To develop a new high-throughput screening model for human high-density lipoprotein （HDL） receptor （CD36 and LIMPⅡ analogous-1, CLA-1） agonists using CLA-1-expressing insect cells. Methods With the total RNA of h...To develop a new high-throughput screening model for human high-density lipoprotein （HDL） receptor （CD36 and LIMPⅡ analogous-1, CLA-1） agonists using CLA-1-expressing insect cells. Methods With the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA （cDNA） of CLA-1 was amplified by reverse transcription-polymerase chain reaction （RT-PCR）. Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus （AcNPV） into donor vector pFastBacl and recombinant pFastBacl-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacrnid DNA. Recombinant bacrnid-CLA-1 was transfected into Spodopterafrugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA- 1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized. Results Western blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries. Conclusion Human HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.展开更多
There are only eight approved small molecule antiviral drugs for treating COVID-19.Among them,four are nucleotide analogues(remdesivir,JT001,molnupiravir,and azvudine),while the other four are protease inhibitors(nirm...There are only eight approved small molecule antiviral drugs for treating COVID-19.Among them,four are nucleotide analogues(remdesivir,JT001,molnupiravir,and azvudine),while the other four are protease inhibitors(nirmatrelvir,ensitrelvir,leritrelvir,and simnotrelvir-ritonavir).Antiviral resistance,unfavourable drug‒drug interaction,and toxicity have been reported in previous studies.Thus there is a dearth of new treatment options for SARS-CoV-2.In this work,a three-tier cell-based screening was employed to identify novel compounds with anti-SARS-CoV-2 activity.One compound,designated 172,demonstrated broad-spectrum antiviral activity against multiple human pathogenic coronaviruses and different SARS-CoV-2 variants of concern.Mechanistic studies validated by reverse genetics showed that compound 172 inhibits the 3-chymotrypsin-like protease(3CLpro)by binding to an allosteric site and reduces 3CLpro dimerization.A drug synergistic checkerboard assay demonstrated that compound 172 can achieve drug synergy with nirmatrelvir in vitro.In vivo studies confirmed the antiviral activity of compound 172 in both Golden Syrian Hamsters and K18 humanized ACE2 mice.Overall,this study identified an alternative druggable site on the SARS-CoV-23CLpro,proposed a potential combination therapy with nirmatrelvir to reduce the risk of antiviral resistance and shed light on the development of allosteric protease inhibitors for treating a range of coronavirus diseases.展开更多
文摘To develop a new high-throughput screening model for human high-density lipoprotein （HDL） receptor （CD36 and LIMPⅡ analogous-1, CLA-1） agonists using CLA-1-expressing insect cells. Methods With the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA （cDNA） of CLA-1 was amplified by reverse transcription-polymerase chain reaction （RT-PCR）. Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus （AcNPV） into donor vector pFastBacl and recombinant pFastBacl-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacrnid DNA. Recombinant bacrnid-CLA-1 was transfected into Spodopterafrugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA- 1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized. Results Western blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries. Conclusion Human HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.
基金National Natural Science Foundation of China(NSFC)/Research Grants Council(RGC)Joint Research Scheme(N_HKU767/22 and 82261160398)Health and Medical Research Fund(COVID190121)+13 种基金the Food and Health Bureau,The Government of the Hong Kong Special Administrative Regionthe National Natural Science Foundation of China(32322087,32300134,and 82272337)Guangdong Natural Science Foundation(2023A1515012907)Health@-InnoHK,Innovation and Technology Commission,the Government of the Hong Kong Special Administrative Regionthe Collaborative Research Fund(C7060-21G and C7002-23Y)and Theme-Based Research Scheme(T11-709/21-N)of the Research Grants Council,The Government of the Hong Kong Special Administrative RegionPartnership Programme of Enhancing Laboratory Surveillance and Investigation of Emerging Infectious Diseases and Antimicrobial Resistance for the Department of Health of the Hong Kong Special Administrative Region GovernmentSanming Project of Medicine in Shenzhen,China(SZSM201911014)the High Level-Hospital Program,Health Commission of Guangdong Province,Chinathe research project of Hainan Academician Innovation Platform(YSPTZX202004)Emergency Collaborative Project of Guangzhou Laboratory(EKPG22-01)and the National Key R&D Program of China(projects 2021YFC0866100 and 2023YFC3041600)The University of Hong Kong Seed Fund for Collaborative Research(2207101537)and Hunan University(521119400156)donations of Providence Foundation Limited(in memory of the late Lui Hac Minh).
文摘There are only eight approved small molecule antiviral drugs for treating COVID-19.Among them,four are nucleotide analogues(remdesivir,JT001,molnupiravir,and azvudine),while the other four are protease inhibitors(nirmatrelvir,ensitrelvir,leritrelvir,and simnotrelvir-ritonavir).Antiviral resistance,unfavourable drug‒drug interaction,and toxicity have been reported in previous studies.Thus there is a dearth of new treatment options for SARS-CoV-2.In this work,a three-tier cell-based screening was employed to identify novel compounds with anti-SARS-CoV-2 activity.One compound,designated 172,demonstrated broad-spectrum antiviral activity against multiple human pathogenic coronaviruses and different SARS-CoV-2 variants of concern.Mechanistic studies validated by reverse genetics showed that compound 172 inhibits the 3-chymotrypsin-like protease(3CLpro)by binding to an allosteric site and reduces 3CLpro dimerization.A drug synergistic checkerboard assay demonstrated that compound 172 can achieve drug synergy with nirmatrelvir in vitro.In vivo studies confirmed the antiviral activity of compound 172 in both Golden Syrian Hamsters and K18 humanized ACE2 mice.Overall,this study identified an alternative druggable site on the SARS-CoV-23CLpro,proposed a potential combination therapy with nirmatrelvir to reduce the risk of antiviral resistance and shed light on the development of allosteric protease inhibitors for treating a range of coronavirus diseases.
