Abstract
Toll-like receptors (TLRs) are transmembrane proteins that are key regulators of innate and adaptive immune responses, particularly TLR4, and they have been identified as potential drug targets for the treatment of disease. Several low-molecular-weight compounds are being considered as new drug targets for various applications, including as immune modulators. Mygalin, a 417 Da synthetic bis-acylpolyamine, is an analog of spermidine that has microbicidal activity. In this study, we investigated the effect of mygalin on the innate immune response based on a virtual screening (VS) and molecular docking analysis. Bone marrow-derived macrophages and the cell lines J774A.1 and RAW 264.7 stimulated with lipopolysaccharide (LPS) were used to confirm the data obtained in silico. Virtual screening and molecular docking suggested that mygalin binds to TLR4 via the protein myeloid differentiation factor 2 (MD-2) and LPS. Macrophages stimulated by mygalin plus LPS showed suppressed gene expression of tumor necrosis factor (TNF-α), interleukine 6 (IL-6), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as inhibition of signaling protein p65 of the nuclear factor κB (NF-κB), resulting in decreased production of nitric oxide (NO) and TNF-α. These results indicate that mygalin has anti-inflammatory potential, being an attractive option to be explored. In addition, we reinforce the importance of virtual screening analysis to assist in the discovery of new drugs.
Highlights
The main receptors involved in innate immunity to infections are the Toll-like receptors (TLRs), which are expressed on the cell surface (TLR 1, 2, 4, 5, 6, and 10) or on endosomes (TLR 3, 7, 8, and 9) [1]
The recognition of LPS by the TLR4 receptor system is accompanied by a series of sequential steps in which the LPS is linked by different LPS-binding proteins and transferred to myeloid differentiation factor 2 (MD-2)/TLR4, dimerization occurs in the cell membrane, initiating the signaling cascade that leads to the production of pro-inflammatory cytokines and interferons [6]
Mygalin was evaluated in the virtual repertoire of small molecules against immune receptors ImmtorLig_DB, in which the program database counts human immune receptors such as Toll-like receptors (TLR1/TLR2, TLR4/MD-2, TLR2/TLR6) and mincle; MHC-I and MHC-II; costimulatory molecules CD28, CD40, CD80, and CD86; coinhibitory molecules CTLA-4, PD-L1, Tim-3, decoy receptor, Fas ligand, and Fas receptor; cytokines IL-1β, IL-2, IL-4, interleukine 6 (IL-6), IL-17, and IL-23; and cell adhesion molecules ICAM, VCAM, CEACAM1
Summary
The main receptors involved in innate immunity to infections are the Toll-like receptors (TLRs), which are expressed on the cell surface (TLR 1, 2, 4, 5, 6, and 10) or on endosomes (TLR 3, 7, 8, and 9) [1]. TLRs are transmembrane proteins that constitute the first line of defense, and they recognize molecular patterns associated with pathogens (PAMPs) that are expressed by infectious agents such as zymosan, peptidoglycan, lipopolysaccharide (LPS), flagellin, and CpG DNA, and mediate the development of an efficient innate immune response system and inflammatory response [2]. These proteins are key molecules in the inflammatory process, and they can be suppressed with specific drugs, antibodies, or inhibitors to treat diseases, such as cancer, sepsis, and asthma; TLRs are considered a promising future therapeutic strategy for various infectious inflammatory and autoimmune diseases [3,4]. The recognition of LPS by the TLR4 receptor system is accompanied by a series of sequential steps in which the LPS is linked by different LPS-binding proteins and transferred to MD-2/TLR4, dimerization occurs in the cell membrane, initiating the signaling cascade that leads to the production of pro-inflammatory cytokines and interferons [6]
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