Fig. 1: NACE2i selectively targets nuclear ACE2 following SARS-CoV-2 infection and inhibits viral replication in hamster lungs.

a Using NLS prediction software (NLStradamus, Revision r.9), an NLS motif (italics) and critical NLS residues were identified (underlined) at the ACE2 C-terminus. A peptide inhibitor targeting this region was designed (red). AlphaFold docked the NLS region of ACE2 in the same location observed in the crystal structure⁴, demonstrating accessibility between the transmembrane region and the cytoplasmic NLS. b Electrophoresis mobility shift assay confirming the IMP1α and ACE2 interaction via the C-terminal domain. The ACE2 C-terminal domain is FITC-labeled. n = 2 experimental replicates. c Microscale thermophoresis and fluorescence polarization to assess NACE2i inhibition of binding between IMP1α and ACE2. n = 3 experimental replicates, KD shown represent the mean ± SD. d Cells immunostained with NFACT1, C-Rel, ACE2, pPKC-zeta, Nurr1/Nur77, HDAC1 or NFKB p50. Nuclear fluorescent intensity (NFI) was determined using ImageJ analysis; n = 2 technical replicates (n = 20 cells analyzed from 1 field of view (FOV) per group). Data are mean ± SEM. One-way ANOVA with Bonferroni post hoc test. e ACE2 activity was measured in Caco2 cell lysates ± NACE2i. Data represent mean ± SEM, n = 3 technical replicates. f Scheme of SARS-CoV-2 infection in golden Syrian hamsters. All the hamsters were sacrificed at day 2 post-infection (dpi), lung tissues were collected (n = 8 hamsters/group in a single experiment). g qRT-PCR analysis of SARS-CoV-2 RNA in infected lungs from golden Syrian hamsters treated ± NACE2i. RNA yield is presented as log10 TCID₅₀ eq./ml. Data represent mean ± SEM, n = 7 animals, i.v. NACE2i; n = 8 animals, Ctrl, i.p. NACE2i. One-way ANOVA with Tukey’s post hoc test. h TCID₅₀ assay to measure infectious viral titers in lungs of infected hamsters. Data represent mean ± SEM, n = 5 animals/group. One-way ANOVA with Tukey’s post hoc test. i Representative images of lungs from SARS-CoV-2-infected hamsters treated with either vehicle control, i.p. NACE2i, or i.v. NACE2i. SARS-CoV-2 spike protein (green), DAPI (blue) for nuclei. Graphs depict the population dynamics of SARS-CoV-2 spike positive cells and the fluorescent intensity of the SARS-CoV-2 spike protein (5 FOVs/animal from n = 8 animals, Ctrl, i.v. NACE2i; n = 3 animals, i.p. NACE2i). Data represent mean ± SEM. One-way ANOVA with Tukey’s post hoc test.