Novel Drug Repurposing Strategy as an Alternative Therapeutic Concept for Scrub Typhus Using Computational Studies

Scrub typhus is one of the most underdiagnosed and unreported febrile illnesses caused by an obligate intracellular bacterium named Orientia tsutsugamushi and the antibiotics were the commonly prescribed drugs to treat the condition . Due to the widespread development of antimicrobial resistance to the standard drugs, the new therapeutic approach is warranted. The drug repurposing approach plays a novel concept in identifying alternative therapies to fight against pathogens. To investigate the anti-scrub typhus activity of nine newly FDA-approved antibiotics from 2018-2019 against Orientia tsutsugamushi deubiquitylase (OtDUB) compared with standard drugs. The structure of ligands was retrieved from the PubChem database and the crystal structure of target OtDUB (PDB ID: 6UPU) with a resolution of 2.2 A o was retrieved from the Protein data bank. Molecular docking studies were performed using PyRx version 0.8 and the amino acid interactions were visualized using BIOVIA Discovery studio and the pharmacokinetic properties of the drugs were analysed by SWISS ADME software. The binding affinity of the drugs to deubiquitylase and amino acids was determined using the In silico approach, the drug Omadacycline shows superior activity when compared with other drugs. Based on our preliminary in-silico docking studies, we conclude that Omadacycline may be repurposed for the treatment of scrub typhus as it shows a higher binding affinity of -8.6 kcal/mol when compared to the standard drugs. For the further advancement of the study, in vitro and in vivo studies should be performed.


INTRODUCTION
In recent years, Rickettsia disease is re-emerging infection found to be the second most common origin of non-malarial febrile illness mainly in the South East Asia after dengue.Recently, there has been a high risk of morbidity and mortality due to infection in different parts of India and neighbouring countries. 1crub typhus is caused by the gramnegative obligate bacterium Orientia tsutsugamushi in the order Rickettsia which is transmitted by the larval stage of the infected vector, a Leptotrombidium mite in the family Trombiculidae.Scrub typhus is usually underdiagnosed because of the non-specific clinical manifestations, lack of access to specific and sensitive diagnostic tests and low suspicion among clinicians.However early treatment helps in shortening the disease course and minimises fatalities.Many states in India have reported a high number of cases of this disease.According to the current estimates, the mortality of Scrub typhus in the present day is in the range of 40% to 45% for cases not treated with antibiotics. 2crub typhus is an endemic disease that affects the Tsutsugamushi Triangle extending from South and East Asia to the Asian Pacific to Northern Australia, where the Tsutsugamushi Triangle is a region of around 13,000,000 Km 2 area of Asia. 3 The pathogenesis of scrub typhus includes where the host gets infected by Orientia tsutsugamushi, then it takes 10-12 days for incubation in the host and develops an eschar-like lesion at the site of the larval bite.The onset of the disease is identified by an unknown fever origin, headache, cough, myalgia and gastrointestinal symptoms. 4Orientia tsutsugamushi enters endothelial cells to cause perivascular inflammatory lesions and disseminated vasculitis that leads to increased vascular permeability and end-organ injury to organs such as pulmonary, heart, and kidney and in some cases, it may cause grievous complications like renal failure, hepatitis, acute respiratory distress syndrome (ARDS) and meningoencephalitis and myocarditis. 5There were numerous cytokines formed such as granulocyte colony-stimulating factor (CSF), macrophages -CSF, tumour necrosis factor -α and interferon γ, where NK T-cells and the cytotoxic T-lymphocytes serve a major aspect in attacking the defiled host cells.The defence mechanism of the host was downregulated by the organism, where GP-96 is downregulating on the endothelial cells and the macrophages, which play a major part in antigen presentation, antibody production and dendritic cell functioning. 6he most frequently observed target cells for this bacterium include endothelial cells, fibroblasts, macrophages, neutrophils and lymphocytes, where endothelial cells are one of the major cellular targets of O. tsutsugamushi during systemic infection. 7,8acterial pathogens often manipulate the host cell membrane-trafficking pathways for the benefit of pathogens to enter the cell, to humiliate immune responses or to gain nutrients. 9ne such protein OtDUB reserved from Orientia tsutsugamushi contains a deubiquitylase (DUB) and additional domains, where the expression of OtDUB disturbs the trafficking of membranes through various mechanisms.The presence of protein plays a major role in infection because the ubiquitin-proteasome system performs an important role in host cell defence. 10,11OtDUB contains a Ubiquitin binding domain (OtDUB UBD ), a guanine nucleotide exchange factor domain (OtDUB GEF ) , Phosphatidyl serine-binding domain and a domain attached to clathrin adaptor protein complexes 1 & 2 (AP-1, AP-2) without intermediary.During infection, OtDUB affiliates with the endosomal system through its preferential binding with AP-2 complexes. 12Thus, ubiquitination and DUBs play a significant role in the host immune response during infections, so these could be chosen as potential targets in therapeutics and effective treatments.
Because of non-specific symptoms and signs of scrub typhus, there may be overlap with other tropical infections such as dengue, typhoid, malaria and leptospirosis. 13Most commonly antimicrobials such as beta-lactam antibiotics are recommended for the management of common infections including typhoid fever. 14linically Scrub typhus has been treated with chloramphenicol or doxycycline.In recent decades, chloramphenicol usage has been minimized because of its toxicity problem, hence making azithromycin, a preferential drug of choice for the initial stage of scrub typhus.Due to the development of antimicrobial resistance leading to minimal utilization of these agents, 16 the drug repurposing approach using computational tools provides an opportunity to find a potential drug to combat resistance development in serious infectious diseases. 17This approach provides several merits compared with the development of new drugs, due to its faster and low-cost process and while the drugs were tested in clinical trials for safety and toxicity studies before introducing the drug into the market. 18,19he current work focuses on the drug repurposing approach using newly FDA-approved antibiotics for their anti-scrub typhus activity targeting Orientia tsutsugamushi deubiquitylase (OtDUB) through molecular docking studies.The outcome of the study will help clinicians and pharmaceutical experts to investigate and further validate these drugs as potent anti-scrub typhus against Orientia tsutsugamushi infection.

In silico docking studies Protein preparation
The crystal structure of the deubiquitylase (Orientia tsutsugamushi OtDUB in complex with three molecules of ubiquitin) of PDB ID-6UPU with a resolution of 2.2 A o is used as a target and retrieved through Protein Data Bank.The Protein was prepared using the Biovia Discovery Studio tool, where the water molecules, heteroatoms and the chains B, C, D, F, and G were removed from the macromolecule.Finally, polar hydrogen was added to the target protein and saved in '.pdb' format.The prepared protein is shown in Figure 1.

Ligand preparation
Nine new FDA-approved antibiotic drugs from 2018-2019 and standard drugs for scrub typhus were chosen for this study.Chemical structures were retrieved from the PubChem Database and shown in Table 1.By using Biovia Discovery Studio, the three-dimensional geometry of the compounds was generated and saved in '.pdb' format for further interaction with the target.

Molecular docking studies
The ligands were docked against deubiquitylase (DUB) and were performed by using the Auto dock tool complied in Pyrx software which is an open-source virtual screening tool.The prepared target has been opened in PyRx software and the macromolecule is converted to PDBQT and then the prepared ligand is imported and minimized, finally converted to PDBQT format.
After the ligand and target are ready for docking, click vina wizard, then select the target and molecules for docking, and click forward results in the auto grid generated were predetermined around the active site of the protein.When the docking process is completed, the results are saved as a '.csv' file for future purposes and the docked ligand is analysed for interactions.

Visualization and analysis of ligand interactions
The protein-ligand interactions were analysed by using Discovery studio visualizer 4.0 a free viewer software.The protein molecule in the form of '.pdbqt' form was imported on the graphical interface of the molecule window followed by the '.pdbqt' file output of ligand.The 2D and 3D structures of compounds with the highest affinity were analysed and visualized.

Physiochemical and pharmacokinetic properties
The screened ligand molecules were further evaluated for their physicochemical and pharmacokinetic properties using the SWISS ADME 20 and PREADMET server.The SMILE format of the ligand molecules was used as input data and uploaded to the server.
The drug Ceftobiprole forms six hydrogen bonding interactions with leu 124, Ile 121, Gln 125, Gly 118, Ala 119, and Leu 41 of the active site of the target, where these amino acids form hydrogen bonding with nitrogen atoms of the ligand, pi-alkyl interactions with Lys 120 and carbon-hydrogen bond with Thr 126 and shown in Figure 2. Eravacycline forms one hydrogen bonding interaction between the oxygen atom and with the hydrogen atom of Glu 156 an unfavourable acceptor-acceptor bond with Glu 156, alkyl interaction with Val 106 and carbon-hydrogen bond with Asp 103, Asp 96, Thr 72 as shown in Figure 3. Omadacycline forms four hydrogen bonding interactions between the oxygen atom and with the hydrogen atom of Asn 122, Met 98, Ser 101, Gly 99, carbon-hydrogen bond with Gln 125, Ile 152 and pi-alkyl interaction with Leu 124, Val 100, Pro 151 were depicted in Figure 4. Rifamycin forms one hydrogen bonding interaction between the oxygen atom and with hydrogen atom of Gln 125 as shown in Figure 5, where the standard drug doxycycline showed three hydrogen bonding interactions with Ile 152, Thr 126, Leu 124 and pi-alkyl interaction with Ile 121 of the target site as shown in Figure 6.The binding affinity for the New FDA drugs and standard drugs are shown in Table 2.

Physiochemical and pharmacokinetic analysis
To assess the bioavailability of orally administered drugs, it should obey the Lipinski rule of five, which states that the molecular weight < 500, hydrogen bond donor < 5, hydrogen bond acceptor <10 and log p <5. 21 Early analysis of ADME in the drug discovery phases minimize the pharmacokinetic-related problem during the clinical phase. 22Based on the criteria, all the ligand molecules do not obey the Lipinski rule, except Lefamulin, Ozenoxacin, Sarecycline, and Doxycycline, but they still fit into the FDA-approved drugs.Except for ozenoxacin, most of the drugs showed poor gastrointestinal absorption and all the drugs did not penetrate the blood-brain barrier; the results are shown in Table 3.

CONCLUSION
In recent decades, Scrub typhus has remained an important health issue in endemic places but antibiotic resistance for scrub typhus is still existing.So, drug repurposing is the new concept to discover alternative therapy for Scrub typhus.In this study, we conclude that Omadacycline may be repurposed for the treatment of scrub typhus as it shows a higher binding affinity of -8.6 kcal/mol to OtDU and other amino acids when compared to the standard drug and other new FDA-approved drugs.For the further advancement of the study, in vitro and in vivo studies should be performed.

Table 1 .
Chemical structures of the FDA-approved new drugs