key: cord-1051826-xopmqgtl
authors: Bomediano Camillo, Lívia de Moraes; Sasaki, Sergio Daishi
title: Molecular docking between human TMPRSS2 and the serine protease Kunitz-type inhibitor rBmTI-A
date: 2022-03-14
journal: bioRxiv
DOI: 10.1101/2022.03.13.484191
sha: 53d6d8bf768cebe9dce64c8862169410c2e89592
doc_id: 1051826
cord_uid: xopmqgtl

SARS-CoV-2 entrance into host cells is dependent of ACE2 receptor and viral protein S initiation by serine protease TMPRSS2. Cleavage of coronavirus protein S at the junctions Arg685/Ser686 and Arg815/Ser816 leads to the production of the S1/S2 and S2’ fragments needed for the fusion of viral and cell membranes. Studying and identifying serine protease inhibitors is an important step towards the development of candidate drugs to prevent SARS-CoV-2 infection. It has already been stablished that camostat mesylate, a serine protease inhibitor, is capable of blocking TMPRSS2 activity and prevent SARS-CoV-2 entrance into host cells. In this work, the interaction between the two domains of Kunitz-type serine protease inhibitor rBmTI-A and TMPRSS2 was studied through molecular docking. rBmTI-A domain 2 (P1 site Leu84) had the best complex results with predicted binding affinity of -12 Kcal.mol-1 and predicted dissociation constant at 25°C of 1.6 nM. The results suggest that rBmTI-A is capable of binding TMPRSS2 cleavage site at the junction Arg815/Ser816 using essentially the same residues that camostat mesylate.

enzyme 2 (ACE2, angiotensin-converting enzyme 2). Protein S has a receptorbinding domain (RBD) that specifically recognizes ACE2 as its receptor [18, 19] .

SARS-CoV-2 entry into host cells also depends on the ACE2 receptor and viral protein S initiation by serine protease TMPRSS2 [10, 11, 13] . Other studies have demonstrated and characterized the interaction of SARS-CoV-2 with the ACE2 receptor [20, 21] . Furthermore, Hoffmann et al., 2021 showed that the camostat mesylate drug, a serine protease inhibitor used to treat chronic pancreatitis [23] was able to block the action of TMPRSS2 and, therefore, block the entry of the virus into host cells. Baughn et al., 2020 and Wettstein et al., 2021 demonstrated that serine protease inhibitor alpha-1 antitrypsin is also capable to inhibit the action of TMPRSS2 and stop the development of the SARS-CoV-2 infection. These results reflect the importance of studying and identifying more serine protease inhibitors as potential drug candidates to prevent SARS-CoV-2 infection [10, 24] .

rBmTI-A is a recombinant Kunitz-BPTI serine protease inhibitor with inhibitory action against trypsin, human plasma kallikrein (HuPK), HNE and human plasmin [25, 26] . The inhibitor was initially obtained from the extract of larvae of the bovine tick Rhipicephalus microplus [25] [26] [27] and it has two Kunitz-BPTI domains, with a total molecular weight of 13.87 kDa. Domain 1 has the amino acid Arg at its P1 site with inhibitory action against trypsin and HuPK. Domain 2 has the amino acid Leu in its reactive site with inhibitory action against HNE [26] .

The serine protease inhibitor rBmTI-A has shown important experimental results in the context of pulmonary emphysema and lung tissue inflammation [27] [28] [29] . Given the health emergency imposed by SARS-CoV-2, responsible for a disease that mainly affects the lung tissue and the relationship between the interaction of its protein S with the ACE2 receptor through an initialization of protein S by the serine protease TMPRSS2, the study of the interaction of TMPRSS2 and serine protease inhibitor rBmTI-A can lead to development of a therapeutic potential drug against COVID-19 disease.

TMPRSS2 structure was previous modeled by Hussein et al., 2020 [30] and made publicly available by the authors. rBmTI-A structure was modeled by our group in previous work [31] . Molecular dynamic simulations were performed to flexibilize both structures in water as solvent for 100 nanoseconds using NAMD v.2.14 [32] under default parameters.

The prediction of the interaction interface between TMPRSS2 and rBmTI-A was made using HADDOCK v.2.2 webserver [33, 34] . TMPRSS2 catalytic triad (His296, Asp345 and Ser441) and amino acids already described as involved in the substrate binding sites (Asp435, Ser460 and Gly462) [30] were defined as active residues. rBmTI-A domain 1 P1 site Arg24 and domain 2 P1 site Leu84 were defined as active residues. The passive residues were defined as the surrounded amino acids in both cases. The cluster with the best HADDOCK score was chosen for further pose analysis. PRODIGY webserver v.1.0 [35] and Ligplot + v.2.2 [36] were used to assess binding affinity energy and different [37] .

The interaction complexes between TMPRSS2 and P1 sites 1 (Arg24) and 2 (Leu84) are shown in Figure 1 and TMPRSS2 interface residues involved in proteolytic cleavage of the Sars-CoV-2 spike protein have been determined and the proteolytic cleavage sites has been mapped at the junction of Arg685/Ser686 and Arg815/Ser816 [30] . Table 1 shows the comparison between TMPRSS2 interface residues found in the interaction with SARS-CoV-2 cleavage sites 1 and 2 [30] with camostat mesylate inhibitor [38] and rBmTI-A inhibitor P1 sites 1 and 2.

Tables 2 and 3 show the main type of interactions between rBmTI-A P1 sites and TMPRSS2 residues. Figure 3 shows the comparison between rBmTI-A and Furthermore, these findings shows that rBmTI-A P1 site Leu84 has more interfacial contacts with TMPRSS2, with better energy binding affinity and dissociation constant. Table 1 . TMPRSS2 interface residues in the interaction with SARS-CoV-2 cleavage sites 1 and 2, with camostat mesylate inhibitor and rBmTI-A inhibitor P1 sites 1 and 2. Table 3 . Corresponding residues and main interactions type between rBmTI-A P1 site Leu84 and TMPRSS2. Retrieved from www.who.int/health-topics/severe-acute-respiratory-syndrome). receptor for SARS-CoV as the angiotensin-converting enzyme 2 (ACE2) [18] .

ACE2 is a type I membrane protein mainly expressed in lungs, heart, kidneys and intestine) [40, 41] . ACE2 primary physiological role is the maturation of angiotensin, a peptide hormone that controls vasoconstriction and blood pressure [40] . Li et al and Wu et al demonstrated how ACE2 is capable of efficiently bind the S1 domain of the SARS-CoV spike protein (S) and enable host and viral membranes fusion [16, 18] .

The entry of SARS-CoV into the host cell also depends on the initialization of viral protein S by cellular proteases which implies cleavage of protein S at the junctions Arg685/Ser686 and Arg815/Ser816, which leads to the production of the S1/S2 and S2' fragments needed for the fusion of viral and cell membranes [14] [15] [16] [17] 30] . The initialization of viral protein S is done by TMPRSS2 [8, 9, 42, 43] .

The TMPRSS2 serine protease is a type II transmembrane serine protease that are expressed in many human tissues, such as prostate, stomach, salivary gland, mammary gland, pancreas, kidney, liver, small intestine, lung, trachea, fetal kidney, fetal lung, eye and airway epithelial cells [44, 45] . SARS-CoV-2, responsible for the ongoing disease outbreak, is closely related to SARS-CoV and both share the same mechanisms of host cell fusion and entrance [19, 30, 46] .

The coronavirus spike protein is a 1273 amino acid long homo-trimeric glycoprotein that has two major subunits: S1, involved in receptor recognition and binding and membrane anchored S2, involved in the mediation of the fusion of the viral and cell host membranes [47] [48] [49] . S protein is first cleaved at S1/S2 site, assuming a prefusion conformation. Then, a second cleavage occurs at S2' site which leads to an irreversible conformational change that result in residues, a typical pattern of disulfide bond [61, 62] . The Kunitz domain has a secondary alpha + beta-type structural fold, where alpha helices and beta sheets occur separately along the skeleton. The domain is characterized by the presence of three highly conserved disulfide bonds that are necessary for stabilization of these inhibitors native conformation [61, 62] . The bovine pancreatic trypsin inhibitor (Bovine Pancreatic Trypsin Inhibitor -BPTI) is the typical model of Kunitz-BPTI type inhibitors. BPTI has broad specificity, being able to inhibit trypsin, chymotrypsin and elastase-type serine proteases [63] .

Tanaka et al characterized a Kunitz-type inhibitor from tick larvae [64] . BmTI-A is an 18 kDa inhibitor with activity on several serine proteases, such as trypsin, HNE, human plasma kallikrein, human plasmin and chymotrypsin [25, 26, 64] .

BPTI type with inhibitory action against trypsin, human plasma kallikrein, HNE and human plasmin in nM range [26] . Like the BmTI-A inhibitor, it has two Kunitz-BPTI domains and a total molecular weight of 13.87 kDa. Domain 1 has the amino acid Arg at its P1 site with an inhibitory action against trypsin and HuPK and domain 2 has the amino acid Leu at its P1 site with an inhibitory action against HNE [26] .

rBmTI-A domains have 51-64 residues that form a central anti-parallel beta sheet and a short C-terminal helix. Each domain has six cysteine residues that form three disulfide bonds, resulting in a double loop structure. The active inhibitory binding loop is located between the N-terminal region and the first beta sheet [62] . The structure and thermostability of the rBmTI-A inhibitor were demonstrated by Bomediano Camillo et al [31] . rBmTI-A has been studied by different groups with positive results regarding its effects on chronic obstructive Studies have shown that TMPRSS2 is mainly expressed in the lung tissue, salivary gland, thyroid, gastrointestinal tract, pancreas, kidney, and liver according to RNA and protein expression data available [65] . Matusiak et al studied the differential expression of TMPRSS2 and ACE2 in publicly available data and investigated the effect of smoking behaviour, infections and pollutants in the lung, nasal tissue, bronchial tissue and cell lines [66, 67] . TMPRSS2 expression in airway epithelia is highly upregulated by IL-13, a cytokine involved in allergic airway inflammation response, asthma and COPD development [68, 69] . It has been shown that IL-13 is also highly expressed in the plasma of COVID-19 patients that require intensive care [70] . These results indicate that TMPRSS2 is greatly expressed in lung tissue inflammation processes and, therefore, COPD contributes to aggravate COVID-19 severity. Since rBmTI-A treatment has shown positive outcome in COPD and lung tissue inflammation [26] [27] [28] [29] , the study of the interaction and inhibition of TMPRSS2 by rBmTI-A is an important first step in the development of a drug candidate for COVID-19 treatment. 

None. 

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We are grateful to Federal University of ABC for providing the infrastructural and funding support. Also, we are grateful by financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 2018/11874-5 and CAPES, financial code 001.