Publications by authors named "Guangshun Zhang"

13 Publications

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A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes.

Nat Commun 2021 05 11;12(1):2623. Epub 2021 May 11.

Shanghai Synchrotron Radiation Facility, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai, People's Republic of China.

COVID-19 pandemic caused by SARS-CoV-2 constitutes a global public health crisis with enormous economic consequences. Monoclonal antibodies against SARS-CoV-2 can provide an important treatment option to fight COVID-19, especially for the most vulnerable populations. In this work, potent antibodies binding to SARS-CoV-2 Spike protein were identified from COVID-19 convalescent patients. Among them, P4A1 interacts directly with and covers majority of the Receptor Binding Motif of the Spike Receptor-Binding Domain, shown by high-resolution complex structure analysis. We further demonstrate the binding and neutralizing activities of P4A1 against wild type and mutant Spike proteins or pseudoviruses. P4A1 was subsequently engineered to reduce the potential risk for Antibody-Dependent Enhancement of infection and to extend its half-life. The engineered antibody exhibits an optimized pharmacokinetic and safety profile, and it results in complete viral clearance in a rhesus monkey model of COVID-19 following a single injection. These data suggest its potential against SARS-CoV-2 related diseases.
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http://dx.doi.org/10.1038/s41467-021-22926-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113581PMC
May 2021

Structural basis for SARS-CoV-2 neutralizing antibodies with novel binding epitopes.

PLoS Biol 2021 05 7;19(5):e3001209. Epub 2021 May 7.

MOE Key Laboratory of Protein Science & Collaborative Innovation Center of Biotherapy, School of Medicine, Tsinghua University, Beijing, China.

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) threatens global public health and economy unprecedentedly, requiring accelerating development of prophylactic and therapeutic interventions. Molecular understanding of neutralizing antibodies (NAbs) would greatly help advance the development of monoclonal antibody (mAb) therapy, as well as the design of next generation recombinant vaccines. Here, we applied H2L2 transgenic mice encoding the human immunoglobulin variable regions, together with a state-of-the-art antibody discovery platform to immunize and isolate NAbs. From a large panel of isolated antibodies, 25 antibodies showed potent neutralizing activities at sub-nanomolar levels by engaging the spike receptor-binding domain (RBD). Importantly, one human NAb, termed PR1077, from the H2L2 platform and 2 humanized NAb, including PR953 and PR961, were further characterized and subjected for subsequent structural analysis. High-resolution X-ray crystallography structures unveiled novel epitopes on the receptor-binding motif (RBM) for PR1077 and PR953, which directly compete with human angiotensin-converting enzyme 2 (hACE2) for binding, and a novel non-blocking epitope on the neighboring site near RBM for PR961. Moreover, we further tested the antiviral efficiency of PR1077 in the Ad5-hACE2 transduction mouse model of COVID-19. A single injection provided potent protection against SARS-CoV-2 infection in either prophylactic or treatment groups. Taken together, these results shed light on the development of mAb-related therapeutic interventions for COVID-19.
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http://dx.doi.org/10.1371/journal.pbio.3001209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8133496PMC
May 2021

Screening of Botanical Drugs against Lassa Virus Entry.

J Virol 2021 Feb 3. Epub 2021 Feb 3.

State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan 430071, China

Lassa virus (LASV) belongs to the Old World genus (family ). At present, there are no approved drugs or vaccines specific for LASV. In this study, high-throughput screening of a botanical drug library was performed against LASV entry using a pseudotype virus bearing the LASV envelope glycoprotein complex (GPC). Two hit compounds, bergamottin and casticin, were identified as micromolar range inhibitors of LASV entry. A mechanistic study revealed that casticin inhibited LASV entry by blocking low pH-induced membrane fusion. Analysis of adaptive mutants demonstrated that the F446L mutation, located in the transmembrane domain of GP2, conferred resistance to casticin. Furthermore, casticin antiviral activity extends to the New World (NW) pathogenic mammarenaviruses, and mutation of the conserved F446 also conferred resistance to casticin in these viruses. Unlike casticin, bergamottin showed little effect on LASV GPC-mediated membrane fusion, instead inhibiting LASV entry by blocking endocytic trafficking. Notably, both compounds showed inhibitory effects on authentic lymphocytic choriomeningitis virus. Our study shows that both casticin and bergamottin are candidates for LASV therapy and that the conserved F446 in LASV GPC is important in drug resistance in mammarenaviruses. Currently, there is no approved therapy to treat Lassa fever (LASF). Our goal was to identify potential candidate molecules for LASF therapy. Herein, we screened a botanical drug library and identified two compounds, casticin and bergamottin, that inhibited LASV entry via different mechanisms.
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http://dx.doi.org/10.1128/JVI.02429-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8103687PMC
February 2021

Characterizing the Lassa Virus Envelope Glycoprotein Membrane Proximal External Region for Its Role in Fusogenicity.

Virol Sin 2021 Apr 8;36(2):273-280. Epub 2020 Sep 8.

State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, 430071, China.

The membrane-proximal external region (MPER) of Lassa virus (LASV) glycoprotein complex (GPC) is critical in modulating its functionality. Till now, the high-resolution structure of the intact GPC, including MPER is not available. In this study, we used alanine substitution to scan all 16 residues located in LASV MPER. Western blotting and quantification fusion assay showed that the residues located at the C terminus of the HR2 (M414 and L415) and N terminus of the MPER (K417 and Y419) are critical for GPC-mediated membrane fusion function. Furthermore, cell surface biotinylation experiments revealed that M414A, K417A and Y419A expressed similar levels as WT, whereas L415A mutant led to a reduction of mature GPC on the cell surface. Moreover, substitution of these residues with the similar residue such as M414L, L415I, K417R and Y419F would partly compensate the loss of the fusion activity caused by the alanine mutant in these sites. Results from this study showed that several key residues in the MPER region are indispensable to promote the conformational changes that drive fusion events and shed light on the structure analysis of LASV GPC and anti-LASV therapeutics.
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http://dx.doi.org/10.1007/s12250-020-00286-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087742PMC
April 2021

Structure of African swine fever virus p15 reveals its dual role for membrane-association and DNA binding.

Protein Cell 2020 08;11(8):606-612

State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, 300350, People's Republic of China.

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http://dx.doi.org/10.1007/s13238-020-00731-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7381529PMC
August 2020

Crystal Structure of African Swine Fever Virus pS273R Protease and Implications for Inhibitor Design.

J Virol 2020 05 4;94(10). Epub 2020 May 4.

State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin, People's Republic of China.

African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs that is responsible for serious economic and production losses. It is caused by the African swine fever virus (ASFV), a large and complex icosahedral DNA virus of the family. Currently, there is no effective treatment or approved vaccine against the ASFV. pS273R, a specific SUMO-1 cysteine protease, catalyzes the maturation of the pp220 and pp62 polyprotein precursors into core-shell proteins. Here, we present the crystal structure of the ASFV pS273R protease at a resolution of 2.3 Å. The overall structure of the pS273R protease is represented by two domains named the "core domain" and the N-terminal "arm domain." The "arm domain" contains the residues from M1 to N83, and the "core domain" contains the residues from N84 to A273. A structure analysis reveals that the "core domain" shares a high degree of structural similarity with chlamydial deubiquitinating enzyme, sentrin-specific protease, and adenovirus protease, while the "arm domain" is unique to ASFV. Further, experiments indicated that the "arm domain" plays an important role in maintaining the enzyme activity of ASFV pS273R. Moreover, based on the structural information of pS273R, we designed and synthesized several peptidomimetic aldehyde compounds at a submolar 50% inhibitory concentration, which paves the way for the design of inhibitors to target this severe pathogen. African swine fever virus, a large and complex icosahedral DNA virus, causes a deadly infection in domestic pigs. In addition to Africa and Europe, countries in Asia, including China, Vietnam, and Mongolia, were negatively affected by the hazards posed by ASFV outbreaks in 2018 and 2019, at which time more than 30 million pigs were culled. Until now, there has been no vaccine for protection against ASFV infection or effective treatments to cure ASF. Here, we solved the high-resolution crystal structure of the ASFV pS273R protease. The pS273R protease has a two-domain structure that distinguishes it from other members of the SUMO protease family, while the unique "arm domain" has been proven to be essential for its hydrolytic activity. Moreover, the peptidomimetic aldehyde compounds designed to target the substrate binding pocket exert prominent inhibitory effects and can thus be used in a potential lead for anti-ASFV drug development.
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http://dx.doi.org/10.1128/JVI.02125-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7199414PMC
May 2020

Temporal lobe injury patterns following intensity modulated radiotherapy in a large cohort of nasopharyngeal carcinoma patients.

Oral Oncol 2018 10 10;85:8-14. Epub 2018 Aug 10.

Department of Radiation Oncology, Duke University, Durham, NC 27710, USA. Electronic address:

Objectives: To analyze the correlation between dose-volume-histograms (DVHs) with three patterns (edema, enhancement, and necrosis) of temporal lobe injury (TLI) in patients receiving intensity modulated radiation therapy (IMRT) for nasopharyngeal carcinoma (NPC) and to determine optimal thresholds to predict the incidence of each TLI pattern, with particular emphasis on the relationship between edema volume and the risk of enhancement and necrosis.

Materials And Methods: A cohort of 4186 NPC patients treated with IMRT was retrospectively reviewed with TLI presenting in 188 patients. The atlases of complication incidence (ACI) for each pattern were constructed using DVH curves of temporal lobes. Optimal threshold for predicting incidence of each pattern was determined using the point closest to top-left of the plot. The accuracy of using edema volume to predict enhancement and necrosis incidence was evaluated via area under curve (AUC) of receiver operator characteristics (ROC).

Results: All DVH parameters, D, D, D, D, D, D, D, V, V, V, V, V, and V, except D showed statistically significant differences between subgroups of each pattern (p < 0.05). For predicting incidence of each pattern, optimal DVH thresholds over the range of D-D D and V-V were derived. The optimal thresholds of edema volume for predicting enhancement were 0.96 and 2.2cc and for predicting necrosis were 0.94 and 11.5cc.

Conclusion: Optimal DVH thresholds were generated for limiting risk of each injury pattern. Edema volume was a strong predictor for risk of enhancement and necrosis, which could potentially be reduced by lowering edema volume below threshold.
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http://dx.doi.org/10.1016/j.oraloncology.2018.07.020DOI Listing
October 2018

Comparison of 3D and 2D gamma passing rate criteria for detection sensitivity to IMRT delivery errors.

J Appl Clin Med Phys 2018 Jul 15;19(4):230-238. Epub 2018 Jun 15.

Department of Radiation Oncology, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China.

This study compared three-dimensional (3D) and two-dimensional (2D) percentage gamma passing rates (%GPs) for detection sensitivity to IMRT delivery errors and investigated the correlation between two kinds of %GP. Eleven prostate IMRT cases were selected, and errors in multileaf collimator (MLC) bank sag, MLC leaf traveling, and machine output were simulated by recalculating the dose distributions in patients. 2D doses were extracted from the 3D doses at the isocenter position. The 3D and 2D %GPs with different gamma criteria were then obtained by comparing the recalculated and original doses in specific regions of interest (ROI), such as the whole body, the planning target volume (PTV), the bladder, and the rectum. The sensitivities to simulated errors of the two types of %GP were compared, and the correlation between the 2D and 3D %GPs for different ROIs were analyzed. For the whole-body evaluation, both the 2D and 3D %GPs with the 3%/3 mm criterion were above 90% for all tested MLC errors and for MU deviations up to 4%, and the 3D %GP was higher than the 2D %GP. In organ-specific evaluations, the PTV-specific 2D and 3D %GP gradients were -4.70% and -5.14% per millimeter of the MLC traveling error, and -17.79% and -20.50% per percentage of MU error, respectively. However, a stricter criterion (2%/1 mm) was needed to detect the tested MLC sag error. The Pearson correlation analysis showed a significant strong correlation (r > 0.8 and P < 0.001) between the 2D and 3D %GPs in the whole body and PTV-specific gamma evaluations. The whole-body %GP with the 3%/3 mm criterion was inadequate to detect the tested MLC and MU errors, and a stricter criterion may be needed. The PTV-specific gamma evaluation helped to improve the sensitivity of the error detection, especially using the 3D GP%.
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http://dx.doi.org/10.1002/acm2.12389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6036388PMC
July 2018

Screening and Identification of Lassa Virus Entry Inhibitors from an FDA-Approved Drug Library.

J Virol 2018 08 31;92(16). Epub 2018 Jul 31.

University of the Chinese Academy of Sciences, Beijing, China

(LASV) belongs to the genus (family ) and causes severe hemorrhagic fever in humans. At present, there are no Food and Drug Administration (FDA)-approved drugs or vaccines specific for LASV. Here, high-throughput screening of an FDA-approved drug library was performed against LASV entry by using pseudotype virus bearing LASV envelope glycoprotein (GPC). Two hit compounds, lacidipine and phenothrin, were identified as LASV entry inhibitors in the micromolar range. A mechanistic study revealed that both compounds inhibited LASV entry by blocking low-pH-induced membrane fusion. Accordingly, lacidipine showed virucidal effects on the pseudotype virus of LASV. Adaptive mutant analyses demonstrated that replacement of T40, located in the ectodomain of the stable-signal peptide (SSP), with lysine (K) conferred LASV resistance to lacidipine. Furthermore, lacidipine showed antiviral activity against LASV, the closely related Mopeia virus (MOPV), and the New World arenavirus Guanarito virus (GTOV). Drug-resistant variants indicated that V36M in the ectodomain of the SSP mutant and V436A in the transmembrane domain of the GP2 mutant conferred GTOV resistance to lacidipine, suggesting the interface between SSP and GP2 is the target of lacidipine. This study shows that lacidipine is a candidate for LASV therapy, reinforcing the notion that the SSP-GP2 interface provides an entry-targeted platform for arenavirus inhibitor design. Currently, there is no approved therapy to treat Lassa fever; therefore, repurposing of approved drugs will accelerate the development of a therapeutic stratagem. In this study, we screened an FDA-approved library of drugs and identified two compounds, lacidipine and phenothrin, which inhibited Lassa virus entry by blocking low-pH-induced membrane fusion. Additionally, both compounds extended their inhibition against the entry of Guanarito virus, and the viral targets were identified as the SSP-GP2 interface.
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http://dx.doi.org/10.1128/JVI.00954-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6069169PMC
August 2018

Diterpenoid Tanshinones, the extract from Danshen (Radix Salviae Miltiorrhizae) induced apoptosis in nine human cancer cell lines.

J Tradit Chin Med 2016 08;36(4):514-21

Objective: To identify the active anti-tumor constituents in the extract from Danshen (Radix Salviae Miltiorrhizae) and investigate the mechanisms underlying the actions.

Methods: First, we introduced a two-step counter- current chromatography to extract the therapeutically active diterpenoid, tanshinone from Danshen (Radix Salviae Miltiorrhizae). The cholecystokinin (CCK-8) method was used to evaluate the inhibitory effect of diterpenoid tanshinone in liver cancer QGY-7703, lung cancer PC9, lung cancer A549, gastric cancer MKN-45, gastric cancer HGC-27, colon cancer HCT116, myeloma cellU266/ RPMI8226, and human breast cancer MCF-7 in vitro. Fluorescence staining was used to observe the cytotoxicity ofditerpenoid tanshinone on PC9 cells. The Western blot was used to detect apoptosis- related protein poly ADP-ribose polymerase (PARP), cysteinyl aspartate specific proteinase3/9 (caspase3/9), and cleaved-cysteinyl aspartate specific proteinase3/9 (cleaved-caspase3/9). The endoplasmic reticulum stress-related activating transcription factor 4 (ATF4), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), and phosphorylated jun amino-terminal kinase (p-JNK), and caspase- 12 were also analyzed using the Western blot.

Results: Diterpenoid tanshinone inhibited the nine human tumor cell lines, with an IC50 of 4.37-29 μg/mL, with the PC9 and MCF-7 displaying the lowest values. Fluorescence staining showed a lethal effect of diterpenoid tanshinone on PC9 cells. The Western blot showed that the expression of caspase3/9 protein and ATF-4 protein decreased gradually. However, the PARP, cleaved-caspase 3/9 and the expression of p-eIF2 α, P-JNK, and caspase- 12 increased gradually, in a dose-dependent fashion.

Conclusion: We successfully introduced a two-step counter-current chromatography method to extract diterpenoid tanshinone, and demonstrated its antitumor activity. Diterpenoid tanshinone can induce apoptosis in nine human cancer cell lines.
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http://dx.doi.org/10.1016/s0254-6272(16)30069-3DOI Listing
August 2016

Cryptotanshinone enhances the effect of Arsenic trioxide in treating liver cancer cell by inducing apoptosis through downregulating phosphorylated- STAT3 in vitro and in vivo.

BMC Complement Altern Med 2017 Feb 10;17(1):106. Epub 2017 Feb 10.

College of Basic Medical Science, Zhejiang Chinese Medical University, 548 Bin Wen Road, Hangzhou, 310053, Zhejiang Province, China.

Background: Arsenic trioxide (ATO) is approved for treating terminal-stage liver cancer in China. Cryptotanshinone (CT), a STAT3 inhibitor, has exhibited certain anti-tumor potency; however, the use of CT enhanced ATO for treating liver cancer has not been reported. Here we try to elucidate how CT could enhance the efficacy of ATO for treating liver cancer and its correlation to STAT3 in vitro and in vivo.

Methods: Cell viability of ATO combined with CT was assessed by MTT assay. Cell apoptosis induced by ATO combined with CT was detected by Annexin V/PI staining and apoptosis-related proteins were detected by western blotting. STAT3-related proteins were analysis by western blotting analysis and Immunofluorescence assays. Efficacy evaluation of ATO combined with CT on xenograft was carried in nude mice and related proteins were analysis by Immunohistochemistry assays.

Results: First we evaluated cell vitality, and our data indicated that the ATO combined with CT showed obvious growth inhibition of Bel-7404 cells compared to ATO or CT alone. Next we found that ATO combined with CT induced cell apoptosis in Bel-7404 cells and upregulated the activation of apoptosis-related proteins cleaved-caspase-3, cleaved-caspase-9, and cleaved-poly(ADP-ribose) polymerase in a time-dependent manner. Next, we found that ATO combined with CT not only inhibited the constitutive levels of phosphorylated-JAK2 and phosphorylated-STAT3 but did so in a time-dependent manner. We also found that ATO combined with CT reversed the upregulated expression of phosphorylated-STAT3 stimulated by interleukin-6 and downregulated STAT3 direct target genes and the anti-apoptotic proteins Bcl-2, XIAP, and survivin but obviously upregulated the promoting apoptosis proteins Bak,.In vivo studies showed that ATO combined with CT decreased tumor growth. Tumors from ATO combined with CT-treated mice showed decreased levels of phosphorylated-STAT3 and the anti-apoptotic protein Bcl-2 but an increased level of pro-apoptotic protein Bax.

Conclusions: Our study provides strong evidence that CT could enhance the efficacy of ATO in treating liver cancer both in vitro and in vivo. Downregulation of phosphorylated-STAT3 expression may play an important role in inducing apoptosis of Bel-7404 cells.
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http://dx.doi.org/10.1186/s12906-016-1548-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303285PMC
February 2017