Anti-Coronavirus Potential of Polyether Ionophores: The New Application of Veterinary Antibiotics in Livestock.

Coronaviruses have consistently posed a major global concern in the field of livestock industry and public health. However, there is currently a lack of efficient drugs with broad-spectrum antiviral activity to address the challenges presented by emerging mutated strains or drug resistance. Additionally, the method for identifying multitarget drugs is also insufficient. Aminopeptidase N (APN) and 3C-like proteinase (3CLpro) represent promising targets for host-directed and virus-directed strategies, respectively, in the development of effective drugs against various coronaviruses. In this study, maduramycin ammonium demonstrated a broad-spectrum antiviral effect by targeting both of the proteins. The binding domains 4 Å from the ligand of both target proteins shared a structural similarity, suggesting that screening and designing drugs based on these domains might exhibit broad-spectrum and highly effective antiviral activity. Furthermore, it was identified that the polyether ionophores' ability to carry zinc ion might be one of the reasons why they were able to target APN and exhibit antiviral effect. The findings of this experiment provide novel perspectives for future drug screening and design, while also offering valuable references for the utilization of polyether ionophores in the management of livestock health.

Design, synthesis, and biological evaluation of novel HDAC/CD13 dual inhibitors for the treatment of cancer.

Aminopeptidase N (APN/CD13) plays a role in tumors progression, but its inhibitor lacks cytotoxicity and is used as an adjuvant drug in cancer treatment. Histone deacetylases (HDACs) are a type of epigenetic targets, and HDAC inhibitors are cytotoxic and exhibit synergistic effects with other anticancer agents. Herein, a novel series of HDAC/CD13 dual inhibitors were rationally designed and synthesized to combine the anti-metastasis and anti-invasion of CD13 inhibitor with the cytotoxic of HDAC inhibitor. The representative compound 12 exhibited more potent inhibitory activity against human CD13, HDAC1-3, and antiproliferative activity than positive controls bestatin and SAHA. Compound 12 effectively induced apoptosis in MV4-11 cells, while arresting A549 cells in G2/M phase. Moreover, 12 exhibited significantly better anti-metastasis and anti-invasion effects than mono-inhibitors 32 and 38, indicating that it is a promising anti-cancer agent for further investigation.

Structure, receptor recognition, and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein.

The isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. We determined the structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and showed that its domain 0 recognizes sialosides. We identified that the CCoV-HuPn-2018 spike binds canine, feline, and porcine aminopeptidase N (APN) orthologs, which serve as entry receptors, and determined the structure of the receptor-binding B domain in complex with canine APN. The introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single-nucleotide polymorphisms might account for viral detection in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 spike-mediated entry, underscoring the cross-neutralizing activity among ɑ-coronaviruses. These data pave the way for vaccine and therapeutic development targeting this zoonotic pathogen representing the eighth human-infecting coronavirus.

CD13 orients the apical-basal polarity axis necessary for lumen formation.

Polarized epithelial cells can organize into complex structures with a characteristic central lumen. Lumen formation requires that cells coordinately orient their polarity axis so that the basolateral domain is on the outside and apical domain inside epithelial structures. Here we show that the transmembrane aminopeptidase, CD13, is a key determinant of epithelial polarity orientation. CD13 localizes to the apical membrane and associates with an apical complex with Par6. CD13-deficient cells display inverted polarity in which apical proteins are retained on the outer cell periphery and fail to accumulate at an intercellular apical initiation site. Here we show that CD13 is required to couple apical protein cargo to Rab11-endosomes and for capture of endosomes at the apical initiation site. This role in polarity utilizes the short intracellular domain but is independent of CD13 peptidase activity.

Identification and Characterization of Aminopeptidase N 1 Gene of the Indian Malaria Vector Anopheles culicifacies (Diptera: Culicidae).

Aminopeptidase N1 (APN) is one of the important enzymes involved in blood digestion and is up-regulated along with several other enzymes in response to bloodmeal ingestion. APN is a zinc metalloprotease that cleaves one amino acid residue at a time from the amino terminus of the protein. The APN1 gene of the Indian malaria vector Anopheles culicifacies Giles was cloned and characterized. The An. culicifacies APN1 (AcAPN1) gene has an Open Reading Frame of 3084 basepairs which encodes a putative protein of 1027 amino acids. The coding region of the gene shares 81% and 78% similarity to the APN1 genes found in An. stephensi (Diptera: Culicidae) and An. gambiae (Diptera: Culicidae), respectively. The organization of the APN1 gene was studied in available mosquito genomes and a three-dimensional structure of AcAPN1 modeled using homology structure modeling. The enzymatic active site was predicted to consist of HEYAH and GAMEN amino acid residues, and a comparison of the protein sequences among different genera revealed the conservation of zinc-binding residues. The expression pattern of AcAPN1 showed that the gene was expressed rapidly in response to the ingestion of the bloodmeal and therefore this gene may be used to exploit its promoter region as an antiparasite candidate molecule.

Biochemical and cellular characterisation of the Plasmodium falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP).

The Plasmodium falciparum M1 alanyl aminopeptidase and M17 leucyl aminopeptidase, PfM1AAP and PfM17LAP, are potential targets for novel anti-malarial drug development. Inhibitors of these aminopeptidases have been shown to kill malaria parasites in culture and reduce parasite growth in murine models. The two enzymes may function in the terminal stages of haemoglobin digestion, providing free amino acids for protein synthesis by the rapidly growing intra-erythrocytic parasites. Here we have performed a comparative cellular and biochemical characterisation of the two enzymes. Cell fractionation and immunolocalisation studies reveal that both enzymes are associated with the soluble cytosolic fraction of the parasite, with no evidence that they are present within other compartments, such as the digestive vacuole (DV). Enzyme kinetic studies show that the optimal pH of both enzymes is in the neutral range (pH 7.0-8.0), although PfM1AAP also possesses some activity (< 20%) at the lower pH range of 5.0-5.5. The data supports the proposal that PfM1AAP and PfM17LAP function in the cytoplasm of the parasite, likely in the degradation of haemoglobin-derived peptides generated in the DV and transported to the cytosol.

In silico design of novel aptamers utilizing a hybrid method of machine learning and genetic algorithm.

Aptamers can be regarded as efficient substitutes for monoclonal antibodies in many diagnostic and therapeutic applications. Due to the tedious and prohibitive nature of SELEX (systematic evolution of ligands by exponential enrichment), the in silico methods have been developed to improve the enrichment processes rate. However, the majority of these methods did not show any effort in designing novel aptamers. Moreover, some target proteins may have not any binding RNA candidates in nature and a reductive mechanism is needed to generate novel aptamer pools among enormous possible combinations of nucleotide acids to be examined in vitro. We have applied a genetic algorithm (GA) with an embedded binding predictor fitness function to in silico design of RNA aptamers. As a case study of this research, all steps were accomplished to generate an aptamer pool against aminopeptidase N (CD13) biomarker. First, the model was developed based on sequential and structural features of known RNA-protein complexes. Then, utilizing RNA sequences involved in complexes with positive prediction results, as the first-generation, novel aptamers were designed and top-ranked sequences were selected. A 76-mer aptamer was identified with the highest fitness value with a 3 to 6 time higher score than parent oligonucleotides. The reliability of obtained sequences was confirmed utilizing docking and molecular dynamic simulation. The proposed method provides an important simplified contribution to the oligonucleotide-aptamer design process. Also, it can be an underlying ground to design novel aptamers against a wide range of biomarkers.

Infectious bronchitis virus: Identification of Gallus gallus APN high-affinity ligands with antiviral effects.

Infectious bronchitis virus (IBV) is a coronavirus, causes infectious bronchitis (IB) with high morbidity and mortality, and gives rise to huge economic losses for the poultry industry. Aminopeptidase N (APN) may be one of the IBV functional receptors. In this study, Gallus gallus APN (gAPN) protein was screened by phage-displayed 12-mer peptide library. Two high-affinity peptides H (HDYLYYTFTGNP) and T (TKFSPPSFWYLH) to gAPN protein were selected for in depth characterization of their anti-IBV effects. In vitro, indirect ELISA showed that these two high-affinity ligands could bind IBV S1 antibodies. Quantitative real-time PCR (qRT-PCR) assay, virus yield reduction assay and indirect immunofluorescence assay results revealed 3.125-50 µg/ml of peptide H and 6.25-50 µg/ml of peptide T reduced IBV proliferation in chicken embryo kidney cells (CEKs). In vivo, high-affinity phage-vaccinated chickens were able to induce specific IBV S1 antibodies and IBV neutralizing antibodies. QRT-PCR results confirmed that high-affinity phages reduced virus proliferation in chicken tracheas, lungs and kidneys, and alleviated IBV-induced lesions. By multiple sequence alignment, motif 'YxYY' and 'FxPPxxWxLH' of high-affinity peptides were identified in IBV S1-NTD, while another motif 'YxFxGN' located in S2. These results indicated that high affinity peptides of gAPN could present an alternative approach to IB prevention or treatment.

Synthesis and 4D-QSAR Studies of Alanine Hydroxamic Acid Derivatives as Aminopeptidase N Inhibitors.

BACKGROUND: As a target for anticancer treatment, aminopeptidase N (APN) shows its overexpression on diverse malignant tumor cells and associates with cancer invasion, angiogenesis and metastasis. OBJECTIVE: The objective of the study was the design, synthesis and biological activity evaluation of alanine hydroxamic acid derivatives as APN inhibitors, and investigation of the binding mode of inhibitors in the APN active site. METHODS: Alanine hydroxamic acid derivatives were synthesized and evaluated for their in vitro anti-cancer activity using CCK-8 assay. Molecular docking and 4D-QSAR studies were carried out to suggest the mechanism of biological activity. RESULTS: Compared with Bestatin, compound 9b showed the best APN inhibition activity. The putative binding mode of 9b in the APN active site was also discussed. Moreover, the robust and reliable 4D-QSAR model exhibited the following statistics: R2 = 0.9352, q2 LOO = 0.8484, q2 LNO =0.7920, R2 Pred = 0.8739. CONCLUSION: Newly synthesized compounds exerted acceptable anticancer activity and further investigation of the current scaffold would be beneficial.

Increasing the activity of cell adherent cyclic NGR peptides by optimizing the peptide length and amino acid character.

Cyclic peptides are an attractive modality for the development of therapeutics and the identification of functional cyclic peptides that contribute to novel drug development. The peptide array is one of the optimization methods for peptide sequences and also useful to understand sequence-function relationship of peptides. Cell adherent cyclic NGR peptide which selectively binds to the aminopeptidase N (APN or CD13) is known as an attractive tumor marker. In this study, we designed and screened a library of different length and an amino acid substitution library to identify stronger cell adhesion peptides and to reveal that the factor of higher binding between CD13 and optimized cyclic peptides. Additionally, we designed and evaluated 192 peptide libraries using eight representative amino acids to reduce the size of the library. Through these optimization steps of cyclic peptides, we identified 23 peptides that showed significantly higher cell adhesion activity than cKCNGRC, which was previously reported as a cell adhesion cyclic peptide. Among them, cCRHNGRARC showed the highest activity, that is, 1.65 times higher activity than cKCNGRC. An analysis of sequence and functional data showed that the rules which show higher cell adhesion activity for the three basic cyclic peptides (cCX1 HNGRHX2 C, cCX1 HNGRAX2 C, and cCX1 ANGRHX2 C) are related with the position of His residues and cationic amino acids.

MMP activation-associated aminopeptidase N reveals a bivalent 14-3-3 binding motif.

Aminopeptidase N (APN, CD13) is a transmembrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction and is involved in the activation of matrix metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases, such as fibrosis, rheumatoid arthritis, tumor angiogenesis, and metastasis, as well as viral infections. However, the exact mechanism that leads to APN-driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins bind to APN and thereby induce the transcription of MMPs. As a first step, we sought to identify potential 14-3-3-binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3-binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding fluorescence polarization assays and thermodynamic analysis. In addition, we identified a secondary, noncanonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14-3-3σ bound to mono- and bis-phosphorylated APN-derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and open the field for further investigation of this important signaling pathway.

Synthesis and Inhibitory Studies of Phosphonic Acid Analogues of Homophenylalanine and Phenylalanine towards Alanyl Aminopeptidases.

A library of novel phosphonic acid analogues of homophenylalanine and phenylalanine, containing fluorine and bromine atoms in the phenyl ring, have been synthesized. Their inhibitory properties against two important alanine aminopeptidases, of human (hAPN, CD13) and porcine (pAPN) origin, were evaluated. Enzymatic studies and comparison with literature data indicated the higher inhibitory potential of the homophenylalanine over phenylalanine derivatives towards both enzymes. Their inhibition constants were in the submicromolar range for hAPN and the micromolar range for pAPN, with 1-amino-3-(3-fluorophenyl) propylphosphonic acid (compound 15c) being one of the best low-molecular inhibitors of both enzymes. To the best of our knowledge, P1 homophenylalanine analogues are the most active inhibitors of the APN among phosphonic and phosphinic derivatives described in the literature. Therefore, they constitute interesting building blocks for the further design of chemically more complex inhibitors. Based on molecular modeling simulations and SAR (structure-activity relationship) analysis, the optimal architecture of enzyme-inhibitor complexes for hAPN and pAPN were determined.

Computational study of novel natural inhibitors targeting aminopeptidase N(CD13).

OBJECTIVES: To screen and identify ideal leading compounds from a drug library (ZINC15 database) with potential inhibition of aminopeptidase N(CD13) to contribute to medication design and development. RESULTS: Two novel natural compounds, ZINC000000895551 and ZINC000014820583, from the ZINC15 database were found to have a higher binding affinity and more favorable interaction energy binding with CD13 with less rodent carcinogenicity, Ames mutagenicity, and non-inhibition with cytochrome P-450 2D6. Molecular dynamics simulation analysis suggested that the 2 complexes, ZINC000000895551-CD13 and ZINC000014820583-CD13, have favorable potential energy, and exist stably in the natural circumstances. CONCLUSION: This study discovered that ZINC000000895551 and ZINC000014820583 were ideal leading compounds to be inhibitions targeting to CD13. These compounds were selected as safe drug candidates as CD13 target medication design and improvement. MATERIALS AND METHOD: Potential inhibitors of CD13 were identified using a series of computer-aided structural and chemical virtual screening techniques. Structure-based virtual screening was carried out to calculate LibDock scores, followed by analyzing their absorption, distribution, metabolism, and excretion and toxicity predictions. Molecule docking was employed to reveal binding affinity between the selected compounds and CD13. Molecular dynamics simulation was applied to evaluate stability of the ligand-CD13 complex under natural environment.

Designed Three-in-One Peptides with Anchoring, Antifouling, and Recognizing Capabilities for Highly Sensitive and Low-Fouling Electrochemical Sensing in Complex Biological Media.

Nonspecific adsorption is of great concern for electrochemical biosensors performing in complex biological media, and various antifouling materials have been introduced into the sensing interfaces to improve the antifouling capability of different biosensors. However, for most of the biosensors with antifouling materials and sensing probes coexisting in the sensing interfaces, either the antifouling materials will impair the sensing performances or the sensing probes will affect the antifouling ability. Herein, a facile and efficient antifouling biosensor was developed based on a newly designed three-in-one peptide with anchoring, antifouling, and recognizing capabilities. One end of the designed peptide is a unique anchoring part that is rich in amine groups, and this part can be anchored to the poly(3,4-ethylenedioxythiophene) (PEDOT)-citrate film electrodeposited on a glassy carbon electrode. The other end of the peptide is a recognizing part that can specifically bind to the aminopeptidase N (APN) and human hepatocellular carcinoma cells (HepG2 cells). Meanwhile, the middle part of the peptide, together with the anchoring part, was designed to be antifouling. With this designed multifunctional peptide, highly sensitive and low-fouling biosensors capable of assaying target APN and HepG2 cells in complex biological media can be easily prepared, with detection limits of 0.4 ng·mL-1 and 20 cells·mL-1, respectively. This antifouling biosensor is feasible for practical target detection in real complex samples, and it is highly expected that this peptide designing strategy may be extended to the development of various antifouling biosensors.

Design and synthesis of a novel peptide for selective detection of cancer cells.

Using a minimalist approach, an 11-residue peptide (Peptide 1) tagged with rhodamine fluorophore was designed and synthesized for selective detection of cancer cells. Peptide 1 contains RGD and NGR motifs to bind, respectively, integrins and aminopeptidase CD13, which are over expressed in cancer cells. Surface tension measurements revealed that peptide 1 possess surface-active property owing to the overall hydrophobicity and cationic nature of the peptide. Peptide 1 displays cancer cell-selective binding at ≤5.0 µM concentrations, while peptide 2 (randomized sequence of 1) shows non-selective binding to normal and cancer cells. Fluorescence microscopy and FACS analysis demonstrated the intracellular localization of peptide 1 in three different cancer cell lines, confirming the role of RGD and NGR motifs. Cytotoxicity assay exhibited the viability of normal and cancer cells up to 100 µM concentrations of peptide 1. Steady-state fluorescence measurements disclosed the preferential interactions of the peptide 1 with anionic POPC/POPG bilayers rather than with zwitterionic POPC lipid bilayers. Circular dichroism studies showed minimal changes in the secondary structure of peptide 1 upon binding with the anionic lipid bilayers. Peptide 1 is largely unordered, non-toxic, and useful for identification of cancer cells. Peptide 1 provides a template for designing drug-loaded peptides for targeted delivery into cancer cells.

CD13-specific ligand facilitates Xanthatin nanomedicine targeting dendritic cells for therapy of refractory allergic rhinitis.

Relapse in Allergic Rhinitis (AR) is triggered by various unclear mechanisms. Xanthium strumarium L. as a traditional folk medicine can inhibit inflammatory responses through multiple mechanisms. Xanthatin (XT) is a bioactive compound derived from Xanthium strumarium L, and we developed a polymeric micelle (PM) that is dendritic cells (DCs)-specific targeting delivery system loading XT (NGR-XT-PM) based on a cyclic peptide moiety (NGR) to render DCs maturation-resistant for therapy of refractory AR. A murine model of AR was employed to investigate the in vivo therapeutic efficiency and relapse rate compared with the commercial product Budesonide. The results showed intranasal administration of NGR-XT-PM presented significant anti-allergy effect with no recurrence, in contrast, all mice treatment with Budesonide relapsed. NGR-XT-PM could effectively reverse the Th1/Th2 imbalance by depleting the serum inflammatory levels (IgE, histamine and IL-4) and DCs surface costimulatory molecules (CD80, CD86 and I-A/I-E), and promote immune tolerance by upregulating the level of Treg cells and reducing the levels of Th2, Th9 and Th17 cells. Furthermore, we appealed to virtual screening of inflammatory targets and found XT blocking the COX-2/PGE2 signaling pathway, which is a key effector in immune responses. These indicated CD13-specific NGR could facilitate XT selectively targeting DCs for efficiently ameliorating refractory rhinitis, and NGR-XT-PM should be a potential anti-AR drug.

Inhibition of Porcine Aminopeptidase M (pAMP) by the Pentapeptide Microginins.

Aminopeptidase M (AMP) inhibition is of interest for several diseases, such as highly vascularized cancer types. AMP can be inhibited by linear pentapeptides isolated from Microcystis aeruginosa LTPNA08 (MG7XX). Porcine AMP inhibition-a model for human AMP-activity was spectrophotometrically measured by the formation of p-nitroanilide from L-leucine-p-nitroanilide substrate by AMP. AMP inhibition by MG770 exhibited comparable inhibition levels to amastatin (IC50 values: 1.20 ± 0.1 µM and 0.98 ± 0.1 µM, respectively), while MG756 was slightly less potent (with IC50 values of 3.26 ± 0.5 µM). Molecular modelling suggests a potential binding mode, based on the interaction with the Zn2+ cofactor, where MG770's extra methyl group contributes to the disturbance of the Zn2+ cofactor complex and highlights the importance of hydrophobicity for the site.

The human coronavirus HCoV-229E S-protein structure and receptor binding.

The coronavirus S-protein mediates receptor binding and fusion of the viral and host cell membranes. In HCoV-229E, its receptor binding domain (RBD) shows extensive sequence variation but how S-protein function is maintained is not understood. Reported are the X-ray crystal structures of Class III-V RBDs in complex with human aminopeptidase N (hAPN), as well as the electron cryomicroscopy structure of the 229E S-protein. The structures show that common core interactions define the specificity for hAPN and that the peripheral RBD sequence variation is accommodated by loop plasticity. The results provide insight into immune evasion and the cross-species transmission of 229E and related coronaviruses. We also find that the 229E S-protein can expose a portion of its helical core to solvent. This is undoubtedly facilitated by hydrophilic subunit interfaces that we show are conserved among coronaviruses. These interfaces likely play a role in the S-protein conformational changes associated with membrane fusion.

Fluorogenic l-alanylaminopeptidase substrates derived from 6-amino-2-hetarylquinolines and 7-amino-3-hetarylcoumarins and their potential applications in diagnostic microbiology.

Six novel fluorogenic enzyme substrates for detecting l-alanylaminopeptidase activity in microorganisms have been prepared and evaluated in Columbia agar media. The substrates are l-alanyl derivatives of 6-amino-2-hetarylquinolines and 7-amino-3-hetarylcoumarins. Both the quinoline and coumarin series of substrates produced fluorescence in the presence of Gram-negative microorganisms. In contrast, fluorescence generation in the presence of the Gram-positive microorganisms and yeasts was limited or absent.

Fast and accurate data collection for macromolecular crystallography using the JUNGFRAU detector.

The accuracy of X-ray diffraction data is directly related to how the X-ray detector records photons. Here we describe the application of a direct-detection charge-integrating pixel-array detector (JUNGFRAU) in macromolecular crystallography (MX). JUNGFRAU features a uniform response on the subpixel level, linear behavior toward high photon rates, and low-noise performance across the whole dynamic range. We demonstrate that these features allow accurate MX data to be recorded at unprecedented speed. We also demonstrate improvements over previous-generation detectors in terms of data quality, using native single-wavelength anomalous diffraction (SAD) phasing, for thaumatin, lysozyme, and aminopeptidase N. Our results suggest that the JUNGFRAU detector will substantially improve the performance of synchrotron MX beamlines and equip them for future synchrotron light sources.