The small molecule inhibitor of SARS-CoV-2 3CLpro EDP-235 prevents viral replication and transmission in vivo

Abstract

The COVID-19 pandemic has led to the deaths of millions of people and severe global economic impacts. Small molecule therapeutics have played an important role in the fight against SARS-CoV-2, the virus responsible for COVID-19, but their efficacy has been limited in scope and availability, with many people unable to access their benefits, and better options are needed. EDP-235 is specifically designed to inhibit the SARS-CoV-2 3CLpro, with potent nanomolar activity against all SARS-CoV-2 variants to date, as well as clinically relevant human and zoonotic coronaviruses. EDP-235 maintains potency against variants bearing mutations associated with nirmatrelvir resistance. Additionally, EDP-235 demonstrates a ≥ 500-fold selectivity index against multiple host proteases. In a male Syrian hamster model of COVID-19, EDP-235 suppresses SARS-CoV-2 replication and viral-induced hamster lung pathology. In a female ferret model, EDP-235 inhibits production of SARS-CoV-2 infectious virus and RNA at multiple anatomical sites. Furthermore, SARS-CoV-2 contact transmission does not occur when naïve ferrets are co-housed with infected, EDP-235-treated ferrets. Collectively, these results demonstrate that EDP-235 is a broad-spectrum coronavirus inhibitor with efficacy in animal models of primary infection and transmission.

Fig. 1. EDP-235 inhibits SARS-CoV-2 3CLpro and binds reversibly to 3CLpro’s active site.

a Structure of EDP-235 and its FRET 3CLpro inhibition activity. Representative data are mean ± standard deviation of technical replicates from one experiment with non-linear regression fit (n = 6 independent experiments used to derive IC₅₀ and K_i^app values). b Semi-transparent surface representation of the X-ray crystal structure of SARS-CoV-2 3CLpro dimer (shown in cyan and gray) in complex with EDP-235 solved to 2 Å. EPD-235 is represented by red stick structure. c Detailed cartoon representation of the binding interactions between EDP-235 and residues located in SARS-CoV-2 3 CLpro catalytic active site. Hydrogen bonds are depicted between the NH side chain of H136 with the spyrolactam C = O of EDP-235, the C = O side chain of E166 with the spyrolactam NH of EDP-235, the NH side chain of H41 with amide C = O of EDP-235, and the NH side chain of Q189 with the trifluoroindole F of EDP-235. The catalytic residue C145 is represented as a yellow stick configuration and monomer coloration is matched to (b, d). The surface map of 3CLpro and electron density of EDP-235 was rendered using UCSF Chimera. d A close-up semi-transparent surface representation of EDP-235 in the binding pocket. 3CLpro = 3C-like protease; FRET = fluorescence resonance energy transfer; IC₅₀ = half-maximal inhibitory concentration; K_i^app = apparent inhibition constant; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; UCSF = University of California San Franscisco.

Fig. 2. EDP-235 is efficacious in a prophylactic Syrian hamster model.

a Study design. Hamsters were orally dosed with EDP-235 or vehicle 1 h prior to intranasal inoculation with 6$\times$10³ TCID₅₀/animal SARS-CoV-2 USA/WA1/2020 or PBS for naïve. b Percent body weight change from 0-day baseline (n = 8 per cohort). c IHC staining of terminal lung samples for SARS-CoV-2 N protein (brown) (2X magnification). Minimal focal immunoreactivity seen in 200 mg/kg animal (arrow). Representative images shown (n = 8 per cohort). d Sections of panel C were scored from 1 (minimal/minor) to 5 (severe/overwhelming) (n = 8 per cohort). e H&E stain of terminal lung samples (1.25X magnification). SARS-CoV-2 effects characterized by dark consolidated lung regions (arrows). Representative images shown (n = 8 per cohort). f Left lung composite histopathology score. Microscopic finding categories were alveolar hemorrhage, alveolar/interstitial inflammation, vascular inflammation, bronchial-alveolar hyperplasia, perivascular edema, and immunoreactivity. Each variable scored from 1-5 as in (d) (n = 8 per cohort). g Infectious virus titer in terminal lung samples (n = 8 per cohort). h sgRNA (N protein) in terminal lung samples (n = 8 per cohort). i) gRNA (N protein) in terminal lung samples (n = 8 per cohort). All graphs are mean ± SEM; statistical significance determined versus infected VC by 2-way ANOVA with Tukey’s (b) or 1-way ANOVA with Dunnett’s (d, f, g, h, i) post hoc test. All P values were < 0.0001 unless indicated otherwise in the figure. ANOVA = analysis of variance; g/sgRNA = genomic/sub-genomic RNA; h = hour; H&E = hematoxylin and eosin; IHC = immunohistochemistry; LoD = Limit of Detection; N protein = nucleocapsid protein; PBS = phosphate buffered saline; sac. = sacrifice; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; SEM = standard error of the mean; TCID₅₀ = median tissue culture infectious dose; VC = vehicle control.

Fig. 3. EDP-235 effectively blocks infection and viral transmission in ferrets.

a Study design. Ferrets were infected with 1 × 10⁵ PFU of SARS-CoV-2 USA/WA1/2020 intranasally. 12 hpi, animals were orally dosed with vehicle or EDP-235 QD or BID. 60 hpi uninfected and untreated ferrets were co-housed 1:1 with infected dosed animals. Originally infected animals were sacrificed 4 dpi while the contact ferrets were monitored for an additional 4 days. b Percent body weight change from baseline (0 days) (n = 6 per cohort). c Animal temperature (n = 6 per cohort) (d) Nasal lavage viral load determined by RT-qPCR (nsp9 gene) (n = 6 per cohort). e Nasal lavage infectious virus quantified by TCID₅₀ (n = 6 per cohort). f Rectal swab viral load determined by RT-qPCR (nsp9 gene) (n = 6 per cohort). g Endpoint (day 4 or day 8) nasal turbinate viral load determined by RNA levels of nsp9 (n = 6 per cohort). h Infectious virus in endpoint nasal turbinates determined by TCID₅₀ (n = 6 per cohort). All graphs are mean ± SEM. All P-values were < 0.0001 unless indicated otherwise in the figure. Statistics are for both QD and BID differences versus vehicle and were run using 2-way ANOVA with Tukey’s (b–f) or 1-way ANOVA with Dunnett’s (g, h) post hoc test. In (b–f), both QD and BID treatment resulted in the same statistical significance versus vehicle-treated animals in both primary challenged and contact animals except where noted in (e). ANOVA = analysis of variance; BID = twice daily; dpi = days post-infection; hpi = hours post-infection; LoD = limit of detection; nsp9 = non-structural protein 9; PFU = plaque-forming units; QD = once a day; RT-qPCR = reverse transcription quantitative polymerase chain reaction; sac. = sacrifice; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; SEM = standard error of the mean; TCID₅₀ = median tissue culture infectious dose; VC = vehicle control.