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. 2022 Mar 22;14(4):655.
doi: 10.3390/v14040655.

A Modified Fibronectin Type III Domain-Conjugated, Long-Acting Pan-Coronavirus Fusion Inhibitor with Extended Half-Life

Qianyu Duan  1 , Shuai Xia  1 , Fanke Jiao  1 , Qian Wang  1 , Rui Wang  2 , Lu Lu  1 , Shibo Jiang  1 , Wei Xu  1
Affiliations

A Modified Fibronectin Type III Domain-Conjugated, Long-Acting Pan-Coronavirus Fusion Inhibitor with Extended Half-Life

Qianyu Duan et al. Viruses. .

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by infection of SARS-CoV-2 and its variants has posed serious threats to global public health, thus calling for the development of potent and broad-spectrum antivirals. We previously designed and developed a peptide-based pan-coronavirus (CoV) fusion inhibitor, EK1, which is effective against all human CoVs (HCoV) tested by targeting the HCoV S protein HR1 domain. However, its relatively short half-life may limit its clinical use. Therefore, we designed, constructed, and expressed a recombinant protein, FL-EK1, which consists of a modified fibronectin type III domain (FN3) with albumin-binding capacity, a flexible linker, and EK1. As with EK1, we found that FL-EK1 could also effectively inhibit infection of SARS-CoV-2 and its variants, as well as HCoV-OC43. Furthermore, it protected mice from infection by the SARS-CoV-2 Delta variant and HCoV-OC43. Importantly, the half-life of FL-EK1 (30 h) is about 15.7-fold longer than that of EK1 (1.8 h). These results suggest that FL-EK1 is a promising candidate for the development of a pan-CoV fusion inhibitor-based long-acting antiviral drug for preventing and treating infection by current and future SARS-CoV-2 variants, as well as other HCoVs.

Keywords: SARS-CoV-2; albumin; fusion inhibitor; human coronavirus; long-acting.

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Conflict of interest statement

S.J., L.L., S.X., W.X., Q.W., Q.D. and R.W. are the inventors in the patent or patent application covering the peptides EK1 and FL- EK1. The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Design, construction, and characterization of FL-EK1. (A) Schematic diagram of SARS-CoV-2 S2 subunit and sequences of the peptides HR1P, HR2P, and EK1. The amino acids highlighted in red in EK1 represent amino acids that are different from those in HR2P derived from the SARS-CoV-2 S2 subunit. FP, fusion peptide; HR, heptad repeat; TM, transmembrane region; CP, cytoplasm region. (B) Diagram of FN3-conjugated EK1 (FL-EK1) binding to HSA. (C) SDS-PAGE analysis of the purified FL-EK1 and FN3 proteins. (D) FL-EK1 inhibition of 6-HB formation between HR1P and HR2P, as determined by N-PAGE. **** p < 0.0001. (E) Binding affinity of FL-EK1 to HSA, as evaluated by isothermal titration calorimetry (ITC) assay. Data were analyzed and processed by TA-ITC software.
Figure 2
Figure 2
FL-EK1-mediated inhibition of SARS-CoV-2 infection. (A) FL-EK1-mediated inhibition of SARS-CoV-2 S-mediated cell–cell fusion. (B) FL-EK1-mediated inhibition of pseudotyped SARS-CoV-2. (C) FL-EK1-mediated inhibition of authentic SARS-CoV-2 infection. (D) FL-EK1-mediated inhibition of authentic SARS-CoV-2 Delta variant infection. Samples were tested in triplicate, and the experiment was repeated three times. Data from a representative experiment are presented as mean ± SD.
Figure 3
Figure 3
FL-EK1-mediated inhibition of HCoV-OC43 infection. (A) FL-EK1-mediated inhibition of HCoV-OC43 S-mediated cell–cell fusion. (B) FL-EK1-mediated inhibition of pseudotyped HCoV-OC43. (C) FL-EK1-mediated inhibition of authentic HCoV-OC43 infection. Samples were tested in triplicate, and the experiment was repeated three times. Data from a representative experiment are presented as mean ± SD.
Figure 4
Figure 4
FL-EK1-mediated inhibition of authentic HCoV-OC43 infection in vivo. In vivo prophylactic and protective efficacy of FL-EK1 and EK1 against authentic HCoV-OC43 infection in ICR suckling mice via the intranasal route for FL-EK1 administration and viral challenge. Mouse survival rate (A) and bodyweight changes (B) were recorded. Viral titer in the brain was measured by real-time PCR (C). Samples were tested in triplicate, and the RT-PCR was repeated twice. Data from a representative experiment are presented as mean ± SD. * p < 0.05, ** p < 0.01.
Figure 5
Figure 5
FL-EK1-mediated inhibition of authentic SARS-CoV-2 Delta variant infection in vivo. Prophylactic and protective efficacy of FL-EK1 against SARS-CoV-2 Delta variant infection in Tgtn mice via the intranasal route for FL-EK1 administration and viral challenge. Viral titer in lung (A), brain (B), and intestine (C) was measured by real-time PCR. Samples were tested in triplicate, and the RT-PCR was repeated twice. Data from a representative experiment are presented as mean ± SD. ns, no significance, * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 6
Figure 6
Ex vivo anti-SARS-CoV-2 PsV activity and concentrations of FL-EK1 and EK1 in mouse serum and evaluation of in vitro cytotoxicity and in vivo safety of FL-EK1. (A) Ex vivo anti-SARS-CoV-2 PsV activity of serum samples collected from mice at different time points after intraperitoneal injection of FL-EK1 and EK1 (n = 3). (B) Concentration of FL-EK1 and EK1 in mouse serum samples and the half-life of each were estimated. (C) In vitro cytotoxicity of FL-EK1 at the graded concentration on Huh7, Caco2, RD, and Calu3 cells. (D) Bodyweight changes of mice treated with FL-EK1 via the intranasal route. (E) Creatinine in serum samples of mice treated with FL-EK1 via intranasal administration was measured by using a creatinine assay kit. (F) ALT in serum samples of mice treated with FL-EK1 via intranasal administration was measured by an ALT assay kit. Samples were tested in triplicate, and the experiment was repeated three times. Data from a representative experiment are presented as mean ± SD.
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