TMP1 is an orally active dual inhibitor targeting M pro (IC50= 312.5 nM) and TMPRSS2 (IC50= 1.28 μM, KD= 10.10 μM). It shows broad-spectrum protective effects in vitro against various SARS-CoV-2 variants. In vivo, TMP1 provides cross-protection against highly pathogenic coronaviruses, including SARS-CoV-1, SARS-CoV-2, and MERS-CoV, and effectively blocks the transmission of SARS-CoV-2. Additionally, TMP1 can inhibit the infection of SARS-CoV-2 escape mutants resistant to Nivolumab. TMP1 is useful in coronavirus research.

TMP1 exhibits no cytotoxicity against wild-type SARS-CoV-2 and other variants (VOC) including Alpha, Beta, Delta, and Omicron (BA.1 and JN.1) in VeroE6-TMPRSS2 cells over 48 hours. It reduces viral gene copies and titers in SARS-CoV-2 Omicron subtypes JN.1 and KP.2 infected human nasal epithelial cells (ALI-hNEC). At concentrations of 0-20 µM for 24 hours in the presence of a 2 µM P-gp inhibitor CP-100356, TMP1 decreases viral load and infectious progeny virus titers in VeroE6, VeroE6-TMPRSS2, and LLC-MK2 cells. It also inhibits the replication of seasonal HCoV (HCoV-HKU1, -OC43, -NL63, and -229E) with EC50 values ranging from 0.31 to 2.60 µM and offers cross-protection against highly pathogenic coronaviruses SARS-CoV-1 and MERS-CoV with EC50 values of 0.55 and 5.26 µM, respectively. TMP1 (1-50 µM) inhibits TMPRSS2-dependent cell-cell fusion mediated by wild-type SARS-CoV-2 spike protein. It also inhibits the Mpro of recombinant SARS-CoV-2, SARS-CoV-1, and MERS-CoV with IC50 values of 0.313, 0.516, and 74.6 µM, respectively. TMP1 reduces the replication of SARS-CoV-2, SARS-CoV-1, and MERS-CoV in TMPRSS2-deficient VeroE6 cells at 0-50 µM. In Calu3 cells infected with rSARS-CoV-2 NSP5-E166V, TMP1 decreases the intracellular viral gene copies with an EC50 of 0.449 µM.

TMP1, administered orally at 100 mg/kg twice daily for four days, reduces viral load in the respiratory tracts of SARS-CoV-2-infected heterozygous K18-hACE2 C57BL/6J mice, improves histopathology related to infection, and enhances overall survival. A single dose of TMP1 at 90 mg/kg effectively counters the Omicron lineage prevalent in the upper respiratory epithelium of PFU SARS-CoV-2 Delta-infected Syrian golden hamster models and significantly mitigates SARS-CoV-2 transmission. Broad-spectrum antiviral protection is provided by TMP1 (100-150 mg/kg, orally, twice daily for four days) in heterozygous mice infected with SARS-CoV-1 and in hDPP4-KI mice infected with MERS-CoV MA. Additionally, TMP1 at 100 mg/kg orally twice daily for four days maintains sensitivity in rSARS-CoV-2 NSP5-E166V-infected heterozygous K18-hACE2 C57BL/6J mice due to a mechanism of interaction with M pro that differs from NRV.