Molecular Design of SERTlight: A Fluorescent Serotonin Probe for Neuronal Labeling in the Brain.

The serotonergic transmitter system plays fundamental roles in the nervous system in neurotransmission, synaptic plasticity, pathological processes, and therapeutic effects of antidepressants and psychedelics, as well as in the gastrointestinal and circulatory systems. We introduce a novel small molecule fluorescent agent, termed SERTlight, that specifically labels serotonergic neuronal cell bodies, dendrites, and axonal projections as a serotonin transporter (SERT) fluorescent substrate. SERTlight was developed by an iterative molecular design process, based on an aminoethyl-quinolone system, to integrate structural elements that impart SERT substrate activity, sufficient fluorescent brightness, and a broad absence of pharmacological activity, including at serotonin (5-hydroxytryptamine, 5HT) receptors, other G protein-coupled receptors (GPCRs), ion channels, and monoamine transporters. The high labeling selectivity is not achieved by high affinity binding to SERT itself but rather by a sufficient rate of SERT-mediated transport of SERTlight, resulting in accumulation of these molecules in 5HT neurons and yielding a robust and selective optical signal in the mammalian brain. SERTlight provides a stable signal, as it is not released via exocytosis nor by reverse SERT transport induced by 5HT releasers such as MDMA. SERTlight is optically, pharmacologically, and operationally orthogonal to a wide range of genetically encoded sensors, enabling multiplexed imaging. SERTlight enables labeling of distal 5HT axonal projections and simultaneous imaging of the release of endogenous 5HT using the GRAB5HT sensor, providing a new versatile molecular tool for the study of the serotonergic system.

Identification of Fluorescent Small Molecule Compounds for Synaptic Labeling by Image-Based, High-Content Screening.

Few tools are available for noninvasive imaging of synapses in the living mammalian brain. Current paradigms require the use of genetically modified mice or viral delivery of genetic material to the brain. To develop an alternative chemical approach, utilizing the recognition of synaptic components by organic small molecules, we designed an imaging-based, high-content screen in cultured cortical neurons to identify molecules based on their colocalization with fluorescently tagged synaptic proteins. We used this approach to screen a library of ∼7000 novel fluorescent dyes, and identified a series of compounds in the xanthone family that exhibited consistent synaptic labeling. Follow-up studies with one of these compounds, CX-G3, demonstrated its ability to label acidic organelles and in particular synaptic vesicles at glutamatergic synapses in cultured neurons and murine brain tissue, indicating the potential of this screening approach to identify promising lead compounds for use as synaptic markers, sensors, and targeting devices.

Multiplex quantitative assays indicate a need for reevaluating reported small-molecule TrkB agonists.

Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), have emerged as key regulators of brain plasticity and represent disease-modifying targets for several brain disorders, including Alzheimer's disease and major depressive disorder. Because of poor pharmacokinetic properties of BDNF, the interest in small-molecule TrkB agonists and modulators is high. Several compounds have been reported to act as TrkB agonists, and their increasing use in various nervous system disorder models creates the perception that these are reliable probes. To examine key pharmacological parameters of these compounds in detail, we have developed and optimized a series of complementary quantitative assays that measure TrkB receptor activation, TrkB-dependent downstream signaling, and gene expression in different cellular contexts. Although BDNF and other neurotrophic factors elicited robust and dose-dependent receptor activation and downstream signaling, we were unable to reproduce these activities using the reported small-molecule TrkB agonists. Our findings indicate that experimental results obtained with these compounds must be carefully interpreted and highlight the challenge of developing reliable pharmacological activators of this key molecular target.

Complexes of Chlorin e6 with Pluronics and Polyvinylpyrrolidone: Structure and Photodynamic Activity in Cell Culture.

Polymeric carriers are extensively used in photodynamic therapy (PDT) for increase of efficacy of photosensitizers. Here, we report the influence of nine Pluronic copolymers on phototoxicity of chlorin e6 (Ce6), in particular 5- to 7-fold rise in the phototoxicity caused by hydrophilic Pluronics F127, F108, F68 and F87 and practically no influence on Ce6 of more hydrophobic polymers. The revealed value of 0.2 mg mL(-1) of Pluronic F127 concentration sufficient for half-of-maximal increase of Ce6 photodynamic activity proved to be close to 0.16 mg mL(-1) inherent in well-documented carrier poly(N-vinylpyrrolidone) (PVP). The dissociation constants of Ce6 complexes with Pluronic F127 and PVP that were estimated from UV spectra were 0.252 and 0.036 mg mL(-1) , respectively, indicating higher stability of Ce6 complex with PVP. According to the results of (1) H-NMR studies of Ce6 complexes, the porphyrin interacts not only with hydrophobic regions but also with hydrophilic sides of both polymers.