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Fluorescent chemical probes are used nowadays as a chemical resource to study the physiology and pharmacology of several important endogenous receptors. Different fluorescent groups have been coupled with known ligands of these receptors, allowing the visualization of their localization and trafficking. One of the most important molecular players of innate immunity and inflammation are the Toll-Like Receptors (TLRs). These Pattern-Recognition Receptors (PRR) have as natural ligands microbial-derived pathogen-associated molecular patterns (PAMPs) and also endogenous molecules called danger-associated molecular patterns (DAMPs). These ligands activate TLRs to start a response that will determine the host's protection and overall cell survival but can also lead to chronic inflammation and autoimmune syndromes. TLRs action is tightly related to their subcellular localization and trafficking. Understanding this trafficking phenomenon can enlighten critical molecular pathways that might allow to decipher the causes of different diseases. In this chapter, the study of function, localization and trafficking of TLRs through the use of chemical probes will be discussed. Furthermore, an example protocol of the use of fluorescent chemical probes to study TLR4 trafficking using high-content analysis will be described.

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Mitochondria play important roles in diverse cellular processes such as energy production, cellular metabolism, and apoptosis to promote cell death. To investigate mitochondria-associated biological processes such as structure, dynamics, morphological change, metabolism, and mitophagy, there exists a continuous demand for visualizing and monitoring techniques elucidating mitochondrial biology and disease-relevancy. Due to the advantages of high sensitivity and practicality, fluorescence phenomena have been most widely used as scientific techniques for the visualization of biological phenomena and systems. In this review, we briefly overview the different types of fluorescent materials such as chemical probes, peptide- or protein-based probes, and nanomaterials for monitoring mitochondrial biology.

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Intravital microscopy with multiphoton excitation is a recently developed optical imaging technique for deep tissue imaging without fixation or sectioning, which permits examination of fundamental concepts regarding the dynamic nature of cells under physiological and pathological conditions in living animals. This novel technique also offers exciting opportunities for pharmacological research by providing new platforms for the study of cellular dynamics in response to drugs in vivo. Moreover, fluorescent chemical probes for functional or molecular analysis in single cells in vivo play important roles in pharmacology. For example, we have recently revealed the pharmacodynamic actions of different biological agents for the treatment of rheumatoid arthritis (RA) in vivo by directly visualizing drug-induced cellular behaviors and functions of osteoclasts on bone surfaces. This review focuses on the principles and advantages of intravital imaging for the dissection of pharmacological mechanisms, and discusses how such imaging can contribute to the drug development process, introducing recent trials that evaluated the in vivo pharmacological effects of various agents.

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The cationic glycolipid IAXO-102, a potent TLR4 antagonist targeting both MD-2 and CD14 co-receptors, has been used as scaffold to design new potential TLR4 modulators and fluorescent labels for the TLR4 receptor complex (membrane TLR4.MD-2 dimer and CD14). The primary amino group of IAXO-102, not involved in direct interaction with MD-2 and CD14 receptors, has been exploited to covalently attach a fluorescein (molecules 1 and 2) or to link two molecules of IAXO-102 through diamine and diammonium spacers, obtaining 'dimeric' molecules 3 and 4. The structure-based rational design of compounds 1-4 was guided by the optimization of MD-2 and CD14 binding. Compounds 1 and 2 inhibited TLR4 activation, in a concentration-dependent manner, and signaling in HEK-Blue TLR4 cells. The fluorescent labeling of murine macrophages by molecule 1 was inhibited by LPS and was also abrogated when cell surface proteins were digested by trypsin, thus suggesting an interaction of fluorescent probe 1 with membrane proteins of the TLR4 receptor system.

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