Exploring the action of new FimH inhibitors against CTX– 15 enzyme by enzoinformatics approach: A plausible arsenal against drug-resistant uropathogenic bacterial strains

Purpose: To explore the potency of FimH inhibitors against CTX-M β-lactamase enzyme type 15, in view of the increasing prevalence of CTX-M 15 in uropathogenic strains which has reduced the treatment options to minimal. Method: FimH inhibitors were targeted against CTXM-15 by a molecular docking approach. Thereafter, the best ligand-target confirmation was selected and analyzed using LIGPLOT+ Version v.2.1. The hydrophobic and hydrogen bonding among the catalytic site amino acids of CTXM-15 and the FimH inhibitors were analyzed and 3-D structures were converted into 2-D images by LIGPLOT algorithm. Results: Out of all the FimH inhibitors tested, 3′-chloro-4′(α-D-mannopyranosyloxy) biphenyl-4carbonitrile, para-biphenyl-2-methyl-3′-methylamidemannoside, para-biphenyl-2-methyl-3′,5′dimethylamide-α-D-mannoside, and thiazolylamino mannoside exhibited better interaction with the CTX-M 15 active site than the positive control avibactam. Moreover, in CTX-M 15, the amino acid residues, Ser70, Tyr105, Ser130, Asn132, Thr216, Thr235, Gly236, and Ser237 were commonly interacting with these FimH inhibitors as well as avibactam. Conclusion: The predicted findings suggest that these FimH inhibitors could be explored as potential CTX-M 15 inhibitors to cope-up with resistance issues of uropathogenic bacteria in the form of an alternate strategy.


INTRODUCTION
Globally, around 150 million individuals suffer from urinary tract infections (UTI) each year, and its occurrence is neither gender-specific nor age-dependent [1,2]. The causal bacteria in most UTI cases belong to the Enterobacteriaceae family [3], and the prevalence of extended-spectrum β lactamases (ESBLs) in this family has hindered the treatment options [4][5][6]. ESBLs of the (Cefotaximase-Munich) CTX-M type, particularly CTX-M 15 enzyme, has become more common in clinical samples [5,7]. Thus, CTX-M 15 enzyme was selected as a target for performing the docking analysis in the present study.
On the other hand, in any infection process, adherence of bacteria to the mucosal surface of the host is usually the first crucial step, and this statement holds true in the case of UTI caused by Enterobacteriaceae species [8]. The uropathogenic strains of the Enterobacteriaceae family have type-1 fimbriae as an adhesive organelle. The FimH protein Hung is present at the end of type-1 fimbriae that is utilized by the bacteria for host cell attachment [9,10]. Various studies have suggested that type-1 fimbriae or FimH play a crucial role in the UTI caused by Escherichia coli and Klebsiella pneumoniae [11][12][13][14]. Thus, the advantages of using FimH inhibitors over the currently used antibiotics are their specificity for some bacterial species' particular adherence process, no effect on host microflora, and, most importantly, no resistance issues as it interferes with a bacterial attachment without bactericidal action [9]. The majority of FimH inhibitors structures were formulated using x-ray crystallographic findings such as 8-(methoxycarbonyl)octyl-α-D-mannoside, heptyl α-D-mannopyranoside, para-biphenyl-2-methyl-3′,5′di-methylamide-α-D-mannoside, parabiphenyl-2-methyl-3ꞌ-methylamide mannoside, 3′-chloro-4′-(α-D-mannopyranosyloxy)biphenyl-4carbonitrile, and thiazolylamino mannoside [9,[15][16][17]. In the present study, all these FimH inhibitors were selected to target the CTX-M 15-type ESBLs by applying the enzoinformatics approach. The findings suggested that FimH inhibitors might also be used against uropathogenic CTX-M 15 (ESBLs)-producing resistant bacteria.
They serve two purposes: (1) Hinder the attachment of uropathogenic strains, and (2) Avoid resistance due to CTX-M 15. However, wet-lab experimental analysis is needed to establish the findings of the present study. Nonetheless, these FimH inhibitors could be explored further to transform the status of the antibiotic therapy regimen for UTI treatment.

FimH inhibitors, aviabactam and target protein structure retrieval
FimH inhibitors three-dimensional structure were obtained from the ꞌFimH-inhibitorsꞌ complex present in protein data bank. The FimH inhibitors used for the study were 8-

Physicochemical properties and toxicity potential prediction
The physicochemical properties and toxicity potential of FimH inhibitors and positive control were estimated by using the Osiris Datawarrior property explorer tool.
Initially, different physicochemical parameters such as no. of hydrogen bond acceptors and donors, cLogP value, molecular weight, topological polar surface area, number of rotatable bonds, and the Lipinski's rule violation [18] were calculated. Thereafter, absorption was estimated as in Eq 1 [19].
Absorption % = 109 -(0.345 × TPSA)………(1) Prediction of toxicity was also evaluated by the orisis datawarrior tool, in which predictions are based on comparative analysis of our tested compounds with the pre-estimated set of known structural molecules. Mutagenicity, tumorigeniccity, reproductive effects and irritability features of our tested compounds were predicted for toxicity assessment.

Molecular docking
ꞌFimH inhibitors and avibactamꞌ were considered as ligands and docked to target protein ꞌCTX-M 15ꞌ by following Rizvi et al protocol [20]. Each ligand energy was minimized by applying the MMFF94 force field, followed by gasteiger charges addition. Rotatable bonds were specified after adding non-polar hydrogen atoms. Kollman united atom type charges, solvation parameters, and hydrogen atoms were added using AutoDock 4.2. Autogrid was used to keep 60 x 60 x 60 Å as a grid dimension, with points separated by 0.375 Å. For explicitly targeting the ꞌCTX-M 15 catalytic siteꞌ, the values of x, y, and z coordinates were kept as 6.930, 14.060, 9.920. Electrostatic and Van der Waals parameters were estimated by applying dielectric functions and default parameters of AutoDock 4.2. 'Lamarckian and Solis and Wets localꞌ algorithm was used to perform molecular docking experiments. One hundred different runs were applied for each docking experiment that was further set to end after 2,500,000 energy evaluations, and population size was kept as 150. At the end, final AutoDock 4.2 figures were studied using Discovery Studio 2.5 (Accelrys).

LIGPLOT+ analysis of docked results
After performing the docking, the best ligandtarget confirmation was selected and analyzed using LIGPLOT+ Version v.2.1. The hydrophobic and hydrogen bonding among the catalytic site amino acids of CTXM-15 and the FimH inhibitors were analyzed and 3-D structures were converted into 2-D images by LIGPLOT algorithm.

DISCUSSION
Antibiotic resistance in uropathogenic bacteria due to ESBLs, especially CTX-M 15, has become a significant issue globally [4][5][6]. Thus, finding an alternative solution to this issue is a matter of great concern. Several studies have reported that uropathogenic bacteria belonging to Enterobacteriaceae family have been regarded as the primary causative agents for most UTI cases [3]. In addition, bacterial adherence to the host mucosal surface is a crucial step in any UTI case, and the FimH produced by the Enterobacteriaceae family pathogens helps them attach to the mannosylated glycoproteins present on the epithelial cell surface of the urinary tract [8][9][10]21,22]. Therefore, FimH inhibitors might play an essential role in coping with the issues of antibiotic resistance by targeting cell adhesion without exhibiting bactericidal action. However, in the present study, the new potential of these FimH inhibitors was evaluated by assessing their CTX-M 15 inhibition potency. The present study outcomes might provide an add-on boost to overthe-counter antibiotics available for UTI treatment, by targeting both cell adhesion and ESBLs (CTX-M 15).
Structure-activity relationship studies have shown the importance of Ser70 and Ser130 during the acylation process of CTX-M 15 by their inhibitors [7,24]. In the present work, two amino acid residues, Ser70 and Ser130, found in the CTX-M 15 active site commonly interacted with the FimH inhibitors. Thus, the FimH inhibitors tested in this study could be further explored as CTX-M 15 inhibitors as well. Further experimental validations are needed to ascertain the CTX-M 15 inhibition potential of FimH inhibitors and to convert them as dual inhibitors of FimH and CTX-M 15 enzyme. However, in silico findings have been reported to often correlate well with wet-lab outcomes. The preliminary findings of the present study suggest a plausible alternate strategy for combating the current resistance issues caused by CTX-M 15producing uropathogenic bacterial strains.

CONCLUSION
FimH inhibitors are currently under development for their use against adherence of uropathogenic bacterial strains to host cells. In the present study, FimH inhibitors were docked to CTX-M 15 enzyme to predict their dual targeting potential. Interestingly, they have shown better interaction with the catalytic site of CTX-M 15 than positive control. This would provide an add-on advantage to the ongoing research on FimH inhibitors, in order to develop them as alternative antibacterial therapy candidates. In addition, the findings of this study would help researchers to design more versatile and potent CTX-M 15 inhibitors to cope with the antibiotic resistance issues of uropathogenic bacteria more effectively.