). Electroretinography (ERG) measurements, histopathological and immunohistochemical analysis, and transmission electronic microscopy (TEM) records were performed to check the neuroprotection effect of Neurovespina in the retina of the animals. The angiogenic activity of the peptide was investigated by CAM assay. The results showed that Neurovespina was able to reduce the effects induced by ischemic injury, preventing the reduction of a- and b-waves in the scotopic ERG. Histopathological and immunohistochemistry assays showed that Neurovespina, mainly at 60 μg/ml, protected all layers of the retina. The CAM assay revealed that the peptide promoted the reduction of CAM vessels. So, Neurovespina was able to protect retinal cells from ischemic insult and has an antiangiogenic effect, which can be considered as a promising neuroprotective agent for intravitreal application.

Modulation of co-stimulatory signal from CD2-CD58 proteins by a grafted peptide.

Peptides were designed to inhibit the protein-protein interaction of CD2 and CD58 to modulate the immune response. This work involved the design and synthesis of eight different peptides by replacing each amino acid residue in peptide 6 with alanine as well as grafting the peptide to the sunflower trypsin-inhibitor framework. From the alanine scanning studies, mutation at position 2 of the peptide was shown to result in increased potency to inhibit cell adhesion interactions. The most potent peptide from the alanine scanning was further studied for its detailed three-dimensional structure and binding to CD58 protein using surface plasmon resonance and flow cytometry. This peptide was used to graft to the sunflower trypsin inhibitor to improve the stability of the peptide. The grafted peptide, SFTI-a1, was further studied for its potency as well as its thermal, chemical, and enzymatic stability. The grafted peptide exhibited improved activity compared to our previously grafted peptide and was stable against thermal and enzymatic degradation.

Characterization of Critical Residues in the Extracellular and Transmembrane Domains of the Endothelin Type B Receptor for Propagation of the Endothelin-1 Signal.

We have previously reported the crystal structures of endothelin-1 (ET-1)-bound, ligand-free, and antagonist bosentan-bound forms of the thermostabilized ET type B receptor (ETB). Although other agonist-bound structures of ETB have been determined, the interactions for high-affinity binding and ETB receptor activation, as well as the roles of rearrangement of the hydrogen-bond network surrounding the ligand in G protein activation, remain elusive. ET-1, a 21-amino acid residue peptide, plays fundamental roles in basal vascular tone, sodium balance, cell proliferation, and stress-responsive regulation. We studied the interactions between the ET-1(8-21) peptide and ETB in the ligand binding and activation of ETB using a series of Ala-substituted ET-1(8-21) analogues and the mutated ETB. We found that while D8, L17, D18, I20, and W21 were responsible for high-affinity binding and potent G protein activation, Y13 and F14 in the helical region of ET-1 are prerequisites for the full activation of ETB via interactions near the extracellular side. Furthermore, we introduced the mutation into the residues around the ET-1 binding pocket of ETB. The results showed that while S1843.35, W3366.48, N3787.45, and S3797.46 in a conserved polar network are required for full activation, N1191.50, D1472.50, and N3827.49 are essential for G protein activation via direct interactions after rearrangement upon ET-1 binding. These results demonstrate that both interactions near the extracellular side and within the transmembrane helices with ET-1 play crucial roles in the full activation of the ETB receptor.

Photochemically-Generated Silver Chloride Nanoparticles Stabilized by a Peptide Inhibitor of Cell Division and Its Antimicrobial Properties

The 40 amino acid residues peptide MciZ (Mother Cell Inhibitor of FtsZ) represents a promising antibacterial agent as it is an effective inhibitor of bacterial cell division, Z-ring formation and localization. However, its efficacy is limited to Gram-positive bacteria as MciZ is unable to penetrate into Gram-negative organisms. In this study, we report the synthesis of plasmonic silver chloride nanoparticles stabilized by MciZ (NP/MciZ). NP/MciZ were synthesized using a fast and green route under blue light irradiation. Electron microscopy showed that NP/MciZ were enveloped by a peptide layer responsible for colloidal stability. X-ray diffraction analysis showed that silver chloride nanoparticles were crystalline in nature with small proportion of metallic silver. NP/MciZ showed a dose-dependent cytotoxicity against mammalian VERO cells. However, NP/MciZ exhibited remarkable antibacterial activity against Gram-positive bacterium B. megaterium comparable to MciZ. Furthermore, NP/MciZ showed similar antimicrobial activity against the Gram-negative bacterium E. coli and the yeast C. albicans. The photochemically-generated NP/MciZ presented here is a new organic–inorganic hybrid nanomaterial and has potential for biomedical or other applications.