key: cord-0865307-lq269aty authors: Sonawane, Kailas D.; Barale, Sagar S.; Dhanavade, Maruti J.; Waghmare, Shailesh R.; Nadaf, Naiem H.; Kamble, Subodh A.; Mohammed, Ali Abdulmawjood; Makandar, Asiya M.; Fandilolu, Prayagraj M.; Dound, Ambika S.; Naik, Nitin M.; More, Vikramsinh B. title: Structural insights and inhibition mechanism of TMPRSS2 by experimentally known inhibitors Camostat mesylate, Nafamostat and Bromhexine hydrochloride to control SARS-Coronavirus-2: A molecular modeling approach date: 2021-05-26 journal: Inform Med Unlocked DOI: 10.1016/j.imu.2021.100597 sha: be54a941e980ddc87f3d9fb47e63ea3fe6182530 doc_id: 865307 cord_uid: lq269aty Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been responsible for the cause of global pandemic Covid-19 and to date, there is no effective treatment available. The spike ‘S’ protein of SARS-CoV-2 and ACE2 of the host cell are being targeted to design new drugs to control Covid-19. Similarly, a transmembrane serine protease, TMPRSS2 of the host cell plays a significant role in the proteolytic cleavage of viral ‘S’ protein helpful for the priming of ACE2 receptors and viral entry into human cells. However, three-dimensional structural information and the inhibition mechanism of TMPRSS2 is yet to be explored experimentally. Hence, we have used a molecular dynamics (MD) simulated homology model of TMPRSS2 to study the inhibition mechanism of experimentally known inhibitors Camostat mesylate, Nafamostat and Bromhexine hydrochloride (BHH) using molecular modeling techniques. Prior to docking, all three inhibitors were geometry optimized by semi-empirical quantum chemical RM1 method. Molecular docking analysis revealed that Camostat mesylate and its structural analogue Nafamostat interact strongly with residues His296 and Ser441 present in the catalytic triad of TMPRSS2, whereas BHH binds with Ala386 along with other residues. Comparative molecular dynamics simulations revealed the stable behavior of all the docked complexes. MM-PBSA calculations also revealed the stronger binding of Camostat mesylate to TMPRSS2 active site residues as compared to Nafamostat and BHH. Thus, this structural information could be useful to understand the mechanistic approach of TMPRSS2 inhibition, which may be helpful to design new lead compounds to prevent the entry of SARS-Coronavirus 2 in human cells. N-terminus for TMPRSS2 prior to simulation by using a tool in Gromacs. The TMPRSS2 149 protein was centered into a 6.9 nm from the edge of the cubic box and the system was then showed that the predicted model of TMPRSS2 has a Z score of -7.48 ( Figure 2A ) as compared to 235 the template having a Z score of -6.64, (Supplementary Figure S1 ), which is within the range of showed negative interaction energy suggesting the good quality of the predicted 3-D structure 238 ( Figure 2B ). Further, PROCHECK analysis was carried out in order to check the quality of the 239 predicted TMPRSS2 model [31] . This analysis shows that 99% of residues are present in allowed 240 regions and only 1% residues in the disallowed region (Figure 3 ), suggesting the good quality of 241 the TMPRSS2 model. . TMPRSS2 active site residues were predicted using CASTp online server 246 [36] . The CASTp server showed several pockets, out of which, a pocket was selected in such a 247 way that at least one residue from the catalytic triad remain present in the selected pocket, these 248 resulted in the identification of three binding pockets in TMPRSS2. As TMPRSS2 is a serine 249 protease, hence pockets having Ser, Thr, His, Asp residues, were selected for further study. However, from the selected pockets, we observed that the residues His296, Glu299, Asp435, Table 1 , 2). Docked complex analysis revealed that Camostat mesylate and Nafamostat binds 283 in the same pocket of TMPRSS2. The docked complex analysis of Bromhexine hydrochloride (BHH) with TMPRSS2 285 shows fewer hydrogen bonding interactions as compared to Camostat mesylate and Nafamostat. A nitrogen atom of BHH interacts with Thr393 of TMPRSS2 ( Figure 6C ; Table 1 , 2). However, 287 the residues such as Val278, His279, Val280, His296, Gly391, Lys392, Gln438, Asp440, and 288 Ser441 of TMPRSS2 are providing additional hydrophobic interactions ( Figure 6 , Table 1 ). The Table 1 ). The binding energies of MD simulated TMPRSS2 model with Camostat mesylate, Nafamostat and Bromhexine hydrochloride 298 complexes found as -6.23 kcal/mol, -7.20 kcal/mol, and -5.51 kcal/mol respectively (Table 1) . 299 The docked complexes of TMPRSS2 with Camostat mesylate and Nafamostat show lower 300 binding energy with a minor difference as compared to Bromhexine hydrochloride. The inhibitor 301 constant (Ki) of Camostat mesylate, Nafamostat, and Bromhexine hydrochloride is 26.98 uM, 302 5.25 uM, and 91.26 uM respectively as shown in Table 2 . (Table 4 ) . Ramchandran plot of TMPRSS2 model. 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