key: cord-1045602-owjgs6ja
authors: Liu, Xiaoyan; Li, Zhe; Liu, Shuai; Chen, Zhanghua; Zhao, Zhiyao; Huang, Yi-you; Zhang, Qingling; Wang, Jun; Shi, Yinyi; Xu, Yanhui; Sun, Jing; Xian, Huifang; Fang, Rongli; Bai, Fan; Ou, Changxing; Xiong, Bei; Lew, Andrew M; Cui, Jun; Huang, Hui; Zhao, Jincun; Hong, Xuechuan; Zhang, Yuxia; Zhou, Fulin; Luo, Hai-Bin
title: Therapeutic effects of dipyridamole on COVID-19 patients with coagulation dysfunction
date: 2020-02-29
journal: nan
DOI: 10.1101/2020.02.27.20027557
sha: 2d0285c90976dfa43d58eca6b7d1d7ef29994fc2
doc_id: 1045602
cord_uid: owjgs6ja

The human coronavirus HCoV-19 infection can cause acute respiratory distress syndrome (ARDS), hypercoagulability, hypertension, extrapulmonary multiorgan dysfunction. Effective antiviral and anti-coagulation agents with safe clinical profiles are urgently needed to improve the overall prognosis. We screened an FDA approved drug library and found that an anticoagulant agent dipyridamole (DIP) suppressed HCoV-19 replication at an EC50 of 100 nM in vitro. It also elicited potent type I interferon responses and ameliorated lung pathology in a viral pneumonia model. In analysis of twelve HCoV-19 infected patients with prophylactic anti-coagulation therapy, we found that DIP supplementation was associated with significantly increased platelet and lymphocyte counts and decreased D-dimer levels in comparison to control patients. Two weeks after initiation of DIP treatment, 3 of the 6 severe cases (60%) and all 4 of the mild cases (100%) were discharged from the hospital. One critically ill patient with extremely high levels of D-dimer and lymphopenia at the time of receiving DIP passed away. All other patients were in clinical remission. In summary, HCoV-19 infected patients could potentially benefit from DIP adjunctive therapy by reducing viral replication, suppressing hypercoagulability and enhancing immune recovery. Larger scale clinical trials of DIP are needed to validate these therapeutic effects.

28 other countries, such as Japan, South Korea, Italy, Singapore, Iran, Thailand, and U.S.A; this 69 rapid spread threatens to become a pandemic. To date, no agents have been reported to be effective After viral entry to the host cells, the coronavirus messenger RNA is first translated to yield the 94 polyproteins, which are subsequently cleaved by two viral proteinases, 3C-like protease (3CLP, aka 95 nsp5 or Mpro) and papain-like protease (PLP, or nsp3), to yield non-structural proteins essential for 96 viral replication. 21 Inhibitors that suppress the activity of these proteases may inhibit viral replication 97 and offer a revenue for the HCoV-19 therapy. inflammation and promotes mucosal healing. 32 Third, as a pan-PDE inhibitor, DIP may prevent 105 acute injury and progressive fibrosis of the lung, heart, liver, and kidney. 33 Here we provide three 106 levels of evidence advocating DIP as a therapy. In silico and in vitro, we demonstrated that DIP 107 possessed anti-HCoV-19 effects. In a VSV-induced pneumonia model, we also confirmed that DIP 108 elicited potent antiviral immunity and significantly improved the survival rate. In a small clinical 109 cohort, we found that DIP adjunctive therapy led to increased circulating lymphocyte and platelet 110 counts and lowered D-dimer levels, and markedly improved clinical outcomes.

Free energy perturbation and surface plasmon resonance (SPR) assay 114 We virtually screened an FDA-approved drug database using the HCoV-19 protease Mpro as a target 115 and validated the binding affinity by the SPR assay. DIP (PubChem CID: 3108, Figure S1 ) was 116 identified as a lead drug. In order to obtain the binding pattern and calculate the binding free energy 117 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.27.20027557 doi: medRxiv preprint between DIP and Mpro, DIP was firstly docked onto Mpro by using Glide, and the optimal binding 118 pose was further assessed by absolute binding free energy calculation with free energy perturbation.

119 34 The calculations were carried out in Gromacs 2019, and the thermodynamic cycle and procedure 120 was similar to that used by Matteo et al. 35 In the calculation, the ligand electrostatic and van der 121 Waals interactions were decoupled using a linear alchemical pathway with Δλ = 0.10 for the van der 122 Waals and Δλ = 0.20 for electrostatic interactions. To add the restraints, 12 λ values were used.

Therefore, a total of 28 windows for complex and 16 windows for ligand systems were employed, The pGEX4T1-Mpro plasmid was constructed (Atagenix, Wuhan) and transfected the E. coli strain 132 BL21 (Codonplus, Stratagene). GST-tagged protein was purified by GST-glutathione affinity 133 chromatography and cleaved with FXa. The purity of recombinant protein was greater than 95% as 134 judged by SDS-PAGE. The binding of DIP to Mpro was measured by the Biacore 8K system (GE CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.27.20027557 doi: medRxiv preprint at 10 µL/min for 3 min (1800 response units), which followed by a blocking step using ethanolamine 140 (1 M, pH 8.5) at 10 µL/min for 7 min. Binding studies were performed by passing 5-80 µM of DIP CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.02.27.20027557 doi: medRxiv preprint Patients and study variables 185 As of February 8, 2020, 124 confirmed COVID-19 cases had been identified from Zhongnan 186 Hospital of Wuhan University (Table S1 ). All patients met the diagnostic criteria of "Diagnosis and CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.02.27.20027557 doi: medRxiv preprint 229 We virtually screened an FDA approved drug library and found that DIP bound to the HCoV-19 230 protease Mpro (Figure S1 ). Hydrophobic and hydrogen bond (H-bond) interactions are the main 231 driving forces for the binding between DIP and Mpro. According to the free energy perturbation 232 calculation, the binding free energy of Gpred was -6.34 kcal/mol. An SPR assay was carried out to 233 validate the in silico result. This has revealed that DIP bound to Mpro with an experimentally 234 confirmed affinity of 34 µM (KD,eq) ( Figure 1A-B) .

To demonstrate directly that DIP suppresses HCoV-19 replication, we measured the viral titers using 245 Published studies have shown that DIP possessed broad spectrum activity to a wide range of viruses, 246 indicating that it may elicit antiviral immunity from the host cells. We examined these using two Table 2) .

The baseline temperature was higher and the oxygenation status was worse in the DIP-treated group 285 than those in the control group, however, temperature and oxygenation were stabilized in the mild 286 and severe patients from both groups after one day of treatment ( Figure S2) . Furthermore, we 287 observed a continuously increased trend in lymphocyte counts and significantly increased platelet (Table 1) . We measured dynamic 293 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.02.27.20027557 doi: medRxiv preprint changes in reference to the respective baseline levels for all patients, and found that the levels of 294 D-dimer continuously rose in the control group, whereas they were decreased and stabilized in the 295 DIP-treated group (Figure 3A, Figure S2 ).

We then examined the two critically ill patients who received DIP adjunctive therapy. A 70-year-old 298 man with very low oxygen saturation and has suffered from hypoxia and multiorgan dysfunction at All patients had chest CT scans and showed typical multiple patchy ground-glass shadows in the 310 lungs before the treatment. In the DIP treated patients, the lesions had varied degree of absorption.

Typical findings following DIP treatment was illustrated in one of the severe COVID-19 patient who 312 had taken sequential CT scans (Figure 4) . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

China. Lancet 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.02.27.20027557 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.02.27.20027557 doi: medRxiv preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.02.27.20027557 doi: medRxiv preprint experiments. P values were calculated by Student's t test, *P < 0.05, ** P < 0.01, *** P < 0.001 and 512 **** P < 0.0001. Data in F are presented with 3 to 9 mice per group, P value was calculated by 513 Log-rank (Mantel-Cox) test (conservative).

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.27.20027557 doi: medRxiv preprint Lymphocyte counts were persistently elevated in trend and PLT counts was significantly increased in 520 the DIP group in comparison to the control group. Data are shown as the means ± SE across different 521 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.27.20027557 doi: medRxiv preprint

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