A majority (49%) of the PF32 targets in humans tissues are proteases, which is followed by electrochemical transporters (12%) and family A G protein coupled receptors (11%) (figure 1B). The insilco analysis of the PF32 structure (figure 1A), indicated several drug/lead like features including its oral bioavailability (figure 1C). In the swiss target prediction analysis, 19 proteins showed probability scores ranging from 0.11- 0.12 (figure 1D), which will be referred to as identified targets of PF32. These identified targets were further analysed for their molecular interactions (hydrogen bonds) with PF32 in Chimera software. The number of hydrogen bonds (figure 1E, 2A) between PF32 and its identified targets did not correlate with their respective probability scores, suggesting other molecular interaction (probably Van der Waals forces) may also influence these interactions. The number of hydrogen bonds between PF32 and its identified targets ranged from 0 to 178 (figure 1E). Previous reports have indicated an affinity (IC50) of 73.2±0.5 between PF32 homologue and Fibroblast activation protein alpha (FAP) and this was used as a reference to predict the affinity of PF32 with its identified targets (figure 1f). Affinity (IC50) of PF32 against the various receptors ranged from 26 to 4745 nM.
The following proteins showed higher affinity (4UFA, 1XU9, 3DDU, 1H8D, 1DUZ, 2RA3) with their IC50 values ranging from 26 to 41 nM (table 1). PF32 showed higher affinity with Identified targets 4UFA and 1XU9 and least affinity with 4A5S (table 1). Nevertheless the affinities predicted with 11 of the Identified targets were in pharmacologically relevant concentrations (table 1).
The affinity of PF32 against various SARS-COV2 specific targets were also assessed. Representative images of molecular interaction of PF32 against selected identified and SARS-COV2 specific targets are shown in figure 2A. PF32 showed highest affinity (IC50 45-60 nM) against SARS-COV2 main protease and Replicase polyprotein 1ab (figure 2B). As several 3D structures of Replicase polyprotein 1ab are reported in the PDB databased, a representative of each of the variable 3D structure reported was screened in this study. PF32 did not show any affinity against SARS-COV2 spike protein, RNA polymerase and some variable structures of Replicase polyprotein 1ab (table 2).
The expression pattern of the identified targets of PF32 was evaluated in the human tissues. Expression pattern of representative identified targets (ACE, HSD11B1, PREP and FAP) are shown in figure 3. While some identified targets were expressed only in selected tissues (ACE and HSD11B1) others were ubiquitously expressed (PREP and FAP) (figure 3).