Postulates for validating TLR4 agonists

Abstract

TLRs play a central role in the innate immune response, recognizing a variety of molecular structures characteristic of pathogens. Although TLR4, together with its co-receptor myeloid differentiation-2 (MD-2), recognize bacterial LPS and therefore Gram-negative bacterial infections, it also plays a key role in many other pathophysiological processes, including sterile inflammation and viral infection. Specifically, numerous endogenous agonists of TLR4 of notably diverse nature, ranging from proteins to metal ions, have been reported. Direct activation of a single receptor by such a range of molecular signals is very difficult to explain from a structural and mechanistic point of view. It is likely that only a subset of these directly activate the TLR4-MD-2 complex. We propose three postulates aimed at distinguishing the direct agonists of TLR4 from indirect activators. These postulates are as follows: (i) that the agonist requires the TLR4/MD-2 receptor complex; (ii) that agonist formed synthetically or in situ must activate the receptor complex in order to eliminate artifacts of contamination by other agonists; and (iii) that a specific molecular interaction between the agonist and TLR4/MD-2 must be identified. The same type of postulates can be applied to pattern recognition receptors in general.Keywords: Ligand recognition r Molecular mechanism r TLR4 agonists Molecular understanding of immunological processesAt a fundamental level, we only understand a physiological process once we know in detail the underlying molecular interactions that drive the process. This makes knowledge of the 3D structures of signaling complexes and the molecular mechanisms governing self and nonself-recognition, important in immunology. To resolve interactions at the molecular scale, high-resolution structural analysis, e.g. X-ray crystallography or nuclear magnetic resonance (NMR) can be combined with biochemical and biophysical methods, such as mutagenesis of specific protein residues, guided by molecular modeling, electron microscopy, dynamic light scattering, surface plasmon resonance, and spectroscopy. Together, these provide quantitative information and are able to clarify often Correspondence: Prof. Roman Jerala e-mail: roman.jerala@ki.si conflicting physiological findings, as it may be difficult to replace the widely accepted but incorrect paradigms.An understanding of the physiology of immunological processes often precedes the elucidation of the underlying molecular mechanisms; this is the case for TLR4, the pattern recognition receptor for LPS (endotoxin). While the mouse strains hypo-responsive to LPS have been known for a long time, searches initially identified LPS-binding protein (LBP) and CD14 as its binding partners [1, 2] and were later recognized as the extracellular components essential for coordinating a highly sensitive response to LPS [2, 3]. Actually, the identification of human TLR1 [4] preceded the implication of human TLR4 in innate immunity [5]. The subsequent identifications of human TLR2 to TLR5, as...

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