A general fluorescence off/on strategy for fluorogenic probes: Steric repulsion-induced twisted intramolecular charge transfer (sr-TICT).

Various control strategies are available for building fluorogenic probes to visualize biological events in terms of a fluorescence change. Here, we performed the time-dependent density functional theory (TD-DFT) computational analysis of the twisted intramolecular charge transfer (TICT) process in rhodamine dyes. On the basis of the results, we designed and synthesized a series of rhodamine dyes and established a fluorescence quenching strategy that we call steric repulsion-induced TICT (sr-TICT), in which the fluorescence quenching process is greatly accelerated by simple intramolecular twisting. As proof of concept of this design strategy, we used it to develop a fluorogenic probe, 2-Me PeER (pentyloxyethylrhodamine), for the N-dealkylation activity of CYP3A4. We applied 2-Me PeER for CYP3A4 activity-based fluorescence-activated cell sorting (FACS), providing access to homogeneous, highly functional human-induced pluripotent stem cell (hiPSC)-derived hepatocytes and intestinal epithelial cells. Our results suggest that sr-TICT represents a general fluorescence control method for fluorogenic probes.

Modular Design Platform for Activatable Fluorescence Probes Targeting Carboxypeptidases Based on ProTide Chemistry.

Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins and play indispensable roles in various physiological and pathological processes. However, only a few highly activatable fluorescence probes for CPs have been reported, and there is a need for a flexibly tunable molecular design platform to afford a range of fluorescence probes for CPs for biological and medical research. Here, we focused on the unique activation mechanism of ProTide-based prodrugs and established a modular design platform for CP-targeting florescence probes based on ProTide chemistry. In this design, probe properties such as fluorescence emission wavelength, reactivity/stability, and target CP can be readily tuned and optimized by changing the four probe modules: the fluorophore, the substituent on the phosphorus atom, the linker amino acid at the P1 position, and the substrate amino acid at the P1' position. In particular, switching the linker amino acid at position P1 enabled us to precisely optimize the reactivity for target CPs. As a proof-of-concept, we constructed probes for carboxypeptidase M (CPM) and prostate-specific membrane antigen (also known as glutamate carboxypeptidase II). The developed probes were applicable for the imaging of CP activities in live cells and in clinical specimens from patients. This design strategy should be useful in studying CP-related biological and pathological phenomena.

Discovery of a cystathionine γ-lyase (CSE) selective inhibitor targeting active-site pyridoxal 5'-phosphate (PLP) via Schiff base formation.

D,L-Propargylglycine (PAG) has been widely used as a selective inhibitor to investigate the biological functions of cystathionine γ-lyase (CSE), which catalyzes the formation of reactive sulfur species (RSS). However, PAG also inhibits other PLP (pyridoxal-5'-phosphate)-dependent enzymes such as methionine γ-lyase (MGL) and L-alanine transaminase (ALT), so highly selective CSE inhibitors are still required. Here, we performed high-throughput screening (HTS) of a large chemical library and identified oxamic hydrazide 1 as a potent inhibitor of CSE (IC50 = 13 ± 1 µM (mean ± S.E.)) with high selectivity over other PLP-dependent enzymes and RSS-generating enzymes. Inhibitor 1 inhibited the enzymatic activity of human CSE in living cells, indicating that it is sufficiently membrane-permeable. X-Ray crystal structure analysis of the complex of rat CSE (rCSE) with 1 revealed that 1 forms a Schiff base linkage with the cofactor PLP in the active site of rCSE. PLP in the active site may be a promising target for development of selective inhibitors of PLP-dependent enzymes, including RSS-generating enzymes such as cystathionine ß-synthase (CBS) and cysteinyl-tRNA synthetase 2 (CARS2), which have unique substrate binding pocket structures.

Development of pathway-oriented screening to identify compounds to control 2-methylglyoxal metabolism in tumor cells.

Controlling tumor-specific alterations in metabolic pathways is a useful strategy for treating tumors. The glyoxalase pathway, which metabolizes the toxic electrophile 2-methylglyoxal (MG), is thought to contribute to tumor pathology. We developed a live cell-based high-throughput screening system that monitors the metabolism of MG to generate D-lactate by glyoxalase I and II (GLO1 and GLO2). It utilizes an extracellular coupled assay that uses D-lactate to generate NAD(P)H, which is detected by a selective fluorogenic probe designed to respond exclusively to extracellular NAD(P)H. This metabolic pathway-oriented screening is able to identify compounds that control MG metabolism in live cells, and we have discovered compounds that can directly or indirectly inhibit glyoxalase activities in small cell lung carcinoma cells.

General Design Strategy to Precisely Control the Emission of Fluorophores via a Twisted Intramolecular Charge Transfer (TICT) Process.

Fluorogenic probes for bioimaging have become essential tools for life science and medicine, and the key to their development is a precise understanding of the mechanisms available for fluorescence off/on control, such as photoinduced electron transfer (PeT) and Förster resonance energy transfer (FRET). Here we establish a new molecular design strategy to rationally develop activatable fluorescent probes, which exhibit a fluorescence off/on change in response to target biomolecules, by controlling the twisted intramolecular charge transfer (TICT) process. This approach was developed on the basis of a thorough investigation of the fluorescence quenching mechanism of N-phenyl rhodamine dyes (commercially available as the QSY series) by means of time-dependent density functional theory (TD-DFT) calculations and photophysical evaluation of their derivatives. To illustrate and validate this TICT-based design strategy, we employed it to develop practical fluorogenic probes for HaloTag and SNAP-tag. We further show that the TICT-controlled fluorescence off/on mechanism is generalizable by synthesizing a Si-rhodamine-based fluorogenic probe for HaloTag, thus providing a palette of chemical dyes that spans the visible and near-infrared range.

Molecular design of near-infrared (NIR) fluorescent probes targeting exopeptidase and application for detection of dipeptidyl peptidase 4 (DPP-4) activity.

Monitoring the activities of proteases in vivo is an important requirement in biological and medical research. Near-infrared (NIR) fluorescent probes are particularly useful for in vivo fluorescence imaging, due to the high penetration of NIR and the low autofluorescence in tissue for this wavelength region, but most current NIR fluorescent probes for proteases are targeted to endopeptidase. Here, we describe a new molecular design for NIR fluorescent probes that target exopeptidase by utilizing the >110 nm blueshift of unsymmetrical Si-rhodamines upon amidation of the N atom of their xanthene moiety. Based on this molecular design, we developed Leu-SiR640 as a probe for leucine amino peptidase (LAP). Leu-SiR640 shows a one order of magnitude larger fluorescence increment (669-fold) upon reaction with LAP than existing NIR fluorescent probes. We similarly designed and synthesized EP-SiR640, a NIR fluorescent probe that targets dipeptidyl peptidase 4 (DPP-4). We show that this probe can monitor DPP-4 activity not only in living cells but also in mouse organs and tumors. This probe could also detect esophageal cancer in human clinical specimens, based on the overexpression of DPP-4 activity.

Development of a fluorescent probe library enabling efficient screening of tumour-imaging probes based on discovery of biomarker enzymatic activities.

Fluorescent probes that can selectively detect tumour lesions have great potential for fluorescence imaging-guided surgery. Here, we established a library-based approach for efficient screening of probes for tumour-selective imaging based on discovery of biomarker enzymes. We constructed a combinatorial fluorescent probe library for aminopeptidases and proteases, which is composed of 380 probes with various substrate moieties. Using this probe library, we performed lysate-based in vitro screening and/or direct imaging-based ex vivo screening of freshly resected clinical specimens from lung or gastric cancer patients, and found promising probes for tumour-selective visualization. Further, we identified two target enzymes as novel biomarker enzymes for discriminating between tumour and non-tumour tissues. This library-based approach is expected to be an efficient tool to develop tumour-imaging probes and to discover new biomarker enzyme activities for various tumours and other diseases.

Leading-edge elongation by follower cell interruption in advancing epithelial cell sheets.

Collective cell migration is seen in many developmental and pathological processes, such as morphogenesis, wound closure, and cancer metastasis. When a fish scale is detached and adhered to a substrate, epithelial keratocyte sheets crawl out from it, building a semicircular pattern. All the keratocytes at the leading edge of the sheet have a single lamellipodium, and are interconnected with each other via actomyosin cables. The leading edge of the sheet becomes gradually longer as it crawls out from the scale, regardless of the cell-to-cell connections. In this study, we found leading-edge elongation to be realized by the interruption of follower cells into the leading edge. The follower cell and the two adjacent leader cells are first connected by newly emerging actomyosin cables. Then, the contractile forces along the cables bring the follower cell forward to make it a leader cell. Finally, the original cables between the two leader cells are stretched to tear by the interruption and the lamellipodium extension from the new leader cell. This unique actomyosin-cable reconnection between a follower cell and adjacent leaders offers insights into the mechanisms of collective cell migration.

Discovery of an F-actin-binding small molecule serving as a fluorescent probe and a scaffold for functional probes.

Actin is a ubiquitous cytoskeletal protein, forming a dynamic network that generates mechanical forces in the cell. There is a growing demand for practical and accessible tools for dissecting the role of the actin cytoskeleton in cellular function, and the discovery of a new actin-binding small molecule is an important advance in the field, offering the opportunity to design and synthesize of new class of functional molecules. Here, we found an F-actin­binding small molecule and introduced two powerful tools based on a new class of actin-binding small molecule: One enables visualization of the actin cytoskeleton, including super-resolution imaging, and the other enables highly specific green light­controlled fragmentation of actin filaments, affording unprecedented control of the actin cytoskeleton and its force network in living cells.

Establishment of live-cell-based coupled assay system for identification of compounds to modulate metabolic activities of cells.

In this study, we present a live-cell-based fluorometric coupled assay system to identify the compounds that can regulate the targeted metabolic pathways in live cells. The assay is established through targeting specific metabolic pathways and using "input" and "output" metabolite pairs. The changes in the extracellular output that are generated and released into the extracellular media from the input are assessed as the activity of the pathway. The screening for the glycolytic pathway and amino acid metabolism reveals the activities of the present drugs, 6-BIO and regorafenib, that regulate the metabolic fate of tumor cells.

Nongenetic control of receptor signaling dynamics using a DNA-based optochemical tool.

Optochemical tools that can modulate the activity of the target protein provide an opportunity for studying and regulating the related biological processes. Here we present a DNA-based nongenetic optochemical tool that can control the dynamics of growth factor signaling. This photo-caged mimicry of growth factor can be a promising tool for elucidating a linkage between the dynamics of signaling and the resulting biological outcomes, as well as for manipulating cellular functions and the fate of living cells.

Amino BODIPY-Based Blue Fluorescent Probes for Aldehyde Dehydrogenase 1-Expressing Cells.

Aldehyde dehydrogenase 1 (ALDH1) plays an important role as a stem cell marker. In the field of stem cell biology, a green fluorescent ALDH1 probe has been principally used, but there is a need for more options in probe color. We designed and synthesized two blue fluorescent ALDH1 probes using 8-amino BODIPY and aminomethylbenzaldehyde. These probes can be simultaneously used with other color probes. Here, we demonstrate successful examples of the simultaneous use of these probes with green fluorescent protein.

Design of spontaneously blinking fluorophores for live-cell super-resolution imaging based on quantum-chemical calculations.

Spontaneously blinking fluorophores are powerful tools for live-cell super-resolution imaging under physiological conditions. Here we show that quantum-chemical calculations can predict key parameters for fluorophore design. We applied this methodology to develop a spontaneously blinking fluorophore with yellow fluorescence for super-resolution imaging of microtubules in living cells.

A cytosolically localized far-red to near-infrared rhodamine-based fluorescent probe for calcium ions.

Ca2+ is one of the most important second messengers in cells. A far-red to near-infrared (NIR) Ca2+ fluorescent probe is useful for multi-color imaging in GFP or YFP-expressing biosamples. Here we developed a cytosolically localized far-red to NIR rhodamine-based fluorescent probe for Ca2+, CaSiR-2 AM, while rhodamine dyes are basically localized to mitochondria or lysosomes in cells.

Antibody Clicking as a Strategy to Modify Antibody Functionalities on the Surface of Targeted Cells.

We established a methodology for initiating cross-linking of antibodies selectively on the cell surface through intermolecular copper-free click reactions facilitated by increased effective concentrations of antibodies binding to target antigens. Upon cross-linking of tetrazine- and bicyclononyne-modified trastuzumab on the surface of HER2-overexpressing cells, increased antibody uptake and activation of intracellular signaling were observed. Our findings demonstrate that the cross-linking reaction can significantly alter the biophysical properties of proteins, activating their unique functionalities on targeted cells to realize an increased cargo delivery and synthetic manipulation of cellular signaling.

Rational Design of a Near-infrared Fluorescence Probe for Ca2+ Based on Phosphorus-substituted Rhodamines Utilizing Photoinduced Electron Transfer.

Fluorescence imaging in the near-infrared (NIR) region (650-900 nm) is useful for bioimaging because background autofluorescence is low and tissue penetration is high in this range. In addition, NIR fluorescence is useful as a complementary color window to green and red for multicolor imaging. Here, we compared the photoinduced electron transfer (PeT)-mediated fluorescence quenching of silicon- and phosphorus-substituted rhodamines (SiRs and PRs) in order to guide the development of improved far-red to NIR fluorescent dyes. The results of density functional theory calculations and photophysical evaluation of a series of newly synthesized PRs confirmed that the fluorescence of PRs was more susceptible than that of SiRs to quenching via PeT. Based on this, we designed and synthesized a NIR fluorescence probe for Ca2+ , CaPR-1, and its membrane-permeable acetoxymethyl derivative, CaPR-1 AM, which is distributed to the cytosol, in marked contrast to our previously reported Ca2+ far-red to NIR fluorescence probe based on the SiR scaffold, CaSiR-1 AM, which is mainly localized in lysosomes as well as cytosol in living cells. CaPR-1 showed longer-wavelength absorption and emission (up to 712 nm) than CaSiR-1. The new probe was able to image Ca2+ at dendrites and spines in brain slices, and should be a useful tool in neuroscience research.

A Fluorescent Probe for Rapid, High-Contrast Visualization of Folate-Receptor-Expressing Tumors In†Vivo.

Folate receptors (FRs) are membrane proteins involved in folic acid uptake, and the alpha isoform (FR-α) is overexpressed in ovarian and endometrial cancer cells. For fluorescence imaging of FRs in vivo, the near-infrared (NIR) region (650-900 nm), in which tissue penetration is high and autofluorescence is low, is optimal, but existing NIR fluorescent probes targeting FR-α show high non-specific tissue adsorption, and require prolonged washout to visualize tumors. We have designed and synthesized a new NIR fluorescent probe, FolateSiR-1, utilizing a Si-rhodamine fluorophore having a carboxy group at the benzene moiety, coupled to a folate ligand moiety through a negatively charged tripeptide linker. This probe exhibits very low background fluorescence and afforded a tumor-to-background ratio (TBR) of up to 83 in FR-expressing tumor-bearing mice within 30 min. Thus, FolateSiR-1 has the potential to contribute to the research in the field of biology and the clinical medicine.

Metabolic-Pathway-Oriented Screening Targeting S-Adenosyl-l-methionine Reveals the Epigenetic Remodeling Activities of Naturally Occurring Catechols.

Methyl transfer reactions play important roles in many biological phenomena, wherein the methylation cofactor S-adenosyl-l-methionine (SAM) serves as the important currency to orchestrate those reactions. We have developed a fluorescent-probe-based high-throughput screening (HTS) system to search for the compounds that control cellular SAM levels. HTS with a drug repositioning library revealed the importance of catechol-O-methyltransferase (COMT) and its substrates in controlling the SAM concentrations and histone methylation levels in colorectal tumor cells.

Development of ratiometric carbohydrate sensor based on boron dipyrromethene (BODIPY) scaffold.

We designed a ratiometric carbohydrate sensor consisting of the boron dipyrromethene fluorophore substituted with boronic acid at the 2-position, based upon the strong substituent dependency of the absorbance/fluorescence wavelengths of BODIPY. The substituent is in equilibrium between the boronic acid B(OH)2 and boronate (B(OH)3-) forms, which have different absorbance/fluorescence wavelengths in the visible region. Reaction of the boronic acid moiety with hydroxy groups of carbohydrate affords a cyclic ester and shifts the equilibrium in favor of the boronate (B(OR)3-) form, resulting in a carbohydrate-concentration-dependent change of the fluorescence ratio. Thus, the sensor, BA-BODIPY, can ratiometrically detect carbohydrate at a pH near the pKa of cyclic ester formation.

Separation-Based Enzymomics Assay for the Discovery of Altered Peptide-Metabolizing Enzymatic Activities in Biosamples.

We have developed a novel method to globally monitor the enzymatic activities of biological samples based on performing the global activity analysis on a proteome separated by native electrophoresis. The study of the alteration in peptide-metabolizing enzymatic activity in colorectal tumor specimens led us to the discovery of elevated thimet oligopeptidase activity, which contributed to the faster consumption of immune-stimulating peptide neurotensin.