Mechanistic Evaluation of Lipopolysaccharide–Alexidine Interaction Using Spectroscopic and in Silico Approaches

Abstract

The increasing problem of multidrug resistance (MDR) in bacteria calls for discovery of new molecules and diagnostic methodologies that are effective against a wide range of microbial pathogens. We have studied the role of alexidine dihydrochloride (alex) as a bioaffinity ligand against lipopolysaccharide (LPS), a pathogen-associated surface marker universally present on all Gram-negative bacteria. While the activity of alex against bacteria is biologically known, little information exists on its mechanism of action or binding stoichiometry. We have used nuclear magnetic resonance (NMR), fluorescence, and surface plasmon resonance (SPR) spectroscopies to probe the binding characteristics of alex and LPS molecules. Our results indicate that LPS:alex stoichiometry lies between 1:2 and 1:4 and has a dissociation constant ( K) of 38 μM that is mediated through electrostatic interactions between the negatively charged phosphate groups present on LPS and the positively charged guanidinium groups present in alex. Further, molecular dynamics (MD) simulations performed to determine the conformational interaction between the two molecules show good agreement with the experimental results, which substantiate the potential of alex molecule for LPS neutralization and hence, development of efficient in vitro diagnostic assays.

Fighting Against Bacterial Lipopolysaccharide-Caused Infections through Molecular Dynamics Simulations: A Review

“…Therefore, the development of strategies to diminish the exaggerated and detrimental LPS-induced immune response is of outstanding importance. 21 , 22 , 24 , 79 , 81 , 82 , 86 , 87 , 90 , 91 In this regard, several MD studies have focused on gaining an improved understanding about key steps in the TLR4 activation by LPS. Moreover, the rational design of TLR4–MD2 antagonists that inhibit TLR4 signaling by competing with LPS in the binding to MD2, as illustrated in Figure 4 , has evolved into a hot research topic.…”

“…Therefore, LPS is understood as a target molecule of outstanding interest. 20 , 91 , 94 − 97 These tactics for fighting against LPS-caused infections have been schematized in Figure 5 , and MD studies that address such investigations have been reviewed in Table 3 .…”

“…Particularly, MD simulations were carried out to gain molecular insights and investigate the dynamics of the interaction of (+)-naltrexone, its derivates, and lipid A with MD2; moreover, the computationally calculated binding free energies were in agreement with their TLR4 antagonistic activities and binding affinities determined experimentally. On the other hand, Jagtap et al 91 combined several experimental techniques and MD simulations to explore the mechanism of interaction between alex and E. coli LPS. Thereby, they experimentally determined the binding stoichiometry, the binding sites, and the thermodynamic and kinetic binding and dissociation constants, whereas MD simulations were carried out to support experimental data and to gain insights into the conformational interaction between these molecules.…”

A Single Step in vitro Bioassay Mimicking TLR4-LPS Pathway and the Role of MD2 and CD14 Coreceptors

“…Four ligands were chosen for this purpose. Alexidine and sushi peptide were selected as positive controls due to their specific affinity toward LPS as shown in our recent studies (3,4). Vancomycin and bacitracin were used as negative controls for their lack of reported interaction with LPS (41,42).…”

“…Among the TLRs, TLR4 is a type I integral membrane glycoprotein that is specifically produced against Gram-negative bacterial endotoxins lipopolysaccharides (LPS). These LPS molecules exist in million copies per cell and are released into the bloodstream, in the form of random clusters or outer membrane vesicles (OMVs), as the cells multiply or die in response to antibiotic treatment (3,4). It is now well-established that activation of TLR4 is one of the two innate immune pathways triggered during Gram-negative bacterial sepsis in immunocompromised patients (the other one is caspase 11 activation in macrophages) (5,6).…”