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Melittin, a natural antimicrobial peptide, has broad-spectrum antimicrobial activity. This has resulted in it gaining increasing attention as a potential antibiotic alternative; however, its practical use has been limited by its weak antimicrobial activity, high hemolytic activity, and low proteolytic stability. In this study, N-terminal fatty acid conjugation was used to develop new melittin-derived lipopeptides (MDLs) to improve the characteristics of melittin. Our results showed that compared with native melittin, the antimicrobial activity of MDLs was increased by 2 to 16 times, and the stability of these MDLs against trypsin and pepsin degradation was increased by 50 to 80%. However, the hemolytic activity of the MDLs decreased when the length of the carbon chain of fatty acids exceeded 10. Among the MDLs, the newly designed analog Mel-C8 showed optimal antimicrobial activity and protease stability. The antimicrobial mechanism studied revealed that the MDLs showed a rapid bactericidal effect by interacting with lipopolysaccharide (LPS) or lipoteichoic acid (LTA) and penetrating the bacterial cell membrane. In conclusion, we designed and synthesized a new class of MDLs with potent antimicrobial activity, high proteolytic stability, and low hemolytic activity through N-terminal fatty acid conjugation. [ABSTRACT FROM AUTHOR]

Melittin, a natural antimicrobial peptide, has broad-spectrum antimicrobial activity. This has resulted in it gaining increasing attention as a potential antibiotic alternative; however, its practical use has been limited by its weak antimicrobial activity, high hemolytic activity, and low proteolytic stability. In this study, N-terminal fatty acid conjugation was used to develop new melittin-derived lipopeptides (MDLs) to improve the characteristics of melittin. Our results showed that compared with native melittin, the antimicrobial activity of MDLs was increased by 2 to 16 times, and the stability of these MDLs against trypsin and pepsin degradation was increased by 50 to 80%. However, the hemolytic activity of the MDLs decreased when the length of the carbon chain of fatty acids exceeded 10. Among the MDLs, the newly designed analog Mel-C8 showed optimal antimicrobial activity and protease stability. The antimicrobial mechanism studied revealed that the MDLs showed a rapid bactericidal effect by interacting with lipopolysaccharide (LPS) or lipoteichoic acid (LTA) and penetrating the bacterial cell membrane. In conclusion, we designed and synthesized a new class of MDLs with potent antimicrobial activity, high proteolytic stability, and low hemolytic activity through N-terminal fatty acid conjugation.

Targeting effectual epitopes is essential for therapeutic antibodies to accomplish their desired biological functions. This study developed a competitive dual color fluorescence-activated cell sorting (FACS) to maturate a matrix metalloprotease 14 (MMP-14) inhibitory antibody. Epitope-specific screening was achieved by selection on MMP-14 during competition with N-terminal domain of tissue inhibitor of metalloproteinase-2 (TIMP-2) (nTIMP-2), a native inhibitor of MMP-14 binding strongly to its catalytic cleft. 3A2 variants with high potency, selectivity, and improved affinity and proteolytic stability were isolated from a random mutagenesis library. Binding kinetics indicated that the affinity improvements were mainly from slower dissociation rates. In vitro degradation tests suggested the isolated variants had half lives 6-11-fold longer than the wt. Inhibition kinetics suggested they were competitive inhibitors which showed excellent selectivity toward MMP-14 over highly homologous MMP-9. Alanine scanning revealed that they bound to the vicinity of MMP-14 catalytic cleft especially residues F204 and F260, suggesting that the desired epitope was maintained during maturation. When converted to immunoglobulin G, B3 showed 5.0 nM binding affinity and 6.5 nM inhibition potency with in vivo half-life of 4.6 days in mice. In addition to protease inhibitory antibodies, the competitive FACS described here can be applied for discovery and engineering biosimilars, and in general for other circumstances where epitope-specific modulation is needed.

Radiolabeled peptides play a key role in nuclear medicine to selectively deliver radionuclides to malignancies for diagnosis (imaging) and therapy. Yet, their efficiency is often compromised by low metabolic stability. The use of 1,4-disubstituted 1,2,3-triazoles (1,4-Tzs) as stable amide bond bioisosteres can increase the half-life of peptides in vivo while maintaining their biological properties. Previously, the amide-to-triazole substitution strategy was used for the stabilization of the pansomatostatin radioligand [ 111 In]In-AT2S, resulting in the mono-triazolo-peptidomimetic [ 111 In]In-XG1, a radiotracer with moderately enhanced stability in vivo and retained ability to bind multiple somatostatin receptor (SSTR) subtypes. However, inclusion of additional 1,4-Tz led to a loss of affinity towards SST 2 R, the receptor overexpressed by most SSTR-positive cancers. To enhance further the stability of [ 111 In]In-XG1, alternative modifications at the enzymatically labile position Thr 10 -Phe 11 were employed. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-peptide conjugates were synthesized with a 1,4-Tz (Asn 5 -Ψ[Tz]-Phe 6 ) and either a β-amino acid (β-Phe 11 ), reduced amide bond (Thr 10 -Ψ[NH]-Phe 11 ), or N-methylated amino acid (N-Me-Phe 11 ). Two of the new peptidomimetics were more stable in blood plasma in vitro than [ 111 In]In-XG1. Yet none of them retained high affinity towards SST 2 R. We demonstrate for the first time the combination of the amide-to-triazole substitution strategy with alternative stabilization methods to improve the metabolic stability of tumor-targeting peptides., (© 2024 The Author(s). Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

The poly-arginine peptides R18D and R18 represent novel potential neuroprotective treatments for acute ischaemic stroke. Here we examined whether R18D and R18 had any significant effects on the thrombolytic activity of alteplase (tPA) and tenecteplase (TNK) on clots formed from whole blood in an in vitro thrombolysis plate assay. R18D and R18 were examined at concentrations of 0.25, 0.5, 1, 2, 4, 8 and 16 µM during the 1-h thrombolytic assay. We also included the well-characterised neuroprotective NA-1 peptide as a control. R18D, R18 and NA-1 all reduced tPA or TNK percentage clot lysis by 0–9.35%, 0–3.44% and 0–4.8%, respectively. R18D, R18 and NA-1 had a modest and variable effect on the lag time, increasing the time to the commencement of thrombolysis by 0–9.9 min, 0–5.53 min and 0–7.16 min, respectively. Lastly, R18 and NA-1 appeared to increase the maximal activity of the thrombolysis reaction. In addition, the in vitro anti-excitotoxic neuroprotective efficacy of R18D and R18 was not affected by pre-incubation for 1–2 h or overnight with tPA or TNK, whereas only R18D retained high anti-excitotoxic neuroprotective efficacy when pre-incubated in a synthetic trypsin (TrypLE Express). The present in vitro findings suggest that neither R18D or R18 when co-administered with the thrombolytic inducing agents tPA or TNK are likely to have a significant impact when used clinically during clot thrombolysis and confirm the superior proteolytic stability of the R18D peptide. [ABSTRACT FROM AUTHOR]

Abstract SAMP-5 is a short histidine-derived antimicrobial peptidomimetic with pendant dialkylated tail. In this study, we evaluated the potential of SAMP-5 as an antimicrobial agent to combat multidrug-resistant gram-negative bacteria. SAMP-5 showed potent antimicrobial activity (minimum inhibitory concentration 16-64 μg/ml) comparable to melittin against multidrug-resistant Escherichia coli (MDREC) and multidrug-resistant (MDRPA). SAMP-5 displayed no cytotoxicity against three mammalian cells such as mouse macrophage RAW264.7, mouse embryonic fibroblast NIH-3T3, and human bone marrow SH-SY5Y cells at the concentration of 128 μg/ml. SAMP-5 showed resistance to proteolytic degradation with pepsin, trypsin, α-chymotrypsin, and proteinase K. Importantly, unlike ciprofloxacin, no antibiotic resistance against SAMP-5 arose for Pseudomonas aeruginosa during 7 days of serial passage at 0.5 × MIC. Moreover, SAMP-5 showed synergy or additive effects against MDRPA and MDREC, when it combined with chloramphenicol, ciprofloxacin, and oxacillin. Collectively, our results suggested that SAMP-5 is a promising alternative and adjuvant to treat infections caused by multidrug-resistant gram-negative bacteria.

Cyclic peptides are an attractive option as therapeutics due to their ability to disrupt crucial protein–protein interactions and their flexibility in display type screening strategies, but they come with their own bioanalytical challenges in metabolite identification. Initial amide hydrolysis of a cyclic peptide results in a ring opening event in which the sequence is linearized. Unfortunately, the mass of the singly hydrolyzed sequence is the same (M + 18.0106 Da) irrespective of the initial site of hydrolysis, or soft spot. Soft spot identification at this point typically requires time-consuming manual interpretation of the tandem mass spectra, resulting in a substantial bottleneck in the hit to lead process. To overcome this, derivatization using 2-pyridine carboxaldehyde, which shows high selectivity for the alpha amine on the N-terminus, was employed. This strategy results in moderate- to high-efficiency derivatization with a unique mass tag and diagnostic ions that serve to highlight the first amino acid in the newly linearized peptide. The derivatization method and analytical strategy are demonstrated on a whole cell lysate digest, and the soft spot identification strategy is shown with two commercially available cyclic peptides: JB1 and somatostatin. Effective utilization of the automated sample preparation and interpretation of the resulting spectra shown here will serve to reduce the hit-to-lead time for generating promising proteolytically stable peptide candidates. [ABSTRACT FROM AUTHOR]

An increase in the frequency of mycoses and spreading of multidrug-resistant fungal pathogens necessitates the search for new antifungal agents. Earlier, we isolated the novel defensin from lentil Lensculinaris seeds, designated as Lc-def, which inhibited the growth of phytopathogenic fungi. Here, we studied an antifungal activity of Lc-def against human pathogenic Candida species, structural stability of the defensin, and its immunomodulatory effects that may help to prevent fungal infection. We showed that Lc-def caused 50% growth inhibition of clinical isolates of Candida albicans, C. krusei, and C. glabrata at concentrations of 25–50 μM, but was not toxic to different human cells. The lentil defensin was resistant to proteolysis by C. albicans and was not cleaved during simulated gastroduodenal digestion. By using the multiplex xMAP assay, we showed for the first time for plant defensins that Lc-def increased the production of such essential for immunity to candidiasis pro-inflammatory cytokines as IL-12 and IL-17 at the concentration of 2 μM. Thus, we hypothesized that the lentil Lc-def and plant defensins in general may be effective in suppressing of mucocutaneous candidiasis due to their antifungal activity, high structural stability, and ability to activate a protective immune response.

SAMP-5 is a short histidine-derived antimicrobial peptidomimetic with pendant dialkylated tail. In this study, we evaluated the potential of SAMP-5 as an antimicrobial agent to combat multidrug-resistant gram-negative bacteria. SAMP-5 showed potent antimicrobial activity (minimum inhibitory concentration 16-64 μg/ml) comparable to melittin against multidrug-resistant Escherichia coli (MDREC) and multidrug-resistant (MDRPA). SAMP-5 displayed no cytotoxicity against three mammalian cells such as mouse macrophage RAW264.7, mouse embryonic fibroblast NIH-3T3, and human bone marrow SH-SY5Y cells at the concentration of 128 μg/ml. SAMP-5 showed resistance to proteolytic degradation with pepsin, trypsin, α-chymotrypsin, and proteinase K. Importantly, unlike ciprofloxacin, no antibiotic resistance against SAMP-5 arose for Pseudomonas aeruginosa during 7 days of serial passage at 0.5 × MIC. Moreover, SAMP-5 showed synergy or additive effects against MDRPA and MDREC, when it combined with chloramphenicol, ciprofloxacin, and oxacillin. Collectively, our results suggested that SAMP-5 is a promising alternative and adjuvant to treat infections caused by multidrug-resistant gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Cyclic peptides are an attractive option as therapeutics due to their ability to disrupt crucial protein–protein interactions and their flexibility in display type screening strategies, but they come with their own bioanalytical challenges in metabolite identification. Initial amide hydrolysis of a cyclic peptide results in a ring opening event in which the sequence is linearized. Unfortunately, the mass of the singly hydrolyzed sequence is the same (M + 18.0106 Da) irrespective of the initial site of hydrolysis, or soft spot. Soft spot identification at this point typically requires time-consuming manual interpretation of the tandem mass spectra, resulting in a substantial bottleneck in the hit to lead process. To overcome this, derivatization using 2-pyridine carboxaldehyde, which shows high selectivity for the alpha amine on the N-terminus, was employed. This strategy results in moderate- to high-efficiency derivatization with a unique mass tag and diagnostic ions that serve to highlight the first amino acid in the newly linearized peptide. The derivatization method and analytical strategy are demonstrated on a whole cell lysate digest, and the soft spot identification strategy is shown with two commercially available cyclic peptides: JB1 and somatostatin. Effective utilization of the automated sample preparation and interpretation of the resulting spectra shown here will serve to reduce the hit-to-lead time for generating promising proteolytically stable peptide candidates.

Therapeutic hypothermia represents a brain-protective strategy for multiple emergency situations, such as stroke or traumatic injury. Neurotensin (NT), which exerts its effects through activation of two G protein-coupled receptors, namely NTS1 and NTS2, induces a strong and long-lasting decrease in core body temperature after its central administration. Growing evidence demonstrates that NTS1 is the receptor subtype mediating the hypothermic action of NT. As such, potent NTS1 agonists designed on the basis of the minimal C-terminal NT(8-13) bioactive fragment have been shown to produce mild hypothermia and exert neuroprotective effects under various clinically relevant conditions. The high susceptibility of NT(8-13) to protease degradation (half-life 24 h; 16). Among them, the NT(8-13) analogs harboring the reduced amine bond combined with the unnatural amino acids TMSAla13 (4) and Sip10 (6) or the di-substitution Lys11 - TMSAla13 (12), D-Trp11-TMSAla13 (14), and Dmt11-Tle12 (16) produced sustained hypothermic effects (−3°C for at least 1 h). Importantly, we observed that hypothermia was mainly driven by the increased stability of the NT(8-13) derivatives, instead of the high binding-affinity at NTS1. Altogether, these results reveal the importance of the reduced amine bond in optimizing the metabolic properties of the NT(8-13) peptide and support the development of stable NTS1 agonists as first drug candidate in neuroprotective hypothermia.

[Display omitted] The permeation enhancers (PEs) sodium caprate (C10) and sodium N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC) have been utilized for the intestinal and gastric delivery of macromolecules, respectively. However, the potential of C10 for the gastric delivery of a peptide and the ability of SNAC to deliver other peptides to the stomach beyond semaglutide have not been investigated. In this study, we have developed and evaluated C10 and SNAC-containing erodible tablets for the gastricdelivery of a glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GIP/GLP1) dual agonist peptide (LY) in cynomolgus monkeys. We also evaluated the impact of release rates on the in vivo performance of C10 and SNAC. Furthermore, we compared the oral exposure of the LY peptide and semaglutide with different proteolytic stabilities using a SNAC erodible tablet. Additionally, we investigated the mechanism of action of SNAC for improving gastric absorption of the LY peptide via tissue distribution in monkey. C10 and SNAC tablets released the peptide and PE by erosion from the tablet surface with 100 % release within 60 min at pH 6.8. Following a single oral administration to monkeys, C10 and SNAC erodible tablets at 300 mg exhibited similar LY mean absolute oral bioavailability of 5.7 % and 4.2 %, respectively. The C10 immediate release capsule (500 mg) with faster dissolution profile (10 min) showed a decrease in the LY oral bioavailability; however, a faster dissolution profile (15 min) with erodible SNAC tablet resulted in a relatively higher LY oral bioavailability compared to the slow-release erodible tablets (60 min). Using SNAC as the PE, the combination of slow-release tablet design and LY peptide with higher pepsin stability resulted in about 4-fold higher mean oral bioavailability in the monkeys than semaglutide (4.2 % vs 1.2 %, respectively). In the monkey gastric tissue, SNAC was found to reduce tight junction protein levels and increase the peptide uptake into the gastric epithelium suggesting its permeation enhancing mechanism via both paracellular and transcellular pathways. Taking these data altogether, the enhanced proteolytic stability of the LY peptide combined with the optimal erodible tablets enabled the gastric delivery of the LY peptide with a higher oral bioavailability than semaglutide. [ABSTRACT FROM AUTHOR]

Natural backbone-cyclized proteins have an increased thermostability and resistance towards proteases, characteristics that have sparked interest in head-to-tail cyclization as a method to stability-enhance proteins used in diagnostics and therapeutic applications, for example. In this proof-of principle study, we have produced and investigated a head-to-tail cyclized and HER2-specific ZHER2:342 Affibody dimer. The sortase A-mediated cyclization reaction is highly efficient (>95%) under optimized conditions, and renders a cyclic ZHER3:342-dimer with an apparent melting temperature, Tm, of 68 °C, which is 3 °C higher than that of its linear counterpart. Circular dichroism spectra of the linear and cyclic dimers looked very similar in the far-UV range, both before and after thermal unfolding to 90 °C, which suggests that cyclization does not negatively impact the helicity or folding of the cyclic protein. The cyclic dimer had an apparent sub-nanomolar affinity (Kd ~750 pM) to the HER2-receptor, which is a ~150-fold reduction in affinity relative to the linear dimer (Kd ~5 pM), but the anti-HER2 Affibody dimer remained a high-affinity binder even after cyclization. No apparent difference in proteolytic stability was detected in an endopeptidase degradation assay for the cyclic and linear dimers. In contrast, in an exopeptidase degradation assay, the linear dimer was shown to be completely degraded after 5 min, while the cyclic dimer showed no detectable degradation even after 60 min. We further demonstrate that a site-specifically DyLight 594-labeled cyclic dimer shows specific binding to HER2-overexpressing cells. Taken together, the results presented here demonstrate that head-to-tail cyclization can be an effective strategy to increase the stability of an Affibody dimer.

The development of biomolecule-derived biocompatible scaffolds for drug delivery applications is an emerging research area. Herein, we have synthesized a series of nucleobase guanine (G) functionalized amino acid conjugates having different chain lengths to study their molecular self-assembly in the hydrogel state. The gelation properties have been induced by the correct choice of chain lengths of fatty acids present in nucleobase functionalized molecules. The effect of alkali metal cations, pH, and the concentration of nucleobase functionalized amino acid conjugates in the molecular self-assembly process has been explored. The presence of Hoogsteen hydrogen bonding interaction drives the formation of a G-quadruplex functionalized hydrogel. The DOSY nuclear magnetic resonance is also performed to evaluate the self-assembling behavior of the newly formed nucleobase functionalized hydrogel. The nanofibrillar morphology is responsible for the formation of a hydrogel, which has been confirmed by various microscopic experiments. The mechanical behaviors of the hydrogel were evaluated by rheological experiments. The in vitro biostability of the synthesized nucleobase amino acid conjugate is also investigated in the presence of hydrolytic enzymes proteinase K and chymotrypsin. Finally, the nucleobase functionalized hydrogel has been used as a drug delivery platform for the control and sustained pH-responsive release of vitamins B 2 and B 12 . This synthesized nucleobase functionalized hydrogel also exhibits noncytotoxic behavior, which has been evaluated by their in vitro cell viability experiment using HEK 293 and MCF-7 cell lines.

To improve the proteolytic stability of the lipase LIP2 from Yarrowia lipolytica , the peptide bonds susceptible to trypsin in LIP2 were analyzed by tandem mass spectrometry and redesigned by site-directed mutagenesis. Different variants of the enzyme were expressed in Pichia pastoris GS115 and their biochemical properties were subsequently investigated. Although most of the variants were still cleaved by trypsin, some of them did show an evident increase of resistance against proteolytic degradation. The most stable mutant was LIP2-C5, in which five trypsin-cleavage sites were replaced by non-preferred amino acids. Upon incubation with human trypsin for 80 min at 37°C, the mutant LIP2-C5 was found to retain >70% of its initial activity, compared to only 10% for the wild-type. [ABSTRACT FROM AUTHOR]

Playing pivotal roles in tumor growth and metastasis, matrix metalloproteinase‐14 (MMP‐14) is an important cancer target. Potent inhibitory Fab 3A2 with therapy‐desired high selectivity has been isolated from a synthetic antibody library carrying long CDR‐H3s. However, like many standard mechanism protease inhibitors, Fab 3A2 can be cleaved by high concentrations of MMP‐14 after extended incubation at acidic pH. Edman sequencing of generated 3A2 fragments indicated that cleavage occurred within its CDR‐H3 between residues N100h (P1) and L100i (P1'). To improve proteolytic stability of 3A2, three positions adjacent to its cleavage site (P1, P1', and P3') were subjected to site‐saturation mutagenesis (SSM). Mutations at P1' (L100i) resulted in loss of inhibition function, while screening of 3A2 Fab mutants at P1 (N100h) or P3' (A100k) positions identified four clones exhibiting improvements in both stability and inhibition potency. The majority of these mutants with improved stability were substitutions to either hydrophobic (Lue, Trp) or basic residues (Arg, Lys, His). Combinations of these beneficial mutations resulted in a double mutant N100hR/A100kR, which prolonged half‐life twofold with an inhibition potency KI of 6.6 nM. Enzyme kinetics and competitive ELISA suggested that N100hR/A100kR was a competitive inhibitor overlapping its binding epitope with that of nTIMP‐2. This study demonstrated that site‐directed mutagenesis at or near the cleavage position reduced proteolytic liability of standard mechanism protease inhibitors especially inhibitory antibodies. [ABSTRACT FROM AUTHOR]

A method for the solid-phase synthesis of the apelin-12 analog has been developed using the Fmoc methodology in combination with the temporary protection of the guanidine function of the arginine residues by protonation (salt formation) during the formation of the amide bond. Proton magnetic resonance spectroscopy has been used to compare the proteolytic stability of apelin-12 and its structural analog in human blood plasma. The half-life of the analog in plasma has been shown to be about three times longer than that of the natural peptide. [ABSTRACT FROM AUTHOR]

We recently designed a series of cationic deoxythymidine-based amphiphiles that mimic the cationic amphipathic structure of antimicrobial peptides (AMPs). Among these amphiphiles, ADG-2e and ADL-3e displayed the highest selectivity against bacterial cells. In this study, ADG-2e and ADL-3e were evaluated for their potential as novel classes of antimicrobial, antibiofilm, and anti-inflammatory agents. Minimum inhibitory concentrations of ADG-2e and ADL-3e against bacteria were determined using the broth microdilution method. Proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K was determined by radial diffusion and HPLC analysis. Biofilm activity was investigated using the broth microdilution and confocal microscopy. The antimicrobial mechanism was investigated by membrane depolarization, cell membrane integrity analysis, scanning electron microscopy (SEM), genomic DNA influence and genomic DNA binding assay. Synergistic activity was evaluated using checkerboard method. Anti-inflammatory activity was investigated using ELISA and RT-PCR. ADG-2e and ADL-3e showed good resistance to physiological salts and human serum, and a low incidence of drug resistance. Moreover, they exhibit proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K. ADG-2e and ADL-3e were found to kill bacteria by an intracellular target mechanism and bacterial cell membrane-disrupting mechanism, respectively. Furthermore, ADG-2e and ADL-3e showed effective synergistic effects when combined with several conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Importantly, ADG-2e and ADL-3e not only suppressed MDRPA biofilm formation but also effectively eradicated mature MDRPA biofilms. Furthermore, ADG-2e and ADL-3e drastically decreased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) gene expression and protein secretion in lipopolysaccharide (LPS)-stimulated macrophages, implying potent anti-inflammatory activity in LPS-induced inflammation. Our findings suggest that ADG-2e and ADL-3e could be further developed as novel antimicrobial, antibiofilm, and anti-inflammatory agents to combat bacterial infections. [Display omitted] [ABSTRACT FROM AUTHOR]

Four bis-lactam [i, i+4]-stapled peptides with d- or l-α-methyl-thialysines were constructed on a model peptide sequence derived from p110α[E545K] and subjected to circular dichroism (CD) and proteolytic stability assessment, alongside the corresponding bis-lactam [i, i+4]-stapled peptide with l-thialysine. The % α-helicity values of these four stapled peptides were found to be largely comparable to each other yet greater than that of the stapled peptide with l-thialysine. An l-α-methyl-thialysine-stapled peptide built on a model peptide sequence derived from ribonuclease A (RNase A) was also found to exhibit a greater % α-helicity than its l-thialysine-stapled counterpart. Moreover, a greater proteolytic stability was demonstrated for the l-α-methyl-thialysine-stapled p110α[E545K] and RNase A peptides than that of their respective l-thialysine-stapled counterparts.

Resistance to high temperature, acidic pH and proteolytic degradation during the pelleting process and in the digestive tract are important features of phytases as animal feed. The integration of insights from structural and in silico analyses into factors affecting thermostability, acid stability, proteolytic stability, catalytic efficiency and specific activity, as well as N-glycosylation, could improve the limitations of marginal stable biocatalysts with trade-offs between stability and activity. Synergistic mutations give additional benefits to single substitutions. Rigidifying the flexible loops or inter-molecular interactions by reinforcing non-bonded interactions or disulfide bonds, based on structural and roof mean square fluctuation (RMSF) analyses, are contributing factors to thermostability. Acid stability is normally achieved by targeting the vicinity residue at the active site or at the neighboring active site loop or the pocket edge adjacent to the active site. Extending the positively charged surface, altering protease cleavage sites and reducing the affinity of protease towards phytase are among the reported contributing factors to improving proteolytic stability. Remodeling the active site and removing steric hindrance could enhance phytase activity. N-glycosylation conferred improved thermostability, proteases degradation and pH activity. Hence, the integration of structural and computational biology paves the way to phytase tailoring to overcome the limitations of marginally stable phytases to be used in animal feeds.

The pathological role of zinc in Alzheimer's disease (AD) is not yet fully elucidated, but there is strong evidence that zinc homeostasis is impaired in the AD brain and that this contributes to disease pathogenesis. In this study we examined the effects of zinc on the proteolysis of synthetic Apolipoprotein E (ApoE), a protein whose allelic variants differentially contribute to the onset/progression of disease. We have demonstrated that zinc promotes the proteolysis (using plasma kallikrein, thrombin and chymotrypsin) of synthetic ApoE in an isoform-specific way (E4>E2 and E3), resulting in more ApoE fragments, particularly for ApoE4. In the absence of exogenous proteases there was no effect of metal modulation on either lipidated or non-lipidated ApoE isoforms. Thus, increased zinc in the complex milieu of the ageing and AD brain could reduce the level of normal full-length ApoE and increase other forms that are involved in neurodegeneration. We further examined human plasma samples from people with different ApoE genotypes. Consistent with previous studies, plasma ApoE levels varied according to different genotypes, with ApoE2 carriers showing the highest total ApoE levels and ApoE4 carriers the lowest. The levels of plasma ApoE were not affected by either the addition of exogenous metals (copper, zinc or iron) or by chelation. Taken together, our study reveals that zinc may contribute to the pathogenesis of AD by affecting the proteolysis of ApoE, which to some extent explains why APOE4 carriers are more susceptible to AD.

The recent approval of antibody-based therapy for targeting the clearance of amyloid plaques fuels the research in designing small molecules and peptide inhibitors to target the aggregation of Aβ-peptides. Here, we report that the 15-residue ααγ-hybrid peptide not only inhibits the aggregation of soluble Aβ 42 into fibrils but also disintegrates the aggregated Aβ 42 fibrils into smaller assemblies. Further, the hybrid peptide completely rescues neuronal cells from the toxicity of Aβ 42 at equimolar concentrations. The shorter 10- and 12-mer peptides showed weak aggregation inhibition activity, while the fully hydrophobic 15-mer ααγ-hybrid peptide analogue showed no aggregation inhibition activity. Further, the 15-mer ααγ-hybrid peptide showed resistance against trypsin digestion and also nontoxic to the neuronal cells. The CD revealed that the peptide upon interaction induces a helix-type conformation in the Aβ 42 . This is in sharp contrast to the β-sheet conformation of Aβ 42 upon incubation. The two-dimensional-NMR (2D-NMR) analysis revealed a large perturbation in the chemical shifts of residues at the N-terminus. The presence of 15-mer peptide at an equimolar concentration of Aβ 42 showed less tendency for aggregation and also exhibited nontoxicity to the neuronal cells. The results reported here may be useful in designing new therapeutics for Alzheimer's disease.

The focus of the presented work was on improvement of spirulina based phycocyanin (PhyC) extracts as a potential natural blue colorant by enhancement of color stability via polysaccharide complexation. Beet pectin (β-pectin), guar gum and soluble soy polysaccharides (SSPS) were assessed for their ability to protect the color of a commercial phycocyanin (PhyC) extract from thermal and proteolytic degradation. Both the beet pectin and guar gum studied formed stable and homogenous solutions with the PhyC extract; SSPS did not. Color change was assessed throughout by measurement of L, a, and b color space values (Hunter, 1948) before and after thermal or proteolytic treatment. Beet pectin improved color stability, over PhyC alone, with respect to heating at 65 °C (20 min) and proteolysis by Alcalase 2.4L, papain and bromelain. For these treatments beet pectin reduced color change values (ΔE ab ) from 13.0 ± 0.1 to 6.2 ± 0.4 following thermal treatment and from 12.7 ± 0.2 to 5.9 ± 0.7, 19.6 ± 1.3 to 12.5 ± 0.8, and 10.8 ± 0.4 to 4.6 ± 0.6 for the Alcalase, papain and bromelain treatments, respectively. Intrinsic fluorescence (excitation at 285 nm, emission at 325 nm) of beet pectin complexed PhyC indicates an increased presence of exposed tryptophan residues compared to PhyC alone. Furthermore, measured zeta-potentials following complex heating generally diverged to slightly more negative values for PhyC-beet pectin complexes at elevated temperatures compared to PhyC alone, suggesting that maintenance of a negatively charged environment may be important to pigment color stability in PhyC complexes. [ABSTRACT FROM AUTHOR]

To follow up on our recent discovery of the 18-amino acid all-hydrocarbon [i, i + 4]-stapled p110α[E545K] peptide 1  that was shown to potently block the intracellular p110α[E545K]-IRS1 interaction (a protein-protein interaction uniquely present in cancer cells expressing p110α[E545K]) and the growth of the xenograft tumors formed by cancers harboring this mutation, in the current study we prepared and examined six derivatives of 1 , i.e. stapled peptides 2-A , 2-B , 3-A , 3-B , 4-A , 4-B . We found that 2-A , 2-B , 4-A , and 4-B had higher % α-helicity than 1 ; moreover, the enhanced % α-helicity also led to an enhanced proteolytic stability. When compared with 1 , the structurally simplified 14-amino acid 4-A and 4-B were found to more potently deactivate the AKT phosphorylation at Ser473 in the p110α[E545K]-expressing colon cancer cells, whose activation was previously demonstrated by us to be specifically derived from the p110α[E545K]-IRS1 interaction. The preliminary findings from the current study have laid a foundation for future more extensive studies on the stapled p110α[E545K] peptides newly identified in the current study. [ABSTRACT FROM AUTHOR]

[Display omitted] • An engineered peptide zp80r showed bioactivity against all ESKAPE strains. • d -arginine substitution increased proteolytic stability of the synthetic peptide. • Peptide zp80r suppressed growth of persisters and cellular swarming. • Peptide zp80r inhibited bacterial contamination in chilled fresh pork. The co-existence of various pathogenic bacteria on the surface of pork products exacerbates difficulties in food safety control. Developing broad-spectrum and stable antibacterial agents that are not antibiotics is an unmet need. To address this issue, all l -arginine residues of a reported peptide (IIRR) 4 -NH 2 (zp80) were substituted with the corresponding D enantiomers. This novel peptide (IIrr) 4 -NH 2 (zp80r) was expected to maintain favourable bioactivity against ESKAPE strains and have enhanced proteolytic stability compared with zp80. In a series of experiments, zp80r maintained favourable bioactivities against starvation-induced persisters. Electron microscopy and fluorescent dye assays were used to verify the antibacterial mechanism of zp80r. Importantly, zp80r reduced bacterial colonies in chilled fresh pork contaminated with multiple bacterial species. This newly designed peptide is a potential antibacterial candidate to combat problematic foodborne pathogens during storage of pork. [ABSTRACT FROM AUTHOR]

Proteolytic stability of nisin-loaded pectin and nisin-loaded pectin-inulin particles, previously developed for food preservation, was investigated and compared with free nisin. The acid protease from Aspergillus saitoi and trypsin were used. The hydrolysis of free nisin by the mentioned proteases resulted in four and three peaks of degradation products, respectively, in the analysis by capillary zone electrophoresis method. The complexation of nisin with biopolymers increased its proteolytic stability. The areas of detected peaks significantly decreased. The stability of particles was dependent on the degree of pectin esterification. The particles prepared using pectic acid or the pectin with low degree of esterification exhibited the highest proteolytic stability. Overall, nisin-loaded pectin-inulin particles were more stable than nisin-loaded pectin particles. [ABSTRACT FROM AUTHOR]

Recently, we reported on the design of a multimodal peptide conjugate useful as delivery platform for targeting hypoxic cells. A nitroimidazole (2-(2-nitroimidazol-1-yl)acetic acid, NIA) moiety, which is selectively entrapped in hypoxic cells, was coupled to a cell-penetrating peptide serving as the transporter. Furthermore, attachment of a bifunctional linker allowed the introduction of a diagnostic or therapeutic radiometal. However, although selective tumor accumulation could be detected in vivo, a fast renal clearance of the compound was observed. The present study aims to improve the system by using the more proteolytically stable all- d version of the peptide carrier (DsC18), by attaching two NIA moieties instead of one (DsC18(NIA)2) to enhance the tumor uptake, and by incorporating the bifunctional chelator NODAGA instead of DOTA (NODAGA-DsC18(NIA)2) to optimize labeling chemistry. First, we characterized in vitro the novel all- d peptide compared with its parent l-version. Then, in order to investigate and compare the pharmacological profiles of the peptides, these were radiolabeled with 64CuII and 68GaIII, and the biodistribution and kinetics were evaluated in vivo. Our results show the versatility of the d-peptide as cell-penetrating peptide and transporter. However, attaching two NIA groups modified the system in such a way that no selective tumor uptake could be observed compared with the peptide without NIA moieties. Still, this work highlights new pharmacokinetic data on the biodistribution of such compounds in vivo. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Peptide materials are promising for various biomedical applications; however, a significant concern is their lack of stability and rapid degradation in vivo due to non-specific proteolysis. For materials specifically designed to respond to disease-specific proteases, it would be desirable to retain high susceptibility to target proteases while minimizing the impact of non-specific proteolysis. We describe N-terminal acetylation as a simple synthetic modification of amphiphilic self-assembling peptides that contain an MMP-9-cleavable segment and form soluble, nanoscale filaments. We found that the N-terminus capping of these peptides did not significantly impact their self-assembly behavior, critical aggregation concentration, or ability to encapsulate hydrophobic payloads. By contrast, their proteolytic stability in human plasma (especially for anionic peptide sequences) was considerably increased while susceptibility to hydrolysis by MMP-9 was retained when compared to non-acetylated peptides, especially during the first 12 h. We note, however, that due to the longer time scale required for in vitro studies (72 h), non-specific proteolysis of both anionic acetylated peptides leads to similar activity in vitro despite differing MMP-9 kinetics during the early stages. Overall, the enhanced stability against non-specific proteases, combined with the ability of these nanofilaments to enhance the effectiveness of gold-based drugs toward cancerous cells compared to healthy cells, brings these acetylated peptide filaments a step closer toward clinical translation.

Recently, utilization of the enzyme Chondroitinase ABC I (cABC I) has received considerable attention in treatment of spinal cord injury. cABC I removes chondroitin sulfate proteoglycans which are inhibitory to axon growth and enhances nerve regeneration. Therefore, determination of cABC I resistance to proteolysis and oxidation provides valuable information for optimizing its clinical application. In this work, proteolytic stability of cABC I to trypsin and chymotrypsin as well as its oxidative resistance to H 2 O 2 was measured. Moreover, the effect of cosolvents glycerol, sorbitol and trehalose on cABC I proteolytic and oxidative stability was determined. The results indicated that cABC I is highly susceptible to proteolysis and oxidation. Comparison of proteolytic patterns demonstrated a high degree of similarity which confirmed the exposure of specific regions of cABC I to proteolysis. However, proteolytic degradation was significantly reduced in the presence of cosolvents. In addition, cosolvents decreased the rate of both cABC I proteolytic and oxidative inactivation. Notably, the degree of stabilization provided by these cosolvents varied greatly. These findings indicated the high potential of cosolvents in protein stabilization to proteolysis and oxidative inactivation. [ABSTRACT FROM AUTHOR]

Due to the alarming developing rate of multidrug-resistant bacterial pathogens, the development and modification of antimicrobial peptides (AMPs) are unprecedentedly active. Despite the fact that considerable efforts have been expended on the discovery and design strategies of AMPs, the clinical translation of peptide antibiotics remains inadequate. A large number of articles and reviews credited the limited success of AMPs to their poor stability in the biological environment, particularly their poor proteolytic stability. In the past forty years, various design strategies have been used to improve the proteolytic stability of AMPs, such as sequence modification, cyclization, peptidomimetics, and nanotechnology. Herein, we focus our discussion on the progress made in improving the proteolytic stability of AMPs and the principle, successes, and limitations of various anti-proteolytic design strategies. It is of prospective significance to extend current insights into the degradation-related inactivation of AMPs and also alleviate/overcome the problem. [ABSTRACT FROM AUTHOR]

The pathological role of zinc in Alzheimer's disease (AD) is not yet fully elucidated, but there is strong evidence that zinc homeostasis is impaired in the AD brain and that this contributes to disease pathogenesis. In this study we examined the effects of zinc on the proteolysis of synthetic Apolipoprotein E (ApoE), a protein whose allelic variants differentially contribute to the onset/progression of disease. We have demonstrated that zinc promotes the proteolysis (using plasma kallikrein, thrombin and chymotrypsin) of synthetic ApoE in an isoform-specific way (E4 > E2 and E3), resulting in more ApoE fragments, particularly for ApoE4. In the absence of exogenous proteases there was no effect of metal modulation on either lipidated or non-lipidated ApoE isoforms. Thus, increased zinc in the complex milieu of the ageing and AD brain could reduce the level of normal full-length ApoE and increase other forms that are involved in neurodegeneration. We further examined human plasma samples from people with different ApoE genotypes. Consistent with previous studies, plasma ApoE levels varied according to different genotypes, with ApoE2 carriers showing the highest total ApoE levels and ApoE4 carriers the lowest. The levels of plasma ApoE were not affected by either the addition of exogenous metals (copper, zinc or iron) or by chelation. Taken together, our study reveals that zinc may contribute to the pathogenesis of AD by affecting the proteolysis of ApoE, which to some extent explains why APOE4 carriers are more susceptible to AD. [ABSTRACT FROM AUTHOR]

ABSTRACT Adiponectin, an adipose tissue-excreted adipokine plays protective roles in metabolic and cardiovascular diseases and exerts anti-cancer activities, partially by interfering with leptin-induced signaling. Previously we identified the active site in the adiponectin protein, and generated both a nanomolar monomeric agonist of the adiponectin receptor (10-mer ADP355) and an antagonist (8-mer ADP400) to modulate various adiponectin receptor-mediated cellular functions. As physiologically circulating adiponectin forms multimeric complexes, we also generated an agonist dimer with improved biodistribution and in vitro efficacy. In the current report, we attempted to optimize the monomeric agonist structure. Neither extension of the peptide up to 14-mer analogs nor reinstallation of native residues in permissible positions enhanced significantly the activity profile. The only substitutions that resulted in 5-10-fold improved agonistic activity were the replacement of turn-forming Gly4 and Tyr7 residues with Pro and Hyp, respectively, yielding the more active native β-sheet structure. All peptides retained good stability in human serum exhibiting half-lives >2 h. The cellular efficacy and stability rankings among the peptides followed expected structure-activity relationship trends. To investigate whether simultaneous activation of adiponectin pathways and inhibition of leptin-induced signals can result in cytostatic and anti-oncogenic signal transduction processes, we developed a chimera of the leptin receptor antagonist peptide Allo-aca (placed to the N-terminus) and ADP355 (at the C-terminus). The in vitro anti-tumor activity and intracellular signaling of the chimera were dominated by the more active Allo-aca component. The ADP355 part, however, reversed unfavorable in vivo metabolic effects of the leptin receptor antagonist. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 104: 156-166, 2015. [ABSTRACT FROM AUTHOR]

Cecropin A–melittin (CAM), a chimeric antimicrobial peptide with potent antimicrobial activity, is threatened by some special extracellular proteases when used to deal with certain drug-resistant pathogenic microbes in the gastrointestinal tract. Thus, a four-tryptophan-substitution mutant (CAM-W) from CAM was developed via the replacement of special amino acid residues to enhance the antimicrobial potency and to improve the proteolytic stability of this agent. The pharmaceutical index of CAM-W was investigated, with a focus on biological potency, cytotoxicity, and proteolytic stability, as well as pH and thermal resistance. CAM-W exhibited potent antimicrobial activity and was approximately 3–12 times higher than that of CAM. CAM-W also exhibited a strong antifungal activity against a series of common pathogenic fungi, in a lower IC50 range between 2.1 mg/L and 3.3 mg/L than that of its reference CAM ranging from 9.8 mg/L to 14.2 mg/L. Besides, CAM-W showed moderate cytotoxicity (IC 50 > 300 mg/L) in erythrocyte lysis test. In addition, CAM-W overcame challenges under various conditions, including specific temperatures (20, 30, 40, 50, 60, 70, 80, and 90 °C), pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0), and proteases (trypsin, pepsin, human neutrophil elastase, Pseudomonas aeruginosa elastase, and Staphylococcus aureus V8 protease) that are commonly present in human gastrointestinal tract. These results suggest that the four-tryptophan-substitution can confer CAM-W with a high pharmaceutical index, which is important for CAM-W to become a potential alternative to conventional antibiotics against bacteria and fungi associated with gastroenteritis. [ABSTRACT FROM AUTHOR]

An increase in the frequency of mycoses and spreading of multidrug-resistant fungal pathogens necessitates the search for new antifungal agents. Earlier, we isolated the novel defensin from lentil Lens culinaris seeds, designated as Lc-def, which inhibited the growth of phytopathogenic fungi. Here, we studied an antifungal activity of Lc-def against human pathogenic Candida species, structural stability of the defensin, and its immunomodulatory effects that may help to prevent fungal infection. We showed that Lc-def caused 50% growth inhibition of clinical isolates of Candida albicans , C. krusei , and C. glabrata at concentrations of 25-50 μM, but was not toxic to different human cells. The lentil defensin was resistant to proteolysis by C. albicans and was not cleaved during simulated gastroduodenal digestion. By using the multiplex xMAP assay, we showed for the first time for plant defensins that Lc-def increased the production of such essential for immunity to candidiasis pro-inflammatory cytokines as IL-12 and IL-17 at the concentration of 2 μM. Thus, we hypothesized that the lentil Lc-def and plant defensins in general may be effective in suppressing of mucocutaneous candidiasis due to their antifungal activity, high structural stability, and ability to activate a protective immune response.

Highlights: [•] Legume Bowman-Birk inhibitors contain seven conserved disulfide bonds and are stable to heat. [•] Two intradomain disulfides in the trypsin subdomain contribute profoundly to thermal stability. [•] A disulfide in the trypsin subdomain plays a key role in structural and functional stability. [•] Overall the study demonstrates that the two subdomains in BBIs are not independent of each other. [ABSTRACT FROM AUTHOR]

Abstract: Since the bacterial resistance to antibiotics is increasing rapidly, numerous studies have contributed to the design and synthesis of potent synthetic mimics of antimicrobial peptides (AMPs). In an attempt to find the pharmacophore of short antimicrobial peptidomimetics through systematic tuning of hydrophobic and hydrophilic patterns, we have identified a set of short histidine-derived antimicrobial peptides (SAMPs) with potent and broad-spectrum activity. A combination of high antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), without hemolytic activity and proteolytic stability makes these molecules promising candidates for novel antimicrobial therapeutics. [Copyright &y& Elsevier]

The oral delivery of protein and peptide drugs is limited by their proteolytic degradation and the poor absorption across the intestinal epithelia. In this work, we exposed a phage library of small bicyclic peptides (<1.5 kDa) to a pancreatic extract of proteases prior to affinity selection to enrich binders with higher stability in the intestinal environment. Panning with the therapeutic target plasma kallikrein yielded potent inhibitors (Kis between 5.6 and 336 nM) wherein bicyclic peptides isolated with proteolytic pressure were more stable. A proline residue found in a specific position of several resistant bicyclic peptides proved to be a ‘protective mark’, rendering the bicyclic peptides resistant to significantly higher concentrations of intestinal proteases while retaining essentially their inhibitory activity. [ABSTRACT FROM AUTHOR]

Salt-bridge interactions between acidic and basic amino acids contribute to the structural stability of proteins and to protein-protein interactions. A conserved salt-bridge is a canonical feature of the α-defensin antimicrobial peptide family, but the role of this common structural element has not been fully elucidated. We have investigated mouse Paneth cell α-defensin cryptdin-4 (Crp4) and peptide variants with mutations at Arg or Glu residue positions to disrupt the salt-bridge and assess the consequences on Crp4 structure, function, and stability. NMR analyses showed that both (R7G)-Crp4 and (E15G)-Crp4 adopt native-like structures, evidence of fold plasticity that allows peptides to reshuffle side chains and stabilize the structure in the absence of the salt-bridge. In contrast, introduction of a large hydrophobic side chain at position 15, as in (E15L)-Crp4 cannot be accommodated in the context of the Crp4 primary structure. Regardless of which side of the salt-bridge was mutated, salt-bridge variants retained bactericidal peptide activity with differential microbicidal effects against certain bacterial cell targets, confirming that the salt-bridge does not determine bactericidal activity per se. The increased structural flexibility induced by salt-bridge disruption enhanced peptide sensitivity to proteolysis. Although sensitivity to proteolysis by MMP7 was unaffected by most Arg and Glu substitutions, every salt-bridge variant was degraded extensively by trypsin. Moreover, the salt-bridge facilitates adoption of the characteristic α-defensin fold as shown by the impaired in vitro refolding of (E15D)-proCrp4, the most conservative salt-bridge disrupting replacement. In Crp4, therefore, the canonical α-defensin salt-bridge facilitates adoption of the characteristic α-defensin fold, which decreases structural flexibility and confers resistance to degradation by proteinases. [ABSTRACT FROM AUTHOR]