Holding-Oriented versus Gating-Oriented Live-Cell Distinction: Highlighting the Role of Transporters in Cell Imaging Probe Development

Abstract

Conspectus Small molecule imaging probes are powerful tools to understand complex biological systems. The mainstreams of imaging probe developments have been focused on the target holding of the probes; the holding targets are often cell-type-specific biomarkers. This type of the probe mechanism can be designated as holding-oriented live-cell distinction (HOLD). Our group has worked on the development of cell-type-selective probes using a diversity-oriented fluorescence library approach (DOFLA), where unbiased phenotypic screening is employed using fluorescent library compounds. Through the conventional target identification methods such as an affinity-based analysis, we elucidated that some of the probe mechanisms are HOLD. However, we also realized that sometimes there is no specific holding target for probes or the holding targets are ubiquitous. The observation led us to test an alternative mechanism of cell-type-specific probes as gating-oriented live-cell distinction (GOLD). We started to examine the gating mechanism of probes, which is mainly based on transporters but which does not necessarily require probe holding to cellular targets. Transporters can control the in and out movement of various nutrients and chemicals. Different expression levels of transporters in various cell types could provide the molecular mechanism of differential staining of cells by regulating the intracellular accumulation of a certain specific probe. A number of GOLD probes have been developed by modifying or mimicking endogenous substrates of transporters such as inorganic ions, glucose, amino acids, or neurotransmitters, utilizing broad substrate specificity of transporters. The radiolabeled or fluorophore-conjugated substrate mimetics have been widely used for live cell distinction and various applications such as disease-related cell or tissue imaging. In humans, there are about 400 solute carrier (SLC) transporters and 50 ATP-binding cassette (ABC) transporters. Since some transporters have broad substrate specificity, they can transport not only derivatives of endogenous natural substrates but also totally synthetic diverse imaging probes, such as DOFLA probes. Without preconsidering the structure of endogenous substrates, we recently demonstrated a series of live-cell imaging probes and elucidated their molecular mechanism as a gating one, either by SLC or ABC transporters. Transporter inhibitor panel and CRISPR-based transporter libraries could provide a systematic gating target elucidation platform. Considering the generality of DOFLA and the CRISPR-based genomic tool for transporter systems (>450 in humans), the GOLD approach will offer new insight and promise for unprecedented levels of novel cell imaging probe development.

Target identification of mouse stem cell probe CDy1 as ALDH2 and Abcb1b through live-cell affinity-matrix and ABC CRISPRa library

“…The transporter-based mechanism has been described as a Gating-Oriented Live-cell Distinction (GOLD). 9 In CDy1 case, it seems both POLD and GOLD play important roles together to increase the cell staining selectivity in mESC ( Fig. 5 ).…”

“…Then, we hypothesized that transporters on plasma membrane may also have an essential role for the kinetic flow of CDy1 in mESC and MEF. 9,10 There are hundreds of transporters for molecules to be imported or exported, and we mainly focused on ABC transporters, which generally work as efflux gate proteins. 11 We constructed CRISPR activation (CRISPRa) library 12,13 targeting 153 kinds of transporters including all 48 human ABC transporters 8,14 for systematic screening of the specific ABC transporters to efflux CDy1 from the cells.…”

“…Then, we hypothesized that transporters on the plasma membrane may also have an essential role for the kinetic flow of CDy1 in mESC and MEF. 9,10 …”

“…In summary, we identified ALDH2 as the binding target of CDy1 in mESC. Following the mechanism classification, 9 this may be a case of Holding Oriented Live-cell Distinction (HOLD). In HOLD, there are mainly two subcategories of protein and carbohydrate targeting.…”

Recent advances in construction of small molecule-based fluorophore-drug conjugates

“…Amongst the various monitoring methods, fluorescence-based approaches are quite promising owing to their non-invasiveness, real-time detection, high resolution, qualitative/quantitative capability, and can be employed in various biological systems, e.g., animal models, tissue-based samples and single cells. Moreover, low-molecular-weight fluorescent dyes (small-molecule dyes) are particularly attractive because they are more convenient in achieving fruitful structures through chemical synthesis and can cover a wide range of wavelength and brightness [ 5 ]. In recent years, small molecule-based bioimaging has been firmly established as an attractive tool for accurate monitoring of drug delivery and distribution.…”