Comparative Docking Studies of Potential Candidates from Kokum and Cranberry as Anti-Adhesins Against UTI

Most frequently affecting women and those with diabetes, urinary tract infections (UTI) are a prevalent illness. Inappropriate management of the acute stage of the illness leads to pyelonephritis, which makes the condition chronic. Multiple medications are ineffective against the microorganisms that cause UTIs, due to multi-drug resistance. Escherichia coli fimbria contains the bacterial adhesin protein FimH, which is responsible for adhering bacteria to the host urinary tract's epithelial cells. Therefore, FimH becomes a crucial target for the development of drugs since it plays a key role in bacterial infections like UTIs. In the context, phytochemical intervention using Garcinia indica (Kokum) and Vaccinia macrocarpon (Cranberry) proves to be an effective alternative. Garcinia indica is a tropical plant endemic to India, particularly in the Karnataka, Kerala, and Maharashtra coastal regions. The fruit is abundant in anthocyanins and benzophenones, which have antibacterial properties against a variety of pathogens. Because the fruit includes antiadhesive flavonoids and proanthocyanins, Vaccinia macrocarpon fruit syrup is currently utilized as a treatment for UTIs and the fruit is native to America. The current study contrasts the inhibitory effects of secondary metabolites from Vaccinia macrocarpon and Garcinia indica on E. coli FimH protein. According to the study, garcinol and kaempferol from the plants’ Garcinia indica and Vaccinia macrocarpon, respectively, showed the highest affinities for the protein FimH.

Urinary tract infection (UTI) is one of the prevalent diseases that affects females and people suffering from type II diabetes 1 . Around 50-60% of the women have suffered from UTI at least once in their lifetime 2 . Uropathogenic E. coli (UPEC) is considered to be responsible for the majority of the infection, can form the biofilm, and invade the host immune response 3 . Apart from E. coli which causes around 80% of the total infection, other pathogens like Klebsiella spp., Staphylococcus saprophyticus, Enterobacters and Proteus spp. can also contribute to the same 4 . The threat posed by the uropathogens' due to the rise in antibiotic resistance makes therapy challenging and expensive. This increases the likelihood of recurring infection 5 . Genetic polymorphism in the pathogens' has made them resistant to multiple drugs 6 . Another worrying trend is the rise in the prevalence of multidrugresistant infections among pregnant women 7 .
Pathogens causing UTI possess type-1 fimbriae that facilitates attachment of the bacterial cell to the epithelial cells of the urethra. Due to its interaction with the mannosylated receptors expressed by urothelial cells, type-1 pili is considered to be mannose sensitive. This peculiarity of the pili is due to the activity of the adhesin protein FimH located at the tip of type-1 fimbriae 8,9 .
Several attempts are being carried out to find a potent drug that can inhibit the action of pathogens without altering the host immunity. As the protein FimH is mannose sensitive, D-Mannose was used as vaccine to mask the FimH but it was found to be ineffective 10 . Drugs that target the protein FimH and its mannose specificity are currently the subject of experiments to cure UTI because the treatment functions without killing the pathogen and uropathogens are unlikely to develop resistance. Moreover drugs targeting FimH will be selective in their effect, leaving host microflora intact 11 . Even though there are many alternative medical treatments for UTI, current research on the condition is concentrating on the antiadhesive properties of various secondary metabolites from herbal sources because they are linked to a lower risk of side effects 12,13 .

Flavonols and Proanthocyanidins from cranberry and kokum as antimicrobial agents
The production of flavonoids varies from plant to plant and depends on the environment, type of soil, and other physiological parameters. Flavonoids are generated in different sections of plants at varied amounts. They guard against biotic and abiotic stressors on the plants. Plants produce flavonoids to help them defend themselves 14 .
Berries include flavonols and their derivatives, which are known for their antibacterial properties among other health advantages 15 . Their antiadhesive properties are proving to be an efficient defense mechanism against pathogens that can withstand antibiotic action 16 .
Proanthocyanidins, also known as condensed flavonols, are extensively dispersed in different areas of the plant and have a number I3-II8-Biapigenin -6.6 2 1 3.
Garcinol -8.5 12 3 of positive health effects, such as anticancer, neuroprotective, and antibacterial capabilities 17 . They have a very low Minimum Inhibitory Concentration (MIC) and can suppress microbial growth 18 . Even nanoparticles synthesized from chitosan and proanthocyanidins along with gentamicin are proven to be bactericidal 19 . The antiadhesive properties polyphenols and flavanols (kaempferol, quercetin, and myricetin) of cranberry make them effective against a variety of infections 20 .
The flavones apigenin, i3-ii8-biapigenin, and luteolin are among the promising anti-adhesive chemicals because they exhibit a notable antiadhesive action on uropathogens 21 . Cranberry syrup is a highly suggested alternative to antibiotics among the different herbal remedies for UTI since it hinders the pathogen's adherence to the urinary system 22 . Several studies on antiadhesive property of secondary metabolites of cranberry shows that flavonoids and proanthocyanidins inhibit the FimH mediated interaction of uropathogens with the epithelial cells of the urethra and bladder 23 Garcinia indica is a plant that is commonly found in the coastal region of India specially in the coastal areas of Karnataka, Maharashtra and Kerala 24 . It is endemic to Western and Eastern Ghats and also to North-eastern regions of India 25 . The fruit is used for domestic purpose in the preparation of curry, pickles and juice. The secondary metabolites of G. indica is believed to have health benefits that can be considered as efficient herbal drug for future therapeutics 26,27 . Kokum extracts are also being studied for various biological activities. Studies have revealed that Kokum extracts have antibacterial property against several pathogens and Benzophenones of kokum especially garcinol, isogarcinol and xanthochymol that are included in the study contributes to the antimicrobial property of kokum 28,29 . Benzophenones derivatives from Garcinia species have shown chemotherapeutic activities against bacteria, fungi and mycobacteria 30 . Kokum fruit rinds are also rich in flavonoids and proanthocyanidins 31 . Cyanidin 3 sambubioside and cyanidin 3 glucoside are the anthocyanins that are present in Kokum that make kokum as a promising agent for therapeutics (28). Pathogens that were resistant to antibiotics have been susceptible to the synergistic effect when the same antibiotics are combined 32 .
In the field of drug discovery molecular Studios Visualizer 2021, a GUI based software for the screening for best conformation was done, based on their binding affinity, non-boding interactions, and total number of hydrogen bonds.

Molecular dynamics (MD) simulation
To get the better understanding of conformational changes and their stability over a period of time, MD study was conducted using the GROMACS biological software package. The prerequisite and the MD experiments were performed based on previous work 39 . Using the XMGRACE software 40 , the trajectory plots were analysed.

Binding free energy (MMPBSA) calculations
The calculation of binding free energies was performed based on work 41,20 . The study was performed to understand formation of energy after dynamic study. The calculation was done using, g_mmpbsa programme, by using GROMACS trajectories as input 43 .

Molecular docking simulation
Based on virtual screening results, garcinol from G. indica showed a higher (most negative) binding affinity of -8.5 kcal/mol with 12 non-bonded interactions, out of which 3 were found to be hydrogen bonds (Table 1). However, kaempferol from V. macrocarpon showed the binding affinity of -7.9 kcal/mol with 7 nonbonded interactions and 3 of them were found to be hydrogen bonds ( Table 2). Visualization of binding interactions garcinol and kaempferol with FimH protein has been given in Figure 1, whereas the details of binding residues along with their respective distances have been depicted in Table 3. docking is having a great importance and application as the study is directed towards computer aided drug designing. Protein ligand interaction study is increasing possibilities of discovering new drugs with greater accuracy. Considering the antimicrobial properties and antiadhesive property of proanthocyanidins, flavonoids, anthocyanins and benzophenones, the present study compares the inhibitory binding interactions of proanthocyanidins and flavonols from cranberry with anthocyanins, benzophenones and other potential secondary metabolites of G. indica while binding with the protein FimH 33 . For selected compounds, molecular dynamics simulation was also done to The virtual screening helps to identify the potential drug candidates from diverse nature of sources 34,35 .

Molecular docking simulation
AutoDock Vina 1.1.2, a command-based software was employed for the docking studies 36,37 . The pre-preparation of protein and ligand required for docking study was conducted based on authors previous work 38 . By using BIOVIA Discovery

Molecular dynamics simulation
The molecular dynamics simulation studies were employed for validation of the docked structure of garcinol bound FimH complex and kaempferol bound FimH complex. The result was analysed by plotting the graphs for protein-ligand complex RMSD, RMSF, Rg, SASA, and ligand hydrogen bonds (Figure 2). Based on the RMSD Further, based on the Rg plot and SASA analysis shows the similar pattern, thus it can be said that the complexes are stable. Finally, based on the hydrogen bond evaluation, structural re-agreement in protein-ligand complex could be expected, as the number of hydrogen bonds is increased compared to docked structure. Binding free energy (MMPBSA) calculations Free-binding energy was calculated based on the MM-PBSA method. The predicted values of the calculation along with the energy being formed during the last 50 ns are given in Table 4. Since more negative energy reflects more stability, from the obtained results it can be predicted that proteingarcinol complex shows better stability compared to protein-kaempferol complex.
The study reveals that among the compounds taken for the study kaempferol from cranberry and garcinol from kokum have shown better inhibitory binding action against FimH. Kaempferol derivatives of cranberry are antibiofilm agents that inhibit colonization of microbes 44,45 . Kaempferol is one of the bioactive substances found in cranberry extract that has the ability to function as an anti-adhesin and prevent the development of biofilm by interfering with the anchoring of microbial cells. Despite not being a biocidal agent, kaempferol's antiadhesion activity can stop microbial development 46 . Garcinol demonstrated a higher affinity to bind FimH than the other benzophenones and anthocyanins of kokum included in the study. When the antiadhesive properties of kaempferol and garcinol were evaluated, the results revealed that garcinol's potential to be an anti-adhesin is equal to that of kaempferol. Garcinol can considerably inhibit FimH and can be prospected for drug discovery against UTI. So further studies are required to check for synergism of secondary metabolites with antibiotics and confirm the action.
Urinary Tract Infection is becoming more prevalent among various age groups and the risk of recurrent infection is increasing due to the resistance of pathogens to various antibiotics. Females and diabetic patients are more susceptible to infection (Yu and Pieper, 2019; Sekhar et al., 2020). There are several studies being carried out to develop drug against UTI without altering the host immunity. Being an antiadhesive agent, cranberry extract is coming out to be effective for treating UTI. The fruit comes from a native American plant that grows in wetlands 48 and currently it is being used as a herbal drug against UTI. 49 G. indica is considered as an underutilized fruit and it is being used traditionally 50 . According to the findings of this study, G. indica can be used as an antiadhesive agent and may be investigated for therapeutic development against UTI because Garcinol has shown to have the same affinity for FimH as Kaempferol.
Further studies have to confirm the same as cranberry has shown synergistic effect against uropathogens in combination with few natural borne-antimicrobials 51 . Because of its health benefits, garcinol is a metabolite of interest that is being examined for drug development 52,53 . More research into this area could turn an underutilized fruit into a local bio resource.

COnCLUSIOn
Cranberry and kokum are potent plants to treat uropathogens as they contain secondary metabolites which are having antiadhesive property. Inhibition of the protein FimH of uropathogenic E. coli by the bioactive compound can be considered for drug discovery as alternative to the antibiotic treatment. The present study has indicated that both garcinol and kaempferol from kokum and cranberry, respectively could act as the potential FimH inhibitors (anti-adhesins). The compounds have been reported with extensive binding efficiency and stability. With these outcomes, in silico investigations prove that both the compounds could be taken for biological assays. Therefore, in vitro and in vivo studies can be used to validate on the mode of action of these anti-adhesins.