Covalent activity-based probes for imaging of serine proteases.

Serine proteases are one of the largest mechanistic classes of proteases. They regulate a plethora of biochemical pathways inside and outside the cell. Aberrant serine protease activity leads to a wide variety of human diseases. Reagents to visualize these activities can be used to gain insight into the biological roles of serine proteases. Moreover, they may find future use for the detection of serine proteases as biomarkers. In this review, we discuss small molecule tools to image serine protease activity. Specifically, we outline different covalent activity-based probes and their selectivity against various serine protease targets. We also describe their application in several imaging methods.

A rationally designed nuclei-targeting FAPI 04-based molecular probe with enhanced tumor uptake for PET/CT and fluorescence imaging.

PURPOSE: Fibroblast activation protein inhibitor (FAPI) -based probes have been widely studied in the diagnosis of various malignant tumors with positron emission tomography/computed tomography (PET/CT). However, current imaging studies of FAPI-based probes face challenges in rapid clearance rate and potential false-negative results. Furthermore, FAPI has been rarely explored in optical imaging. Considering this, further modifications are imperative to improve the properties of FAPI-based probes to address existing limitations and broaden their application scenarios. In this study, we rationally introduced methylene blue (MB) to FAPIs, thereby imparting nuclei-targeting and fluorescence imaging capabilities to the probes. Furthermore, we evaluated the added value of FAPI-based fluorescence imaging to traditional PET/CT, exploring the potential application of FAPI-based probes in intraoperative fluorescence imaging. METHODS: A new FAPI-based probe, namely NOTA-FAPI-MB, was designed for both PET/CT and fluorescence imaging by conjugation of MB. The targeting efficacy of the probe was evaluated on fibroblast activation protein (FAP)-transfected cell line and human primary cancer-associated fibroblasts (CAFs). Subsequently, PET/CT and fluorescence imaging were conducted on tumor-bearing mice. The tumor detection and boundary delineation were assessed by fluorescence imaging of tissues from hepatocellular carcinoma (HCC) patients. RESULTS: NOTA-FAPI-MB demonstrated exceptional targeting ability towards FAP-transfected cells and CAFs in comparison to NOTA-FAPI. This benefit arises from the cationic methylene blue (MB) affinity for anionic nucleic acids. PET/CT imaging of tumor-bearing mice revealed significantly higher tumor uptake of [18F]F-NOTA-FAPI-MB (standard uptake value of 2.20 ± 0.31) compared to [18F]F-FDG (standard uptake value of 1.66 ± 0.14). In vivo fluorescence imaging indicated prolonged retention at the tumor site, with retention lasting up to 24 h. In addition, the fluorescent probes enabled more precise lesion detection and tumor margin delineation than clinically used indocyanine green (ICG), achieving a 100.0% (6/6) tumor-positive rate for NOTA-FAPI-MB while 33.3% (2/6) for ICG. These findings highlighted the potential of NOTA-FAPI-MB in guiding intraoperative surgical procedures. CONCLUSIONS: The NOTA-FAPI-MB was successfully synthesized, in which FAPI and MB simultaneously contributed to the targeting effect. Notably, the nuclear delivery mechanism of the probes improved intracellular retention time and targeting efficacy, broadening the imaging time window for fluorescence imaging. In vivo PET/CT demonstrated favorable performance of NOTA-FAPI-MB compared to [18F]F-FDG. This study highlights the significance of fluorescence imaging as an adjunct technique to PET/CT. Furthermore, the encouraging results obtained from the imaging of human HCC tissues hold promise for the potential application of NOTA-FAPI-MB in intraoperative fluorescent surgery guidance within clinical settings.

A NIR-II Photoacoustic Probe for High Spatial Quantitative Imaging of Tumor Nitric Oxide in Vivo.

Nitric oxide (NO) exhibits both pro- and anti-tumor effects. Therefore, real-time in vivo imaging and quantification of tumor NO dynamics are essential for understanding the conflicting roles of NO played in pathophysiology. The current molecular probes, however, cannot provide high-resolution imaging in deep tissues, making them unsuitable for these purposes. Herein, we designed a photoacoustic probe with an absorption maximum beyond 1000 nm for high spatial quantitative imaging of in vivo tumor NO dynamics. The probe exhibits remarkable sensitivity, selective ratiometric response behavior, and good tumor-targeting abilities, facilitating ratiometric imaging of tumor NO throughout tumor progression in a micron-resolution level. Using the probe as the imaging agent, we successfully quantified NO dynamics in tumor, liver and kidney. We have pinpointed an essential concentration threshold of around 80 nmol/cm3 for NO, which plays a crucial role in the "double-edged-sword" function of NO in tumors. Furthermore, we revealed a reciprocal relationship between the NO concentration in tumors and that in the liver, providing initial insights into the possible NO-mediated communication between tumor and the liver. We believe that the probe will help resolve conflicting aspects of NO biology and guide the design of imaging agents for tumor diagnosis and anti-cancer drug screening.

Molecular Rotors: Fluorescent Sensors for Microviscosity and Conformation of Biomolecules.

The viscosity and crowding of biological environment are considered vital for the correct cellular function, and alterations in these parameters are known to underly a number of pathologies including diabetes, malaria, cancer and neurodegenerative diseases, to name a few. Over the last decades, fluorescent molecular probes termed molecular rotors proved extremely useful for exploring viscosity, crowding, and underlying molecular interactions in biologically relevant settings. In this review, we will discuss the basic principles underpinning the functionality of these probes and will review advances in their use as sensors for lipid order, protein crowding and conformation, temperature and non-canonical nucleic acid structures in live cells and other relevant biological settings.

The linkage-type and the exchange molecule affect the protein-labeling efficiency of iminoboronate probes.

Reversible bioorthogonal conjugation reactions have been exploited in the chemoproteomic field to prepare protein labeling reagents and to visualize labeled proteins. We recently demonstrated that reversible iminoboronates can be used to prepare probes from fragment libraries and that the linkage subsequently can be used to detect the labeled proteins. In this study, we determined the effect of the stability of the iminoboronate linkage on the efficiency of the labeling protocol. Our study reveals that the linkage should be stable enough to allow for efficient targeting, but should be labile enough to detect the labeled protein. Acyl hydrazides were identified as the most suitable handles for the probe synthesis step. Anthranilic hydrazides and N-hydroxy semicarbazides were found to be the most efficient read-out molecules. With these novel exchange molecules, native probe-labeled proteins could be visualized under physiological conditions.

Design, synthesis, and evaluation of a mitoxantrone probe (MXP) for biological studies.

Mitoxantrone (MX) is a robust chemotherapeutic with well-characterized applications in treating certain leukemias and advanced breast and prostate cancers. The canonical mechanism of action associated with MX is its ability to intercalate DNA and inhibit topoisomerase II, giving it the designation of a topoisomerase II poison. Years after FDA approval, investigations have unveiled novel protein-binding partners, such as methyl-CpG-binding domain protein (MBD2), PIM1 serine/threonine kinase, RAD52, and others that may contribute to the therapeutic profile of MX. Moreover, recent proteomic studies have revealed MX's ability to modulate protein expression, illuminating the complex cellular interactions of MX. Although mechanistically relevant, the differential expression across the proteome does not address the direct interaction with potential binding partners. Identification and characterization of these MX-binding cellular partners will provide the molecular basis for the alternate mechanisms that influence MX's cytotoxicity. Here, we describe the design and synthesis of a MX-biotin probe (MXP) and negative control (MXP-NC) that can be used to define MX's cellular targets and expand our understanding of the proteome-wide profile for MX. In proof of concept studies, we used MXP to successfully isolate a recently identified protein-binding partner of MX, RAD52, in a cell lysate pulldown with streptavidin beads and western blotting.

Chemical synthesis of on demand-activated SUMO-based probe by a photocaged glycine-assisted strategy.

The transiently-activated SUMO probes are conducive to understand the dynamic control of SENPs activity. Here, we developed a photocaged glycine-assisted strategy for the construction of on demand-activated SUMO-ABPs. The light-sensitive groups installed at G92 and G64 backbone of SUMO-2 can temporarily block probes activity and hamper aspartimide formation, respectively, which enabled the efficient synthesis of inert SUMO-2 propargylamide (PA). The probe could be activated to capture SENPs upon photo-irradiation not only in vitro but also in intact cells, providing opportunities to further perform intracellular time-resolved proteome-wide profiling of SUMO-related enzymes.

A molecular rotor FLIM probe reveals dynamic coupling between mitochondrial inner membrane fluidity and cellular respiration.

The inner mitochondrial membrane (IMM), housing components of the electron transport chain (ETC), is the site for respiration. The ETC relies on mobile carriers; therefore, it has long been argued that the fluidity of the densely packed IMM can potentially influence ETC flux and cell physiology. However, it is unclear if cells temporally modulate IMM fluidity upon metabolic or other stimulation. Using a photostable, red-shifted, cell-permeable molecular-rotor, Mitorotor-1, we present a multiplexed approach for quantitatively mapping IMM fluidity in living cells. This reveals IMM fluidity to be linked to cellular-respiration and responsive to stimuli. Multiple approaches combining in vitro experiments and live-cell fluorescence (FLIM) lifetime imaging microscopy (FLIM) show Mitorotor-1 to robustly report IMM 'microviscosity'/fluidity through changes in molecular free volume. Interestingly, external osmotic stimuli cause controlled swelling/compaction of mitochondria, thereby revealing a graded Mitorotor-1 response to IMM microviscosity. Lateral diffusion measurements of IMM correlate with microviscosity reported via Mitorotor-1 FLIM-lifetime, showing convergence of independent approaches for measuring IMM local-order. Mitorotor-1 FLIM reveals mitochondrial heterogeneity in IMM fluidity; between-and-within cells and across single mitochondrion. Multiplexed FLIM lifetime imaging of Mitorotor-1 and NADH autofluorescence reveals that IMM fluidity positively correlates with respiration, across individual cells. Remarkably, we find that stimulating respiration, through nutrient deprivation or chemically, also leads to increase in IMM fluidity. These data suggest that modulating IMM fluidity supports enhanced respiratory flux. Our study presents a robust method for measuring IMM fluidity and suggests a dynamic regulatory paradigm of modulating IMM local order on changing metabolic demand.

Visualize intracellular ß-galactosidase using an asymmetric near-infrared fluorescent probe with a large Stokes shift.

ß-galactosidase (ß-Gal) is an important biomarker of cell senescence and primary ovarian cancer. Therefore, it is of great significance to construct a near-infrared fluorescent probe with deep tissue penetration and a high signal-to-noise ratio for visualization of ß-galactosidase in biological systems. However, most near-infrared probes tend to have small Stokes shifts and low signal-to-noise ratios due to crosstalk between excitation and emission spectra. Using d-galactose residues as specific recognition units and near-infrared dye TJ730 as fluorophores, a near-infrared fluorescence probe SN-CR with asymmetric structure was developed for the detection of ß-Gal. The probe has a fast reaction equilibrium time (<12 min) with ß-Gal, excellent biocompatibility, near-infrared emission (738 nm), low detection limit (0.0029 U/mL), and no crosstalk between the excitation spectrum and emission spectrum (Stokes shifts 142 nm) of the probe. Cell imaging studies have shown that SN-CR can visually trace ß-Gal in different cells and distinguish ovarian cancer cells from other cells.

Development of a novel chitosan-based two-photon fluorescent nanoprobe with enhanced stability for the specific detection of endogenous H2O2.

Hydrogen peroxide (H2O2) acts as an important reactive oxygen species (ROS) and maintains the redox equilibrium in organisms. Imbalance of H2O2 concentration is associated with the development of many diseases. Traditional small molecular based fluorescent probes often show drawbacks of cytotoxicity and easily metabolic clearance. Herein, a chitosan-based two-photon fluorescent nanoprobe (DC-BI) was constructed and applied for H2O2 detection in live organisms. DC-BI was composed by chitosan nanoparticles and a two-photon fluorophore of naphthalimide analogues (BI) with H2O2-responsive property. The structure of DC-BI was characterized by NMR, FTIR, XPS, XRD, DLS and MLS analyses. As study shown, the nanoprobe DC-BI exhibited improved distribution stability and smaller cytotoxicity. In the presence of H2O2, both the absorption and emission spectra show dramatic changes, the fluorescence intensity at 580 nm obviously enhanced. Furthermore, fluorescence imaging results indicate that DC-BI is capable of imaging endogenous H2O2 in cells and zebrafish. The design and development of chitosan-based nanoprobe DC-BI has provided a general example of nanoprobe construction with excellent distribution stability, two-photon property, and biocompatibility.

An activatable near-infrared molecular reporter for fluoro-photoacoustic imaging of liver fibrosis.

Noninvasive and accurate detection of liver fibrosis is extremely significant for well-timed intervention and treatment to prevent or reverse its progression. Fluorescence imaging probes hold great potential for imaging of liver fibrosis, but they always encounter the inherent limitation of shallow penetration depth, which compromises their ability of in vivo detection. To overcome this issue, an activatable fluoro-photoacoustic bimodal imaging probe (IP) is herein developed for specific visualization of liver fibrosis. The probe IP is constructed on a near-infrared thioxanthene-hemicyanine dye that is caged with gamma-glutamyl transpeptidase (GGT) responsive substrate and linked with integrin-targeted peptide (cRGD). Such molecular design permits IP to effectively accumulate in the liver fibrosis region through specific recognition of cRGD towards integrin and activate its fluoro-photoacoustic signal after interaction with overexpressed GGT to precisely monitor the liver fibrosis. Thus, our study presents a potential strategy to design dual-target fluoro-photoacoustic imaging probes for noninvasive detection of early-stage liver fibrosis.

HiBiT-SpyTag: A Minimal Tag for Covalent Protein Capture and Degrader Development.

The ability to rapidly and selectively modulate cellular protein levels using small molecules is essential for studying complex biological systems. Degradation tags, such as dTAG, allow for selective protein removal with a specific degrader molecule, but their utility is limited by the large tag size (>12 kDa) and the low efficiency of fusion product gene knock-in. Here, we describe the development of a short 24 amino acid peptide tag that enables cell-based quantification and covalent functionalization of proteins to which it is fused. The minimalistic peptide, termed HiBiT-SpyTag, incorporates the HiBiT peptide for protein level quantification and SpyTag, which forms a spontaneous isopeptide bond in the presence of the SpyCatcher protein. Transient expression of dTAG-SpyCatcher efficiently labels HiBiT-SpyTag-modified BRD4 or IRE1α in cells, and subsequent treatment with the dTAG13 degrader results in efficient protein removal without the need for full dTAG knock-in. We also demonstrate the utility of HiBiT-SpyTag for validating the degradation of the endoplasmic reticulum (ER) stress sensor IRE1α, which led to the development of the first PROTAC degrader of the protein. Our modular HiBiT-SpyTag system represents a valuable tool for the efficient development of degraders and for studying other proximity-induced pharmacology.

Synthetic Approaches of Epoxysuccinate Chemical Probes.

Synthetic chemical probes are powerful tools for investigating biological processes. They are particularly useful for proteomic studies such as activity-based protein profiling (ABPP). These chemical methods initially used mimics of natural substrates. As the techniques gained prominence, more and more elaborate chemical probes with increased specificity towards given enzyme/protein families and amenability to various reaction conditions were used. Among the chemical probes, peptidyl-epoxysuccinates represent one of the first types of compounds used to investigate the activity of the cysteine protease papain-like family of enzymes. Structurally derived from the natural substrate, a wide body of inhibitors and activity- or affinity-based probes bearing the electrophilic oxirane unit for covalent labeling of active enzymes now exists. Herein, we review the literature regarding the synthetic approaches to epoxysuccinate-based chemical probes together with their reported applications, from biological chemistry and inhibition studies to supramolecular chemistry and the formation of protein arrays.

Deep Annotation of Donated Chemical Probes (DCP) in Organotypic Human Liver Cultures and Patient-Derived Organoids from Tumor and Normal Colorectum.

Well-characterized small molecules are essential tools for studying the biology and therapeutic relevance of a target protein. However, many compounds reported in the literature and routinely studied in biomedical research lack the potency and selectivity required for mechanistic cellular studies on the function of a given protein. Furthermore, commercially available compounds often do not include useful tools developed by industry as part of their research and development efforts, as they frequently remain proprietary. The freely available donated chemical probe (DCP) library, fueled by generous donations of compounds from industry and academia, enables easy access to a steadily growing collection of these valuable and well-characterized tools. Here, we provide a systematic description of the current DCP library collection and their associated comprehensive characterization data, including a variety of in vitro and cellular assays. Of note, we characterized the set in relevant human primary models by employing hepatotoxicity screening in primary human liver spheroids and viability screening in patient-derived colorectal cancer organoids and matched normal-adjacent epithelium. Taken together, the DCP library represents a well-annotated, openly available collection of tool compounds for studying a wide range of targets, including kinases, G-protein-coupled receptors, and ion channels. As such, it represents a unique resource for the biomedical research community.

Enzyme-Triggered Transforming of Assembly Peptide-Modified Magnetic Resonance-Tuned Probe for Highly Sensitive Imaging of Bacterial Infection In Vivo.

Confirming bacterial infection at an early stage and distinguishing between sterile inflammation and bacterial infection is still highly needed for efficient treatment. Here, in situ highly sensitive magnetic resonance imaging (MRI) bacterial infection in vivo based on a peptide-modified magnetic resonance tuning (MRET) probe (MPD-1) that responds to matrix metallopeptidase 2 (MMP-2) highly expressed in bacteria-infected microenvironments is achieved. MPD-1 is an assembly of magnetic nanoparticle (MNP) bearing with gadolinium ion (Gd3+ ) modified MMP-2-cleavable self-assembled peptide (P1 ) and bacteria-targeting peptide (P), and it shows T2 -weighted signal due to the assemble of MNP and MRET ON phenomenon between MNP assembly and Gd3+ . Once MPD-1 accumulates at the bacterially infected site, P1 included in MPD-1 is cleaved explicitly by MMP-2, which triggers the T2 contrast agent of MPD-1 to disassemble into the monomer of MNP, leading the recovery of T1 -weighted signal. Simultaneously, Gd3+ detaches from MNP, further enhancing the T1 -weighted signal due to MRET OFF. The sensitive MRI of Staphylococcus aureus (low to 104 CFU) at the myositis site and accurate differentiation between sterile inflammation and bacterial infection based on the proposed MPD-1 probe suggests that this novel probe would be a promising candidate for efficiently detecting bacterial infection in vivo.

Triubiquitin Probes for Identification of Reader and Eraser Proteins of Branched Polyubiquitin Chains.

The important roles played by branched polyubiquitin chains were recently uncovered in proteasomal protein degradation, mitotic regulation, and NF-κB signaling. With the new realization of a wide presence of branched ubiquitin chains in mammalian cells, there is an urgent need of identifying the reader and eraser proteins of the various branched ubiquitin chains. In this work, we report the generation of noncleavable branched triubiquitin probes with combinations of K11-, K48-, and K63-linkages. Through a pulldown approach using the branched triUb probes, we identified human proteins that recognize branched triubiquitin structures including ubiquitin-binding proteins and deubiquitinases (DUBs). Proteomics analysis of the identified proteins enriched by the branched triubiquitin probes points to possible roles of branched ubiquitin chains in cellular processes including DNA damage response, autophagy, and receptor endocytosis. In vitro characterization of several identified UIM-containing proteins demonstrated their binding to branch triubiquitin chains with moderate to high affinities. Availability of this new class of branched triubiquitin probes will enable future investigation into the roles of branched polyubiquitin chains through identification of specific reader and eraser proteins, and the modes of branched ubiquitin chain recognition and processing using biochemical and biophysical methods.

Menin "reads" H3K79me2 mark in a nucleosomal context.

Methylation of histone H3 lysine-79 (H3K79) is an epigenetic mark for gene regulation in development, cellular differentiation, and disease progression. However, how this histone mark is translated into downstream effects remains poorly understood owing to a lack of knowledge about its readers. We developed a nucleosome-based photoaffinity probe to capture proteins that recognize H3K79 dimethylation (H3K79me2) in a nucleosomal context. In combination with a quantitative proteomics approach, this probe identified menin as a H3K79me2 reader. A cryo-electron microscopy structure of menin bound to an H3K79me2 nucleosome revealed that menin engages with the nucleosome using its fingers and palm domains and recognizes the methylation mark through a π-cation interaction. In cells, menin is selectively associated with H3K79me2 on chromatin, particularly in gene bodies.

HIV-1 gp120-CXCR4 recognition probed with synthetic nanomolar affinity D-peptides containing fragments of gp120 V3 loop.

The human immunodeficiency virus type 1 (HIV-1) recognizes one of its principal coreceptors, the CXC chemokine receptor 4 (CXCR4) on the host cell via the third variable loop (V3 loop) of HIV-1 envelope glycoprotein gp120 during the viral entry process. Here, we investigated the stereochemical mechanism of the molecular recognition of HIV-1 gp120 V3 loop with coreceptor CXCR4 by using peptide probes containing important fragments of the V3 loop. The tip and base/stem fragments of the V3 loop critical for V3 loop function were linked individually with the fragment derived from another CXCR4's chemokine ligand, vMIP-II to generate nanomolar affinity peptide probes of the interactions of CXCR4-V3 loop fragments. When the amino acid residues of the V3 loop fragments in these combinational peptides were changed from L-to D-configurations, the resulting peptides remarkably retained or had even enhanced recognition by CXCR4 as shown by competitive ligand-receptor binding. The ability of these peptides, regardless of the different l- or d-amino acids used, in binding CXCR4 and antagonizing CXCR4 functions was demonstrated by their blockade of calcium influx, cell migration, and CXCR4 internalization triggered by the activation of CXCR4 signaling by its endogenous ligand SDF-1α. The structural mechanisms of CXCR4 interactions with these peptides were examined with site-directed mutagenesis and molecular modeling. These results indicate that CXCR4's interface with key segments of HIV-1 gp120 V3 loop is flexible in terms of stereospecificity of ligand-receptor interaction which may have implication on understanding the viral entry mechanism and how the virus evades immune detection with V3 loop mutations and retains effective recognition of the host cell's coreceptor.

Development of a nitroreductase-dependent theranostic payload for antibody-drug conjugate.

Antibody-drug conjugates are gradually revolutionizing anticancer therapy. Payload is one of the most crucial components of ADC for high antitumor activity. However, there is no direct and real-time monitoring method for the intracellular release mechanism of the payload. Herein, we developed a theranostic payload that possessed dual functions of therapy and imaging. This payload consisted of the classic payload MMAE and the novel nitro-coumarin probe reported for the first time, which has the dual characteristics of electron transfer ability and the on-off fluorescence property. In this paper, the theranostic property of the novel payload has been preliminarily demonstrated. The fluorescence intensity of the payload in target cells greatly increased approximately 9 times in 120 min through the high content analysis, and the intracellular distribution of the payload could be directly monitored by a confocal microscope. In in vitro cytotoxicity assays, the payload showed broad-spectrum and high antitumor activity (0.09 nM to 1.2 nM), which was equivalent to the MMAE (0.06 nM to 1.1 nM). Moreover, the ADC loaded with L-233 maintained the theranositc property. In conclusion, our work developed a theranostic payload for the first time and provides a new direct and real-time monitoring method for intracellular studies of ADC payloads.

Design and Characterization of Hyperpolarized 15N-BBCP as a H2O2-Sensing Probe.

Hydrogen peroxide (H2O2) is a type of reactive oxygen species that regulates essential biological processes. Despite the central role of H2O2 in pathophysiological states, available molecular probes for assessing H2O2 in vivo are still limited. This work develops hyperpolarized 15N-boronobenzyl-4-cyanopyridinium (15N-BBCP) as a rationally designed molecular probe for detecting H2O2. The 15N-BBCP demonstrated favorable physicochemical and biochemical properties for H2O2 detection and dynamic nuclear polarization, allowing noninvasive detection of H2O2. In particular, 15N-BBCP and the products possessed long spin-lattice relaxation times and spectrally resolvable 15N chemical shift differences. The performance of hyperpolarized 15N-BBCP was demonstrated both in vitro and in vivo with time-resolved 15N-MRS. This study highlights a promising approach to designing a reaction-based 15N-labeled molecular imaging agent for detecting oxidative stress in vivo.