X-Ray-Induced Drug Release for Cancer Therapy.

Drug delivery systems (DDSs) are designed to deliver therapeutic agents to specific target sites while minimizing systemic toxicity. Recent developments in drug-loaded DDSs have demonstrated promising characteristics and paved new pathways for cancer treatment. Light, a prevalent external stimulus, is widely utilized to trigger drug release. However, conventional light sources primarily concentrate on the ultraviolet (UV) and visible light regions, which suffer from limited biological tissue penetration. This limitation hinders applications for deep-tissue tumor drug release. Given their deep tissue penetration and well-established application technology, X-rays have recently received attention for the pursuit of controlled drug release. With precise spatiotemporal and dosage controllability, X-rays stand as an ideal stimulus for achieving controlled drug release in deep-tissue cancer therapy. This article explores the recent advancements in using X-rays for stimulus-triggered drug release in DDSs and delves into their action mechanisms.

A hydrogel-based mechanical metamaterial for the interferometric profiling of extracellular vesicles in patient samples.

The utility of mechanical metamaterials for biomedical applications has seldom been explored. Here we show that a metamaterial that is mechanically responsive to antibody-mediated biorecognition can serve as an optical interferometric mask to molecularly profile extracellular vesicles in ascites fluid from patients with cancer. The metamaterial consists of a hydrogel responsive to temperature and redox activity functionalized with antibodies to surface biomarkers on extracellular vesicles, and is patterned into micrometric squares on a gold-coated glass substrate. Through plasmonic heating, the metamaterial is maintained in a transition state between a relaxed form and a buckled state. Binding of extracellular vesicles from the patient samples to the antibodies on the hydrogel causes it to undergo crosslinking, induced by free radicals generated via the activity of horseradish peroxidase conjugated to the antibodies. Hydrogel crosslinking causes the metamaterial to undergo fast chiral re-organization, inducing amplified changes in its mechanical deformation and diffraction patterns, which are detectable by a smartphone camera. The mechanical metamaterial may find broad utility in the sensitive optical immunodetection of biomolecules.

Blood-brain barrier (BBB)-on-a-chip: a promising breakthrough in brain disease research.

The blood-brain barrier (BBB) represents a key challenge in developing brain-penetrating therapeutic molecules. BBB dysfunction is also associated with the onset and progression of various brain diseases. The BBB-on-a-chip (µBBB), an organ-on-chip technology, has emerged as a powerful in vitro platform that closely mimics the human BBB microenvironments. While the µBBB technology has seen wide application in the study of brain cancer, its utility in other brain disease models ("µBBB+") is less appreciated. Based on the advances of the µBBB technology and the evolution of in vitro models for brain diseases over the last decade, we propose the concept of a "µBBB+" system and summarize its major promising applications in pathological studies, personalized medical research, drug development, and multi-organ-on-chip approaches. We believe that such a sophisticated "µBBB+" system is a highly tunable and promising in vitro platform for further advancement of the understanding of brain diseases.

Extracellular vesicle drug occupancy enables real-time monitoring of targeted cancer therapy.

Current technologies to measure drug-target interactions require complex processing and invasive tissue biopsies, limiting their clinical utility for cancer treatment monitoring. Here we develop an analytical platform that leverages circulating extracellular vesicles (EVs) for activity-based assessment of tumour-specific drug-target interactions in patient blood samples. The technology, termed extracellular vesicle monitoring of small-molecule chemical occupancy and protein expression (ExoSCOPE), utilizes bio-orthogonal probe amplification and spatial patterning of molecular reactions within matched plasmonic nanoring resonators to achieve in situ analysis of EV drug dynamics. It measures changes in drug occupancy and protein composition in molecular subpopulations of EVs. When used to monitor various targeted therapies, the ExoSCOPE revealed EV signatures that closely reflected cellular treatment efficacy. We further applied the technology for clinical cancer diagnostics and treatment monitoring. Using a small volume of blood, the ExoSCOPE accurately classified disease status and rapidly distinguished between targeted treatment outcomes, within 24 h after treatment initiation.

Expanding the "minimalist" small molecule tagging approach to different bioactive compounds.

"Minimalist" small molecule tagging (MSMT) is a promising approach that easily converts bioactive compounds into affinity-based probes (AfBPs) for proteomic studies. In this work, seven bioactive compounds targeting diversified protein classes were installed with "minimalist" linkers through common reactions to generate the corresponding AfBPs. These probes were evaluated for cell-based protein profiling and target validation. Among them, the entinostat-derived probe EN and the camptothecin-derived probe CA were further utilized in cellular imaging and SILAC-based large-scale target identification. Our extensive studies suggest that the "minimalist" small molecule tagging approach could be expanded to different classes of bioactive compounds for modification into AfBPs as a dual functional tool for both proteomics and cellular imaging.

A Vinyl Sulfone-Based Fluorogenic Probe Capable of Selective Labeling of PHGDH in Live Mammalian Cells.

Chemical probes are powerful tools for interrogating small molecule-target interactions. With additional fluorescence Turn-ON functionality, such probes might enable direct measurements of target engagement in live mammalian cells. DNS-pE (and its terminal alkyne-containing version DNS-pE2) is the first small molecule that can selectively label endogenous 3-phosphoglycerate dehydrogenase (PHGDH) from various mammalian cells. Endowed with an electrophilic vinyl sulfone moiety that possesses fluorescence-quenching properties, DNS-pE/DNS-pE2 became highly fluorescent only upon irreversible covalent modification of PHGDH. With an inhibitory property (in vitro Ki =7.4 µm) comparable to that of known PHGDH inhibitors, our probes thus offer a promising approach to simultaneously image endogenous PHGDH activities and study its target engagement in live-cell settings.

Simultaneous Imaging of Endogenous Survivin mRNA and On-Demand Drug Release in Live Cells by Using a Mesoporous Silica Nanoquencher.

The design of multifunctional drug delivery systems capable of simultaneous target detection, imaging, and therapeutics in live mammalian cells is critical for biomedical research. In this study, by using mesoporous silica nanoparticles (MSNs) chemically modified with a small-molecule dark quencher, followed by sequential drug encapsulation, MSN capping with a dye-labeled antisense oligonucleotide, and bioorthogonal surface modification with cell-penetrating poly(disulfide)s, the authors have successfully developed the first mesoporous silica nanoquencher (qMSN), characterized by high drug-loading and endocytosis-independent cell uptake, which is able to quantitatively image endogenous survivin mRNA and release the loaded drug in a manner that depends on the survivin expression level in tumor cells. The authors further show that this novel drug delivery system may be used to minimize potential cytotoxicity encountered by many existing small-molecule drugs in cancer therapy.

Protein-Protein Interaction Inhibitors of BRCA1 Discovered Using Small Molecule Microarrays.

Microarray screening technology has transformed the life sciences arena over the last decade. The platform is widely used in the area of mapping interaction networks, to molecular fingerprinting and small molecular inhibitor discovery. The technique has significantly impacted both basic and applied research. The microarray platform can likewise enable high-throughput screening and discovery of protein-protein interaction (PPI) inhibitors. Herein we demonstrate the application of microarray-guided PPI inhibitor discovery, using human BRCA1 as an example. Mutations in BRCA1 have been implicated in ~50 % of hereditary breast cancers. By targeting the (BRCT)2 domain, we showed compound 15a and its prodrug 15b inhibited BRCA1 activities in tumor cells. Unlike previously reported peptide-based PPI inhibitors of BRCA1, the compounds identified could be directly administered to tumor cells, thus making them useful in targeting BRCA1/PARP-related pathways involved in DNA damage and repair response, for cancer therapy.

In Situ Proteome Profiling and Bioimaging Applications of Small-Molecule Affinity-Based Probes Derived From DOT1L Inhibitors.

DOT1L is the sole protein methyltransferase that methylates histone H3 on lysine 79 (H3K79), and is a promising drug target against cancers. Small-molecule inhibitors of DOT1L such as FED1 are potential anti-cancer agents and useful tools to investigate the biological roles of DOT1L in human diseases. FED1 showed excellent in vitro inhibitory activity against DOT1L, but its cellular effect was relatively poor. In this study, we designed and synthesized photo-reactive and "clickable" affinity-based probes (AfBPs), P1 and P2, which were cell-permeable and structural mimics of FED1. The binding and inhibitory effects of these two probes against DOT1L protein were extensively investigated in vitro and in live mammalian cells (in situ). The cellular uptake and sub-cellular localization properties of the probes were subsequently studied in live-cell imaging experiments, and our results revealed that, whereas both P1 and P2 readily entered mammalian cells, most of them were not able to reach the cell nucleus where functional DOT1L resides. This offers a plausible explanation for the poor cellular activity of FED1. Finally with P1/P2, large-scale cell-based proteome profiling, followed by quantitative LC-MS/MS, was carried out to identify potential cellular off-targets of FED1. Amongst the more than 100 candidate off-targets identified, NOP2 (a putative ribosomal RNA methyltransferase) was further confirmed to be likely a genuine off-target of FED1 by preliminary validation experiments including pull-down/Western blotting (PD/WB) and cellular thermal shift assay (CETSA).

A small-molecule protein-protein interaction inhibitor of PARP1 that targets its BRCT domain.

Poly(ADP-ribose)polymerase-1 (PARP1) is a BRCT-containing enzyme (BRCT = BRCA1 C-terminus) mainly involved in DNA repair and damage response and a validated target for cancer treatment. Small-molecule inhibitors that target the PARP1 catalytic domain have been actively pursued as anticancer drugs, but are potentially problematic owing to a lack of selectivity. Compounds that are capable of disrupting protein-protein interactions of PARP1 provide an alternative by inhibiting its activities with improved selectivity profiles. Herein, by establishing a high-throughput microplate-based assay suitable for screening potential PPI inhibitors of the PARP1 BRCT domain, we have discovered that (±)-gossypol, a natural product with a number of known biological activities, possesses novel PARP1 inhibitory activity both in vitro and in cancer cells and presumably acts through disruption of protein-protein interactions. As the first known cell-permeable small-molecule PPI inhibitor of PAPR1, we further established that (-)-gossypol was likely the causative agent of PARP1 inhibition by promoting the formation of a 1:2 compound/PARP1 complex by reversible formation of a covalent imine linkage.

Multiplex imaging and cellular target identification of kinase inhibitors via an affinity-based proteome profiling approach.

MLN8237 is a highly potent and presumably selective inhibitor of Aurora kinase A (AKA) and has shown promising antitumor activities. Like other kinase inhibitors which target the ATP-binding site of kinases, MLN8237 might be expected to have potential cellular off-targets. Herein, we report the first photoaffinity-based, small molecule AKA probe capable of both live-cell imaging of AKA activities and in situ proteome profiling of potential off-targets of MLN8237 (including AKA-associating proteins). By using two mutually compatible, bioorthogonal reactions (copper-catalyzed azide-alkyne cycloaddition chemistry and TCO-tetrazine ligation), we demostrate small molecule-based multiplex bioimaging for simultaneous in situ monitoring of two important cell-cycle regulating kinases (AKA and CDK1). A broad range of proteins, as potential off-targets of MLN8237 and AKA's-interacting partners, is subsequently identified by affinity-based proteome profiling coupled with large-scale LC-MS/MS analysis. From these studies, we discover novel AKA interactions which were further validated by cell-based immunoprecipitation (IP) experiments.

Discovery of cell-permeable inhibitors that target the BRCT domain of BRCA1 protein by using a small-molecule microarray.

BRCTs are phosphoserine-binding domains found in proteins involved in DNA repair, DNA damage response and cell cycle regulation. BRCA1 is a BRCT domain-containing, tumor-suppressing protein expressed in the cells of breast and other human tissues. Mutations in BRCA1 have been found in ca. 50% of hereditary breast cancers. Cell-permeable, small-molecule BRCA1 inhibitors are promising anticancer agents, but are not available currently. Herein, with the assist of microarray-based platforms, we have discovered the first cell-permeable protein-protein interaction (PPI) inhibitors against BRCA1. By targeting the (BRCT)2 domain, we showed compound 15 a and its prodrug 15 b inhibited BRCA1 activities in tumor cells, sensitized these cells to ionizing radiation-induced apoptosis, and showed synergistic inhibitory effect when used in combination with Olaparib (a small-molecule inhibitor of poly-ADP-ribose polymerase) and Etoposide (a small-molecule inhibitor of topoisomerase II). Unlike previously reported peptide-based PPI inhibitors of BRCA1, our compounds are small-molecule-like and could be directly administered to tumor cells, thus making them useful for future studies of BRCA1/PARP-related pathways in DNA damage and repair response, and in cancer therapy.

"Minimalist" cyclopropene-containing photo-cross-linkers suitable for live-cell imaging and affinity-based protein labeling.

Target identification of bioactive compounds within the native cellular environment is important in biomedical research and drug discovery, but it has traditionally been carried out in vitro. Information about how such molecules interact with their endogenous targets (on and off) is currently highly limited. An ideal strategy would be one that recapitulates protein-small molecule interactions in situ (e.g., in living cells) and at the same time enables enrichment of these complexes for subsequent proteome-wide target identification. Similarly, small molecule-based imaging approaches are becoming increasingly available for in situ monitoring of a variety of proteins including enzymes. Chemical proteomic strategies for simultaneous bioimaging and target identification of noncovalent bioactive compounds in live mammalian cells, however, are currently not available. This is due to a lack of photoaffinity labels that are minimally modified from their parental compounds, yet chemically tractable using copper-free bioorthogonal chemistry. We have herein developed novel minimalist linkers containing both an alkyl diazirine and a cyclopropene. We have shown chemical probes (e.g., BD-2) made from such linkers could be used for simultaneous in situ imaging and covalent labeling of endogenous BRD-4 (an important epigenetic protein) via a rapid, copper-free, tetrazine-cyclopropene ligation reaction (k2 > 5 M(-1) s(-1)). The key features of our cyclopropenes, with their unique C-1 linkage to BRD-4-targeting moiety, are their tunable reactivity and solubility, relative stability, and synthetic accessibility. BD-2, which is a linker-modified analogue of (+)-JQ1 (a recently discovered nanomolar protein-protein-interaction inhibitor of BRD-4), was subsequently used in a cell-based proteome profiling experiment for large-scale identification of potential off-targets of (+)-JQ1. Several newly identified targets were subsequently confirmed by preliminary validation experiments.