Antidepressants enter cells, organelles, and membranes.

We begin by summarizing several examples of antidepressants whose therapeutic actions begin when they encounter their targets in the cytoplasm or in the lumen of an organelle. These actions contrast with the prevailing view that most neuropharmacological actions begin when drugs engage their therapeutic targets at extracellular binding sites of plasma membrane targets-ion channels, receptors, and transporters. We review the chemical, pharmacokinetic, and pharmacodynamic principles underlying the movements of drugs into subcellular compartments. We note the relationship between protonation-deprotonation events and membrane permeation of antidepressant drugs. The key properties relate to charge and hydrophobicity/lipid solubility, summarized by the parameters LogP, pKa, and LogDpH7.4. The classical metric, volume of distribution (Vd), is unusually large for some antidepressants and has both supracellular and subcellular components. A table gathers structures, LogP, PKa, LogDpH7.4, and Vd data and/or calculations for most antidepressants and antidepressant candidates. The subcellular components, which can now be measured in some cases, are dominated by membrane binding and by trapping in the lumen of acidic organelles. For common antidepressants, such as selective serotonin reuptake inhibitors (SSRIs) and serotonin/norepinephrine reuptake inhibitors (SNRIs), the target is assumed to be the eponymous reuptake transporter(s), although in fact the compartment of target engagement is unknown. We review special aspects of the pharmacokinetics of ketamine, ketamine metabolites, and other rapidly acting antidepressants (RAADs) including methoxetamine and scopolamine, psychedelics, and neurosteroids. Therefore, the reader can assess properties that markedly affect a drug's ability to enter or cross membranes-and therefore, to interact with target sites that face the cytoplasm, the lumen of organelles, or a membrane. In the current literature, mechanisms involving intracellular targets are termed "location-biased actions" or "inside-out pharmacology". Hopefully, these general terms will eventually acquire additional mechanistic details.

Fluorescence Screens for Identifying Central Nervous System-Acting Drug-Biosensor Pairs for Subcellular and Supracellular Pharmacokinetics.

Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)-acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the plasma membrane and in organelles. We describe screening and validation protocols for identifying hit pairs comprising a drug and biosensor, with each screen including 13-18 candidate biosensors and 44-84 candidate drugs. After a favorable hit pair is identified and validated via these protocols, the biosensor is then optimized, as described in other papers, for sensitivity and selectivity to the drug. We also show sample hit pair data that may lead to future intensity-based drug-sensing fluorescent reporters (iDrugSnFRs). These protocols will assist scientists to use fluorescence responses as criteria in identifying favorable fluorescent biosensor variants for CNS-acting drugs that presently have no corresponding biosensor partner. This protocol was validated in: eLife (2022), DOI: 10.7554/eLife.74648 Graphical abstract.

Metal nanoparticle alters adenine induced charge transfer kinetics of vitamin K3 in magnetic field.

In this article, we highlight the alterations in the photoinduced electron transfer (ET) and hydrogen atom transfer (HAT) pathways between an anti-tumor drug vitamin-K3 (MQ) and a nucleobase adenine (ADN) in the presence of gold (Au) and iron (Fe) nanoparticles (NPs). Inside the confined micellar media, with laser flash photolysis corroborated with an external magnetic field (MF), we have detected the transient geminate radicals of MQ and ADN, photo-generated through ET and HAT. We observe that the presence of AuNP on the MQ-ADN complex (AuMQ-ADN) assists HAT by limiting the ET channel, on the other hand, FeNP on the MQ-ADN complex (FeMQ-ADN) mostly favors a facile PET. We hypothesize that through selective interactions of the ADN molecules with AuNP and MQ molecules with FeNP, a preferential HAT and PET process is eased. The enhanced HAT and PET have been confirmed by the escape yields of radical intermediates by time-resolved transient absorption spectroscopy in the presence of MF.

Biosensors Show the Pharmacokinetics of S-Ketamine in the Endoplasmic Reticulum.

The target for the "rapid" (<24 h) antidepressant effects of S-ketamine is unknown, vitiating programs to rationally develop more effective rapid antidepressants. To describe a drug's target, one must first understand the compartments entered by the drug, at all levels-the organ, the cell, and the organelle. We have, therefore, developed molecular tools to measure the subcellular, organellar pharmacokinetics of S-ketamine. The tools are genetically encoded intensity-based S-ketamine-sensing fluorescent reporters, iSKetSnFR1 and iSKetSnFR2. In solution, these biosensors respond to S-ketamine with a sensitivity, S-slope = delta(F/F0)/(delta[S-ketamine]) of 0.23 and 1.9/µM, respectively. The iSKetSnFR2 construct allows measurements at <0.3 µM S-ketamine. The iSKetSnFR1 and iSKetSnFR2 biosensors display >100-fold selectivity over other ligands tested, including R-ketamine. We targeted each of the sensors to either the plasma membrane (PM) or the endoplasmic reticulum (ER). Measurements on these biosensors expressed in Neuro2a cells and in human dopaminergic neurons differentiated from induced pluripotent stem cells (iPSCs) show that S-ketamine enters the ER within a few seconds after appearing in the external solution near the PM, then leaves as rapidly after S-ketamine is removed from the extracellular solution. In cells, S-slopes for the ER and PM-targeted sensors differ by <2-fold, indicating that the ER [S-ketamine] is less than 2-fold different from the extracellular [S-ketamine]. Organelles represent potential compartments for the engagement of S-ketamine with its antidepressant target, and potential S-ketamine targets include organellar ion channels, receptors, and transporters.

Development of a Next-Generation Fluorescent Turn-On Sensor to Simultaneously Detect and Detoxify Mercury in Living Samples.

Strategies for simultaneous detection and detoxification of Hg2+ using a single sensor from biological and environmental samples are limited and have not been realized in living organisms so far. We report a highly selective, small molecule "turn-on" fluorescent sensor, PYDMSA, based on the cationic dye Pyronin Y (PY) and chelating agent meso-2,3-dimercaptosuccinic acid (DMSA) for the simultaneous detection and detoxification of inorganic mercury (Hg2+). After Hg2+ detection, concomitant detoxification was carried out with sufficient efficacy in living samples, which makes the sensor unique. PYDMSA exhibits high selectivity for Hg2+ over other competing metal ions with an experimental detection limit of ∼300 pM in aqueous buffer solution. When PYDMSA reacts with Hg2+, the CS-C9 bond in the sensor gets cleaved. This results in the "turn-on" response of the fluorescence probe with a concomitant release of one equivalent of water-soluble Hg2+-DMSA complex which leads to a synchronous detoxifying effect. The sensor by itself is nontoxic to cells in culture and has been used to monitor the real-time uptake of Hg2+ in live cells and zebrafish larvae. Thus, PYDMSA is a unique sensor which can be used to detect and detoxify mercury at the same time in living samples.

Determining the pharmacokinetics of nicotinic drugs in the endoplasmic reticulum using biosensors.

Nicotine dependence is thought to arise in part because nicotine permeates into the endoplasmic reticulum (ER), where it binds to nicotinic receptors (nAChRs) and begins an "inside-out" pathway that leads to up-regulation of nAChRs on the plasma membrane. However, the dynamics of nicotine entry into the ER are unquantified. Here, we develop a family of genetically encoded fluorescent biosensors for nicotine, termed iNicSnFRs. The iNicSnFRs are fusions between two proteins: a circularly permutated GFP and a periplasmic choline-/betaine-binding protein engineered to bind nicotine. The biosensors iNicSnFR3a and iNicSnFR3b respond to nicotine by increasing fluorescence at [nicotine] <1 µM, the concentration in the plasma and cerebrospinal fluid of a smoker. We target iNicSnFR3 biosensors either to the plasma membrane or to the ER and measure nicotine kinetics in HeLa, SH-SY5Y, N2a, and HEK293 cell lines, as well as mouse hippocampal neurons and human stem cell-derived dopaminergic neurons. In all cell types, we find that nicotine equilibrates in the ER within 10 s (possibly within 1 s) of extracellular application and leaves as rapidly after removal from the extracellular solution. The [nicotine] in the ER is within twofold of the extracellular value. We use these data to run combined pharmacokinetic and pharmacodynamic simulations of human smoking. In the ER, the inside-out pathway begins when nicotine becomes a stabilizing pharmacological chaperone for some nAChR subtypes, even at concentrations as low as ∼10 nM. Such concentrations would persist during the 12 h of a typical smoker's day, continually activating the inside-out pathway by >75%. Reducing nicotine intake by 10-fold decreases activation to ∼20%. iNicSnFR3a and iNicSnFR3b also sense the smoking cessation drug varenicline, revealing that varenicline also permeates into the ER within seconds. Our iNicSnFRs enable optical subcellular pharmacokinetics for nicotine and varenicline during an early event in the inside-out pathway.

DNA Damage and Apoptosis Induction in Cancer Cells by Chemically Engineered Thiolated Riboflavin Gold Nanoassembly.

Herein we have engineered a smart nuclear targeting thiol-modified riboflavin-gold nano assembly, RfS@AuNPs, which accumulates selectively in the nucleus without any nuclear-targeting peptides (NLS/RGD) and shows photophysically in vitro DNA intercalation. A theoretical model using Molecular Dynamics has been developed to probe the mechanism of formation and stability as well as dynamics of the RfS@AuNPs in aqueous solution and within the DNA microenvironment. The RfS@AuNPs facilitate the binucleated cell formation that is reflected in the significant increase of DNA damage marker, γ-H2AX as well as the arrest of most of the HeLa cells at the pre-G1 phase indicating cell death. Moreover, a significant upregulation of apoptotic markers confirms that the cell death occurs through the apoptotic pathway. Analyses of the microarray gene expression of RfS@AuNPs treated HeLa cells show significant alterations in vital biological processes necessary for cell survival. Taken together, our study reports a unique nuclear targeting mechanism through targeting the riboflavin receptors, which are upregulated in cancer cells and induce apoptosis in the targeted cells.

Fluorogenic Detection of Monoamine Neurotransmitters in Live Cells.

Monoamine neurotransmission is key to neuromodulation, but imaging monoamines in live neurons has remained a challenge. Here we show that externally added ortho-phthalaldehyde (OPA) can permeate live cells and form bright fluorogenic adducts with intracellular monoamines (e.g., serotonin, dopamine, and norepinephrine) and with L-DOPA, which can be imaged sensitively using conventional single-photon excitation in a fluorescence microscope. The peak excitation and emission wavelengths (λex = 401 nm and λem = 490 nm for serotonin; λex = 446 nm and λem = 557 nm for dopamine; and λex = 446 nm and λem = 544 nm for norepinephrine, respectively) are accessible to most modern confocal imaging instruments. The identity of monoamine containing structures (possibly neurotransmitter vesicles) in serotonergic RN46A cells is established by quasi-simultaneous imaging of serotonin using three-photon excitation microscopy. Mass spectrometry of cell extracts and of in vitro solutions helps us identify the chemical nature of the adducts and establishes the reaction mechanisms. Our method has low toxicity, high selectivity, and the ability to directly report the location and concentration of monoamines in live cells.

Intermolecular disulfide bond formation promotes immunoglobulin aggregation: investigation by fluorescence correlation spectroscopy.

Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, additional mechanisms arising from specific interactions, such as intermolecular disulfide bond formation, may also contribute to the process. The latter is proposed to be the initiating pathway for aggregation of immunoglobulin (IgG), which is essential for triggering its immune response. To test the veracity of this hypothesis, we have employed fluorescence correlation spectroscopy to measure the kinetics of aggregation of IgG in separate experiments either allowing or inhibiting disulfide formation. Fluorescence correlation spectroscopy measurements yielded a diffusion time (τ(D)) of ∼200 µsec for Rhodamine-labeled IgG, corresponding to a hydrodynamic radius (R(H)) of 56 Å for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of τ(D) under conditions in which its cysteine residues were either free or blocked. In both cases, the progress curves confirmed that aggregation proceeded via the nucleation-dependent polymerization pathway. However, for aggregation in the presence of free cysteines, the lag times were shorter, and the aggregate sizes bigger, than their respective counterparts for aggregation in the presence of blocked cysteines. This result clearly demonstrates that formation of intermolecular disulfide bonds represents a preferred pathway in the aggregation process of IgG. Fluorescence spectroscopy showed that aggregates formed in experiments where disulfide formation was prevented denatured at lower concentration of guanidine hydrochloride than those obtained in experiments where the disulfides were free to form, indicating that intermolecular disulfide bridging is a valid pathway for IgG aggregation.

Development of a rhodamine-rhodanine-based fluorescent mercury sensor and its use to monitor real-time uptake and distribution of inorganic mercury in live zebrafish larvae.

We introduce a new rhodamine-rhodanine-based "turn-on" fluorescent sensor (RR1) and describe its application for detection of mercury, including in solution, in live cells, and in a living vertebrate organism. The sensor RR1, which is a one-pot synthesis from rhodamine B, undergoes a rapid and irreversible 1:1 stoichiometric reaction with Hg(2+) in aqueous medium. Using fluorescence correlation spectroscopy (FCS), RR1 was shown to detect the presence of as low as a 0.5 pM concentration of Hg(2+). It may also lend itself to tagging with biomolecules and nanoparticles, leading to the possibility of organelle-specific Hg detection. Results of experiments with mammalian cells and zebrafish show that RR1 is cell and organism permeable and that it responds selectively to mercury ions over other metal ions. In addition, real-time monitoring of inorganic mercury ion uptake by cells and live zebrafish using this chemosensor shows that saturation of mercury ion uptake occurs within 20-30 min in cells and organisms. We also demonstrate the acquisition of high-resolution real-time distribution maps of inorganic mercury (Hg(2+)) in the zebrafish brain by using a simple fluorescence confocal imaging technique.

Sensing of hydrophobic cavity of serum albumin by an adenosine analogue: fluorescence correlation and ensemble spectroscopic studies.

Adenosine is a naturally occurring purine nucleoside that plays important role in various biochemical processes. We have studied the binding of TNP-Ado (trinitrophenylated-adenosine), a fluorescent analogue of adenosine (which itself is a weak fluorophore), with a model transport protein, bovine serum albumin (BSA). The binding affinity was determined using Fluorescence correlation spectroscopy (FCS) and compared with its value obtained from macroscopic fluorescence spectroscopic studies. Fluorescence and circular dichroism (CD) spectroscopies were employed together with molecular docking study to locate the probable binding site of TNP-Ado on BSA and its effect on the conformation and stability of BSA. Fluorescence studies showed that TNP-Ado binds to BSA in 1:1 stoichiometry via an entropically favoured process. Induced CD spectra revealed that a chiro-optical switching of TNP-Ado occurs upon binding to BSA. Results on urea-induced denaturation of BSA and docking study suggested that the binding site for the ligand is in the hydrophobic subdomain IIA of BSA, consistent with the results of other measurements. This study establishes TNP-Ado as a sensor of hydrophobic regions in proteins like serum albumin, having the capability of detecting a minimum concentration of 140ng/ml protein. FCS measurement of binding interaction of rhodamine-labeled TNP-Ado (RTNP-Ado) with BSA yielded an association constant of KFCS=(1.03±0.06) × 10(4)M(-1). The association constants (Ka) obtained for binding of BSA with rhodamine-free (i.e. TNP-Ado) and rhodamine-labeled (RTNP-Ado) ligands, obtained using the ensemble spectroscopic technique, were (2.3±0.06) × 10(5)M(-1) and (3.4±0.03) × 10(4)M(-1), respectively. The difference between the values of Ka for the free and labeled ligands suggests that fluorescent labeling of small molecules perceptibly interferes with the binding process. On the other hand, the difference in Ka obtained by FCS and ensemble techniques is due to the fact that while the former measures the change in the diffusion constant (i.e. size) of RTNP-Ado upon binding to BSA, the latter focuses on the change of tryptophan emission properties of BSA due to the presence of bound RTNP-Ado.