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Identification of TLR2 modulators and exploration of possible binding modes

초록/요약

Toll-like receptor 2 (TLR2) antagonists are key therapeutic targets because they inhibit several inflammatory diseases caused by surplus TLR2 activation. We identified two novel TLR2 antagonists, C11 and C13, through pharmacophore-based virtual screening. At 10 µM, C13 inhibited almost 67%, and C11 inhibited more than 44%, of Interleukin-8 production in human embryonic kidney TLR2 overexpressing cells. Moreover, these two compounds bind directly to the human recombinant TLR2 ectodomain during surface plasmon resonance analysis, and did not affect the cell viability in MTT assay. In total, two virtually screened molecules, C11 and C13, were experimentally proven to be effective, and thus, will provide new understanding into the structure of TLR2 antagonists and help for the development of TLR2 drug molecules. Plasmodium falciparum, a protozoan pathogen causes malaria via glycosylphosphatidylinositols (GPIs). GPIs activate TLR2 signaling to protect humans via the induction of proinflammatory cytokines. Biological studies provided us the evidence of TLR2-GPIs interactions. However, the binding modes of the molecules that is key to therapeutics were unknown. To unravel this, we used computational approaches such as computational docking, MD simulations and essential dynamics. We identified the GPIs-induced conformational changes in the ligand binding region and C-term of TLR2 subfamily members, and based on the results we proposed that GPIs binding are similar to already known synthetic ligands and the acyl chains in GPIs may mainly involve in dimerization of TLR2.

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목차

TOLL-LIKE RECEPTOR 2 1
 Historical Background 1
 Structure of TLR2 2
 Ligand Recognition by TLR2 3
 MyD88-dependent TLR2 Signaling 6
 Role of TLR2 in Diseases 8

TOLL-LIKE RECEPTOR 2 ANTAGONISTS IDENTIFIED THROUGH VIRTUAL SCREENING AND EXPERIMENTAL VALIDATION 10
 Introduction 10
 Results 12
 Discussion 27

RESULTS (EXTENDED ABSTRACTS OF MANUSCRIPTS) 29
Structure and dynamic behavior of Toll-like receptor 2 subfamily triggered by malarial glycosylphosphatidylinositols of Plasmodium falciparum 29

MATERIALS AND METHODS 30
Computational Mutation Scanning and Pharmacophore Modeling 30
Pharmacophore-based Database Search and Isolation of Drug-like Molecules 31
Structure Preparation and Molecular Docking 32
MD Simulations, Binding Free Energy Calculations and Principal component analysis 33
Surface Plasmon Resonance Analysis 34
Cell Culture and Treatments 35
Western Blot 36
Interleukin-8 Secretion Assay 47
Cell Viability Assay 37
Statistical Analysis 38

FUTURE WORKS 38

REFERENCES 39

APPENDIX 45
Structure and dynamic behavior of Toll-like receptor 2 subfamily triggered by malarial glycosylphosphatidylinositols of Plasmodium falciparum 45

SUPPORTING INFORMATION 63

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