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1.
Elife ; 132024 Jul 01.
Article in English | MEDLINE | ID: mdl-38949655

ABSTRACT

Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid-liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.


Subject(s)
Chemokine CCL5 , Chemotaxis , Cricetulus , Heparitin Sulfate , Chemokine CCL5/metabolism , Chemokine CCL5/genetics , Animals , Heparitin Sulfate/metabolism , Humans , CHO Cells , Mice , Heparin/metabolism , Heparin/pharmacology , Phase Separation
2.
Anal Chem ; 96(15): 5940-5950, 2024 Apr 16.
Article in English | MEDLINE | ID: mdl-38562013

ABSTRACT

Peptide-based supramolecules exhibit great potential in various fields due to their improved target recognition ability and versatile functions. However, they still suffer from numerous challenges for the biopharmaceutical analysis, including poor self-assembly ability, undesirable ligand-antibody binding rates, and formidable target binding barriers caused by ligand crowding. To tackle these issues, a "polyvalent recognition" strategy employing the CD20 mimotope peptide derivative NBD-FFVLR-GS-WPRWLEN (acting on the CDR domains of rituximab) was proposed to develop supramolecular nanofibers for target antibody recognition. These nanofibers exhibited rapid self-assembly within only 1 min and robust stability. Their binding affinity (179 nM) for rituximab surpassed that of the monomeric peptide (7 µM) by over 38-fold, highlighting that high ligand density and potential polyvalent recognition can efficiently overcome the target binding barriers of traditional supramolecules. Moreover, these nanofibers exhibited an amazing "instantaneous capture" rate (within 15 s), a high recovery (93 ± 3%), and good specificity for the target antibody. High-efficiency enrichment of rituximab was achieved from cell culture medium with good recovery and reproducibility. Intriguingly, these peptide nanofibers combined with bottom-up proteomics were successful in tracking the deamidation of asparagine 55 (from 10 to 16%) on the rituximab heavy chain after 21 day incubation in human serum. In summary, this study may open up an avenue for the development of versatile mimotope peptide supramolecules for biorecognition and bioanalysis of biopharmaceuticals.


Subject(s)
Biological Products , Nanofibers , Humans , Rituximab , Nanofibers/chemistry , Ligands , Reproducibility of Results , Peptides/chemistry
3.
Chem Biol Drug Des ; 101(6): 1406-1415, 2023 06.
Article in English | MEDLINE | ID: mdl-36862057

ABSTRACT

Antibody-directed drugs for targeted cancer treatment have become a hot topic in new anticancer drug development; however, antibody-fused therapeutic peptides were rarely documented. Herein, we designed a fusion protein with a cetuximab-derived single-chain variable fragment targeting epidermal growth factor receptor (anti-EGFR scFv) and the anticancer lytic peptide (ACLP) ZXR2, connected by a linker (G4 S)3 and MMP2 cleavage site. The anti-EGFR scFv-ZXR2 recombinant protein showed specific anticancer activity on EGFR-overexpressed cancer cell lines in a concentration- and time-dependent manner, as it can bind to EGFR on cancer cell surfaces. This fusion protein caused cell membrane lysis as ZXR2, and showed improved stability in serum compared with ZXR2. These results suggest that scFv-ACLP fusion proteins may be potential anticancer drug candidates for targeted cancer treatment, which also provide a feasible idea for targeted drug design.


Subject(s)
Antineoplastic Agents , Neoplasms , Single-Chain Antibodies , Humans , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cetuximab/therapeutic use , Neoplasms/drug therapy , Neoplasms/metabolism , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/therapeutic use , Recombinant Proteins/therapeutic use , Single-Chain Antibodies/pharmacology , Single-Chain Antibodies/therapeutic use
4.
ACS Nano ; 16(9): 13783-13799, 2022 09 27.
Article in English | MEDLINE | ID: mdl-36099446

ABSTRACT

Anticancer peptides are promising drug candidates for cancer treatment, but the short circulation time and low delivery efficiency limit their clinical applications. Herein, we designed several lasso-like self-assembling anticancer peptides (LASAPs) integrated with multiple functions by a computer-aided approach. Among these LASAPs, LASAP1 (CRGDKGPDCGKAFRRFLGALFKALSHLL, 1-9 disulfide bond) was determined to be superior to the others because it can self-assemble into homogeneous nanoparticles and exhibits improved stability in serum. Thus, LASAP1 was chosen for proving the design idea. LASAP1 can self-assemble into nanoparticles displaying iRGD on the surface because of its amphiphilic structure and accumulate to the tumor site after injection because of the EPR effect and iRGD targeting to αVß3 integrin. The nanoparticles could disassemble in the acidic microenvironment of the solid tumor, and cleaved by the overexpressed hK2, which was secreted by prostate tumor cells, to release the effector peptide PTP-7b (FLGALFKALSHLL), which was further activated by the acidic pH. Therefore, LASAP1 could target the orthotopic prostate tumor in the model mice after intraperitoneal injection and specifically inhibit tumor growth, with low systematic toxicity. Combining the multiple targeting functions, LASAP1 represents a promising design of self-delivery of peptide drugs for targeted cancer treatments.


Subject(s)
Antineoplastic Agents , Nanoparticles , Prostatic Neoplasms , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Computer-Aided Design , Disulfides , Drug Delivery Systems , Humans , Integrins , Male , Mice , Nanoparticles/chemistry , Peptides/chemistry , Prostatic Neoplasms/drug therapy , Tumor Microenvironment
5.
Eur J Pharm Biopharm ; 179: 137-146, 2022 Oct.
Article in English | MEDLINE | ID: mdl-36096399

ABSTRACT

Water insolubility poses a significant challenge in the clinical applications of many small molecule drugs. To improve the drug delivery efficiency, two branched amphiphilic peptides (BAPs) were designed in a computer-aided manner, for drug-loading through peptide self-assembling. The structures of the two BAPs, bis(LVFFA)-K-RGD (PepV-1) and bis(FHF)-K-RGD (PepV-2), were inspired by phospholipids, containing the RGD sequence as the hydrophilic head and two hydrophobic sequences as the hydrophobic tails. PepV-1 could self-assemble into nano-fibrils with a hydrophobic core and the RGD moiety on the surface. Its drug-loading efficiency (DE%) of three small molecule anticancer drugs (doxorubicin, camptothecin and curcumin) ranged from 9.90% to 11.74%, and entrapment efficiency (EE%) ranged from 37.30% to 43.00%. Pep-V2 could self-assemble into bilayer delimited nano-vesicles. The DE% of PepV-2 for these drugs ranged from 15.87% to 18.55%, and the EE% ranged from 60.45% to 73.23%. Both BAP carriers could prolong the release of the small molecule drugs, and the PepV-2 vesicles also showed pH-triggered increase of drug release due to the histidine residues. Bothe BAP carriers could increase the cytotoxicity against cancer cells, which might be due to the targeting on the cancer overexpressed integrins. The designed BAP carriers represent promising functional drug carriers for targeted drug delivery, and will be useful for improving the clinical use of small molecule drugs, especially for those with poor water solubility.


Subject(s)
Antineoplastic Agents , Curcumin , Antineoplastic Agents/chemistry , Camptothecin , Doxorubicin/chemistry , Drug Carriers/chemistry , Histidine , Hydrophobic and Hydrophilic Interactions , Integrins , Oligopeptides , Peptides/chemistry , Water/chemistry
6.
Emerg Microbes Infect ; 11(1): 2658-2669, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36153659

ABSTRACT

The binding of the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein onto human angiotensin-converting enzyme 2 (ACE2) is considered as the first step for the virus to adhere onto the host cells during the infection. Here, we investigated the adhesion of spike proteins from different variants and ACE2 using single-molecule and single-cell force spectroscopy. We found that the unbinding force and binding probability of the spike protein from Delta variant to the ACE2 were the highest among the variants tested in our study at both single-molecule and single-cell levels. As the most popular variants, the Omicron variants have slightly higher unbinding force to the ACE2 than wild type. Molecular dynamics simulation showed that ACE2-RBD (Omicron BA.1) complex is destabilized by the E484A and Y505H mutations and stabilized by S477N and N501Y mutations, when compared with Delta variant. In addition, a neutralizing antibody, produced by immunization with wild type spike protein, could effectively inhibit the binding of spike proteins from wild type, Delta and Omicron variants (BA.1 and BA.5) onto ACE2. Our results provide new insight for the molecular mechanism of the adhesive interactions between spike protein and ACE2 and suggest that effective monoclonal antibody can be prepared using wild type spike protein against different variants.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Humans , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/genetics , SARS-CoV-2/genetics , Peptidyl-Dipeptidase A/metabolism , Protein Binding , Mutation
7.
iScience ; 25(1): 103720, 2022 Jan 21.
Article in English | MEDLINE | ID: mdl-35005526

ABSTRACT

It is unknown whether antibody-mediated enhancement (ADE) contributes to the pathogenesis of COVID-19, and the conditions for ADE needs to be elucidated. We demonstrated that without inducing an ACE2-independent ADE on Raji cells, the neutralizing antibody CB6, a mouse anti-S1 serum and convalescent plasma, induced ADE on cells expressing FcγRIIA/CD32A and low levels of endogenous ACE2. ADE occurred at sub-neutralizing antibody concentrations, indicating that unneutralized S protein was required for ADE. The enhanced infectivity of 614G variant was higher than that of 614D wildtype in the presence of antibodies, further suggesting that ADE may be influenced by virus strains with different ACE2-binding affinity. Finally, knockdown of ACE2 or treatment with a fusion-inhibition peptide EK1C4 significantly reduced ADE. In conclusion, we identified an ADE mechanism mediated by neutralizing antibodies against SARS-CoV-2. ACE2 may act as a secondary receptor required for the antibody- and FcγR-mediated enhanced entry of SARS-CoV-2.

9.
Brief Bioinform ; 22(6)2021 11 05.
Article in English | MEDLINE | ID: mdl-34180984

ABSTRACT

Targeting the interaction between severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)-receptor-binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) is believed to be an effective strategy for drug design to inhibit the infection of SARS-CoV-2. Herein, several ultrashort peptidase inhibitors against the RBD-ACE2 interaction were obtained by a computer-aided approach based on the RBD-binding residues on the protease domain (PD) of ACE2. The designed peptides were tested on a model coronavirus GX_P2V, which has 92.2 and 86% amino acid identity to the SARS-CoV-2 spike protein and RBD, respectively. Molecular dynamics simulations and binding free energy analysis predicted a potential binding pocket on the RBD of the spike protein, and this was confirmed by the specifically designed peptides SI5α and SI5α-b. They have only seven residues, showing potent antiviral activity and low cytotoxicity. Enzyme-linked immunosorbent assay result also confirmed their inhibitory ability against the RBD-ACE2 interaction. The ultrashort peptides are promising precursor molecules for the drug development of Corona Virus Disease 2019, and the novel binding pocket on the RBD may be helpful for the design of RBD inhibitors or antibodies against SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , COVID-19 Drug Treatment , Peptides/chemistry , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Antiviral Agents/chemistry , Binding Sites/drug effects , COVID-19/genetics , COVID-19/virology , Drug Design , Humans , Molecular Dynamics Simulation , Peptides/genetics , Peptides/therapeutic use , Protein Binding/drug effects , Protein Domains/drug effects , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
10.
Amino Acids ; 52(1): 1-14, 2020 Jan.
Article in English | MEDLINE | ID: mdl-31797056

ABSTRACT

Biofilm poses a serious challenge for the treatment of bacterial infections, as it endows bacteria a pronounced resistance to traditional antibiotics. Antimicrobial peptides (AMPs) are considered potential substitutes for antibiotics. Combinational use of AMPs with other compounds to exert antibiofilm effects has been proved to be an effective means to reduce their toxicity and maximize their antimicrobial activity. However, the combination of various AMPs with different action mechanisms is rarely investigated. A newly designed lytic AMP ZXR-2.3 combined with antibiofilm peptide IDR-1018 or KT2 was tested for the antibiofilm effect on mature Streptococcus mutans biofilms. In general, the combination of ZXR-2.3 + IDR-1018 displayed synergistic effect on both biofilm eradication and bacterial killing, while ZXR-2.3 + KT2 showed no obvious synergism. The confocal images of preformed S. mutans biofilms confirmed the effective bactericidal activity of ZXR-2.3 + IDR-1018. A tube system was applied to investigate the biofilm infection under a flow of medium and SEM images indicated the biofilm disruption and bacterial killing effects of ZXR-2.3 + IDR-1018. Quantitative RT-PCR analysis showed that IDR-1018 induced dramatic changes in the mRNA expressions of the quorum sensing (QS) related genes comC, comD, vicR, and vicK of S. mutans in mature biofilms, whereas the other peptides and ciprofloxacin did not cause obvious changes in these genes. This might explain the better synergism of ZXR-2.3 and IDR-1018. The results of this study provide a potential application using the combination of different AMPs in the treatment of mature biofilm infection.


Subject(s)
Anti-Infective Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Biofilms/drug effects , Streptococcus mutans/drug effects , Anti-Bacterial Agents/adverse effects , Anti-Bacterial Agents/therapeutic use , Drug Resistance, Bacterial/drug effects , Humans , Microbial Sensitivity Tests , Streptococcus mutans/pathogenicity
11.
Infect Drug Resist ; 12: 3943-3953, 2019.
Article in English | MEDLINE | ID: mdl-31920348

ABSTRACT

BACKGROUND: Bacterial infections cause a serious public health crisis due to the emergence of resistance towards multiple conventional antibacterial drugs. In particular, multidrug-resistant (MDR) Enterococcus faecium which belongs to "ESKAPE" organisms is causing significant problems worldwide. Hence, there is an urgent need to find alternative therapies. Recently, substituted benzene guanidine compounds have been used as lead structures to discover new promising drugs in both synthetic and medicinal chemistry. PURPOSE: Here we investigated the antimicrobial activity of a new substituted benzene guanidine analog, isopropoxy benzene guanidine, against Enterococci. MATERIAL AND METHODS: The isopropoxy benzene guanidine was synthesized by Guangzhou Insighter Biotechnology Co., Ltd and tested on both reference bacterial strain and 32 clinical MDR Enterococci strains. The in vitro antibacterial activity was evaluated by microdilution method and kill kinetic assays. The potential antibacterial mechanism was measured by fluorescence spectrometry using fluorescent membrane potential probe 3, 3-diethyloxacarbocyanine iodide (DiOC2 (3)). RESULTS: Isopropoxy benzene guanidine exhibited potent bactericidal activity against both reference strain and MDR Enterococci isolates. The minimum inhibitory concentration (MIC) range for isopropoxy benzene guanidine was 1-4 µg/mL. Minimum bactericidal concentration (MBC) was about 2-8-fold of its MIC values. Time-kill studies showed that isopropoxy benzene guanidine provided superior bactericidal effect against reference and MDR strains within 12 hrs at 2×MIC. Furthermore, isopropoxy benzene guanidine could cause a large reduction in the magnitude of the generated membrane potential compared to that of the untreated cells. CONCLUSION: The present study highlights the potent bactericidal activity of isopropoxy benzene guanidine on Enterococci by disrupting the cell membrane potential. These findings demonstrate that isopropoxy benzene guanidine may be a good chemical lead for further medicinal chemistry and pharmaceutical development and could be used as a therapeutic agent for infectious diseases caused by MDR Enterococci.

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