Xanthene-Based Dyes for Photoacoustic Imaging and their Use as Analyte-Responsive Probes.

Developing imaging tools that can report on the presence of disease-relevant analytes in multicellular organisms can provide insight into fundamental disease mechanisms as well as provide diagnostic tools for the clinic. Photoacoustic imaging (PAI) is a light-in, sound-out imaging technique that allows for high resolution, deep-tissue imaging with applications in pre-clinical and point-of-care settings. The continued development of near-infrared (NIR) absorbing small-molecule dyes promises to improve the capabilities of this emerging imaging modality. For example, new dye scaffolds bearing chemoselective functionalities are enabling the detection and quantification of disease-relevant analytes through activity-based sensing (ABS) approaches. Recently described strategies to engineer NIR absorbing xanthenes have enabled development of analyte-responsive PAI probes using this classic dye scaffold. Herein, we present current strategies for red-shifting the spectral properties of xanthenes via bridging heteroatom or auxochrome modifications. Additionally, we explore how these strategies, coupled with chemoselective spiroring-opening approaches, have been employed to create ABS probes for in vivo detection of hypochlorous acid, nitric oxide, copper (II), human NAD(P)H: quinone oxidoreductase isozyme 1, and carbon monoxide. Given the versatility of the xanthene scaffold, we anticipate continued growth and development of analyte-responsive PAI imaging probes based on this dye class.

Standardized Parts for Activation of Small GTPase Signaling in Living Cells.

Small GTPases comprise a superfamily of over 167 proteins in the human genome and are critical regulators of a variety of pathways including cell migration and proliferation. Despite the importance of these proteins in cell signaling, a standardized approach for controlling small GTPase activation within living cells is lacking. Herein, we report a split-protein-based approach to directly activate small GTPase signaling in living cells. Importantly, our fragmentation site can be applied across the small GTPase superfamily. We highlight the utility of these standardized parts by demonstrating the ability to directly modulate the activity of four different small GTPases with user-defined inputs, providing a plug and play system for direct activation of small GTPases in living cells.

Azaphosphinate Dyes: A Low Molecular Weight Near-Infrared Scaffold for Development of Photoacoustic or Fluorescence Imaging Probes.

Near-infrared (NIR) dyes are desirable for biological imaging applications including photoacoustic (PA) and fluorescence imaging. Nonetheless, current NIR dyes are often plagued by relatively large molecular weights, poor water solubility, and limited photostability. Herein, we provide the first examples of azaphosphinate dyes which display desirable properties such as low molecular weight, absorption/emission above 750 nm, and remarkable water solubility. In PA imaging, an azaphosphinate dye exhibited a 4.1-fold enhancement in intensity compared to commonly used standards, the ability to multiplex with existing dyes in whole blood, imaging depths of 2.75 cm in a tissue model, and contrast in mice. An improved derivative for fluorescence imaging displayed a >10-fold reduction in photobleaching in water compared to the FDA-approved indocyanine green dye and could be visualized in mice. This new dye class provides a robust scaffold for the development of photoacoustic or NIR fluorescence imaging agents.

Single Atom Stabilization of Phosphinate Ester-Containing Rhodamines Yields Cell Permeable Probes for Turn-On Photoacoustic Imaging.

Photoacoustic imaging (PAI) is an emerging imaging technique that uses pulsed laser excitation with near-infrared (NIR) light to elicit local temperature increases through non-radiative relaxation events, ultimately leading to the production of ultrasound waves. The classical xanthene dye scaffold has found numerous applications in fluorescence imaging, however, xanthenes are rarely utilized for PAI since they do not typically display NIR absorbance. Herein, we report the ability of Nebraska Red (NR) xanthene dyes to produce photoacoustic (PA) signal and provide a rational design approach to reduce the hydrolysis rate of ester containing dyes, affording cell permeable probes. To demonstrate the utility of this approach, we construct the first cell permeable rhodamine-based, turn-on PAI imaging probe for hypochlorous acid (HOCl) with maximal absorbance within the range of commercial PA instrumentation. This probe, termed SNR700 -HOCl, is capable of detecting exogenous HOCl in mice. This work provides a new set of rhodamine-based PAI agents as well as a rational design approach to stabilize esterified versions of NR dyes with desirable properties for PAI. In the long term, the reagents described herein could be utilized to enable non-invasive imaging of HOCl in disease-relevant model systems.

Kamuvudine-9 Protects Retinal Structure and Function in a Novel Model of Experimental Rhegmatogenous Retinal Detachment.

Purpose: Rhegmatogenous retinal detachment (RRD) is a vision-threatening event that benefits from surgical intervention. While awaiting surgical reattachment, irreversible hypoxic and inflammatory damage to the retina often occurs. An interim therapy protecting photoreceptors could improve functional outcomes. We sought to determine whether Kamuvudine-9 (K-9), a derivative of nucleoside reverse transcriptase inhibitors (NRTIs) that inhibits inflammasome activation, and the NRTIs lamivudine (3TC) and azidothymidine (AZT) could protect the retina following RRD. Methods: RRD was induced in mice via subretinal injection (SRI) of 1% carboxymethylcellulose (CMC). To simulate outcomes following the clinical management of RRD, we determined the optimal conditions by which SRI of CMC induced spontaneous retinal reattachment (SRR) occurs over 10 days (RRD/SRR). K-9, 3TC, or AZT was administered via intraperitoneal injection. Inflammasome activation pathways were monitored by abundance of cleaved caspase-1, IL-18, and cleaved caspase-8, and photoreceptor death was assessed by TUNEL staining. Retinal function was assessed by full-field scotopic electroretinography. Results: RRD induced retinal inflammasome activation and photoreceptor death in mice. Systemic administration of K-9, 3TC, or AZT inhibited retinal inflammasome activation and photoreceptor death. In the RRD/SRR model, K-9 protected retinal electrical function during the time of RRD and induced an improvement following retinal reattachment. Conclusions: K-9 and NRTIs exhibit anti-inflammatory and neuroprotective activities in experimental RRD. Given its capacity to protect photoreceptor function during the period of RRD and enhance retinal function following reattachment, K-9 shows promise as a retinal neuroprotectant and warrants study in RRD. Further, this novel RRD/SRR model may facilitate experimental evaluation of functional outcomes relevant to RRD.

Experimentally Calibrated Computational Prediction Enables Accurate Fine-Tuning of Near-Infrared Rhodamines for Multiplexing.

A significant barrier inhibiting multiplexed imaging in the near-infrared (NIR) is the extensive trial and error associated with fine-tuning NIR dyes. In particular, the need to synthesize and experimentally evaluate dye derivatives in order to empirically identify those that can be used in multiplexing applications, requires a large investment of time. While coarse-tuning efforts benefit from computational prediction that can be used to identify target dye structures for synthetic campaigns, errors in computational prediction remain too large to accurately parse modifications aimed at fine-tuning changes in dye absorbance and emission. To address this issue, we screened different levels of theory and identified a time-dependent density functional theory (TD-DFT) approach that can rapidly, as opposed to synthesis and experimental evaluation, estimate absorbance and emission. By calibrating these computational estimations of absorbance and emission to experimentally determined parameters for a panel of existing NIR dyes, we obtain calibration curves that can be used to accurately predict the effect of fine-tuning modifications in new dyes. We demonstrate the predictive power of this calibrated dataset using seven previously unreported dyes, obtaining mean percent errors in absorbance and emission of 2.2 and 2.8 %, respectively. This approach provides a significant timesavings, relative to synthesis and evaluation of dye derivatives, and can be used to focus synthetic campaigns on the most promising dye structures. The new dyes described herein can be utilized for multiplexed imaging, and the experimentally calibrated dataset will provide the dye chemistry community with a means to rapidly identify fine-tuned NIR dyes in silico to guide subsequent synthetic campaigns.

A long-wavelength xanthene dye for photoacoustic imaging.

Photoacoustic (PA) imaging is a powerful biomedical imaging modality. We designed KeTMR and KeJuR, two xanthene-based dyes that were readily obtained through a 2-step synthetic route. KeJuR has low molecular weight, good aqueous solubility, and superior chemical stability compared to KeTMR. KeJuR shows a robust PA signal under 860 nm excitation and can be paired with traditional PA dyes for multiplex imaging in blood samples under a tissue-mimicking environment.

Heteroatom-Substituted Xanthene Fluorophores Enter the Shortwave-Infrared Region.

This article is a highlight of the paper by Ivanic and Schnermann et al. in this issue of Photochemistry and Photobiology (Daly et al. Photochem. Photobiol. 2022). The collaborative team utilized computational approaches to investigate the influence of electron-withdrawing groups at the 10' position of tetramethylrhodamine (TMR). Leveraging this information, the team was able to extend the emission of the TMR scaffold into the shortwave-infrared region (SWIR, 1000-2500 nm) by incorporation of a ketone functional group at the 10' position (Daly et al. Photochem. Photobiol. 2022). This work provides the first example of a TMR derivative with peak SWIR emission (λabs : 862 nm, λem : 1058 nm). The authors utilize the ketone rhodamine scaffold to generate fluorogenic, pH-responsive reporters. This work demonstrates the potential of the classic xanthene scaffold for use as a SWIR reporter, an important step in the ultimate expansion of the repertoire of small-molecule organic fluorophore scaffolds available for deep-tissue imaging applications.

Identification of fluoxetine as a direct NLRP3 inhibitor to treat atrophic macular degeneration.

The atrophic form of age-related macular degeneration (dry AMD) affects nearly 200 million people worldwide. There is no Food and Drug Administration (FDA)-approved therapy for this disease, which is the leading cause of irreversible blindness among people over 50 y of age. Vision loss in dry AMD results from degeneration of the retinal pigmented epithelium (RPE). RPE cell death is driven in part by accumulation of Alu RNAs, which are noncoding transcripts of a human retrotransposon. Alu RNA induces RPE degeneration by activating the NLRP3-ASC inflammasome. We report that fluoxetine, an FDA-approved drug for treating clinical depression, binds NLRP3 in silico, in vitro, and in vivo and inhibits activation of the NLRP3-ASC inflammasome and inflammatory cytokine release in RPE cells and macrophages, two critical cell types in dry AMD. We also demonstrate that fluoxetine, unlike several other antidepressant drugs, reduces Alu RNA-induced RPE degeneration in mice. Finally, by analyzing two health insurance databases comprising more than 100 million Americans, we report a reduced hazard of developing dry AMD among patients with depression who were treated with fluoxetine. Collectively, these studies identify fluoxetine as a potential drug-repurposing candidate for dry AMD.

A fluorescent probe for monitoring PTP-PEST enzymatic activity.

Phosphatase non-receptor type 12 (PTPN12 or PTP-PEST) is a critical regulator of cell migration, acting as a tumor suppressor in cancer. Decreases in PTP-PEST expression correlate with aggressive phenotypes in hepatocellular carcinoma (HCC). Despite the importance of PTP-PEST in cellular signaling, methods to directly monitor its enzymatic activity are lacking. Herein, we report the design, synthesis, and optimization of a probe to directly monitor PTP-PEST enzymatic activity via a fluorescent readout. This activity sensor, termed pPEST1tide, is capable of detecting as little as 0.2 nM recombinant PTP-PEST. In addition, we demonstrate that this probe can selectively report on PTP-PEST activity using a panel of potential off-target enzymes. In the long-term, this activity probe could be utilized to identify small molecule modulators of PTP-PEST activity as well as provide a prognostic readout for HCC.

An Evolutionary Strategy for Identification of Higher Order, Green Fluorescent Host-Guest Pairs Compatible with Living Systems.

Engineered miniprotein host-small-molecule guest pairs could be utilized to design new processes within cells as well as investigate fundamental aspects of cell signaling mechanisms. However, the development of host-guest pairs capable of functioning in living systems has proven challenging. Moreover, few examples of host-guest pairs with stoichiometries other than 2:1 exist, significantly hindering the ability to study the influence of oligomerization state on signaling fidelity. Herein, we present an approach to identify host-guest systems for relatively small green fluorescent guests by incorporation into cyclic peptides. The optimal host-guest pair produced a 10-fold increase in green fluorescence signal upon binding. Biophysical characterization clearly demonstrated higher order supramolecular assembly, which could be visualized on the surface of living yeast cells using a turn-on fluorescence readout. This work further defines evolutionary design principles to afford host-guest pairs with stoichiometries other than 2:1 and enables the identification of spectrally orthogonal host-guest pairs.

A Luminescence-Based System for Identification of Genetically Encodable Inhibitors of Protein Aggregation.

Molecules that disrupt protein aggregation represent potential tool compounds for the investigation of numerous human disease states. However, the identification of small molecules capable of disrupting protein aggregation has proven challenging. Larger biomolecules such as antibodies and proteins are promising alternatives due to their increased size. Despite the promise of protein-based inhibitors, generalizable assays are needed to more readily identify proteins capable of inhibiting aggregation. Herein, we utilize our previously reported self-assembling NanoLuc luciferase fragments to engineer a platform in which both detection reagents are expressed from the same plasmid, enabling facile co-transformation with a genetically encodable inhibitor. This streamlined system is capable of detecting changes in the solubility of amylin, huntingtin, and amyloid-ß (Aß) proteins in response to mutations, small-molecule inhibitors, and expression of genetically encodable inhibitors. This improved platform provides a means to begin to identify protein-based inhibitors with improved efficacy.

Synthesis and application of a ratiometric probe for hydrogen peroxide.

Molecular imaging of biological analytes provides detailed insights into signaling processes. Ratiometric probes are particularly attractive due to the ability to quantify analyte production. However, design strategies for ratiometric probes can be hindered by spectral overlap of the product and reactant species. In this chapter, we provide protocols for the synthesis and application of RF620, a ratiometric probe for H2O2 displaying dramatic changes in both excitation and emission wavelengths, designed using an approach we term chemoselective alteration of fluorophore scaffolds. The probe contains a chemoselective functional group within a fluorescent xanthene scaffold, resulting in the in situ synthesis of a new fluorophore upon reaction with H2O2. Under physiological conditions, RF620 exhibits far-red to near-infrared excitation and emission, and upon reaction with H2O2, RF620 is chemically converted into tetramethylrhodamine, producing a significant (~66nm) blue-shift in excitation and emission. RF620 can be used for ratiometric, molecular imaging of endogenous H2O2 production in living cells.

A luminescence-based assay for monitoring changes in alpha-synuclein aggregation in living cells.

Parkinson's disease is characterized by the accumulation of protein aggregates in the brain, termed Lewy bodies. Lewy bodies are predominantly composed of α-synuclein and mutations that increase the aggregation potential of α-synuclein have been associated with early on-set disease. Assays capable of reporting on the solubility of α-synuclein in living cells could provide a means to interrogate the influence of mutations on aggregation as well as identify small molecules capable of modulating the aggregation of α-synuclein. Herein, we repurpose our previously reported self-assembling NanoLuc luciferase fragments to engineer a platform for detecting α-synuclein solubility in living cells. This new assay is capable of reporting on changes in α-synuclein solubility caused by disease-relevant mutations as well as inhibitors of aggregation. In the long term, this new assay platform provides a means to investigate the influence of mutations on α-synuclein solubility as well as identify potential tool compounds capable of modulating α-synuclein aggregation.

Imaging GPCR internalization using near-infrared Nebraska red-based reagents.

Internalization of G protein-coupled receptor (GPCRs) represents a nearly universal pathway for receptor downregulation. Imaging this process provides a means for the identification of pharmaceutical agents as well as potential ligands for orphan receptors. However, there is a need for the further development of near-infrared (NIR) probes capable of monitoring internalization in order to enable multiplexing with existing green fluorescent GPCR activity assays. Our laboratory has recently described a series of near-infrared (NIR) fluorophores in which a phosphinate functionality is inserted at the bridging position of the xanthene scaffold. These fluorophores, termed Nebraska Red (NR) dyes, provide attractive reagents for imaging protein localization. Herein, we disclose the development of NR-based HaloTag ligands for imaging membrane proteins on living cells. These new probes are utilized to image membrane pools of the human orexin type 2 receptor, an established target for the treatment of insomnia. We demonstrate the ability of fetal bovine serum (FBS) to noncovalently associate with a spirolactonized NR probe, enabling no-wash imaging with a 45-fold enhancement of fluorescence. Furthermore, we characterize the utility of NR-based HaloTag ligands for real-time monitoring of receptor internalization upon agonist stimulation. These new reagents enable potential multiplexing with existing GPCR activity assays in order to identify new modulators of GPCR activity as well as ligands for orphan receptors.

A Panel of Protein Kinase Chemosensors Distinguishes Different Types of Fatty Liver Disease.

The worldwide incidence of fatty liver disease continues to rise, which may account for concurrent increases in the frequencies of more aggressive liver ailments. Given the existence of histologically identical fatty liver disease subtypes, there is a critical need for the identification of methods that can classify disease and potentially predict progression. Herein, we show that a panel of protein kinase chemosensors can distinguish fatty liver disease subtypes. These direct activity measurements highlight distinct differences between histologically identical fatty liver diseases arising from diets rich in fat versus alcohol and identify a previously unreported decrease in p38α activity associated with a high-fat diet. In addition, we have profiled kinase activities in both benign (diet-induced) and progressive (STAM) disease models. These experiments provide temporal insights into kinase activity during disease development and progression. Altogether, this work provides the basis for the future development of clinical diagnostics and potential treatment strategies.

Design and synthesis of fluorescent activity probes for protein phosphatases.

Protein phosphatases act in concert with protein kinases to regulate and maintain the phosphoproteome. However, the catalog of chemical tools to directly monitor the enzymatic activity of phosphatases has lagged behind their kinase counterparts. In this chapter, we provide protocols for repurposing the phosphorylation-sensitive sulfonamido-oxine fluorophore known as Sox to afford direct activity probes for phosphatases. With validated activity probes in-hand, inhibitor screens can be conducted with recombinant enzyme and the role of phosphatases in cell signaling can be investigated in unfractionated cell lysates.

Utilizing split-NanoLuc luciferase fragments as luminescent probes for protein solubility in living cells.

Protein misfolding and aggregation is now recognized as a hallmark of numerous human diseases. Standard bioanalytical techniques for monitoring protein aggregation generally rely on small molecules that provide an optical readout of fibril formation. While these methods have been useful for mechanistic studies, additional approaches are required to probe the equilibrium between soluble and insoluble protein within living systems. Such approaches could provide platforms for the identification of inhibitors of protein aggregation as well as a means to investigate the effect of mutations on protein aggregation in model systems. In this chapter, we provide detailed protocols for employing split-NanoLuc luciferase (Nluc) fragments to monitor changes in protein solubility in bacterial and mammalian cells. This sensitive luminesce-based assay can report upon changes in protein solubility induced by inhibitors and disease-relevant mutations.

Phosphinate-containing rhodol and fluorescein scaffolds for the development of bioprobes.

A series of phosphinate-containing rhodol and fluorescein dyes are disclosed. These new fluorophores increase the color palette of phosphinate-based xanthenes in the far-red spectral region. The new chemical functionality of these scaffolds is leveraged to produce a sensitive, no-wash imaging probe for cellular esterase activity. The reported phosphinate-containing dyes provide platforms for the further development of imaging probes and self-reporting delivery vehicles.

A Phosphinate-Containing Fluorophore Capable of Selectively Inducing Apoptosis in Cancer Cells.

Chemotherapeutic agents generally suffer from off-target cytotoxicity in noncancerous cell types, leading to undesired side effects. As a result, significant effort has been put into identifying compounds that are selective for cancerous over noncancerous cell types. Our laboratory has recently developed a series of near-infrared (NIR) fluorophores containing a phosphinate functionality at the bridging position of a xanthene scaffold, termed Nebraska Red (NR) fluorophores. Herein, we report the selective cytotoxicity of one NR derivative, NR744 , against HeLa (cervical cancer) cells versus NIH-3T3 (noncancerous fibroblast) cells. Mechanistic studies based on the NIR fluorescence signal of NR744 showed distinct subcellular localization in HeLa (mitochondrial) versus NIH-3T3 (lysosomal) that resulted from the elevated mitochondrial potential in HeLa cells. This study provides a new, NIR scaffold for the further development of reagents for targeted cancer therapy.