Excellent effects and possible mechanisms of action of a new antibody–drug conjugate against EGFR-positive triple-negative breast cancer

Background:Triple-negative breast cancer(TNBC)is the most aggressive subtype and occurs in approximately 15%–20%of diagnosed breast cancers.TNBC is characterized by its highly metastatic and recurrent features,as well as a lack of specific targets and targeted therapeutics.Epidermal growth factor receptor(EGFR)is highly expressed in a variety of tumors,especially in TNBC.LR004-VC-MMAE is a new EGFR-targeting antibody–drug conjugate produced by our laboratory.This study aimed to evaluate its antitumor activities against EGFR-positive TNBC and further studied its possible mechanism of antitumor action.Methods:LR004-VC-MMAE was prepared by coupling a cytotoxic payload(MMAE)to an anti-EGFR antibody(LR004)via a linker,and the drug-to-antibody ratio(DAR)was analyzed by HIC-HPLC.The gene expression of EGFR in a series of breast cancer cell lines was assessed using a publicly available microarray dataset(GSE41313)and Western blotting.MDA-MB-468 and MDA-MB-231 cells were treated with LR004-VC-MMAE(0,0.0066,0.066,0.66,6.6 nmol/L),and the inhibitory effects of LR004-VC-MMAE on cell proliferation were examined by CCK-8 and colony formation.The migration and invasion capacity of MDA-MB-468 and MDA-MB-231 cells were tested at different LR004-VCMMAE concentrations(2.5 and 5 nmol/L)with wound healing and Transwell invasion assays.Flow cytometric analysis and tumorsphere-forming assays were used to detect the killing effects of LR004-VC-MMAE on cancer stem cells(MDA-MB-468 and MDA-MB-231 cells).The mouse xenograft models were also used to evaluate the antitumor efficacy of LR004-VC-MMAE in vivo.Briefly,BALB/c nude mice were subcutaneously inoculated with MDA-MB-468 or MDAMB-231 cells.Then they were randomly divided into 4 groups(n=6 per group)and treated with PBS,naked LR004(10 mg/kg),LR004-VC-MMAE(10 mg/kg),or doxorubicin,respectively.Tumor sizes and the body weights of mice were measured every 4 d.The effects of LR004-VC-MMAE on apoptosis and cell cycle distribution were analyzed by flow cytometry.Western blotting was used to detect the effects of LR004-VC-MMAE on EGFR,ERK,MEK phosphorylation and tumor stemness marker gene expression.Results:LR004-VC-MMAE with a DAR of 4.02 were obtained.The expression of EGFR was found to be significantly higher in TNBC cells compared with non-TNBC cells(P<0.01).LR004-VC-MMAE inhibited the proliferation of EGFRpositive TNBC cells,and the ICvalues of MDA-MB-468 and MDA-MB-231 cells treated with LR004-VC-MMAE for 72 h were(0.13±0.02)nmol/L and(0.66±0.06)nmol/L,respectively,which were significantly lower than that of cells treated with MMAE[(3.20±0.60)nmol/L,P<0.01,and(6.60±0.50)nmol/L,P<0.001].LR004-VC-MMAE effectively inhibited migration and invasion of MDA-MB-468 and MDA-MB-231 cells.Moreover,LR004-VC-MMAE also killed tumor stem cells in EGFR-positive TNBC cells and impaired their tumorsphere-forming ability.In TNBC xenograft models,LR004-VC-MMAE at 10 mg/kg significantly suppressed tumor growth and achieved complete tumor regression on day 36.Surprisingly,tumor recurrence was not observed until the end of the experiment on day 52.In a mechanistic study,we found that LR004-VC-MMAE significantly induced cell apoptosis and cell cycle arrest at G/M phase in MDAMB-468[(34±5)%vs.(12±2)%,P<0.001]and MDA-MB-231[(27±4)%vs.(18±3)%,P<0.01]cells.LR004-VC-MMAE also inhibited the activation of EGFR signaling and the expression of cancer stemness marker genes such as Oct4,Sox2,KLF4 and EpCAM.Conclusions:LR004-VC-MMAE showed effective antitumor activity by inhibiting the activation of EGFR signaling and the expression of cancer stemness marker genes.It might be a promising therapeutic candidate and provides a potential therapeutic avenue for the treatment of EGFR-positive TNBC.

Development of a novel multi-functional integrated bioconjugate effectively targeting K-Ras mutant pancreatic cancer

Folate receptor(FR)overexpression occurs in a variety of cancers,including pancreatic cancer.In addition,enhanced macropinocytosis exists in K-Ras mutant pancreatic cancer.Furthermore,the occurrence of intensive desmoplasia causes a hypoxic microenvironment in pancreatic cancer.In this study,a novel FR-directed,macropinocytosis-enhanced,and highly cytotoxic bioconjugate folate(F)-human serum albumin(HSA)-apoprotein of lidamycin(LDP)-active enediyne(AE)derived from lidamycin was designed and prepared.F-HSA-LDP-AE consisted of four moieties:F,HSA,LDP,and AE.F-HSA-LDP presented high binding efficiency with the FR and pancreatic cancer cells.Its uptake in wild-type cells was more extensive than in K-Ras mutant-type cells.By in vivo optical imaging,F-HSA-LDP displayed prominent tumor-specific biodistribution in pancreatic cancer xenograft-bearing mice,showing clear and lasting tumor localization for 360 h.In the MTT assay,F-HSA-LDP-AE demonstrated potent cytotoxicity in three types of pancreatic cancer cell lines.It also induced apoptosis and caused G2/M cell cycle arrest.F-HSALDP-AE markedly suppressed the tumor growth of AsPc-1 pancreatic cancer xenografts in athymic mice.At well-tolerated doses of 0.5 and 1 mg/kg,(i.v.,twice),the inhibition rates were 91.2%and 94.8%,respectively(P<0.01).The results of this study indicate that the F-HSA-LDP multi-functional bioconjugate might be effective for treating K-Ras mutant pancreatic cancer.

Multi-Omics-Guided Discovery of Omicsynins Produced by Streptomyces sp.1647:Pseudo-Tetrapeptides Active Against Influenza A Viruses and Coronavirus HCoV-229E

Many microorganisms have mechanisms that protect cells against attack from viruses.The fermentation components of Streptomyces sp.1647 exhibit potent anti-influenza A virus(IAV)activity.This strain was isolated from soil in southern China in the 1970s,but the chemical nature of its antiviral substance(s)has remained unknown until now.We used an integrated multi-omics strategy to identify the antiviral agents from this streptomycete.The antibiotics and Secondary Metabolite Analysis Shell(antiSMASH)analysis of its genome sequence revealed 38 biosynthetic gene clusters(BGCs)for secondary metabolites,and the target BGCs possibly responsible for the production of antiviral components were narrowed down to three BGCs by bioactivity-guided comparative transcriptomics analysis.Through bioinformatics analysis and genetic manipulation of the regulators and a biosynthetic gene,cluster 36 was identified as the BGC responsible for the biosynthesis of the antiviral compounds.Bioactivity-based molecular networking analysis of mass spectrometric data from different recombinant strains illustrated that the antiviral compounds were a class of structural analogues.Finally,18 pseudo-tetrapeptides with an internal ureido linkage,omicsynins A1–A6,B1–B6,and C1–C6,were identified and/or isolated from fermentation broth.Among them,11 compounds(omicsynins A1,A2,A6,B1–B3,B5,B6,C1,C2,and C6)are new compounds.Omicsynins B1–B4 exhibited potent antiviral activity against IAV with the 50%inhibitory concentration(IC_(50))of approximately 1μmol·L^(-1)and a selectivity index(SI)ranging from 100 to 300.Omicsynins B1–B4 also showed significant antiviral activity against human coronavirus HCoV-229E.By integrating multi-omics data,we discovered a number of novel antiviral pseudo-tetrapeptides produced by Streptomyces sp.1647,indicating that the secondary metabolites of microorganisms are a valuable source of novel antivirals.

Carrimycin inhibits coronavirus replication by decreasing the efficiency of programmed–1 ribosomal frameshifting through directly binding to the RNA pseudoknot of viral frameshift-stimulatory element

The pandemic of SARS-CoV-2 worldwide with successive emerging variants urgently calls for small-molecule oral drugs with broad-spectrum antiviral activity.Here,we show that carrimycin,a new macrolide antibiotic in the clinic and an antiviral candidate for SARS-CoV-2 in phase III trials,decreases the efficiency of programmed–1 ribosomal frameshifting of coronaviruses and thus impedes viral replication in a broad-spectrum fashion.Carrimycin binds directly to the coronaviral frameshift-stimulatory element(FSE)RNA pseudoknot,interrupting the viral protein translation switch from ORF1a to ORF1b and thereby reducing the level of the core components of the viral replication and transcription complexes.Combined carrimycin with known viral replicase inhibitors yielded a synergistic inhibitory effect on coronaviruses.Because the FSE mechanism is essential in all coronaviruses,carrimycin could be a new broad-spectrum antiviral drug for human coronaviruses by directly targeting the conserved coronaviral FSE RNA.This finding may open a new direction in antiviral drug discovery for coronavirus variants.

Phomaketals A and B,pentacyclic meroterpenoids from a eupC overexpressed mutant strain of Phoma sp.

Phomaketals A(1)and B(2),two tropolonic meroterpenoids with the unprecedented pentacyclic skeletons,were isolated from the solid-substrate fermentation cultures of a eupC overexpressed mutant strain of the fungus Phoma sp.,together with a biogenetically related secondary metabolite pughiinin B(3),and the known one noreupenifeldin B(4).The structures of 1–3 were elucidated primarily by nuclear magnetic resonance(NMR)experiments.The absolute configurations of 1 and 2 were assigned by electronic circular dichroism calculations and the calculated NMR with DP4+analysis,while that of 3 was established by single-crystal X-ray diffraction analysis using Cu Kαradiation.Biogenetically,phomaketals A(1)and B(2)could be derived from the hypothetical tropolonic sesquiterpene intermediates neosetophomone B(6)and 9-R-neosetophomone B(6),respectively,via different reactions cascades.Compound 1 showed antiproliferative effect only against the SUPB15 cells,with an 50%inhibitory concentration(IC50)value of 4.85μmol/L,while the co-isolated known meroterpenoid 4 displayed potent effects against three tumor cell lines,SUPB15,EL4,and H9,showing IC50values of 0.36–27.08μmol/L.

Medicinal chemistry strategies in the discovery and optimization of HBV core protein allosteric modulators(2018-2022 update)

Despite the improving coverage of preventative vaccines,hepatitis B remains a severe global public health problem,with more than 250 million patients living with hepatitis B virus(HBV)infection.Current available therapies,including nucleos(t)ide analogs and peginterferon,can control HBV replication but fail to eliminate covalently closed circular DNA(cccDNA)and achieve a cure.The HBV core protein(Cp)is a well-conserved structural protein,self-assembling to form the viral capsid.It involves in or modulates almost every stage of the HBV lifecycle,which makes it an attractive target for the development of new anti-HBV therapies.HBV core protein allosteric modulators(CpAMs)have become a hotspot in recent years.Herein,we provide a concise report focusing on the various medicinal chemistry strategies involved in the latest research(2018-2022)of HBV CpAMs,including high throughput screening(HTS),virtual screening(VS),drug repositioning,natural products,substitution decorating approach,scaffold hopping,molecular hybridization,prodrug strategy and conformational constraint strategy,to provide guidance for further development of new and effective anti-HBV drugs.

Recent Progress on Functional Genomics Research of Enterovirus 71

Enterovirus 71(EV71) is one of the main pathogens that causes hand-foot-and-mouth disease(HFMD). HFMD caused by EV71 infection is mostly self-limited; however, some infections can cause severe neurological diseases, such as aseptic meningitis, brain stem encephalitis, and even death. There are still no effective clinical drugs used for the prevention and treatment of HFMD. Studying EV71 protein function is essential for elucidating the EV71 replication process and developing anti-EV71 drugs and vaccines. In this review, we summarized the recent progress in the studies of EV71 noncoding regions(50 UTR and 30 UTR) and all structural and nonstructural proteins, especially the key motifs involving in viral infection, replication, and immune regulation. This review will promote our understanding of EV71 virus replication and pathogenesis, and will facilitate the development of novel drugs or vaccines to treat EV71.

Biosynthesis of antibiotic chuangxinmycin from Actinoplanes tsinanensis

Chuangxinmycin is an antibiotic isolated from Actinoplanes tsinanensis CPCC 200056 in the1970 s with a novel indole-dihydrothiopyran heterocyclic skeleton. Chuangxinmycin showed in vitro antibacterial activity and in vivo efficacy in mouse infection models as well as preliminary clinical trials.But the biosynthetic pathway of chuangxinmycin has been obscure since its discovery. Herein, we report the identification of a stretch of DNA from the genome of A. tsinanensis CPCC 200056 that encodes genes for biosynthesis of chuangxinmycin by bioinformatics analysis. The designated cxn cluster was then confirmed to be responsible for chuangxinmycin biosynthesis by direct cloning and heterologous expressing in Streptomyces coelicolor M1146. The cytochrome P450 CxnD was verified to be involved in the dihydrothiopyran ring closure reaction by the identification of seco-chuangxinmycin in S. coelicolor M1146 harboring the cxn gene cluster with an inactivated cxn D. Based on these results, a plausible biosynthetic pathway for chuangxinmycin biosynthesis was proposed, by hijacking the primary sulfur transfer system for sulfur incorporation. The identification of the biosynthetic gene cluster of chuangxinmycin paves the way for elucidating the detail biochemical machinery for chuangxinmycin biosynthesis, and provides the basis for the generation of novel chuangxinmycin derivatives by means of combinatorial biosynthesis and synthetic biology.

Immunomodulatory activity of polysaccharides from Brassica rapa by activating Akt/NF-κB signaling

Objective:To investigate the immunomodulatory activity of polysaccharides from the roots of Brassica rapa.Methods:The crude polysaccharide from roots of B.rapa(BRP)was extracted and purified to further investigate the active fraction of BRT for inducing macrophage phagocytosis.Results:Effects on RAW264.7 cells demonstrated that BRP behaved better phagocytic capacity and had potent immunomodulatory activity,including increasing production of nitric oxide(NO),tumor necrosis factor a(TNFa)and upregulating mRNA levels of inducible NO synthase(iNOS)and TNFa.Furthermore,modulation of macrophage by BRP was indicated to be mediated via the activation of Akt and nuclear factor-kappa B(NF-κB).Conclusion:The beneficial effects of BRP could be used as an immunotherapeutic adjuvant in treatment of inflammatory diseases.

Effect of ebosin on modulating interleukin-1β-induced nflammatory responses in rat fibroblast-like synoviocytes

The interleukin-1β-mitogen-activated protein kinase （MAPK） and NF-κB signaling pathways are involved in the pathogenesis of rheumatoid arthritis. Ebosin, a novel exopolysaccharide （EPS）, exhibits anti-inflammatory activity in rat collagen-induced arthritis by suppressing the production of tumor necrosis factor-s, interleukin-6 and interleukin-1β. The aim of the present study was to assess the effects of ebosin on NF-KB and MAPK signaling pathways mediated through interleukin-1β. in rat fibroblast-like synoviocytes （FLSs）. Western blotting showed decreased production of phosphorylated p38, JNK1, JNK2, IKKa, IKKa, and IκB in the cytoplasm and NF-κB in the nucleus upon ebosin treatment. The DNA-binding activity of NF-KB in the cell nucleus was also inhibited by ebosin treatment, as demonstrated using an electrophoresis mobility gel shift assay. Analysis of the results of the immunofluorescence assay also showed a reduced amount of NF-KB in the nucleus of cells affected by ebosin. These results provided evidence for the effects of ebosin on both interleukin-1β-mediated MAPK and NF-κB signaling pathways in rat FLSs. In addition, enzyme-linked immunosorbent assay demonstrated that ebosin reduces the levels of matrix metalloproteinases MMP-1 and MMP-3 and the chemokines, interleukin-8 and RANTES. Thus, the results of the present study provide further evidence for understanding the medicinal activity of ebosin at a molecular level, therefore nominating this EPS as a potential therapeutic candidate for the treatment of rheumatic arthritis.

Treating Chronic Diseases by Regulating the Gut Microbiota

Chronic diseases comprise a wide range of abnormal conditions and illnesses that impair patients’physical and/or mental functioning,and last for a long time.Largely a contemporary plague,chronic diseases are responsible for the observed morbidity and mortality in developed countries as well as in some developing countries [1,2].

Enzymatic synthesis of myricetin 3-O-galactoside through a whole-cell biocatalyst

Objective:Myricetin 3-O-galactoside is an active compound with pharmaceutical potential.The insufficient supply of this compound becomes a bottleneck in the druggability study of myricetin 3-Ogalactoside.Thus,it is necessary to develop a biosynthetic process for myricetin 3-O-galactoside through metabolic engineering.Methods:Two genes OcSUS1 and OcUGE1 encoding sucrose synthase and UDP-glucose 4-epimerase were introduced into BL21(DE3)to reconstruct a UDP-D-galactose(UDP-Gal)biosynthetic pathway in Escherichia coli.The resultant chassis strain was able to produce UDP-Gal.Subsequently,a flavonol 3-O-galactosyltransferase DkFGT gene was transformed into the chassis strain producing UDP-Gal.An artificial pathway for myricetin 3-O-galactoside biosynthesis was thus constructed in E.coli.Results:The obtained engineered strain was demonstrated to be capable of producing myricetin 3-Ogalactoside,reaching 29.7 mg/L.Conclusion:Biosynthesis of myricetin 3-O-galactoside through engineered E.coli could be achieved.This result lays the foundation for the large-scale preparation of myricetin 3-O-galactoside.

Yangxin Dawayimixike honey paste inhibits atherosclerosis in ApoE^(-/-)mice by attenuating blood lipids and exerting anti-inflammatory activity

Background:Yangxin Dawayimixike honey paste(YDHP)is a representative traditional Chinese medicine,and its main function is curing angina pectoris,palpitation and neurasthenia.However,it is unclear whether YDHP can suppress the development of atherosclerosis.The aim of this study was to validate the potential application of YDHP in atherosclerosis therapy and search for its potential mechanisms.Methods:Seven-week-old ApoE^(-/-)mice were randomly divided into a normal group fed a normal diet,an atherosclerosis model group fed a high-fat diet,YDHP groups fed a high-fat diet mixed with different doses of YDHP and positive control groups fed a high-fat diet mixed with atorvastatin or rosuvastatin.After feeding for 10 weeks,body weight,blood lipids,liver and kidney function indexes,serum inflammatory cytokines and atherosclerotic plaque areas were measured.Serum metabolic profiles were detected by an automatic biochemical analyser.Serum inflammatory cytokines were quantified by enzyme-linked immunosorbent assay kits.Atherosclerotic plaque areas were analysed using Oil Red O staining.Results:The YDHP(200,400 and 800 mg/kg/d)treated groups showed reduced serum levels of low density lipoprotein-cholesterol(P<0.05,when 200 or 400 mg/kg/d of YDHP group compared with the atherosclerosis model group;P<0.01,when 800 mg/kg/d of YDHP group compared with the atherosclerosis model group),total cholesterol(P<0.05,when 200 mg/kg/d of YDHP group compared with the atherosclerosis model group;P<0.01,when 400 or 800 mg/kg/d of YDHP group compared with the atherosclerosis model group)and triglyceride(P<0.01),however,elevated serum levels of high-density lipoprotein cholesterol(P<0.01)compared to the atherosclerosis model group.YDHP inhibited the area of atherosclerotic lesions.In addition,YDHP suppressed the levels of serum inflammatory cytokines such as interleukin 6(P<0.05,when 200 mg/kg/d of YDHP group compared with the atherosclerosis model group;P<0.01,when 400 or 800 mg/kg/d of YDHP group compared with the atherosclerosis model group)and tumor necrosis factorα(P<0.05,when 200 mg/kg/d of YDHP group compared with the atherosclerosis model group;P<0.01,when 400 or 800 mg/kg/d of YDHP group compared with the atherosclerosis model group).Conclusion:Our study demonstrated that YDHP showed considerable activity in alleviating the formation of atherosclerotic plaques in ApoE^(-/-)mice by reducing blood lipids and exerting anti-inflammatory activity.

PML Suppresses Influenza Virus Replication by Promoting FBXW7 Expression

Influenza A viruses(IAV)are responsible for seasonal flu epidemics,which can lead to high morbidity and mortality each year.Like other viruses,influenza virus can hijack host cellular machinery for its replication.Host cells have evolved diverse cellular defense to resist the invasion of viruses.As the main components of promyelocytic leukemia protein nuclear bodies(PML-NBs),PML can inhibit the replication of many medically important viruses including IAV.However,the mechanism of PML against IAV is unclear.In the present study,we found PML was induced in response to IAV infection and ectopic expression of PML could inhibit IAV replication,whereas knockdown of endogenous PML expression could enhance IAV replication.Further studies showed that PML increased the expression of FBXW7 by inhibiting its K48-linked ubiquitination and enhanced the interaction between FBXW7 and SHP2,which negatively regulated IAV replication during infection.Moreover,PML stabilized RIG-I to promote the production of typeⅠIFN.Collectively,these data indicated that PML inhibited IAV replication by enhancing FBXW7 expression in the antiviral immunity against influenza virus and extended the mechanism of PML in antiviral immunity.

Ebosin: a potential therapeutic agent for rheumatoid arthritis and autoinflammatory syndromes

Rheumatoid arthritis(RA)is a chronic autoimmune disease that has a worldwide prevalence of 1%.The symptoms of RA are pain,stiffness and symmetrical synovitis of diarthrodial joints,which result in articular destruction,functional decline,and substantial comorbidity in the cardiovascular,neurologic and metabolic systems.1 In RA,fibroblast-like synoviocytes(FLS,Type B synoviocytes)and osteoclasts are the main cells that contribute to bone and cartilage degradation,respectively.2 Pro-inflammatory cytokines,including interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and IL-6,play an essential role in the activation of FLS and the differentiation of osteoclasts,which have been implicated in the pathogenesis of RA.3 Targeting pro-inflammation cytokines and blocking their signal pathways have become an important strategy in RA treatment.The major functions of TNF-αin RA are associated with the production of pro-inflammation cytokines,inflamed synovium,chemokine amplification,Treg cells and osteoclast recruitment.4 As a TNF inhibitor,etanercept5 is a human recombinant version of the soluble p75 TNF receptor that is linked to the Fc of human immunoglobulin G subclass 1(IgG1),and it acts as a competitive inhibitor of the binding of TNF-αto cell-surface TNF receptors and thereby inhibits TNF-α-induced pro-inflammatory activity in the joints of RA patients.