Biomarker-driven molecular imaging probes in radiotherapy.

Background: Biomarker-driven molecular imaging has emerged as an integral part of cancer precision radiotherapy. The use of molecular imaging probes, including nanoprobes, have been explored in radiotherapy imaging to precisely and noninvasively monitor spatiotemporal distribution of biomarkers, potentially revealing tumor-killing mechanisms and therapy-induced adverse effects during radiation treatment. Methods: We summarized literature reports from preclinical studies and clinical trials, which cover two main parts: 1) Clinically-investigated and emerging imaging biomarkers associated with radiotherapy, and 2) instrumental roles, functions, and activatable mechanisms of molecular imaging probes in the radiotherapy workflow. In addition, reflection and future perspectives are proposed. Results: Numerous imaging biomarkers have been continuously explored in decades, while few of them have been successfully validated for their correlation with radiotherapeutic outcomes and/or radiation-induced toxicities. Meanwhile, activatable molecular imaging probes towards the emerging biomarkers have exhibited to be promising in animal or small-scale human studies for precision radiotherapy. Conclusion: Biomarker-driven molecular imaging probes are essential for precision radiotherapy. Despite very inspiring preliminary results, validation of imaging biomarkers and rational design strategies of probes await robust and extensive investigations. Especially, the correlation between imaging biomarkers and radiotherapeutic outcomes/toxicities should be established through multi-center collaboration involving a large cohort of patients.

Development of Peptide-Based Probes for Molecular Imaging of the Postsynaptic Density in the Brain.

The postsynaptic density (PSD) comprises numerous scaffolding proteins, receptors, and signaling molecules that coordinate synaptic transmission in the brain. Postsynaptic density protein 95 (PSD-95) is a master scaffold protein within the PSD and one of its most abundant proteins and therefore constitutes a very attractive biomarker of PSD function and its pathological changes. Here, we exploit a high-affinity inhibitor of PSD-95, AVLX-144, as a template for developing probes for molecular imaging of the PSD. AVLX-144-based probes were labeled with the radioisotopes fluorine-18 and tritium, as well as a fluorescent tag. Tracer binding showed saturable, displaceable, and uneven distribution in rat brain slices, proving effective in quantitative autoradiography and cell imaging studies. Notably, we observed diminished tracer binding in human post-mortem Parkinson's disease (PD) brain slices, suggesting postsynaptic impairment in PD. We thus offer a suite of translational probes for visualizing and understanding PSD-related pathologies.

Small-molecule probe for IBD risk variant GPR65 I231L alters cytokine signaling networks through positive allosteric modulation.

The proton-sensing heterotrimeric guanine nucleotide-binding protein-coupled receptor GPR65 is expressed in immune cells and regulates tissue homeostasis in response to decreased extracellular pH, which occurs in the context of inflammation and tumorigenesis. Genome-wide association studies linked GPR65 to several autoimmune and inflammatory diseases such as multiple sclerosis and inflammatory bowel disease (IBD). The loss-of-function GPR65 I231L IBD risk variant alters cellular metabolism, impairs protective tissue functions, and increases proinflammatory cytokine production. Hypothesizing that a small molecule designed to potentiate GPR65 at subphysiological pH could decrease inflammatory responses, we found positive allosteric modulators of GPR65 that engage and activate both human and mouse orthologs of the receptor. We observed that the chemical probe BRD5075 alters cytokine and chemokine programs in dendritic cells, establishing that immune signaling can be modulated by targeting GPR65. Our investigation offers improved chemical probes to further interrogate the biology of human GPR65 and its clinically relevant genetic variants.

Exploring the Antiangiogenic and Anti-Inflammatory Potential of Homoisoflavonoids: Target Identification Using Biotin Probes.

Chemical proteomics using biotin probes of natural products have significantly advanced our understanding of molecular targets and therapeutic potential. This review highlights recent progress in the application of biotin probes of homoisoflavonoids for identifying binding proteins and elucidating mechanisms of action. Notably, homoisoflavonoids exhibit antiangiogenic, anti-inflammatory, and antidiabetic effects. A combination of biotin probes, pull-down assays, mass spectrometry, and molecular modeling has revealed how natural products and their derivatives interact with several proteins such as ferrochelatase (FECH), soluble epoxide hydrolase (sEH), inosine monophosphate dehydrogenase 2 (IMPDH2), phosphodiesterase 4 (PDE4), and deoxyhypusine hydroxylase (DOHH). These target identification approaches pave the way for new therapeutic avenues, especially in the fields of oncology and ophthalmology. Future research aimed at expanding the repertoire of target identification using biotin probes of homoisoflavonoids promises to further elucidate the complex mechanisms and develop new drug candidates.

Assessing Seed Germination Response of Parasitic Plant Striga hermonthica with Small-Molecule Probes.

Seed germination of a parasitic plant Striga hermonthica is elicited by strigolactones which are exuded from roots of host plants. Here, we describe a high-throughput germination assay and a method for visualizing in vivo strigolactone receptor functions with a fluorogenic probe.

Quantification of binding capacity of natural products to target proteins by sensors integrating SERS labeling and photocrosslinked molecular probes.

Natural products-based screening of active ingredients and their interactions with target proteins is an important ways to discover new drugs. Assessing the binding capacity of target proteins, particularly when multiple components are involved, presents a significant challenge for sensors. As far as we know, there is currently no sensor that can accomplish high-throughput quantitative analysis of natural product-target protein binding capacity based on Raman spectroscopy. In this study, a novel sensor model has been developed for the quantitative analysis of binding capacity based on Surface-Enhanced Raman Spectroscopy (SERS) and Photocrosslinked Molecular Probe (PCMP) technology. This sensor, named SERS-PCMP, leverages the high throughput of molecular probe technology to investigate the active ingredients in natural products, along with the application of SERS labelling technology for target proteins. Thus it significantly improves the efficiency and accuracy of target protein identification. Based on the novel strategy, quantitative analysis of the binding capacity of 20 components from Shenqi Jiangtang Granules (SJG) to α-Glucosidase were completed. Ultimately, the binding capacity of these active ingredients was ranked based on the detected Raman Intensity. The compounds with higher binding capacity were Astragaloside IV (Intensity, 138.17), Ginsenoside Rh2 (Intensity, 87.46), Ginsenoside Rg3 (Intensity, 73.92) and Ginsenoside Rh1 (Intensity, 64.37), which all exceeded the binding capacity of the positive drug Acarbose (Intensity, 28.75). Furthermore, this strategy also performed a high detection sensitivity. The limit of detection for the enzyme using 0.1 mg of molecular probe magnetic nanoparticles (MP MNPs) was determined to be no less than 0.375 µg/mL. SERS-PCMP sensor integrating SERS labeling and photocrosslinked molecular probes which offers a fresh perspective for future drug discovery studies. Such as high-throughput drug screening and the exploration of small molecule-target protein interactions in vitro.

Discovery of Two Highly Selective Structurally Orthogonal Chemical Probes for Activin Receptor-like Kinases 1 and 2.

Activin receptor-like kinases 1-7 (ALK1-7) regulate a complex network of SMAD-independent as well as SMAD-dependent signaling pathways. One of the widely used inhibitors for functional investigations of these processes, in particular for bone morphogenetic protein (BMP) signaling, is LDN-193189. However, LDN-193189 has insufficient kinome-wide selectivity complicating its use in cellular target validation assays. Herein, we report the identification and comprehensive characterization of two chemically distinct highly selective inhibitors of ALK1 and ALK2, M4K2234 and MU1700, along with their negative controls. We show that both MU1700 and M4K2234 efficiently block the BMP pathway via selective in cellulo inhibition of ALK1/2 kinases and exhibit favorable in vivo profiles in mice. MU1700 is highly brain penetrant and shows remarkably high accumulation in the brain. These high-quality orthogonal chemical probes offer the selectivity required to become widely used tools for in vitro and in vivo investigation of BMP signaling.

The Human Ganglioside Interactome in Live Cells Revealed Using Clickable Photoaffinity Ganglioside Probes.

Gangliosides, sialic acid bearing glycosphingolipids, are components of the outer leaflet of plasma membranes of all vertebrate cells. They contribute to cell regulation by interacting with proteins in their own membranes (cis) or their extracellular milieu (trans). As amphipathic membrane constituents, gangliosides present challenges for identifying their ganglioside protein interactome. To meet these challenges, we synthesized bifunctional clickable photoaffinity gangliosides, delivered them to plasma membranes of cultured cells, then captured and identified their interactomes using proteomic mass spectrometry. Installing probes on ganglioside lipid and glycan moieties, we captured cis and trans ganglioside-protein interactions. Ganglioside interactomes varied with the ganglioside structure, cell type, and site of the probe (lipid or glycan). Gene ontology revealed that gangliosides engage with transmembrane transporters and cell adhesion proteins including integrins, cadherins, and laminins. The approach developed is applicable to other gangliosides and cell types, promising to provide insights into molecular and cellular regulation by gangliosides.

A CAIX Dual-Targeting Small-Molecule Probe for Noninvasive Imaging of ccRCC.

Carbonic anhydrase IX (CAIX), a zinc metal transmembrane protein, is highly expressed in 95% of clear cell renal cell carcinomas (ccRCCs). A positron emission tomography (PET) probe designed to target CAIX in nuclear medicine imaging technology can achieve precise positioning, is noninvasive, and can be used to monitor CAIX expression in lesions in real time. In this study, we constructed a novel acetazolamide dual-targeted small-molecule probe [68Ga]Ga-LF-4, which targets CAIX by binding to a specific amino acid sequence. After attenuation correction, the radiolabeling yield reached 66.95 ± 0.57% (n = 5) after 15 min of reaction and the radiochemical purity reached 99% (n = 5). [68Ga]Ga-LF-4 has good in vitro and in vivo stability, and in vivo safety and high affinity for CAIX, with a Kd value of 6.62 nM. Moreover, [68Ga]Ga-LF-4 could be quickly cleared from the blood in vivo. The biodistribution study revealed that the [68Ga]Ga-LF-4 signal was concentrated in the heart, lung, and kidney after administration, which was the same as that observed in the micro-PET/CT study. In a ccRCC patient-derived xenograft (PDX) model, the signal significantly accumulated in the tumor after administration, where it was retained for up to 4 h. After competitive blockade with LF-4, uptake at the tumor site was significantly reduced. The SUVmax of the probe [68Ga]Ga-LF-4 at the ccRCC tumor site was three times greater than that in the PC3 group with low CAIX expression at 30 min (ccRCC vs PC3:1.86 ± 0.03 vs 0.62 ± 0.01, t = 48.2, P < 0.0001). These results indicate that [68Ga]Ga-LF-4 is a novel small-molecule probe that targets CAIX and can be used to image localized and metastatic ccRCC lesions.

Unveiling a new strategy for PDIA1 inhibition: Integration of activity-based probes profiling and targeted degradation.

The overexpression of PDIA1 in cancer has spurred the quest for effective inhibitors. However, existing inhibitors often bind to only one active site, limiting their efficacy. In our study, we developed a PROTAC-mimetic probe dPA by combining PACMA31 (PA) analogs with cereblon-directed pomalidomide. Through protein profiling and analysis, we confirmed dPA's specific interaction with PDIA1's active site cysteines. We further synthesized PROTAC variants with a thiophene ring and various linkers to enhance degradation efficiency. Notably, H4, featuring a PEG linker, induced significant PDIA1 degradation and inhibited cancer cell proliferation similarly to PA. The biosafety profile of H4 is comparable to that of PA, highlighting its potential for further development in cancer therapy. Our findings highlight a novel strategy for PDIA1 inhibition via targeted degradation, offering promising prospects in cancer therapeutics. This approach may overcome limitations of conventional inhibitors, presenting new avenues for advancing anti-cancer interventions.

N-Acyl-N-Alkyl Sulfonamide Probes for Ligand-Directed Covalent Labeling of GPCRs: The Adenosine A2B Receptor as Case Study.

Small molecular tool compounds play an essential role in the study of G protein-coupled receptors (GPCRs). However, tool compounds most often occupy the orthosteric binding site, hampering the study of GPCRs upon ligand binding. To overcome this problem, ligand-directed labeling techniques have been developed that leave a reporter group covalently bound to the GPCR, while allowing subsequent orthosteric ligands to bind. In this work, we applied such a labeling strategy to the adenosine A2B receptor (A2BAR). We have synthetically implemented the recently reported N-acyl-N-alkyl sulfonamide (NASA) warhead into a previously developed ligand and show that the binding of the A2BAR is not restricted by NASA incorporation. Furthermore, we have investigated ligand-directed labeling of the A2BAR using SDS-PAGE, flow cytometric, and mass spectrometry techniques. We have found one of the synthesized probes to specifically label the A2BAR, although detection was hindered by nonspecific protein labeling most likely due to the intrinsic reactivity of the NASA warhead. Altogether, this work aids the future development of ligand-directed probes for the detection of GPCRs.

Enzymes in Synergy: Bacteria Specific Molecular Probe for Locoregional Imaging of Urinary Tract Infection in vivo.

Uropathogenic Escherichia coli (UPECs) is a leading cause for urinary tract infections (UTI), accounting for 70-90 % of community or hospital-acquired bacterial infections owing to high recurrence, imprecision in diagnosis and management, and increasing prevalence of antibiotic resistance. Current methods for clinical UPECs detection still rely on labor-intensive urine cultures that impede rapid and accurate diagnosis for timely UTI therapeutic management. Herein, we developed a first-in-class near-infrared (NIR) UPECs fluorescent probe (NO-AH) capable of specifically targeting UPECs through its collaborative response to bacterial enzymes, enabling locoregional imaging of UTIs both in vitro and in vivo. Our NO-AH probe incorporates a dual protease activatable moiety, which first reacts with OmpT, an endopeptidase abundantly present on the outer membrane of UPECs, releasing an intermediate amino acid residue conjugated with a NIR hemicyanine fluorophore. Such liberated fragment would be subsequently recognized by aminopeptidase (APN) within the periplasm of UPECs, activating localized fluorescence for precise imaging of UTIs in complex living environments. The peculiar specificity and selectivity of NO-AH, facilitated by the collaborative action of bacterial enzymes, features a timely and accurate identification of UPECs-infected UTIs, which could overcome misdiagnosis in conventional urine tests, thus opening new avenues towards reliable UTI diagnosis and personalized antimicrobial therapy management.

Fluorinated 1,7-DO2A-Based Iron(II) Complexes as Sensitive 19F MRI Molecular Probes for Visualizing Renal Dysfunction in Living Mice.

Kidney diseases have become an important global health concern due to their high incidence, inefficient diagnosis, and poor prognosis. Devising direct methods, especially imaging means, to assess renal function is the key for better understanding the mechanisms of various kidney diseases and subsequent development of effective treatment. Herein, we developed a fluorinated ferrous chelate-based sensitive probe, 1,7-DO2A-Fe(II)-F18 (Probe 1), for 19F magnetic resonance imaging (MRI). This highly fluorinated probe (containing 18 chemically equivalent 19F atoms with a fluorine content at 35 wt %) achieves a 15-time enhancement in signal intensity compared with the fluorine-containing ligand alone due to the appropriately regulated 19F relaxation times by the ferrous ion, which significantly increases imaging sensitivity and reduces acquisition time. Owing to its high aqueous solubility, biostability, and biocompatibility, this probe could be rapidly cleared by kidneys, which provides a means for monitoring renal dysfunction via 19F MRI. With this probe, we accomplish in vivo imaging of the impaired renal dysfunction caused by various kidney diseases including acute kidney injury, unilateral ureteral obstruction, and renal fibrosis at different stages. Our study illustrates the promising potential of Probe 1 for in vivo real-time visualization of kidney dysfunction, which is beneficial for the study, diagnosis, and even stratification of different kidney diseases. Furthermore, the design strategy of our probe is inspiring for the development of more high-performance 19F MRI probes for monitoring various biological processes.

Development, validation and application of single molecule molecular inversion probe based novel integrated genetic screening method for 29 common lysosomal storage disorders in India.

BACKGROUND: Current clinical diagnosis pathway for lysosomal storage disorders (LSDs) involves sequential biochemical enzymatic tests followed by DNA sequencing, which is iterative, has low diagnostic yield and is costly due to overlapping clinical presentations. Here, we describe a novel low-cost and high-throughput sequencing assay using single-molecule molecular inversion probes (smMIPs) to screen for causative single nucleotide variants (SNVs) and copy number variants (CNVs) in genes associated with 29 common LSDs in India. RESULTS: 903 smMIPs were designed to target exon and exon-intron boundaries of targeted genes (n = 23; 53.7 kb of the human genome) and were equimolarly pooled to create a sequencing library. After extensive validation in a cohort of 50 patients, we screened 300 patients with either biochemical diagnosis (n = 187) or clinical suspicion (n = 113) of LSDs. A diagnostic yield of 83.4% was observed in patients with prior biochemical diagnosis of LSD. Furthermore, diagnostic yield of 73.9% (n = 54/73) was observed in patients with high clinical suspicion of LSD in contrast with 2.4% (n = 1/40) in patients with low clinical suspicion of LSD. In addition to detecting SNVs, the assay could detect single and multi-exon copy number variants with high confidence. Critically, Niemann-Pick disease type C and neuronal ceroid lipofuscinosis-6 diseases for which biochemical testing is unavailable, could be diagnosed using our assay. Lastly, we observed a non-inferior performance of the assay in DNA extracted from dried blood spots in comparison with whole blood. CONCLUSION: We developed a flexible and scalable assay to reliably detect genetic causes of 29 common LSDs in India. The assay consolidates the detection of multiple variant types in multiple sample types while having improved diagnostic yield at same or lower cost compared to current clinical paradigm.

Bifunctional glycosphingolipid (GSL) probes to investigate GSL-interacting proteins in cell membranes.

Glycosphingolipids (GSLs) are abundant glycolipids on cells and essential for cell recognition, adhesion, signal transduction, and so on. However, their lipid anchors are not long enough to cross the membrane bilayer. To transduce transmembrane signals, GSLs must interact with other membrane components, whereas such interactions are difficult to investigate. To overcome this difficulty, bifunctional derivatives of II3-ß-N-acetyl-D-galactosamine-GA2 (GalNAc-GA2) and ß-N-acetyl-D-glucosamine-ceramide (GlcNAc-Cer) were synthesized as probes to explore GSL-interacting membrane proteins in live cells. Both probes contain photoreactive diazirine in the lipid moiety, which can crosslink with proximal membrane proteins upon photoactivation, and clickable alkyne in the glycan to facilitate affinity tag addition for crosslinked protein pull-down and characterization. The synthesis is highlighted by the efficient assembly of simple glycolipid precursors followed by on-site lipid remodeling. These probes were employed to profile GSL-interacting membrane proteins in HEK293 cells. The GalNAc-GA2 probe revealed 312 distinct proteins, with GlcNAc-Cer probe-crosslinked proteins as controls, suggesting the potential influence of the glycan on GSL functions. Many of the proteins identified with the GalNAc-GA2 probe are associated with GSLs, and some have been validated as being specific to this probe. The versatile probe design and experimental protocols are anticipated to be widely applicable to GSL research.

Molecular probes for monitoring pyroptosis: design, imaging and theranostic application.

Pyroptosis is a recently discovered process of programmed cell death that is linked with tumor progression and potential treatment strategies. Unlike other forms of programmed cell death, such as apoptosis or necrosis, pyroptosis is associated with pore-forming proteins gasdermin D (GSDMD), which are cleaved by caspase enzymes to form oligomers. These oligomers are then inserted into the cell surface membrane, causing pores to consequently result in rapid cell death. Pyroptosis, in conjunction with immunotherapy, represents a promising avenue for prognostication and antitumor therapy, providing a more precise direction for disease treatment. To gain deeper insight into the mechanisms underlying pyroptosis in real-time, non-invasive and live cell imaging techniques are urgently needed. Non-invasive imaging techniques can enhance future diagnostic and therapeutic approaches for inflammatory diseases, including different types of tumors. This review article discusses various non-invasive molecular probes for detecting pyroptosis, including genetic reporters and nanomaterials. These strategies can enhance scientists' understanding of pyroptosis and help discover personalized and effective ways to treat inflammatory diseases, particularly tumors.

Molecular probes for super-resolution imaging of drug dynamics.

Super-resolution molecular probes (SRMPs) are essential tools for visualizing drug dynamics within cells, transcending the resolution limits of conventional microscopy. In this review, we provide an overview of the principles and design strategies of SRMPs, emphasizing their role in accurately tracking drug molecules. By illuminating the intricate processes of drug distribution, diffusion, uptake, and metabolism at a subcellular and molecular level, SRMPs offer crucial insights into therapeutic interventions. Additionally, we explore the practical applications of super-resolution imaging in disease treatment, highlighting the significance of SRMPs in advancing our understanding of drug action. Finally, we discuss future perspectives, envisioning potential advancements and innovations in this field. Overall, this review serves to inform and practitioners about the utility of SRMPs in driving innovation and progress in pharmacology, providing valuable insights for drug development and optimization.

Organic fluorophores-based molecular probes with dual-fluorescence ratiometric responses to in-vitro/in-vivo pH for biosensing, bioimaging and biotherapeutics applications.

In recent years, organic fluorophores-based molecular probes with dual-fluorescence ratiometric responses to in-vitro/in-vivo pH (DFR-MPs-pH) have been attracting much interest in fundamental application research fields. More and more scientific publications have reported the exploration of various DFR-MPs-pH systems that have unique dual-fluorescence ratiometry as the signal output, in-built and signal self-calibration functions to improve precise detection of targets. DFR-MPs-pH systems possess high-performance applications in biosensing, bioimaging and biomedicine fields. This review has comprehensively summarized recent advances of DFR-MPs-pH for the first time. First of all, the compositions and types of DFR-MPs-pH are introduced by summarizing different organic fluorophores-based molecule systems. Then, construction strategies are analyzed based on specific components, structures, properties and functions of DFR-MPs-pH. Afterward, biosensing and bioimaging applications are discussed in detail, primarily referring to pH sensing and imaging detection at the levels of living cells and small animals. Finally, biomedicine applications are fully summarized, majorly involving bio-toxicity evaluation, bio-distribution, biomedical diagnosis and therapeutics. Meanwhile, the current status, challenges and perspectives are rationally commented after detailed discussions of representative and state-of-the-art studies. Overall, this present review is comprehensive, in-time and in-depth, and can facilitate the following further exploration of new and versatile DFR-MPs-pH systems toward rational design, facile preparation, superior properties, adjustable functions and highly efficient applications in promising fields.

Hemin as a Molecular Probe for Nitric Oxide Detection in Physiological Solutions: Experimental and Theoretical Assessment.

Given its pivotal role in modulating various pathological processes, precise measurement of nitric oxide (●NO) levels in physiological solutions is imperative. The key techniques include the ozone-based chemiluminescence (CL) reactions, amperometric ●NO sensing, and Griess assay, each with its advantages and drawbacks. In this study, a hemin/H2O2/luminol CL reaction was employed for accurately detecting ●NO in diverse solutions. We investigated how the luminescence kinetics was influenced by ●NO from two donors, nitrite and peroxynitrite, while also assessing the impact of culture medium components and reactive species quenchers. Furthermore, we experimentally and theoretically explored the mechanism of hemin oxidation responsible for the initiation of light generation. Although both hemin and ●NO enhanced the H2O2/luminol-based luminescence reactions with distinct kinetics, hemin's interference with ●NO/peroxynitrite- modulated their individual effects. Leveraging the propagated signal due to hemin, the ●NO levels in solution were estimated, observing parallel changes to those detected via amperometric detection in response to varying concentrations of the ●NO-donor. The examined reactions aid in comprehending the mechanism of ●NO/hemin/H2O2/luminol interactions and how these can be used for detecting ●NO in solution with minimal sample size demands. Moreover, the selectivity across different solutions can be improved by incorporating certain quenchers for reactive species into the reaction.

Carboxylesterase Activatable Molecular Probe for Personalized Treatment Guidance by Analyte-Induced Molecular Transformation.

Accurate visualization of tumor microenvironment is of great significance for personalized medicine. Here, we develop a near-infrared (NIR) fluorescence/photoacoustic (FL/PA) dual-mode molecular probe (denoted as NIR-CE) for distinguishing tumors based on carboxylesterase (CE) level by an analyte-induced molecular transformation (AIMT) strategy. The recognition moiety for CE activity is the acetyl unit of NIR-CE, generating the pre-product, NIR-CE-OH, which undergoes spontaneous hydrogen atom exchange between the nitrogen atoms in the indole group and the phenol hydroxyl group, eventually transforming into NIR-CE-H. In cellular experiments and in vivo blind studies, the human hepatoma cells and tumors with high level of CE were successfully distinguished by both NIR FL and PA imaging. Our findings provide a new molecular imaging strategy for personalized treatment guidance.