Identification of anti-pathogenic activity among in silico predicted small-molecule inhibitors of Pseudomonas aeruginosa LasR or nitric oxide reductase (NOR)

Introduction

Antimicrobial resistance (AMR) among infectious bacteria has emerged as a healthcare challenge of global concern (Online). The World Health Organization (WHO) has also published a global action plan on AMR in 2015. As per the Indian National Action Plan on Antimicrobial Resistance (NAP-AMR: 2017-2021), India is among the nations with the highest burden of bacterial infections. The crude mortality from infectious diseases in India today is 417 per 100,000 persons. The situation in other developing countries is equally grave. Murray et al estimated 1.27 million deaths attributable to bacterial AMR in 2019 (1). Of the six leading pathogens identified by them responsible for maximum death toll, one is Pseudomonas aeruginosa. It is among the most notorious pathogenic bacteria, and its carbapenem-resistant phenotype has been listed by the WHO among priority pathogens for the development of new antibiotics (Online). Antibiotic-resistant P. aeruginosa has been listed as an important pathogen by the U.S. Centers for Disease Control and Prevention (CDC) (Online) as well as the Department of Biotechnology, India (DBT) (Online) against which new antimicrobials are urgently required.

The pipeline for new antibiotics does not contain sufficient number of promising candidates (2). There is a dearth of novel antibacterial leads as well as targets (3,4). Identification and validation of new targets in important pathogens is of utmost significance (5,6). Conventional bactericidal antibiotics attack a very narrow range of targets in susceptible bacteria, for example, cell wall synthesis, protein synthesis, nucleic acid synthesis, etc. Since the past decade, there has been much interest in the research community regarding discovery of anti-virulence molecules, which may attenuate the bacterial virulence without necessarily killing them. Such ‘pathoblockers’ are expected to compromise the ability of the pathogen to damage the host, by exerting their effect on nonessential targets such as bacterial quorum sensing (QS) (7), stress-response machinery (8), metal homeostasis (9,10), etc. The next-generation antibacterials preferably should be attacking such hitherto unexplored/underexplored targets in pathogenic bacteria (11-13).

Proper functioning of the chemical signal-based intercellular communication, known as QS, is crucial for sufficient expression of virulence in pathogens like P. aeruginosa, as QS is an effective mechanism for regulating expression of multiple genes associated with a multitude of functions (14). Interrupting bacterial QS can be an effective strategy to combat pathogens (15,16). QS consists of two components: signal generation and signal response, respectively, encoded by LuxI and LuxR homologues (17,18). Inhibiting the ‘signal response’ component of QS (e.g. LasR in P. aeruginosa) can notably compromise their ability to exert collective behaviour in response to environmental changes or host defence. P. aeruginosa regulates its drug resistance and pathogenicity through multiple QS mechanisms including the LasI/R, RhlI/R, and PQS/MvfR systems. Targeting one or more of these QS systems may prove an effective way of dealing with P. aeruginosa infections (19). Owing to the important role of Las system in overall QS circuit of P. aeruginosa, its receptor protein LasR is believed to be a plausible anti-virulence target (20). LasR is a transcriptional activator of various virulence-associated genes in P. aeruginosa, which recognizes a specific signal molecule, namely N-(3-oxo-dodecanoyl)-L-homoserine lactone (3O-C12-HSL) (21). The LasR-3O-C12-HSL complex triggers the expression of multiple QS-regulated genes. Potential LasR inhibitors either may prevent its binding with its natural ligand or compromise its ability to affect expression of target genes (22). QS not being essential for bacterial survival, its inhibitors are expected to exert lesser selection pressure on bacterial population with respect to development of resistant phenotypes (23). Additionally, due to the overlap in QS systems among various gram-negative bacteria (24-25), inhibitors effective against one gram-negative species may have broad-spectrum activity against multiple other gram-negative pathogens.

Pathogens striving to survive inside a host body are forced to face a variety of stresses such as iron deprivation, oxidative stress, nitrosative stress, etc. Bacteria employ antioxidant enzymes to counter reactive oxygen species, and similarly certain other enzymes to counter reactive nitrogen species (26). From the work done in our lab as well as literature survey, we consider the components of P. aeruginosa genome involved in responding to nitrosative stress to be potential targets. Among the components of nitrosative stress response in P. aeruginosa, one important enzyme is nitric oxide reductase (NOR). This protein was one of the major targets of an anti-infective polyherbal formulation (Panchvalkal) investigated by us in the recent past (6,27). NOR also emerged among the top differentially expressed genes in P. aeruginosa treated by us with other anti-virulence polyherbal formulations Enteropan (SRX15248092) or colloidal silver (SRX14392191) at sub-lethal levels.

NOR is an important detoxifying enzyme in P. aeruginosa, which is crucial to its ability to withstand nitrosative stress (e.g. in the form of nitric oxide [NO]). NOR has also been reported (28,29) to be important for virulence expression of this pathogen, and thus can be a plausible target for novel anti-virulence agents. Molecules capable of inhibiting NOR can be expected to compromise the pathogen’s ability to detoxify NO, not allowing its virulence traits (e.g. biofilm formation, as NO has been indicated to act as a biofilm-dispersal signal) to be expressed fully (30). The test molecules capable of inhibiting NOR may emerge as novel anti-biofilm agents not only against P. aeruginosa but against multiple pathogens as NO is reported to be perceived as a dispersal signal by various gram-negative and gram-positive bacteria (31). Thus, NOR inhibitors may be expected to have a broad-spectrum activity against multiple pathogens. Major function of NOR is to detoxify NO generated by nitrite reductase (NIR). NO is a toxic by-product of anaerobic respiration in P. aeruginosa. NO-derived nitrosative species can damage deoxyribonucleic acid (DNA) and compromise protein function. Intracellular accumulation of NO is likely to be lethal for the pathogen (32). It can be logically anticipated that P. aeruginosa’s ability to detoxify NO will be compromised under the influence of a potent NOR inhibitor. Since NO seems to have a broad-spectrum anti-biofilm effect, NOR activity is essential for effective biofilm formation by the pathogens. NOR activity and NO concentration can modulate cellular levels of cyclic di-GMP, which is a secondary messenger molecule recognized as a key bacterial regulator of multiple processes such as virulence, differentiation, and biofilm formation (33). In mammalian pathogens, the host’s macrophages are a likely source of NO. NOR expressed by the pathogen provides protection against the host defence mechanism. Since NOR activity is known to be important in multiple pathogenic bacteria (e.g. P. aeruginosa, Staphylococcus aureus, Serratia marcescens) for biofilm formation, virulence expression, combating nitrosative stress, and evading hose defence, NOR seems to be an important target for novel anti-pathogenic agents. Any molecule capable of interfering with bacterial NOR activity is likely to be an effective anti-pathogenic agent, since bacterial populations require NOR for various purposes including detoxification and evasion of host defences (34). A potential NOR inhibitor besides troubling the pathogen directly may also boost its clearance by the host macrophages.

Proteins such as LasR and NOR important to the pathogens and whose structure is known can be useful starting point for a drug discovery programme. In silico virtual screening tools can be used to screen large chemical libraries to predict inhibitors of the target proteins. In the recent past, quite a few potent anti-pathogenic compounds have been identified using this approach. For example, one such virtual screening study by Abelyan et al (35) identified benzamides, synthetic derivatives of flavones, as potential inhibitors of LasR. Another in silico effort by Narayanaswamy et al (36) identified potent inhibitors of enzymes involved in nitrogen metabolism in various bacteria including P. aeruginosa, nitrous oxide reductase (N₂OR), and NIR, from among a library of synthetic and natural compounds.

This study aimed at screening 96 compounds identified through a virtual screen as potential LasR inhibitors, and 85 compounds predicted to be NOR inhibitors in silico for their possible anti-virulence activity against P. aeruginosa in the model host Caenorhabditis elegans. Any such potent NOR or LasR inhibitors identified through this study may prove to be useful lead(s) for novel anti-pathogenic drug development. They can be useful either as standalone therapy or in combination with conventional antibiotics.

Methods

Screening for NOR binding ligands

The structure of P. aeruginosa NOR bound to an antibody fragment has been determined by crystallography (PDBID 3O0R). This structure reveals NOR composed of two subunits: NORB containing 12 transmembrane helices and NORC with a transmembrane helix and hydrophilic periplasmic domain. Three haem cofactors are complexed in NOR. Two haems are deep in the NORB transmembrane region, and the third haem spans the NORB-NORC interface. No drug-like small-molecule inhibitors have been reported for NOR, and no druggable pocket is obvious from the reported structure. To identify potential targetable sites, the structure of NOR was analysed by fPocket. This analysis revealed a pocket at the interface of NORB and NORC and near one of the NORB haem cofactors. This positioning suggests that small-molecule binding to this pocket may disrupt norB-norC interactions and/or inhibit haem-mediated electrochemistry. The virtual screen therefore sought to identify small molecules that bind to this pocket in NOR and potentially disrupt enzymatic function. A library of approximately 3 million compounds was screened for identifying compounds capable of virtual binding at the selected target site on NOR (Fig. 2) using AtomNet^®. Top scoring compounds were clustered and filtered to arrive at a final subset of 85 deliverable compounds.

Test compounds

Ninety-six compounds showing in silico affinity to LasR (Supplementary table S1, List of predicted LasR inhibitors subjected to wet-lab assay) and 85 compounds showing in silico affinity to NOR (Supplementary table S2, List of predicted NOR inhibitors subjected to wet-lab assay) were ordered in physical form from Enamine (Kyiv, Ukraine) and Mcule (Budapest, Hungary), respectively. Test compounds were stored in the refrigerator and reconstituted in dimethyl sulfoxide (DMSO; 500-1000 µL) (Merck) on the day of assay.

Bacterial strain

The P. aeruginosa strain used in this study was sourced from our internal culture collection, which has been characterized by us for its antibiotic susceptibility/resistance, pigment production, and certain other virulence traits. Its antibiogram (Online) generated through a disc-diffusion assay performed as per the National Committee for Clinical Laboratory Standards (NCCLS) guidelines revealed it to be resistant to eight antibiotics (cotrimoxazole, augmentin, nitrofurantoin, ampicillin, chloramphenicol, clindamycin, cefixime, and vancomycin) belonging to five different classes. Hence it can be described as a multidrug-resistant (MDR) strain. As reported in our earlier publications (27,38) with this strain, it is a haemolytic strain capable of producing the QS-regulated pigments (pyocyanin and pyoverdine), and also of biofilm formation. We maintained this bacterium on Pseudomonas agar (HiMedia). While culturing the bacteria for in vivo assay, they were grown in Pseudomonas broth (magnesium chloride 1.4 g/L, potassium sulphate 10 g/L, peptic digest of animal tissue 20 g/L, pH 7.0 ± 0.2).

Nematode host

C. elegans (N2 Bristol) was used as the model host in this study. Worms were maintained on nematode growth medium (NGM): 3 g/L NaCl (HiMedia, MB023-500G), 2.5 g/L peptone (HiMedia), 17 g/L agar-agar (HiMedia), 1 M CaCl₂ (HiMedia), 1 M MgSO₄ (Merck), 5 mg/mL cholesterol (HiMedia), 1 M phosphate buffer of pH 6, agar plate with Escherichia coli OP50 (LabTIE B.V., the Netherlands) as food. For synchronization of the worm population, adult worms from a 4- to 5-day-old NGM plate were first washed with distilled water, and then treated with 1 mL of bleaching solution (water + 4% NaOCl [Merck] +1 N NaOH [HiMedia] in 3:1:1 proportion), followed by centrifugation (1,500 rpm at 22°C) for 1 min. Eggs in the resultant pellet were washed multiple times with sterile distilled water, followed by transfer onto a new NGM plate seeded with E. coli OP50. L3-L4 stage worms appearing on this plate after 2-3 days of incubation at 22°C were used for further experimentation.

Virulence assay

P. aeruginosa grown in Pseudomonas broth (at 35±1°C for 21 hours with intermittent shaking) was allowed to attack C. elegans (L3-L4 stage) in a 24-well plate (HiMedia) in the presence or absence of test compounds, and their capacity to kill the worm population was monitored over a period of 5 days. In each well, there were 10 worms in M9 buffer (3 g/L KH₂PO₄, 6 g/L Na₂HPO₄, 5 g/L NaCl), which were challenged with P. aeruginosa by adding 100 µL (OD₇₆₄ = 1.30) of bacterial culture grown in Pseudomonas broth. Appropriate controls, that is, worms, exposed neither to test compound nor to bacteria; worms exposed to test compound, but not to bacterial pathogens (toxicity control); worms challenged with bacteria in the presence of 0.5% v/v DMSO (vehicle control); and worms challenged with bacteria in the presence of 0.5 µg/mL ofloxacin (positive control) were also included in the experiment. Incubation was carried out at 22°C. The number of dead vs. live worms was counted every day for 5 days by putting the plate with lid under a light microscope 4× objective. Straight non-moving worms were considered to be dead. Plates were gently tapped to confirm lack of movement in the apparently dead worms. On the last day of the experiment, when plates could be opened, their death was also confirmed by prodding them with a straight wire, wherein no movement was taken as confirmation of death.

Statistics

Results reported are means of three replicates. Statistical significance was assessed through a t-test performed using Microsoft Excel. Values of p≤0.05 were considered to be significant.

Results

Conclusion

This study is a preliminary demonstration of the utility of virtual screening approach for discovery of potentially novel anti-pathogenic agents. Virtual screening can reduce the number of compounds required to be actually subjected to wet-lab assays, and thus reducing the investment of labour, time, and money. Among the top 181 compounds predicted through virtual screening to be capable of binding to NOR or LasR of P. aeruginosa, we could detect in vivo anti-P. aeruginosa activity in 19 (i.e. 10.4% of all compounds tested) of them in the model host C. elegans. As per our search on PubChem on 1 June 2023, these 19 compounds have yet not been reported to possess any kind of biological activity, and hence we believe this to be the first report of anti-pathogenic activity in these compounds. Further investigation on these active compounds with respect to their mode of action is warranted, which besides confirming their antibacterial activity will also provide additional validation to the targetability of NOR and LasR.

Limitations

The anti-virulence assay performed in this study is not specific to LasR or NOR, hence precise mode of action of active anti-pathogenic compounds warrants further confirmatory assays. We could not carry out in vitro MIC/MBC assay for active compounds owing to limited quantity at our disposal, hence it was not possible to distinguish between growth-inhibitory and virulence-inhibitory (i.e. non-antibiotic action) activity. Further, anti-pathogenic activity of some of the compounds might be masked by their anthelmintic activity (Tabs. II-IV).

Acknowledgements

Authors thank Nirma Education and Research Foundation (NERF), Ahmedabad, for infrastructural support. GG acknowledges scholarship from the Government of Gujarat through their SHODH scheme.

Disclosures

Conflict of interest: The authors declare no conflict of interest.

Financial support: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Authors contribution: GG: Carried out experiments; participated in manuscript preparation; literature survey; NH and BC: Executed in silico part of the study; arranged procurement of test compounds in physical form; VK: Conceptualization, data analysis, and manuscript preparation