Drug Design Targeting Key Proteins Of HIV-1 And SARS-CoV-2

Viral infection is the main infectious disease in the world at present,accounting for more than three-quarters of infectious diseases,which poses a serious threat to human health and life.Since 1981,the first case of HIV/AIDS caused by human immunodeficiency virus（HIV）infection was confirmed,which is still difficult to control in worldwild until now.Meanwhile,in 2019,a severe pneumonia outbreak because of the infection of SARS-CoV-2 also continues around the world.Without doubt,both of them certainly bring great challenges to the human medical and health fields.Both SARS-CoV-2 and HIV-1 are single-stranded RNA retroviruses,which are highly infectious and harmful.There have already admitted anti-HIV drugs,with the use of HIV-1 drug for a long time,therapeutic effect of HIV-1 drugs is limited,it’s urgent to develop new drugs.At present,there is no drug for SARS-CoV-2,the SASR-CoV-2 vaccine has been marketed and administered successively,but the safety and efficacy still need to be evaluated.Therefore,we should actively promote the further development of treatment drugs and vaccines while doing a good job of AIDS and COVID-19 protection.The paper mainly focuses on HIV-1 and SARS-CoV-2 viruses from the following three research contents,with the expectation of facilitating the drug development of HIV-1 and SARS-CoV-2:Part Ⅰ:Computational design of anti-HIV-1 monobody.Monobody derived from10th domain of fibronectin type III（～10 k Da,10Fn3）has the versatility.They have applied for multiple potential therapeutic targets treatment,including VEGFR2,HER2,and IGF-1.Monobody has the charactetics of m Abs,which can overcome the drawback that the m Abs can not reach the target accurately due to its large volume and can be developed as inhibitors of HIV-1 fusion and become a new class of drugs for the treatment of HIV-1.In this study,monobodies（Mbs）targeting CD4 protein（4910＿A08,6940＿B01）and virus gp41 protein（4058＿H08,6200＿A08）were selected on the basis of the anti-HIV-1 efficacy of previous experiments,in the hope of using computational simulation to first elucidate the binding mechanism of Mbs and target protein at the molecular level.First,protein structure prediction methods are used to build Mbs structure,then through a variety of protein-docking tools and molecular dynamics simulation from multi-angle assessment to explore the 4910＿A08-CD4,6940＿B01-CD4,4058＿H08-gp41,6200＿A08-gp41 near-native conformations.We hope to design more stability and affinity monobodies on the basis of interaction mechanism between Mbs and target protein at the end identified by MD simulation and energy analysis.A total of 17 Mbs were designed based on the predicted ideal complex by protein redesign,which can provide ideas for the design of protein drugs on other targets.Part Ⅱ:Structure-based discovery of novel nonpeptide inhibitors targeting SARS-CoV-2 M^pro.The major protease(M^pro)plays an important role in the replication process of SARS-CoV-2 and becomes an attractive target for COVID-19 treatment.Here,we propose a multiple conformations-based virtual screening strategy to discover inhibitors against SARS-CoV-2 M^pro.Ensemble docking was first carried out using nine M^prostructures and a protein mimetics library with 8,960 commercially available compounds.Surface plasmon resonance（SPR）assay revealed that 6 out of 49 compounds were able to bind to SARS-CoV-2 M^pro.And Fluorescence resonance energy transfer（FRET）assay showed the six compounds hamper M^pro activities with IC₅₀ ranging from 0.69±0.05μM to 2.05±0.92μM.Evaluation of antiviral activity using cell-based assay indicated that two compounds（Z1244904919 and Z1759961356）could strongly inhibit the cytopathic effect and reduced replication of the living virus in Vero E6 cells with the EC₅₀ of 4.98±1.83μM and 8.52±0.92μM,respectively.The mechanism of the action（MOA）for the two inhibitors were further elucidated at the molecular level by molecular dynamics（MD）simulation followed by binding free energy analysis.As a result,the discovered noncovalent reversible inhibitors with novel scaffolds are promising antiviral drug candidates,which may be used for the development of COVID-19 treatment.Part Ⅲ:Computational design and modeling of nanobodies toward SARS-CoV-2RBD.When SARS-CoV-2 is infected,the receptor binding domain（RBD）on the spikes of the virus will first bind to the receptor angiotensin converting enzyme 2（ACE2）on the host cells,so preventing the binding of RBD to the host（ACE2）can effectively prevent the invasion of SARS-CoV-2.As a single domain antibody,nanobody can prevent the binding of the two,and the complex structure of the binding of nanobody and SARS-CoV-2 RBD has been analyzed.By grafting the complementarity-determining regions（CDRs）of developed SARS-CoV,MERS-CoVs specific neutralizing antibodies（n Abs）include monoclonal antibodies（m Abs）as well as SARS-CoV-2 monoclonal antibodies（m Abs）onto a known stable nanobody（Nb）scaffold,a total of 16 Nbs sequences were designed.CS01,CS02,CS03,CS10 and CS16 were selected based on the free energy landscape of protein docking verified by the recently reported Nbs-RBD cocrystal structures.CS01,CS02 and CS03 occupied the ACE2 binding site of RBD,while CS10and CS16 were proposed to inhibit the interaction between RBD and ACE2 through an allosteric mechanism.Based on the structures of the five Nbs in complex with RBD,seven brandnew Nbs with enhanced binding affinities（CS02＿RD01,CS03＿RD01,CS03＿RD02,CS03＿RD03,CS03＿RD04,CS16＿RD01 and CS16＿RD02）were generated by redesign of residues on the interface of the five Nbs contact with SARS-CoV-2 RBD.In addition,the identified“hot spots”on the interface of each complex provide useful information to understand the binding mechanism of designed Nbs to SARS-CoV-2 RBD.In sum,the predicted stabilities and high binding affinities of the 11（re）designed Nbs indicating the potential of the developed computational framework in this work to design effective agents to block the infection of SARS-CoV-2.In conclusion,for HIV-1 virus,the complexes of monobody binding to host CD4and viral gp41 protein were predicted at the molecular level for the first time,and the molecular mechanism of interaction were explained and novel Mbs was designed.For SARS-CoV-2 virus,small molecules with M^pro inhibitory activity that can be used as drug lead were found,and 11 Nbs that can prevent RBD from binding to ACE2 were designed.All the above results can provide important references for the discovery and design of drugs inhibit HIV-1 and SARS-CoV-2.