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.

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.

Holographically Activatable Nanoprobe via Glutathione/Albumin-Mediated Exponential Signal Amplification for High-Contrast Tumor Imaging.

Glutathione (GSH)-activatable probes hold great promise for in vivo cancer imaging, but are restricted by their dependence on non-selective intracellular GSH enrichment and uncontrollable background noise. Here, a holographically activatable nanoprobe caging manganese tetraoxide is shown for tumor-selective contrast enhancement in magnetic resonance imaging (MRI) through cooperative GSH/albumin-mediated cascade signal amplification in tumors and rapid elimination in normal tissues. Once targeting tumors, the endocytosed nanoprobe effectively senses the lysosomal microenvironment to undergo instantaneous decomposition into Mn2+ with threshold GSH concentration of ≈ 0.12 mm for brightening MRI signals, thus achieving high contrast tumor imaging and flexible monitoring of GSH-relevant cisplatin resistance during chemotherapy. Upon efficient up-regulation of extracellular GSH in tumor via exogenous injection, the relaxivity-silent interstitial nanoprobe remarkably evolves into Mn2+ that are further captured/retained and re-activated into ultrahigh-relaxivity-capable complex by stromal albumin in the tumor, and simultaneously allows the renal clearance of off-targeted nanoprobe in the form of Mn2+ via lymphatic vessels for suppressing background noise to distinguish tiny liver metastasis. These findings demonstrate the concept of holographic tumor activation via both tumor GSH/albumin-mediated cascade signal amplification and simultaneous background suppression for precise tumor malignancy detection, surveillance, and surgical guidance.

Novel zinc(II)-curcumin molecular probes bearing berberine and jatrorrhizine derivatives as potential mitochondria-targeting anti-neoplastic drugs.

Berberine and jatrorrhizine are major bioactive components that are emerging as potential anti-cancer drugs. However, no zinc(II) - berberine/jatrorrhizine - curcumin compounds have been reported in the literature to date. Therefore, the molecular mechanisms associated with their cytotoxicity remain unexplored. To investigate the potential mitochondria-targeting ability, anti-neoplastic activity, and utility in cell imaging of berberine and jatrorrhizine derivates, four novel zinc(II) complexes, [Zn(Ber)(H2O)Cl2] (Zn(Ber)), [Zn(Ber)(Cur)Cl] (Zn(CurBer)), [Zn(Jat)(H2O)Cl2] (Zn(Jat)), and [Zn(Jat)(Cur)Cl] (Zn(CurJat)) bearing the berberine-derived ligand 2,2,2-trifluoroacetate 10-methoxy-9-((9-((2-(pyridin-2-yl)ethyl)amino)nonyl)oxy) -5,6-dihydro- [1,3]dioxolo[4,5-g]isoquinolino [(Torre et al., 2015; de Ruijter et al., 2020) 3,23,2-a]isoquinolin-7-ium (Ber), the jatrorrhizine-derived ligand 2,2,2-trifluoroacetate 2,9,10-trimethoxy-3-((9- ((2-(pyridin-2-yl)ethyl)amino)nonyl)oxy)-5,6-dihydroisoquinolino [(Torre et al., 2015; de Ruijter et al., 2020) 3,23,2-a]isoquinolin-7-ium (Jat), and/or curcumin (H-Cur) were first synthesised in this study. Zn(Ber), Zn(CurBer), Zn(Jat), and Zn(CurJat) showed higher cytotoxicity against human MCF-7 (breast adenocarcinoma) cells than did cisplatin, with IC50 values ranging from 0.21 to 4.45 µM. The anti-neoplastic activities of the zinc(II) - berberine/jatrorrhizine - curcumin complexes were in the following order: Zn(CurBer) > Zn(CurJat) > Zn(Ber) > Zn(Jat) > cisplatin > H-Cur > Ber > Jat > ZnCl2. Among these, Zn(CurBer) displayed the highest cytotoxicity (0.21 ± 0.06 µM). Furthermore, mechanistic investigations revealed that Zn(CurBer) and Zn(CurJat) could accumulate in the mitochondria, exhibit red fluorescence, and trigger mitophagy and apoptosis. In vivo anti-cancer evaluations also suggested that Zn(CurBer) inhibited MCF-7 xenograft tumour growth more effectively than cisplatin and Zn(CurJat). This is the first report describing the synthesis of zinc(II) - berberine/jatrorrhizine - curcumin complexes and their potential use as molecular probes and mitochondria-targeting anti-neoplastic drugs.

Photocaged dicarbonyl probe provides spatiotemporal control over protein glycation.

Protein glycation is a disease associated, non-enzymatic, posttranslational modification generated by endogenous dicarbonyl metabolites. Currently, there is a lack of chemical tools capable of studying protein adducts caused by this class of reactive species. Here, we report a chemical biology platform, termed T-DiP (targetable-dicarbonyl precursor), that releases a physiologically relevant dose of bio-orthogonally functionalized dicarbonyl probe upon irradiation with 365 nm light. This approach enables protein glycation to be controlled with spatiotemporal precision within live cells and expands the chemical toolbox needed to elucidate the roles of glycated proteins across various pathologies.

A Fluorine-19 Magnetic Resonance Probe, Shiga-Y5, Downregulates Thioredoxin-Interacting Protein Expression in the Brain of a Mouse Model of Alzheimer's Disease.

Thioredoxin-interacting protein (TXNIP) is involved in multiple disease-associated functions related to oxidative stress, especially by inhibiting the anti-oxidant- and thiol-reducing activity of thioredoxin (TXN). Shiga-Y5 (SY5), a fluorine-19 magnetic resonance probe for detecting amyloid-ß deposition in the brain, previously showed therapeutic effects in a mouse model of Alzheimer's disease; however, the mechanism of action of SY5 remains unclear. SY5 passes the blood-brain barrier and then undergoes hydrolysis to produce a derivative, Shiga-Y6 (SY6), which is a TXNIP-negative regulator. Therefore, this study investigates the therapeutic role of SY5 as the prodrug of SY6 in the thioredoxin system in the brain of a mouse model of Alzheimer's disease. The intraperitoneal injection of SY5 significantly inhibited TXNIP mRNA (p = 0.0072) and protein expression (p = 0.0143) induced in the brain of APP/PS1 mice. In contrast, the levels of TXN mRNA (p = 0.0285) and protein (p = 0.0039) in the brain of APP/PS1 mice were increased after the injection of SY5. The ratio of TXN to TXNIP, which was decreased (p = 0.0131) in the brain of APP/PS1 mice, was significantly increased (p = 0.0072) after the injection of SY5. These results suggest that SY5 acts as a prodrug of SY6 in targeting the thioredoxin system and could be a potential therapeutic compound in oxidative stress-related diseases in the brain.

Chemical Biology Tools for Modulating and Visualizing Gram-Negative Bacterial Surface Polysaccharides.

Bacterial cells present a wide diversity of saccharides that decorate the cell surface and help mediate interactions with the environment. Many Gram-negative cells express O-antigens, which are long sugar polymers that makeup the distal portion of lipopolysaccharide (LPS) that constitutes the surface of the outer membrane. This review highlights chemical biology tools that have been developed in recent years to facilitate the modulation of O-antigen synthesis and composition, as well as related bacterial polysaccharide pathways, and the detection of unique glycan sequences. Advances in the biochemistry and structural biology of O-antigen biosynthetic machinery are also described, which provide guidance for the design of novel chemical and biomolecular probes. Many of the tools noted here have not yet been utilized in biological systems and offer researchers the opportunity to investigate the complex sugar architecture of Gram-negative cells.

Design and Development of a Chemical Probe for Pseudokinase Ca2+/calmodulin-Dependent Ser/Thr Kinase.

CASK (Ca2+/calmodulin-dependent Ser/Thr kinase) is a member of the MAGUK (membrane-associated guanylate kinase) family that functions as neurexin kinases with roles implicated in neuronal synapses and trafficking. The lack of a canonical DFG motif, which is altered to GFG in CASK, led to the classification as a pseudokinase. However, functional studies revealed that CASK can still phosphorylate substrates in the absence of divalent metals. CASK dysfunction has been linked to many diseases, including colorectal cancer, Parkinson's disease, and X-linked mental retardation, suggesting CASK as a potential drug target. Here, we exploited structure-based design for the development of highly potent and selective CASK inhibitors based on 2,4-diaminopyrimidine-5-carboxamides targeting an unusual pocket created by the GFG motif. The presented inhibitor design offers a more general strategy for the development of pseudokinase ligands that harbor unusual sequence motifs. It also provides a first chemical probe for studying the biological roles of CASK.

Chemical tools for epichaperome-mediated interactome dysfunctions of the central nervous system.

Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer's disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems.

Development of Covalent, Clickable Probes for Adenosine A1 and A3 Receptors.

Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A1 receptor (A1R) and adenosine A3 receptor (A3R) based on an orthosteric antagonist xanthine-based scaffold and on an existing structure-activity relationship. Bifunctional ligands were functional antagonists with nanomolar affinity and irreversible binding at the A1R and A3R. In-depth pharmacological profiling of these bifunctional ligands showed moderate selectivity over A2A and A2B adenosine receptors. Once bound to the receptor, ligands were successfully "clicked" with a cyanine-5 fluorophore containing the complementary "click" partner, enabling receptor detection. These bifunctional ligands are expected to aid in the understanding of A1R and A3R localization and trafficking in native cells and living systems.

Development of a 1,2-difluorofucoside activity-based probe for profiling GH29 fucosidases.

GH29 α-l-fucosidases catalyze hydrolysis of terminal α-l-fucosyl linkages with varying specificity and are expressed by prominent members of the human gut microbiota. Both homeostasis and dysbiosis at the human intestinal microbiota interface have been correlated with altered fucosidase activity. Herein we describe the development of a 2-deoxy-2-fluoro fucosyl fluoride derivative with an azide mini-tag as an activity-based probe (ABP) for selective in vitro labelling of GH29 α-l-fucosidases. Only catalytically active fucosidases are inactivated by this ABP, allowing their functionalization with a biotin reporter group via the CuAAC reaction and subsequent in-gel detection at nanogram levels. The ABP we present here is shown to be active against a GH29 α-l-fucosidase from Bacteroides fragilis and capable of labeling two other GH29 α-l-fucosidases with different linkage specificity, illustrating its broader utility. This novel ABP is a valuable addition to the toolbox of fucosidase probes by allowing identification and functional studies of the wide variety of GH29 fucosidases, including those in the gut microbiota.

Small-molecule probe reveals a kinase cascade that links stress signaling to TCF/LEF and Wnt responsiveness.

Wnt signaling plays a central role in tissue maintenance and cancer. Wnt activates downstream genes through ß-catenin, which interacts with TCF/LEF transcription factors. A major question is how this signaling is coordinated relative to tissue organization and renewal. We used a recently described class of small molecules that binds tubulin to reveal a molecular cascade linking stress signaling through ATM, HIPK2, and p53 to the regulation of TCF/LEF transcriptional activity. These data suggest a mechanism by which mitotic and genotoxic stress can indirectly modulate Wnt responsiveness to exert coherent control over cell shape and renewal. These findings have implications for understanding tissue morphogenesis and small-molecule anticancer therapeutics.

Public resources for chemical probes: the journey so far and the road ahead.

High-quality small molecule chemical probes are extremely valuable for biological research and target validation. However, frequent use of flawed small-molecule inhibitors produces misleading results and diminishes the robustness of biomedical research. Several public resources are available to facilitate assessment and selection of better chemical probes for specific protein targets. Here, we review chemical probe resources, discuss their current strengths and limitations, and make recommendations for further improvements. Expert review resources provide in-depth analysis but currently cover only a limited portion of the liganded proteome. Computational resources encompass more proteins and are regularly updated, but have limitations in data availability and curation. We show how biomedical scientists may use these resources to choose the best available chemical probes for their research.

Identifying novel B-cell targets for chronic inflammatory autoimmune disease by screening of chemical probes in a patient-derived cell assay.

B-cell secretion of autoantibodies drives autoimmune diseases, including systemic lupus erythematosus and idiopathic inflammatory myositis. Few therapies are presently available for treatment of these patients, often resulting in unsatisfactory effects and helping only some of the patients. We developed a screening assay for evaluation of novel targets suspending B-cell maturation into antibody secreting cells, which could contribute to future drug development. The assay was employed for testing 43 high quality chemical probes and compounds inhibiting under-explored protein targets, using primary cells from patients with autoimmune disease. Probes inhibiting bromodomain family proteins and histone methyl transferases demonstrated abrogation of B-cell functions to a degree comparable to a positive control, the JAK inhibitor tofacitinib. Inhibition of each target rendered a specific functional cell and potential disease modifying effect, indicating specific epigenetic protein targets as potential new intervention points for future drug discovery and development efforts.

Advances of aptamer-based clinical applications for the diagnosis and therapy of cancer.

Aptamers are short single-stranded oligonucleotides that have attracted considerable attention due to their favorable biological characteristics. Aptamers can specifically target and bind to proteins or tumor cells, achieving tumor diagnosis and therapy in vitro and in vivo. Following an introduction of methodologies of producing aptamers and the recent advances of aptamers being applied to clinical samples or xenograft tumors, tumor diagnosis using aptamers will be reviewed, including fluorescence imaging, radionuclide-based imaging, MRI, histochemical imaging, and multimodality imaging. Preclinical applications in tumor therapy in vivo will also be discussed, covering different kinds of treatment mechanisms, including aptamer therapeutics, chemotherapy, gene therapy, immunotherapy, and combination therapy. Safety and efficacy of tumor-targeting therapeutics via aptamers, as well as the current challenges and future perspectives about aptamers' clinical applications, will be summarized.

Reactivity-Based Probe of the Iron(II)-Dependent Interactome Identifies New Cellular Modulators of Ferroptosis.

Ferroptosis is an iron-dependent form of cell death resulting from loss or inhibition of cellular machinery that protects from the accumulation of lipid hydroperoxides. Ferroptosis likely serves a tumor suppressing function in normal cellular homeostasis, but certain cancers exploit and become highly dependent on specific nodes of the pathway, presumably to survive under conditions of increased oxidative stress and elevated labile ferrous iron levels. Here we introduce Ferroptosis Inducing Peroxide for Chemoproteomics-1 (FIPC-1), a reactivity-based probe that couples Fenton-type reaction with ferrous iron to subsequent protein labeling via concomitant carbon-centered radical generation. We show that FIPC-1 induces ferroptosis in susceptible cell types and labels cellular proteins in an iron-dependent fashion. Use of FIPC-1 in a quantitative chemoproteomics workflow reproducibly enriched protein targets in the thioredoxin, oxidoreductase, and protein disulfide isomerase (PDI) families, among others. In further interrogating the saturable targets of FIPC-1, we identified the PDI family member P4HB and the functionally uncharacterized protein NT5DC2, a member of the haloacid dehalogenase (HAD) superfamily, as previously unrecognized modulators of ferroptosis. Knockdown of these target genes sensitized cells to known ferroptosis inducers, while PACMA31, a previously reported inhibitor of P4HB, directly induced ferroptosis and was highly synergistic with erastin. Overall, this study introduces a new reactivity-based probe of the ferrous iron-dependent interactome and uncovers new targets for the therapeutic modulation of ferroptosis.

Rapid Solid-Phase Construction of Serine Hydrolase Probes Results in Selective Activity-Based Probes for Acyl Protein Thioesterases-1/2.

Serine hydrolases (SHs) are a large, diverse family of enzymes that play various biomedically important roles. Their study has been substantially advanced by activity-based protein profiling, which makes use of covalent chemical probes for labeling the active site and detection by various methodologies. However, highly selective probes for individual SHs are scarce because probe synthesis usually takes place by time-consuming solution phase chemistry. We here report a general solid-phase synthesis toward SH chemical probes, which will speed up probe library synthesis. It involves the construction of a recognition element ending in a secondary amine followed by capping with different electrophiles. We illustrate the power of this approach by the discovery of selective chemical probes for the depalmitoylating enzymes APT-1/2. Overall, this study reports new methodologies to synthesize SH probes, while providing new reagents to study protein depalmitoylation.

Development of activity-based probes for the protein deacylase Sirt1.

Sirtuins are NAD+-dependent protein deacylases that remove acyl modifications from acyl-lysine residues, resulting in essential cellular signaling. Recognized for their role in lifespan extension, humans encode seven sirtuin isoforms (Sirt1-7), and loss of sirtuin deacylase activity is implicated in many aging-related diseases. Despite being intriguing therapeutic targets, cellular studies of sirtuins are hampered by the lack of chemical probes to measure sirtuin activity independent of sirtuin protein levels. Here, we use a modular, peptide-based approach to develop activity-based probes (ABPs) that directly measure Sirt1 activity in vitro and in cell lysates. ABPs were synthesized containing four elements: (1) thioacetyl-lysine for mechanism-based affinity towards only active sirtuins, (2) either histone H3 lysine-14 (H3K14) or p53 sequences for Sirt1 specificity, (3) a diazirine for covalent labeling upon UV irradiation, and (4) an alkyne for bioorthogonal conjugation to a fluorophore for gel-based detection of active Sirt1. Compared to the H3K14 ABP, the p53 ABP showed increased sensitivity and selective labeling of active Sirt1. Acyl-lysine peptide competition, pharmacological inhibition, and inhibitory post-translational modification of Sirt1 resulted in the loss of p53 ABP labeling both in vitro and in HEK293T cell lysates, consistent with the ABP measuring decreased Sirt1 activity. Furthermore, the p53 ABP measured subcellular Sirt1 activity in MCF7 breast cancer cells. The development of a Sirt1-selective ABP that detects Sirt1 activity with an order of magnitude increased sensitivity compared to previous approaches demonstrates the utility of a modular, peptide-based approach for selective-targeting of the sirtuin protein family and provides a framework for further development of sirtuin-selective chemical probes.

Chemoproteomic Profiling of Itaconation by Bioorthogonal Probes in Inflammatory Macrophages.

Itaconate is an anti-inflammatory metabolite involved in pathogen-macrophage interactions, but the mechanisms underlying its effect are not fully understood. Competitive cysteine profiling has been performed to interrogate itaconate's reactivity in cell lysates, but methods for analyzing targets of itaconation directly in living macrophages are still lacking. In this work, we developed a specific bioorthogonal probe, itaconate-alkyne (ITalk), for quantitative and site-specific chemoproteomic profiling of itaconation in inflammatory macrophages. ITalk recapitulates the anti-inflammatory property of itaconate and enables biochemical evaluation and proteomic analysis of its direct targets. Our study delineates the widespread landscape of itaconate substrates, providing a versatile tool and comprehensive resource for investigating its function.

Functionalized Cannabinoid Subtype 2 Receptor Ligands: Fluorescent, PET, Photochromic and Covalent Molecular Probes.

Cannabinoid subtype 2 receptors (CB2 Rs) are G protein-coupled receptors (GPCRs) belonging to the endocannabinoid system, a complex network of signalling pathways leading to the regulation of key physiological processes. Interestingly, CB2 Rs are strongly up-regulated in pathological conditions correlated with the onset of inflammatory events like cancer and neurodegenerative diseases. Therefore, CB2 Rs represent an important biological target for therapeutic as well as diagnostic purposes. No CB2 R-selective drugs are yet on the market, thus underlining a that deeper comprehension of CB2 Rs' complex activation pathways and their role in the regulation of diseases is needed. Herein, we report an overview of pharmacological and imaging tools such as fluorescent, positron emission tomography (PET), photochromic and covalent selective CB2 R ligands. These molecular probes can be used in vitro as well as in vivo to investigate and explore the unravelled role(s) of CB2 Rs, and they can help to design suitable CB2 R-targeted drugs.