For nearly a century, antibiotics have been used continuously to treat bacterial infections. The consequence of this trend is the resistance of bacteria, especially the Enterobacteriaceae family which has become a global medical crisis (Navon-Venezia et al., 2017). Two mechanisms of genetic and mechanistic basis such as horizontal gene transfer, modification of antibiotic molecule, change in the target site, and decreased antibiotic penetration are the main reasons for the resistance of bacteria to antibiotics (Munita et al., 2016). One of the most mutable and antibiotic-resistant bacteria is Klebsiella pneumoniae. It is a gram-negative, non-motile, encapsulated, lactose-fermenting, facultative anaerobic, rod-shaped, and pathogenic bacteria that naturally exist in the mouth, nose, and throat and on the skin (Doorduijn et al., 2016; Kidd et al., 2017; Li et al., 2014). Naturally, K. pneumoniae is not a pathogenic organism in humans but, in the case of aspiration, it can damage the lung alveoli and causes dangerous pneumonia. It can make a wide range of hospital infections such as pneumonia (Prince, S. E., Dominger, K. A., Cunha, B. A., Klein, N. C., Brook, M., Brook, 2000), urinary tract infection (El Bouamri et al., 2015), wound infection (Chung, 2016), thrombophlebitis (Maffiolo et al., 2006), osteomyelitis (Sanders, 1989), meningitis (Fang et al., 2000), cholecystitis (Capoor et al., 2008), sepsis and blood-stream infection (Girometti et al., 2014) in humans and these infections are worse in children, the elderly, people with the weak immune system and alcoholism (Kidd et al., 2017). Many antibiotics are used to inhibit the activity of K. pneumoniae, the most important of which are ampicillin/sulbactam, piperacillin/tazobactam, ceftazidime, cefepime, levofloxacin, norfloxacin, gatifloxacin, moxifloxacin, and ciprofloxacin (Brisse et al., 1999; Hawser et al., 2011; Hoffman et al., 1992). However, the ability of this organism to counteract antibiotics due to having a wide range of beta-lactamases and carbapenemases is high, as far as some strains isolated in the clinic are resistant to all antibiotics and the treatment of them are very difficult (Brolund et al., 2010; Hirsch et al., 2011). K. pneumoniae resistance to antibiotics has several consequences, including increased mortality, hospitalization, and cost, and according to the World Health Organization (WHO) statement, pneumonia is one of the most important multidrug-resistant (MDRs) diseases that threatens human health (Kidd et al., 2017; Navon-Venezia et al., 2017).
Nowadays, various enzymes including proteases, topoisomerases, transferases, hydrolases, and reductases are targeted to control and treat pathogenic bacteria. Dihydrofolate reductase (DHFR) is one of the most important and validated targets which exists in all cells and, on the other hand, it has a special structure in different species that makes it suitable for designing potent and selective inhibitors (Lamb et al., 2014). Dihydrofolate reductase catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) using coenzyme NADPH and the proton of water molecule respectively as the donor of the hydride ion. The mechanism of enzyme catalysis and the role of individual amino acids in the reaction has always been extensively studied by experimental and quantum chemical methods. The catalytic process involves two steps protonation of dihydrofolate at N5 atom and transfer of hydride ion to positively charged intermediate. This is done with the help of the aspartic acid in prokaryotes. The rate of transfer of hydride ion to the substrate is the key stage of the catalytic process. It was found that the reduction of 7, 8- Dihydrofolate was accompanied by the transfer of hydrogen atom located in 4-pro R position of NADPH to C-6 atom of dihydrofolate. In the end, NADPH is oxidized to NADP + and 5,6,7,8-tetrahydrofolate is produced. The tetrahydrofolate and its metabolites are involved in the biosynthesis of thymidine monophosphate (dTMP), purine bases, and methionine. Due to the role of dihydrofolate reductase in the production of DNA and RNA precursors, inhibition of the enzyme in the S-phase cells reduces the level of tetrahydrofolate, which eventually leads to cell death (Rao & Road, 2013),(H. Wang et al., 2016).
Trimethoprim (TMP) is an inhibitor of DHFR of K. pneumoniae and other micro-organisms. It is usually used in combination with other antibiotics(Lombardo et al., 2016). The combination of trimethoprim-sulfamethoxazole (TMP/SMX) is generally well tolerated but can induce adverse effects such as leukopenia, thrombocytopenia (Gordin, 1984), anemia (Ho & Juurlink, 2011), agranulocytosis, evocative of a disorder of cellular maturation, probably secondary to an inhibition of the human DHFR (Wu et al., 2015). This combination can cause severe but rare dermatological reactions, Stevens-Johnson and Lyell syndromes (Rijal et al., 2014). Trimethoprim can also cause hyperkalemia by decreasing urinary potassium elimination (Nickels et al., 2012; Velázquez et al., 1993). Resistance to antibiotics has prompted researchers to always look for compounds to control pathogenic bacteria. On the other hand, the long-term use of trimethoprim against the K. pneumonia infection has caused some of its strains to encode a TMP-resistant DHFR by expression of the DfrA1 gene (Lam et al., 2014). Mutations in Asp27 and Leu28 to Glu and Gln, Ile50 to Met50 and Ile94 to Ser, and also deletion of proline between residues 54 − 49, reduces the affinity of the DHFR to trimethoprim (Lombardo et al., 2016).
Structure-based drug discovery (SBDD) is a rapidly rising method in molecular biology and drug design. The purpose of this method is to investigate the mechanism of inhibition and activation of different targets at the atomic scale. Designing and developing new drugs for molecular targets is a time-consuming and costly process. On the other hand, drug repurposing is a process to find existing drugs for a novel medical indication. Each of these two methods (SBDD and drug repurposing) has been very successful in introducing new drugs(Batool et al., 2019; Naveja et al., 2011). Computational drug repurposing approach saves cost and time in drug discovery and also the drugs used in this method have known pharmacokinetics and safety profiles. In recent years, computational methods have always been of interest to researchers. In humans, DHFR is a potential target for cancer treatment, and many studies have been conducted on it, both in the field of computational(Rana et al., 2019) and experimental(Kubbies & Stockinger, 1990). Many studies have also been performed to identify new drugs for pathogenic parasitic(Anderson, 2005) and bacterial dihydrofolate reductase, including leishmania donovani(Vadloori et al., 2018), Toxoplasma gondii(Pacheco Homem et al., 2013), and Yersinia pestis(Bastos et al., 2016), using computational methods. On the other hand, no research has been done in this field on Klebsiella pneumoniae trimethoprim-resistant dihydrofolate reductase (DfrA1). Herein, we intend to identify novel inhibitors for the DfrA1 from FDA-approved drugs library using molecular modeling methods such as Structure-Based Virtual Screening (SBVS), Molecular Dynamics (MD) and binding free energy calculations (MMPBSA).