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We report the development of a new electron-rich aniline (ERA)-based cleavable linker. Anilines can be incorporated into peptides during SPPS and are stable to most reaction conditions. ERA-containing peptides can be cleaved rapidly in the presence of oxidants, such as DDQ, CAN, and NaIO 4 , in 30 min at room temperature. The compatibility of these conditions is demonstrated on peptides containing natural and unnatural amino acids. While the cleavages of other oxidatively labile linkers is known to cause decomposition of Tyr and Trp, these sensitive residues are stable to DDQ oxidations. An ERA-linker also enables the capture and release of a biotinylated peptide from immobilized streptavidin. ERA-linkers may serve as an excellent tool for peptide library screening applications., Competing Interests: Declaration of competing interest The authors declare no financial interests or personal relationships that are potential competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Membranes that destroy anticancer peptides can bind to negatively charged cancer cell membranes through electrostatic interactions, destroying their functions and leading to cancer cell necrosis. Temporin-1CEa, obtained from the skin secretions of the Chinese frog Rana chensinensis, is an anticancer peptide with 17 amino acid residues that exhibits concentration-dependent cytotoxicity against a variety of cancer cell lines, although it has no obvious cytotoxicity to normal HUVECs. In this work, we designed and synthesized 12 derivative peptides through double-cysteine scanning of temporin-1CEa-truncated peptides. Most of these peptides had greater anticancer activity than the lead peptide temporin-1CEa. Among these derivative peptides, Nu-7 had the strongest anticancer activity. Nu-7 has a greater α-helicity than does temporin-1CEa. We connected Nu-7 to podophyllotoxin through a reduction-responsive linker to obtain Nu-7-1, which showed better anticancer activity than free podophyllotoxin. Nu-7-1 was less toxic to HUVECs and had low hemolytic activity at therapeutic concentrations (although Nu-7-1 showed hemolytic activity at 100 μM). Nu-7-1 functions through two mechanisms: damage to cell membranes and promotion of cell apoptosis. Nu-7-1 is less toxic to normal HUVECs than is podophyllotoxin and shows better safety. In summary, we carried out a series of modifications on temporin-1CEa, among which the anticancer activity of Nu-7-1 was significantly improved compared with that of the lead peptide temporin-1CEa, providing a useful reference for the structural modification of anticancer peptides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The search for novel cholinesterase inhibitors is essential for advancing treatments for neurodegenerative disorders such as Alzheimer's disease (AD). In this study, we employed the Rosetta pepspec module, originally developed for designing peptides targeting protein-protein interactions, to design de novo peptides targeting the peripheral aromatic site (PAS) of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). A total of nine peptides were designed for human AChE (hAChE), T. californica AChE (TcAChE), and human BChE (hBChE). These peptides were synthesized using Fmoc-SPPS and tested in vitro using Ellman's reaction to evaluate their inhibitory potency. Peptide 11tA, designed for TcAChE, exhibited potent inhibition of hAChE (IC 50  = 1.21 ± 0.25 µM) and demonstrated strong antioxidant activity against DPPH radicals and lipid peroxidation, making it a promising multitherapeutic candidate for AD. Peptide 11hB, designed for hBChE, showed the highest inhibitory activity against hBChE, with a K i of 12.69 ± 1.27 µM, making it the most potent natural amino acid peptide reported against hBChE. The computational protocol effectively distinguished the specific characteristics of each enzyme target. Toxicity assessments, including hemolysis tests and A. salina lethality assays, revealed no toxic effects at low concentrations, further supporting the potential of these peptides for peptide-based drug development in AD. This study underscores the growing potential of peptides as alternatives to small-molecule drugs. It demonstrates that computational protocols for protein-protein interactions can be successfully adapted to design high-affinity peptide inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Glutathione (GSH) exhibits considerable potential in the cosmetic industry for reducing intracellular tyrosinase activity and inhibiting melanin synthesis. However, its efficacy is hindered by limited permeability, restricting its ability to reach the basal layer of the skin where melanin production occurs. The transdermal enhancer peptide TD1 has emerged as a promising strategy to facilitate the transdermal transfer of proteins or peptides by creating intercellular gaps in keratinocytes, providing access to the basal layer. The primary objective of this study is to enhance the transdermal absorption capacity of GSH while augmenting its inhibitory effect on melanin. Two coupling structures were designed for investigation: linear (TD1-linker-GSH) and branched (TD1-GSH). The study examined the impact of the peptide skeleton on melanin inhibition ability. Our findings revealed that the linear structure not only inhibited synthetic melanin production in B16F10 cells through a direct pathway but also through a paracrine pathway, demonstrating a significant tyrosinase inhibition of nearly 70 %, attributed to the paracrine effect of human keratinocyte HaCaT. In pigmentation models of guinea pigs and zebrafish, the application of TD1-linker-GSH significantly reduced pigmentation. Notably, electric two-photon microscopy demonstrated that TD1-linker-GSH exhibited significant transdermal ability, penetrating 158.67 ± 9.28 μm into the skin of living guinea pigs. Molecular docking analysis of the binding activity with tyrosinase revealed that both TD1-linker-GSH and TD1-GSH occupy the same active pocket, with TD1-linker-GSH binding more tightly to tyrosinase. These results provide a potential foundation for therapeutic approaches aimed at enriched pigmentation and advance our understanding of the mechanisms underlying melanogenesis inhibition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Colorectal cancer (CRC) is among the most lethal and prevalent malignancies in the world. Human epidermal growth factor receptor 2 (HER2) is a promising target for the diagnosis and treatment of CRC. In this study, we aimed to design, synthesize and label peptide-based positron emission tomography (PET) tracers targeting HER2-positive CRC, namely [ 68 Ga]Ga-ES-01 and [ 68 Ga]Ga-ES-02. The results show that [ 68 Ga]Ga-ES-01 and [ 68 Ga]Ga-ES-02 possessed hydrophilicity, rapid pharmacokinetic properties and excellent stabilities. [ 68 Ga]Ga-ES-02 demonstrated higher binding affinity (K d  = 24.29 ± 4.95 nM) toward the HER2 in CRC. In HER2-positive HT-29 CRC xenograft mouse model, PET study showed specific tumor uptake after injection of [ 68 Ga]Ga-ES-02 (SUV 15min max  = 0.87 ± 0.03; SUV 30min max  = 0.64 ± 0.02). In biodistribution study, the T/M ratios of 68 Ga-ES-02 at 30 min after injection reached a maximum of 4.07 ± 0.34. In summary, we successfully synthesized and evaluated two novel peptide-based PET tracers. Our data demonstrate that [ 68 Ga]Ga-ES-01/02 is capable of HER2-positive colorectal cancer, with [ 68 Ga]Ga-ES-02 showing superior imaging effect, enhanced targeting, and increased specificity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In this work, a new ratiometric fluorescent probe DKA was synthesized based on the double sides of lysine backbone conjugated with alanine and dansyl groups. DKA exhibited fluorescence ratiometric response for Hg 2+ with high sensitivity (13.4 nM), specific selectivity (only Hg 2+ ), strong anti-interference ability (no interference), fast recognition (within 60 s) and wide pH range (5-10). The stoichiometry of binding of DKA and Hg 2+ was determined to be 1:1 via Job's plot, ESI-HRMS and 1 HNMR titration analysis. Subsequently, the in situ formation of DKA-Hg 2+ complex was used for highly selective detection of S 2- as a novel fluorescence "on-off" probe, and the lowest detection limit for S 2- was 12.9 nM. In addition, DKA possessed excellent cells permeation and low toxicity, and fluorescence imaging of Hg 2+ and S 2- was performed in living Hacat cells. Most importantly, the digital imaging using a smartphone color recognition APP indicated that DKA could semi-quantitatively and visually detected Hg 2+ and S 2- without expensive equipment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Femoral head necrosis is a debilitating disorder that typically caused by impaired blood supply to the hip joint. In this study, a novel injectable hydrogel based on Oxidized Carboxymethyl Cellulose (OCMC)-Carboxymethyl Chitosan (CMCS) polymers containing an angiogenesis stimulator peptide (QK) with a non-toxic crosslinking interaction (Schiff based reaction) was synthesized to enhance angiogenesis following femoral head necrosis in an animal model. The physicochemical features of fabricated injectable hydrogel were analyzed by FTIR, swelling and degradation rate, rheometry, and peptide release. Also, the safety and efficacy were evaluated following an in vitro hydrogel injection study and an avascular necrosis (AVN) animal model. According to the results, the hydrogel exhibited an appropriate swelling ratio and water uptake (>90 %, 24 h) as well as a suitable degradation rate over 21 days accompanied by a continuous peptide release. Also, data showed that hydrogels containing QK peptide boosted the proliferation, differentiation, angiogenesis, and osteogenic potential of both Bone Marrow mesenchymal Stem Cells (BM-MSCs) and human umbilical vein endothelial cells (HUVECs) (****p < 0.0001 and ***p < 0.001, respectively). Furthermore, molecular and histological evaluations significantly demonstrated the overexpression of Runx2, Osteocalcin, Collagen I, VEGF and CD34 genes (**p < 0.01 and ***p < 0.001, respectively), and also femoral head necrosis was effectively prohibited, and more blood vessels were detected in defect area by OCMC-CMCS hydrogel containing QK peptide (bone trabeculae >9000, ***p < 0.001). In conclusion, the findings demonstrate that OCMC-CMCS-QK injectable hydrogel could be considered as an impressive therapeutic construct for femoral head AVN healing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Developing "turn on" fluorescent probes was desirable for the detection of the effective anticoagulant agent heparin in clinical applications. Through combining the aggregation induced emission (AIE) fluorogen tetraphenylethene (TPE) and heparin specific binding peptide AG73, the promising "turn on" fluorescent probe TPE-1 has been developed. Nevertheless, although TPE-1 could achieve the sensitive and selective detection of heparin, the low proteolytic stability and undesirable poor solubility may limit its widespread applications. In this study, seven TPE-1 derived fluorescent probes were rationally designed, efficiently synthesized and evaluated. The stability and water solubility were systematically estimated. Especially, to achieve real-time monitoring of proteolytic stability, the novel Abz/Dnp-based "turn on" probes that employ the internally quenched fluorescent (IQF) mechanism were designed and synthesized. Moreover, the detection ability of synthetic fluorescent probes for heparin were systematically evaluated. Importantly, the performance of d-type peptide fluorescent probe XH-6 indicated that d-type amino acid substitutions could significantly improve the proteolytic stability without compromising its ability of heparin sensing, and attaching solubilizing tag 2-(2-aminoethoxy) ethoxy) acid (AEEA) could greatly enhance the solubility. Collectively, this study not only established practical strategies to improve both the water solubility and proteolytic stability of "turn on" fluorescent probes for heparin sensing, but also provided valuable references for the subsequent development of enzymatic hydrolysis-resistant d-type peptides based fluorescent probes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Due to the strong selectivity and permeability of tumor tissue, anti-cancer peptide-drug conjugates (PDCs) can accumulate high concentration of toxic payloads at the target, effectively killing tumor cells. This approach holds great promise for tumor-targeted treatment. In our previous study, we identified the optimal peptide P1 (NPNWGRSWYNQRFK) targeting HER2 from pertuzumab, a monoclonal antibody that blocks the HER2 signaling pathway. Here, a series of PDCs were constructed through connecting P1 and CPT with different linkers. Among these, Z8 emerged as the optimal compound, demonstrating good antitumor activity and targeting ability in biological activity tests. Z8 exhibited IC 50 values of 1.04 ± 0.24 μM and 1.91 ± 0.71 μM against HER2-positive SK-BR-3 and NCI-N87 cells, respectively. Moreover, superior antitumor activity and higher biosafety of Z8 were observed compared to the positive control CPT in vivo, suggesting a novel idea for the construction of PDCs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The unique interaction between fluorine atoms has been exploited to alter protein structures and to develop synthetic and analytical applications. To expand such fluorous interaction for novel applications, polyproline peptides represent an excellent molecular nanoscaffold for controlling the presentation of perfluoroalkyl groups on their unique secondary structure. We develop approaches to synthesis fluorinated peptides to systematically investigate how the number, location and types of the fluorous groups on polyproline affect the conformation by monitoring the transition between the two major polyproline structures PPI and PPII. This work provides valuable information on how fluorous interaction affects the peptide structure and also benefits the design of functional fluorous molecules., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Isocitrate dehydrogenases 1 (IDH1) catalyzes the oxidative decarboxylation of isocitrate to ɑ-ketoglutaric acid (α-KG). It is the most frequently mutated metabolic gene in human cancer and its mutations interfere with cell metabolism and epigenetic regulation, thus promoting tumorigenesis. In order to discover potent new mutant IDH1 inhibitors, based on the structure of marketed inhibitor AG-120 (Ivosidenib), we designed, synthesized and evaluated a series of linear unnatural peptide analogues via Ugi reaction, as potential mutant IDH1 inhibitors. All these compounds were evaluated for their inhibition on mutant IDH1 enzyme activity. The structure-activity relationship was discussed on the basis of experimental data, with an attempt to pave the way for future studies. Among them, 43 exhibited potent and selective enzyme inhibitory activity, and showed strong binding affinity with mutant IDH1. It can decrease the cellular concentration of 2-HG, and suppress the proliferation of HT1080 and IDH1 mutant-U-87 cells by selectively inhibiting the activity of mutant IDH1., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Using a recombinant protein antigen for antibody testing shows a sum of antibody responses to multiple different immune epitopes existing in the protein antigen. In contrast, the antibody testing to an immunogenic peptide epitope reflects a singular antibody response to the individual peptide epitope. Therefore, using a panel of peptide epitopes provides an advantage for profiling multiple singular antibody responses with potential to estimate recent malaria exposure in human infections. However, transitioning from malaria immune epitope peptide-based ELISA to an all peptide bead-based multiplex Luminex assay presents some challenges including variation in the ability of different peptides to bind beads. The aim of this study was to develop a peptide coupling method while demonstrating the utility of these peptide epitopes from multiple stage antigens of Plasmodium falciparum for measuring antibodies. Successful coupling of peptide epitopes to beads followed three steps: 1) development of a peptide tag appended to the C-terminus of each peptide epitope consisting of beta-alanine-lysine (x 4)--cysteine, 2) bead modification with a high concentration of adipic acid dihydrazide, and 3) use of the peptide epitope as a blocker in place of the traditional choice, bovine serum albumin (BSA). This new method was used to couple 12 peptide epitopes from multiple stage specific antigens of P. falciparum, 1 Anopheles mosquito salivary gland peptide, and 1 Epstein-Barr virus peptide as an assay control. The new method was applied to testing of IgG in pooled samples from 30 individuals with previously repeated malaria exposure in western Kenya and IgM and IgG in samples from 37 U.S. travelers with recent exposure to malaria. The new peptide-bead coupling method and subsequent multiplex Luminex assay showed reliable detection of IgG to all 14 peptides in Kenyan samples. Among 37 samples from U.S. travelers recently diagnosed with malaria, IgM and IgG to the peptide epitopes were detected with high sensitivity and variation. Overall, the U.S. travelers had a much lower positivity rates of IgM than IgG to different peptide epitopes, ranging from a high of 62.2% positive for one epitope to a low of only 5.4% positive for another epitope. In contrast, the travelers had IgG positive rates from 97.3% to 91.9% to various peptide epitopes. Based on the different distribution in IgM and IgG positivity to overall number of peptide epitopes and to the number of pre-erythrocytic, erythrocytic, gametocytic, and salivary stage epitopes at the individual level, four distinct patterns of IgM and IgG responses among the 37 samples from US travelers were observed. Independent peptide-bead coupling and antibody level readout between two different instruments also showed comparable results. Overall, this new coupling method resolves the peptide-bead coupling challenge, is reproducible, and can be applied to any other immunogenic peptide epitopes. The resulting all peptide bead-based multiplex Luminex assay can be expanded to include other peptide epitopes of P. falciparum, different malaria species, or other diseases for surveillance, either in US travelers or endemic areas., (Published by Elsevier B.V.)

The bone morphogenetic protein-2 (BMP-2) is an essential regulator of bone formation and remodeling, which has also been implicated in the pathogenesis of osteoarthritis and its closely related chondrocyte senescence. The BMP-2 uses a conformational wrist epitope and a linear knuckle epitope to interact with type-I (BMPR-I) and type-II (BMPR-II) receptors, respectively. Previously, the knuckle epitope has been intensely studied, but the wrist epitope still remains largely unexplored due to its discontinuous nature. In the present work, the intermolecular interaction of BMP-2 with BMPR-I was investigated systematically at structural, energetic and dynamic levels. Three discrete hotspots that represent the key BMPR-I recognition sites of BMP-2 were identified; they are spatially dispersed over the two monomers of BMP-2 dimer and totally account for 83.5 % binding potency of BMP-2 to BMPR-I (hotspot 1: residues 49-70 in monomer 1; hotspot 2: residues 24-31 in monomer 2; hotspot 3: residues 88-107 in monomer 2). Therefore, we defined the three discrete hotspot sites as the core region of wrist epitope; their contribution to the binding increases in the order: hotspot 2 < hotspot 3 < hotspot 1. We demonstrated that the primary hotspot 1 site has a native U-shaped conformation in the full-length BMP-2 protein context, but it cannot maintain in the native conformation when split from the context to obtain a free hotspot-1 peptide, thus largely impairing its binding potency to BMPR-I. We further employed disulfide-bonded cyclization and head-to-tail cyclization to constrain the peptide conformation, and found that only the former can effectively constrain the peptide into native conformation, thus considerably improving its binding affinity to BMPR-I, whereas the latter totally disorders the native conformation, thus rendering the peptide as a full nonbinder of BMPR-I., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Diabetic retinopathy (DR) remains high incidence and accounts for severe impact on vision in diabetics, but its mechanism is still poorly understood. Abnormal migration and proliferation of endothelial cells (ECs) drive neovascular retinopathies, which has an important role in promoting the occurrence and development of DR. In this study, we designed and synthesized a series of PEDF-derived peptides as angiogenesis inhibitors. Especially, compound G24 significantly inhibited the cell proliferation in VEGF-activated human umbilical vein endothelial cells (HUVECs) with IC 50 values of 2.88 ± 0.19 μM. Further biological evaluation demonstrated that compound G24 exhibited strong inducing-effects on cell apoptosis and internalization of 67LR, and advanced inhibitory potency in cell migration and angiogenesis formed by HUVECs in vitro. In summary, the optimal compound G24 as a novel angiogenesis inhibitor showed the potentiality in the further research for the treatment for DR., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Porcine astrovirus (PAstV) is prevalent in pigs worldwide and could cause clinical symptoms such as diarrhea and encephalitis. The capsid protein (Cap) of PAstV plays a determinant role for virus immunological characteristics. In this study, the major antigenic regions of PAstV1 Cap were expressed through prokaryotic expression systems and immunized to BALB/c mice. Finally, two anti-Cap monoclonal antibodies (named mAb F4-4 and D3F10) were screened by indirect immune-fluorescence assay (IFA). A series of truncated GST-fused or artificially synthesized peptides were used to detect their reactivity with the mAbs and PAstV positive serum. Two novel B cell epitopes ( 120- GNNTFG -125 , 485- RISDPTWFSA -494 ) were identified by using these two mAbs. Moreover, sequence alignment result showed that epitope 120- GNNTFG -125 was highly conserved in type 1 PAstV capsid protein. Cross-reactivity analysis further confirmed the genotype-specificity of mAb F4-4. The results of this study demonstrated to be the first description of monoclonal antibody preparation and B-cell epitope mapping on PAstV capsid protein, which may provide new information on the biological significance of PAstV capsid protein and lay a foundation for the development of PAstV immunological tests and genotype diagnostic methods., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Transcriptional enhanced associate domains (Teads) are the downstream effectors of the hippo signaling pathway and have been recognized as attractive druggable targets of gastric cancer. The biological function of Teads is regulated by diverse cofactors. In this study, the intermolecular interactions of Teads with their cognate cofactors were systematically characterized at structural, thermodynamic and dynamic levels. The Teads possess a double-stranded helical hairpin that is surrounded by three independent structural elements β-sheet, α-helix and Ω-loop of cofactor proteins and plays a central role in recognition and association with cofactors. A number of functional peptides were split from the hairpin region at Tead-cofactor complex interfaces, which, however, cannot maintain in native conformation without the support of protein context and would therefore incur a considerable entropy penalty upon competitively rebinding to the interfaces. Here, we further used disulfide and hydrocarbon bridges to cyclize and staple the hairpin and helical peptides, respectively. The chemical modification strategies were demonstrated to effectively constrain peptide conformation into active state and to largely reduce peptide flexibility in free state, thus considerably improving their affinity. Since the cyclization and stapling only minimize the indirect entropy cost but do not influence the direct enthalpy effect upon peptide binding, the designed conformationally constrained peptides can retain in their native selectivity over different cofactors. This is particularly interesting because it means that the cyclized/stapled, affinity-improved peptides can specifically compete with their parent Teads for the cofactor arrays as they share consistent target specificity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cellular autophagy is an intracellular degradation pathway, which transports damaged, deformed, senescent or non-functional proteins and organelles to lysosome for digestion and degradation. Cellular autophagy is deeply evolutionarily conservedfromyeasttomammaliancells, and many homologous proteins of the autophahgy regulators are found in several species. This physiological process maintains the steady state of cells. Furtheremore, autophagy dysfunction is closely related to various diseases, such as neurodegenerative diseases, inflammation-related diseases, cardiovascular diseases, metabolic diseases, etc. The LC3 and p62 protein protein interaction (PPI) promotes the formation of autophagosomes and delivers polyubiquitinated "cargoes" to autophagic degradation. Therefore, LC3-p62 PPI plays an integral role in the formation of autophagosomes and effectively inhibits autophagy. However, there are still few studies on the LC3-p62 PPI inhibitors for its unclear molecular mechanism. Furthermore, most of these inhibitors are macromolecules with poorly active, and small molecules are particularly scarce. In this article, the computation method was used to identify the hot spot and design peptides as the binder of LC3-p62 PPI. Findings from this work provide a reference for the follow-up research of discovering small molecule inhibitors targeting LC3-p62 PPI., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Over the past few years, amino acids (AA) have emerged as promising biomaterials for the synthesis of functional polymers. Owing to the diversity of functional groups in amino acids, various polymerization methods may be used to make a wide range of well-defined functional amino-acid/peptide-based optically active polymers with varying polymer lengths, compositions, and designs. When incorporated with chirality and self-assembly, they offer a wide range of applications and are particularly appealing in the field of drug delivery, tissue engineering, and biosensing. There are several classes of these polymers that include polyamides (PA), polyesters (PE), poly(ester-amide)s (PEA)s, polyurethanes (PU)s, poly(depsipeptide)s (PDP)s, etc. They offer the ability to control functionality, conjugation, crosslinking, stimuli responsiveness, and tuneable mechanical/thermal properties. In this review, we present the recent advancements in the synthesis strategies for obtaining these amino acid-derived bio-macromolecules, their self-assembly properties, and the wealth of prevalent applications., (Copyright © 2021. Published by Elsevier B.V.)

PnPP-19 peptide has a primary sequence design based on molecular modeling studies of PnTx2-6 toxin. It comprises the amino acid residues that are potentially significant for the pharmacological action of PnTx2-6. Ex vivo and in vivo experiments in normotensive, hypertensive, or diabetic murine models have shown a significant improvement in penile erection after administration of PnPP-19. Given the potential use of PnPP-19 in pharmaceutical formulations to treat erectile dysfunction and the lack of information concerning its mode of action, the present work investigates its activities on the nitrergic system. PnPP-19 induced a significant increase in nitric oxide (NO) and cGMP levels in corpus cavernosum (cc). These effects were inhibited by l-NAME, a non-selective inhibitor of nitric oxide synthase (NOS); were partially inhibited by 7- Nitroindazole, a selective inhibitor of neuronal NOS (nNOS); and were abolished by L-NIL, a selective inhibitor of inducible NOS (iNOS). This potentiating effect was not affected by atropine. PnPP-19 also led to changes in mRNA levels, protein expression and phosphorylation at specific sites of NOS, in cc. Assays using cavernous tissue from knockout mice to endothelial NOS (eNOS), nNOS or iNOS showed that PnPP-19 potentiates relaxation only in eNOS-knockout mice, which suggests an essential role for nNOS. Surprisingly, iNOS enhanced the potentiation of erectile function evoked by PnPP-19. Our results demonstrate that this new synthetic peptide potentiates erectile function via nitric oxide activation and reinforce its role as a new pharmacological tool for the treatment of erectile dysfunction., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The stabilization of helical structures by peptide stapling approaches is now a mature technology capable to provide a variety of biomedical applications. Recently, it was shown that multicomponent macrocyclization is not only an effective way to introduce conformational constraints but it also allows to incorporate additional functionalities to the staple moiety in a one-pot process. This work investigates the scope of the double Ugi multicomponent stapling approach in its capacity to produce helical peptides from unstructured sequences. For this, three different stapling combinations were implemented and the CD spectra of the cyclic peptides were measured to determine the effect of the multicomponent macrocyclization on the resulting secondary structure. A new insight into some structural factors influencing the helicity type and content is provided, along with new prospects on the utilization of this methodology to diversify the molecular tethers linking the amino acid side chains., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Antimicrobial peptides (AMP) comprise a wide range of small molecules with direct antibacterial activity and immunostimulatory role and are proposed as promising substitutes of the antibiotics. Additionally, they also exert a role against other pathogens such as viruses and fungi less evaluated. NK-lysin, a human granulysin orthologue, possess a double function, taking part in the innate immunity as AMP and also as direct effector in the cell-mediated cytotoxic (CMC) response. This molecule is suggested as a pivotal molecule involved in the defence upon nervous necrosis virus (NNV), an epizootic virus provoking serious problems in welfare and health status in Asian and Mediterranean fish destined to human consumption. Having proved that NK-lysin derived peptides (NKLPs) have a direct antiviral activity against NNV in vitro, we aimed to evaluate their potential use as a prophylactic treatment for European sea bass (Dicentrarchus labrax), one of the most susceptible cultured-fish species. Thus, intramuscular injection of synthetic NKLPs resulted in a very low transcriptional response of some innate and adaptive immune markers. However, the injection of NKLPs ameliorated disease signs and increased fish survival upon challenge with pathogenic NNV. Although NKLPs showed promising results in treatments against NNV, more efforts are needed to understand their mechanisms of action and their applicability to the aquaculture industry., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Transcription factors (TFs) have a remarkable role in the homeostasis of the organisms and there is a growing interest in how they recognize and interact with specific DNA sequences. TFs recognize DNA using a variety of structural motifs. Among those, the ribbon-helix-helix (RHH) proteins, exemplified by the MetJ and ARC repressors, form dimers that insert antiparallel β-sheets into the major groove of DNA. A great chemical challenge consists of using the principles of DNA recognition by TFs to design minimized peptides that maintain the DNA affinity and specificity characteristics of the natural counterparts. In this context, a peptide mimic of an antiparallel β-sheet is very attractive since it can be obtained by a single peptide chain folding in a β-hairpin structure and can be as short as 14 amino acids or less. Herein, we designed eight linear and two cyclic dodeca-peptides endowed with β-hairpins. Their DNA binding properties have been investigated using fluorescence spectroscopy together with the conformational analysis through circular dichroism and solution NMR. We found that one of our peptides, peptide 6, is able to bind DNA, albeit without sequence selectivity. Notably, it shows a topological selectivity for the major groove of the DNA which is the interaction site of ARC and many other DNA-binding proteins. Moreover, we found that a type I' β-hairpin folding pattern is a favorite peptide structure for interaction with the B-DNA major groove. Peptide 6 is a valuable lead compound for the development of novel analogs with sequence selectivity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

There is an urgent need for wound dressings to treat partial-thickness burns. Hydrogels are a promising material that can maintain hydration to promote necrotic tissue removal. Tilapia peptides (TP) and hydroxyapatite (HA) were incorporated into chitosan system to prepare new types of hydrogels. The hydrogels were cross-linking by tannin (TA), which were developed to promote rapid wound healing in a New Zealand rabbit partial-thickness burn model. Nanohydroxyapatite (NHA) was synthesized by coprecipitation method, which made hydrogels have a highly porous structure comprised of interconnected pores, excellent water absorption and low hemolysis. Besides, the hydrogels showed excellent antimicrobial activities against both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), as well as the cytocompatibility on endothelial cells. Moreover, the hydrogels promoted epithelial and dermal regeneration, reduce the expression of TNF-α and IL-6 and promote the skin regeneration by enhancing expression of collagen, STAT3, and VEGF., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021. Published by Elsevier B.V.)

Insulin, a peptide hormone and a key regulator of blood glucose level, is routinely administered to type-I diabetic patients to achieve the required glycemic control. Insulin aggregation and ensuing amyloidosis has been observed at repeated insulin injection sites and in injectable formulations. The latter occurs due to insulin agglomeration during shipping and storage. Such insulin amyloid leads to enhanced immunogenicity and allow potential attachment to cell membranes leading to cell permeability and apoptosis. Small molecule inhibitors provide useful interruption of this process and inhibit protein misfolding as well as amyloid formation. In this context, we report the propensity of a palmitoylated peptide conjugate to inhibit insulin aggregation and amyloid-mediated cytotoxicity, via designed interference with polypeptide interfacial interactions., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The development of vaccines is a crucial response against the COVID-19 pandemic and innovative nanovaccines could increase the potential to address this remarkable challenge. In the present study a B cell epitope (S 461-493 ) from the spike protein of SARS-CoV-2 was selected and its immunogenicity validated in sheep. This synthetic peptide was coupled to gold nanoparticles (AuNP) functionalized with SH-PEG-NH 2 via glutaraldehyde-mediated coupling to obtain the AuNP-S 461-493 candidate, which showed in s.c.-immunized mice a superior immunogenicity (IgG responses) when compared to soluble S 461-493 ; and led to increased expression of relevant cytokines in splenocyte cultures. Interestingly, the response triggered by AuNP-S 461-493 was similar in magnitude to that induced using a conventional strong adjuvant (Freund's adjuvant). This study provides a platform for the development of AuNP-based nanovaccines targeting specific SARS-CoV-2 epitopes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Protein O-linked β-D-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation), an essential post-translational as well as cotranslational modification, is the attachment of β-D-N-acetylglucosamine to serine and threonine residues of nucleocytoplasmic proteins. An aberrant O-GlcNAc profile on certain proteins has been implicated in metabolic diseases such as diabetes and cancer. Inhibitors of O-GlcNAc transferase (OGT) are valuable tools to study the cell biology of protein O-GlcNAc modification. In this study we report novel uridine-peptide conjugate molecules composed of an acceptor peptide covalently linked to a catalytically inactive donor substrate analogue that bears a pyrophosphate bioisostere and explore their inhibitory activities against OGT by a radioactive hOGT assay. Further, we investigate the structural basis of their activities via molecular modelling, explaining their lack of potency towards OGT inhibition., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The chemical structure of oligonucleotide analogues dictates the conformation of oligonucleotide analogue oligomers, their ability to hybridize complementary DNA and RNA, their stability to degradation and their pharmacokinetic properties. In a study aimed at investigating new analogues featuring a neutral backbone, we explored the ability of oligomers containing a morpholino-peptide backbone to bind oligonucleotides. Circular Dichroism studies revealed the ability of our oligomers to interact with DNA, molecular modelling studies revealed the interaction responsible for complex stabilization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Emerging evidence revealed peptides within breast milk may be an abundant source of potential candidates for metabolism regulation. Our previous work identified numerous peptides existed in breast milk, but its function has not been validated. Thus, our study aims to screen for novel peptides that have the potential to antagonize obesity and diabetes., Methods: A function screen was designed to identify the candidate peptide and then the peptide effect was validated by assessing lipid storage. Afterwards, the in vivo study was performed in two obese models: high-fat diet (HFD)-induced obese mice and obese ob/ob mice. For mechanism study, a RNA-seq analysis was conducted to explore the pathway that account for the biological function of peptide., Results: By performing a small scale screening, a peptide (AVPVQALLLNQ) termed AOPDM1 (anti-obesity peptide derived from breast milk 1) was identified to reduce lipid storage in adipocytes. Further study showed AOPDM1 suppressed adipocyte differentiation by sustaining ERK activity at later stage of differentiation which down-regulated PPARγ expression. In vivo, AOPDM1 effectively reduced fat mass and improved glucose metabolism in high-fat diet (HFD)-induced obese mice and obese ob/ob mice., Conclusions: We identified a novel peptide AOPDM1 derived from breast milk could restrict adipocyte differentiation and ameliorate obesity through regulating MAPK pathway., General Significance: Our findings may provide a potential candidate for the discovery of therapeutic drugs for obesity and type 2 diabetes., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Active natural productscan be valuable lead compounds and numerous drugs derived from natural products have successfully entered the clinic. Arenobufagin, one of the important active components of toad venom, indicates significant antitumor activities with limited preclinical development for its strong cardiotoxicity. Ten 3-monopeptide substituted arenobufagin derivatives have been designed and synthesized. Antitumor activity and cardiotoxicity assays lead to the discovery of compound ZM226 as a potent antitumor agent with low cardiotoxicity. These findings suggest optimization of arenobufagin on position 3 maybe an efficacious strategy for the development of antitumor drug candidates derived from arenobufagin., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Protein labeling with a functional molecule is a technique widely used for protein research. The covalent reaction of self-labeling peptide tags with synthetic probe-modified small molecules enables tag-fused protein labeling with chemically diverse molecules, including fluorescent probes. We report the discovery, by in vitro directed evolution, of a novel 23-mer dibenzocyclooctyne (DBCO)-reactive peptide (DRP) tag using Systematic Evolution of Ligands by EXponential enrichment (SELEX) with a combination of a reconstituted cell-free translation system (PURE system) and cDNA display. The N- and C-terminal DRP truncations created a shorter 16-mer DBCO-reactive peptide (sDRP) tag without significant reactivity reduction. By fusing the sDRP tag to a model protein, we showed the chemical labeling and in-gel fluorescence imaging of the sDRP-fused protein using a fluorescent DBCO probe. Results showed that sDRP tag-mediated protein labeling has potential for use as a basic molecular tool in a variety of applications for protein research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We have previously demonstrated that the peptide mimicking small extracellular ring domain of CD82 (CD82EC1-mP) could inhibit tumor cell motility and metastasis. However, its acting mechanism is not understood. Here, we reported that the cell motility-inhibitory function of CD82EC1-mP was involved in the downregulation of epithelial-mesenchymal transition (EMT). Both vimentin and E-cadherin are EMT makers. We found that CD82EC1-mP could inhibit the expression of vimentin, but promot the expression of E-cadherin, suggesting that CD82EC1-mP suppressed EMT. Hippo/YAP and Wnt/β-catenin are both key signal pathways that regulate the EMT process. The futher studies showed that CD82EC1-mP couled activate GSK3β, promote the phosphorylation of β-catenin, and inhibit the β-catenin nuclear location. Moreover, CD82EC1-mP couled activate Hipoo kinase cascade, promote the phosphorylation of YAP, and inhibit the YAP nuclear location. These results suggested that CD82EC1-mP inhibited invation and matestasis via inhibiting EMT through downregulating Wnt pathway and upregulating Hippo pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2020 Elsevier Inc. All rights reserved.)

Dipeptidyl peptidase-4 (DPP4) is the prime target for glycemic control by inactivating glucagon-like peptide and decreasing postprandial glucose levels. Food protein-derived peptides have been considered to be capable of inhibiting DPP4. In this study, a novel strategy was developed by coupling in silico gastrointestinal digestion, pharmacophore and three-dimensional quantitative structure-activity relationships (3D-QSAR) analysis to discover DPP4 inhibitory peptide, and in vitro assay was confirmed. Specifically, the simulated gastrointestinal hydrolysis was firstly performed on Largemouth bass (Micropterus salmoides) proteins, the generated peptides were used to establish peptide library. Secondly, 60 DPP4 inhibitors were selected and pharmacophore model was generated; moreover, 40 DPP4 inhibitory tripeptides were collected to construct 3D-QSAR model. Thirdly, the pharmacophore and 3D-QSAR models were employed to screen the above peptide library. Lastly, the in vitro activity assay was performed, which showed that the six tripeptides (VSM, ISW, VSW, ICY, ISD and ISE) exhibited inhibitory activities on DPP4, and ICY was the most active tripeptide with the IC 50 value of 0.73 mM. This is the first identification of Largemouth bass protein-derived peptides as DPP4 inhibitor, which is good for the development of food protein-derived peptides with glucose lowering activity., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The neuronal apoptotic process requires the nuclear translocation of Apoptosis Inducing Factor (AIF) in complex with Cyclophilin A (CypA) with consequent chromatin condensation and DNA degradation events. Targeting CypA by delivering an AIF-blocking peptide (AIF(370-394)) provides a significant neuroprotection, demonstrating the biological relevance of the AIF/CypA complex. To date pharmaceutical compounds targeting this complex are missing., Methods: We designed and synthesized a set of mono and bicyclic AIF(370-394) analogs containing both disulfide and 1,2,3-triazole bridges, in the attempt to both stabilize the peptide conformation and improve its binding affinity to CypA. Peptide structures in solution and in complex with CypA have been studied by circular dichroism (CD), Nuclear Magnetic Resonance (NMR) and molecular modeling. The ability of stapled peptides to interact with CypA was evaluated by using Epic Corning label free technique and Isothermal Titration Calorimetry experiments., Results: We identified a stapled peptide analogue of AIF(370-394) with a ten-fold improved affinity for CypA. Molecular modeling studies reveal that the new peptide acquires β-turn/β-fold structures and shares with the parent molecule the same binding region on CypA., Conclusions: Data obtained provide invaluable assistance in designing new ligand of CypA for therapeutic approaches in neurodegenerative diseases., General Significance: Due to the crucial role of AIF/CypA complex formation in neurodegeneration, identification of selective inhibitors is of high importance for targeted therapies. We describe new bicyclic peptide inhibitors with improved affinity for CypA, investigating the kinetic, thermodynamic and structural effects of conformational constraints on the protein-ligand interaction, and their utility for drug design., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The prevalence of Alzheimer's disease (AD) has become a substantial global concern. Approved AChE inhibitors have been used for symptomatic treatment of AD. Binding of amyloid β (Aβ) to the peripheral anionic site of AChE facilitates the formation of Aβ plaques. Blocking this proposed protein-protein interaction by inhibition of the peripheral anionic site of AChE, in addition to increasing the level of ACh, reduces the Aβ aggregation and might qualify to slow down the progression of disease besides the palliative treatment. Targeting protein-protein interactions consider as one of the most challenging issues in the realm of drug design in which peptides have potentials to excel in. In the present study, we applied two virtual fragment-based and template-based approaches to design peptidic inhibitors of the PAS of AChE. Based on the in silico studies, high scored peptides p2 (WTWYGYWVW) and p10 (NHRMLTRRY) obtained from fragment-based and template-based design respectively. Regarding in vitro results, p2 (IC 50  = 16 ± 3.2 μM) and p10 (IC 50  = 23.6 ± 4.9 μM) showed significant AChE inhibitory effects. The molecular mechanism of inhibition studied by Lineweaver-Burk plots was mixed inhibition for both peptides. The in vitro results conformed to the in silico results and showed that both peptides occupied the CAS and PAS of AChE. The comparison of two peptide-design approaches revealed that the fragment-based design had more chemical diversity and showed priority to the template-based design. According to the obtained results, peptidic inhibitors of AChE designed by the proposed fragment-based approach might be more efficient in comparison to traditional approaches., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Sirtuins are NAD + -dependent protein deacylases that remove acyl modifications from acyl-lysine residues, resulting in essential cellular signaling. Recognized for their role in lifespan extension, humans encode seven sirtuin isoforms (Sirt1-7), and loss of sirtuin deacylase activity is implicated in many aging-related diseases. Despite being intriguing therapeutic targets, cellular studies of sirtuins are hampered by the lack of chemical probes to measure sirtuin activity independent of sirtuin protein levels. Here, we use a modular, peptide-based approach to develop activity-based probes (ABPs) that directly measure Sirt1 activity in vitro and in cell lysates. ABPs were synthesized containing four elements: (1) thioacetyl-lysine for mechanism-based affinity towards only active sirtuins, (2) either histone H3 lysine-14 (H3K14) or p53 sequences for Sirt1 specificity, (3) a diazirine for covalent labeling upon UV irradiation, and (4) an alkyne for bioorthogonal conjugation to a fluorophore for gel-based detection of active Sirt1. Compared to the H3K14 ABP, the p53 ABP showed increased sensitivity and selective labeling of active Sirt1. Acyl-lysine peptide competition, pharmacological inhibition, and inhibitory post-translational modification of Sirt1 resulted in the loss of p53 ABP labeling both in vitro and in HEK293T cell lysates, consistent with the ABP measuring decreased Sirt1 activity. Furthermore, the p53 ABP measured subcellular Sirt1 activity in MCF7 breast cancer cells. The development of a Sirt1-selective ABP that detects Sirt1 activity with an order of magnitude increased sensitivity compared to previous approaches demonstrates the utility of a modular, peptide-based approach for selective-targeting of the sirtuin protein family and provides a framework for further development of sirtuin-selective chemical probes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Prostate-specific membrane antigen (PSMA), a type II membrane glycoprotein, is considered an excellent target for the diagnosis or treatment of prostate cancer. We previously investigated the effect of β- and γ-amino acids with (S)- or (R)-configuration in the S1 pocket on the binding affinity for PSMA. However, comprehensive studies on the effect of α-amino acid with (R)-configuration in the S1' pocket has not been reported yet. We selected ZJ-43 (1) and DCIBzL (5) as templates and synthesized their analogues with (S)- or (R)-configuration in the P1 and P1' regions. The PSMA-inhibitory activities of compounds with altered chirality in the P1' region were dropped dramatically, with their IC 50 values changing from nM to μM ranges. The compounds with (S)-configuration at both P1 and P1' regions were more potent than the others. The findings of this study may provide insights regarding the structural modification of PSMA inhibitor in the S1' binding pocket., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: The epidermal growth factor receptor (EGFR) has emerged as an attractive target in the treatment of various cancers. Radiolabeled small molecules, antibodies, and peptides that specifically target EGFR are promising probes for tumor imaging to guide personalized treatment with EGFR-targeted drugs. This study aimed to radiolabel GE11 (an EGFR-specific targeting peptide) with 18-fluorine to develop a new EGFR-targeting positron emission tomography (PET) probe, [ 18 F]FP-Lys-GE11, for imaging tumors overexpressing EGFR., Methods: [ 18 F]FP-Lys-GE11 was produced by radiolabeling a GE11 peptide with the prosthetic group 4-nitrophenyl-2-[ 18 F]fluoropropionate ([ 18 F]NFP). Stability in PBS and mice serum, affinity for A431 cell line, U87 and PC-3 cells uptake and blocking studies, and biodistribution of [ 18 F]FP-Lys-GE11 were determined. 2 h dynamic and static PET scans of probe for tumor-bearing mice normal and inhibition uptake were performed., Results: [ 18 F]FP-Lys-GE11 was stable in PBS and mice serum. The Kd and Bmax values of probe for A431 were 42.43 ± 3.75 nM and 3383 ± 81.73 CPM, respectively. In cell uptake and blocking experiments, a significant reduction in radioactivity accumulation (over 4-fold) was observed by blocking U87 and PC-3 cells with unlabeled peptide. PET imaging of U87 and PC-3 tumor-bearing mice revealed clear tumor imaging (tumor radioactivity accumulation was 3.48 ± 0.44 and 3.68 ± 0.76%ID/g respectively, tumor-to-muscle ratio was 3.45 ± 0.43 and 3.64 ± 0.76 respectively). Blocking imaging revealed that the U87 tumor uptake was significantly inhibited (2.21 ± 0.41%ID/g). The biodistribution and dynamic PET imaging showed that [ 18 F]FP-Lys-GE11 was mainly excreted by the kidneys and the rest was excreted through the bile and intestines., Conclusion: The current results showed that [ 18 F]FP-Lys-GE11was a good radiolabeled peptide probe for EGFR overexpression tumor's imaging., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

G-quadruplex (G4) is a non-canonical four-stranded nucleic acid structure and the RHAU helicase has been identified to have high specificity for recognition of parallel-stranded G4s. We have designed and synthesized two stapled peptide analogues of the G4-specfic motif of RHAU, which preserve the G4 binding ability. Characterization of these peptides identified the stapled variants to exhibit higher helical formation propensity in aqueous buffer in comparison to the native RHAU sequence. Moreover, the stapled peptides exhibit superior enzymatic stability towards α-chymotrypsin. Our stapled RHAU peptides can serve as a new tool for targeting G4 nucleic acid structures., Competing Interests: Declaration of competing interest No conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The review gives a large overview of the strategies used for protein synthesis by chemoselective peptide segment ligation on a solid support. It discusses also important aspects that remain to be explored to further develop the technology such as the role of the solid support on reactant diffusion rates, on ligation kinetics, as well as on the folding and functionality of the proteins attached to the solid support., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: To investigate the effects of macromolecular crowding on the folding and aggregation of MUC5AC with different levels of glycosylation during refolding., Methods: Part 1:An in vitro catalytic reaction comprising the ppGalNAc T2 enzyme, uridine-5'-diphospho-N-galactosamine (UDP-GalNAc) and an 11-amino acid peptide substrate, was used to assess the enzyme activity of the ppGalNAc T2 enzyme in macromolecular crowding environment respectively with bovine serum albumin (BSA), polyethylene glycol (PEG2000), Dextran70 and Ficoll70 at different concentration and temperature. Part 2: The recombinant MUC5AC was expressed in HEK293 cells and purified by nickel column chromatography. The purified protein was treated with PNGase F, and the degree of glycosylation was analyzed by SDS-PAGE. Macromolecular crowding was simulated using PEG2000 at the concentrations of 50, 100, and 200 g/L. Deglycosylated-MUC5AC (d-MUC5AC) and glycosylated MUC5AC (g-MUC5AC) were denatured by GdnHCl and renatured by dilution in a refolding buffer. Protein aggregation was monitored continuously by absorbance reading at 488 nm using a UV spectrophotometer at 25 °C. The refolded proteins were centrifuged, the protein concentration of the supernatant was measured, and refolding yield in different refolding buffers was determined., Results: Enzyme activityof ppGalNAc T2 was observed to increase with increasing crowding agent concentration, with highest enzyme activity at 200 g/L. Compared with the group in the absence of crowding reagent, the refolding yield of g-MUC5AC and d-MUC5AC were reduced significantly in the presence of different concentrations of PEG2000 (200, 100, and 50 g/L). Compared with the dilute solution, aggregation increased significantly in the presence of PEG2000, especially at 200 g/L. Moreover, in the crowded reagent with the same concentration, the refolding yield of d-MUC5AC was higher than that of g-MUC5AC, whereas the degree of aggregation of d-MUC5AC was lower than that of g-MUC5AC., Conclusion: The crowded intracellular environment reduces the refolding rate of MUC5AC and strongly induces the misfolding and aggregation of glycosylated MUC5AC., Competing Interests: Declaration of competing interest We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Transcription factor EVI1 is essential for normal hematopoiesis in embryos but is aberrantly elevated in bone marrow cells of myelodysplastic syndrome (MDS) patients. EVI1 and its downstream GATA-2 appear to be a possible therapeutic target of MDS. Here we found that treatment of EVI1-expressing K562 cells with arsenite (As(III)) reduced the mRNA and protein levels of EVI1 and GATA-2. A gel shift assay using the nuclear extract of K562 cells showed that As(III) suppressed the DNA-binding activity of EVI1. The DNA-binding activity of the recombinant EVI1 protein was also suppressed by As(III) but was recovered by excess amounts of dithiothreitol, suggesting the involvement of cysteine residues of EVI1. Since the 7th Zn finger domain of EVI1, having a motif of CCHC, is known to be involved in DNA-binding, the synthetic peptide of 7th Zn finger domain was reacted with As(III) and subjected to MALDI-TOF-MS analysis. The results showed that As(III) binds to this peptide via three cysteine residues. As(III)-induced reduction of the DNA-binding activity of the recombinant EVI1 was abolished by the mutations of each of three cysteine residues to alanine in the 7th Zn finger domain. These results demonstrate that As(III) causes the down-regulation of EVI1 and GATA-2 by inhibiting the transcriptional activity of EVI1 through the binding to the cysteine residues of CCHC-type Zn finger domain., Competing Interests: Declaration of competing interest There are no conflicts of interest for any of the authors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In the current study, polyethylene glycol (PEG) was linked to polylactide (PLA) through the synthetic peptide PVGLIG which can be selectively cleaved by the tumor-associated matrix metalloproteinase 2 (MMP-2) enzyme. The synthesized chimeric triblock polymer of PEG-b-PVGLIG-PLA was implemented to form nanoscale self-assemble chimeric polymersomes. The hydrophobic SN38 was loaded into polymersomes with 70.3% ± 3.0% encapsulation efficiency demonstrating monodispersed spherical morphologies with 172 ± 30 nm dimension. The prepared chimeric polymersomal formulation provided controlled release of SN38 at physiological condition while illustrating seven-folds higher release rate when exposed to MMP-2 enzyme. At the next stage, AS1411 aptamer was conjugated onto the surface of MMP-2 responsive polymersomal formulation in order to provide guided drug delivery against nucleolin positive cells. In vitro cellular toxicity assay against C26 cell line (nucleolin positive) demonstrated significantly higher toxicity of targeted formulation in comparison with non-targeted one in low SN38 concentrations (0.15-1.25 μg/mL). In vivo study in mice bearing subcutaneous C26 tumor showed higher therapeutic index for MMP-2 responsive chimeric polymersomal formulation of SN38 in comparison with non-responsive one. On the other hand, AS1411 aptamer-targeted MMP-2 responsive chimeric polymersomal formulation exhibited highest therapeutic index compared to other groups. It could be concluded that the targeted chimeric polymersomes bearing both cleavable peptide sequence between their blocks and targeting ligand on their surface, provide favorable characteristics as an ideal delivery system against cancer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this work, using [Ru(bpy) 2 (pip)] 2+ (bpy = 2,2'-bipyridine, pip = 2-phenyl-1H-imidazo[4,5-f]-[1,10]-phenanthroline) as chromophores and neutral amino acid glycine as spacers, two novel Arg- and Lys-rich Ru(II) polypyridyl metallopeptides as an intermolecular triplex RNA stabilizers, namely [Ru(bpy) 2 (pic-Lys 2 -Gly-Lys 2 -Gly-Lys 2 )] 8+ (Ru1; pic = 2-(4-carboxy-phenyl)imidazo-[4,5-f] [1,10] phenanthroline, Gly = glycine, Lys = lysine) and [Ru(bpy) 2 (pic-Arg 2 -Gly-Arg 2 -Gly-Arg 2 )] 8+ (Ru2; Arg = arginine), have been synthesized and characterized. The binding properties of Ru1 and Ru2 with poly(U)·poly(A)∗poly(U) triplex have been studied by UV-Vis spectroscopy, fluorescence spectroscopy, viscosity measurements as well as circular dichroism and thermal denaturation. The obtained results suggest that attaching cationic peptides to a Ru(II) polypyridyl complex can obviously enhance the triplex stabilization. Considering the structure natures of Ru1 and Ru2, conceivably besides electrostatic interaction, the forces stabilizing the triplex should also involve hydrophobic interaction and hydrogen binding. Compared with the Lys-rich metallopeptide (Ru1), however, the third-strand stabilizating effect of the Arg-rich one (Ru2) is slightly more marked, which may be due to differences in the interactions of arginine and lysine residues with the third strand of the triplex. The results obtained here may be useful for understanding the interaction of triplex RNA poly(U)·poly(A)∗poly(U) with small molecule, particularly ruthenium(II) complexes., Competing Interests: Declaration of competing interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The quest for ligands alternative to Protein A for the purification of monoclonal antibodies (mAbs) has been pursued for almost three decades. Yet, the IgG-binding peptides known to date still fall short of the host cell protein (HCP) logarithmic removal value (LRV) set by Protein A media (2.5-3.1). In this study, we present an integrated computational-experimental approach leading to the discovery of peptide ligands that provide HCP LRVs on par with Protein A. First, the screening of 60,000 peptide variants was performed using a high-throughput search algorithm to identify sequences that ensure IgG affinity binding. Select sequences WQRHGI, MWRGWQ, RHLGWF, and GWLHQR were then negatively screened in silico against a panel of model HCPs to ensure the selection of peptides with high binding selectivity. Candidate ligands WQRHGI and MWRGWQ were conjugated to chromatographic resins and characterized by isothermal binding and breakthrough assays to quantify static and dynamic binding capacity (Q max and DBC 10% ), respectively. The resulting Q max were 52.6 mg of IgG per mL of adsorbent for WQRHGI and 57.48 mg/mL for MWRGWQ, while the DBC 10% (2 minutes residence time) were 30.1 mg/mL for WQRHGI and 36.4 mg/mL for MWRGWQ. Evaluation of the peptides by isothermal titration calorimetry (ITC) confirmed the binding energy predicted in silico, and an amino acid scanning study corroborated the affinity-like binding activity of the peptides. WQRHGI-WorkBeads resin was finally characterized by purification of a monoclonal antibody from a Chinese Hamster Ovary (CHO) cell culture harvest, affording a remarkable HCP LRV of 2.7, and consistent product yield and purity over 100 chromatographic cycles. These results demonstrate the potential of WQRHGI as an effective alternative to Protein A for antibody purification., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020. Published by Elsevier B.V.)

Aspartic proteases are important biomarkers of human disease and interesting targets for modulation of immune response via MHC class II antigen processing inhibition. The lack of inhibitors with sufficient selectivity hampers precise analysis of the role of cathepsin E and napsin A in samples containing the ubiquitous and highly abundant homolog cathepsin D. Grassystatins from marine cyanobacteria show promising selectivity for cathepsin E but contain several ester bonds that make their synthesis cumbersome and thus limit availability of the inhibitors. Herewith, we present grassystatin-derived cathepsin E inhibitors with greatly facilitated synthesis but retained selectivity profile. We demonstrate their affinity and selectivity with both enzyme kinetic assays and streptavidin-based pull-down from cells and mouse organs. Our findings suggest that grassystatin-like inhibitors are useful tools for targeted inhibition of cathepsin E and thus provide a novel approach for cancer and immunology research., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Early diagnosis of Prostate cancer (PCa) plays a vital role in successful treatment increasing the survival rate of patients. Prostate Specific Membrane Antigen (PSMA) is over-expressed in almost all types of PCa. The goal of present study is to introduce new 99m Tc-labeled peptides as a PSMA inhibitor for specific detection of PCa at early stages. Based on published PSMA-targeting compounds, a set of peptides bearing the well-known Glu-Urea-Lys pharmacophore and new non-urea containing pharmacophore were designed and assessed by in silico docking studies. The selected peptides were synthesized and radiolabeled with 99m Tc. The in-vitro tests (log P, stability in normal saline and fresh human plasma, and affinity toward PSMA-positive LNCaP cell line) and in-vivo characterizations of radiopeptides (biodistribution and Single Photon Emission Computed Tomography-Computed Tomography (SPECT-CT) imaging in normal and tumour-bearing mice) were performed. The peptides 1-3 containing Glu-Urea-Lys and Glu-GABA-Asp as pharmacophores were efficiently interacted with crystal structure of PSMA and showed the highest binding energies range from -8 to -11.2 kcal/mol. Regarding the saturation binding test, 99m Tc-labeled peptide 1 had the highest binding affinity (K d  = 13.58 nM) to PSMA-positive cells. SPECT-CT imaging and biodistribution studies showed high kidneys and tumour uptake 1 h post-injection of radiopeptide 1 and 2 (%ID/g tumour = 3.62 ± 0.78 and 1.8 ± 0.32, respectively). 99m Tc-peptide 1 (Glu-urea-Lys-Gly-Ala-Asp-Naphthylalanine-HYNIC- 99m Tc) exhibited the highest binding affinity, high radiochemical purity, the most stability and high specific accumulation in prostate tumour lesions. 99m Tc-peptide 1 being of comparable efficacy and pharmacokinetic properties with the well-known PET tracer ( 68 Ga-PSMA-11) seems to be applied as a promising SPECT imaging agent to early diagnose of PCa and consequently increase survival rate of patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The native extracellular matrix (ECM) is a complex gel-like system with a broad range of structural features and biomolecular signals. Hydrogel platforms that can recapitulate the complexity and signaling properties of this ECM would have enormous impact in fields ranging from tissue engineering to drug discovery. Here, we report on the design, synthesis, and proof-of-concept validation of a microporous and nanofibrous hydrogel exhibiting multiple bioactive epitopes designed to recreate key features of the bone ECM. The material platform integrates self-assembly with orthogonal enzymatic cross-linking to create a supramolecular environment comprising hyaluronic acid modified with tyramine (HA-Tyr) and peptides amphiphiles (PAs) designed to promote cell adhesion (RGDS-PA), osteogenesis (Osteo-PA), and angiogenesis (Angio-PA). Through individual and co-cultures of human adipose derived mesenchymal stem cells (hAMSCs) and human umbilical vascular endothelial cells (HUVECs), we confirmed the capacity of the HA-Tyr/RGDS-PA/Osteo-PA/Angio-PA hydrogel to promote cell adhesion as well as osteogenic and angiogenic differentiation in both 2D and 3D setups. Furthermore, using immunofluorescent staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), we demonstrated co-differentiation and organization of hAMSCs and HUVECs into 3D aggregates resembling vascularized bone-like constructs. STATEMENT OF SIGNIFICANCE: This body of work presents a new approach to develop more complex, yet functional, in vitro environments for cell culture while enabling a high level of control, tuneability, and reproducibility. The multicomponent self-assembling bioactive 2D and 3D hydrogels with nanofibrous architecture designed to recreate key molecular and macromolecular features of the native bone ECM and promote both osteogenesis and angiogenesis. The materials induce endothelial cells towards large vascular lumens and MSCs into bone cells on/within the same platform and form vascularized-bone like construct in vitro. This strategy looks encouraging for lifelike bone tissue engineering in vitro and bone tissue regeneration in vivo., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier Ltd.)

Multistage delivery systems with size reduction capacity have been proposed as a powerful strategy for improving tissue drug penetration. Here we developed a simple and fast supramolecular approach to construct size-shrinkable polyamine-salt aggregates by ionic cross-linking of biodegradable poly-L-lysine dendrigraft with tripolyphosphate anion. The use of a peptide dendrimer as a nanobuilding block (∼7 nm in diameter) allows the formation of supraparticles (SPs) with well-defined dimensions (∼200 nm in diameter), narrow size distribution and great capacity to encapsulate different molecules, including chemotherapeutic agents as Curcumin and Doxorubicin. When exposed to slightly acidic environments, the crosslinked matrix is instantaneously disassembled to free dendrimer units. Subsequently, model cargo molecules entrapped in the dendrimer architecture can be released by the action of trypsin enzyme through peptide biodegradation. Therefore, these SPs with proved sequential pH and enzyme-responsiveness could be exploited as nanocarriers in multistage drug delivery systems., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Increased Pur-alpha (Pura) protein levels in animal models alleviate certain cellular symptoms of the disease spectrum amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). Pura is a member of the Pur family of evolutionarily conserved guanine-rich polynucleotide binding proteins containing a repeated signature PUR domain of 60-80 amino acids. Here we have employed a synthetic peptide, TZIP, similar to a Pur domain, but with sequence alterations based on a consensus of evolutionarily conserved Pur family binding domains and having an added transporter sequence. A major familial form of ALS/FTD, C9orf72 (C9), is due to a hexanucleotide repeat expansion (HRE) of (GGGGCC), a Pur binding element. We show by circular dichroism that RNA oligonucleotides containing this purine-rich sequence consist largely of parallel G-quadruplexes. TZIP peptide binds this repeat sequence in both DNA and RNA. It binds the RNA element, including the G-quadruplexes, with a high degree of specificity versus a random oligonucleotide. In addition, TZIP binds both linear and G-quadruplex repeat RNA to form higher order G-quadruplex secondary structures. This change in conformational form by Pur-based peptide represents a new mechanism for regulating G quadruplex secondary structure within the C9 repeat. TZIP modulation of C9 RNA structural configuration may alter interaction of the complex with other proteins. This Pur-based mechanism provides new targets for therapy, and it may help to explain Pura alleviation of certain cellular pathological aspects of ALS/FTD., Competing Interests: Declaration of competing interest Drs. Daniel and Johnson are co-inventors on a patent entitled Family of Synthetic Polynucleotide-binding Peptides and Uses Thereof., (Copyright © 2020 Elsevier B.V. All rights reserved.)