ABSTRACT
MicroRNAs (miRNAs) are noncoding RNA molecules of 22-24 nucleotides that are estimated to regulate thousands of genes in humans, and their dysregulation has been implicated in many diseases. MicroRNA-122 (miR-122) is the most abundant miRNA in the liver and has been linked to the development of hepatocellular carcinoma and hepatitis C virus (HCV) infection. Its role in these diseases renders miR-122 a potential target for small-molecule therapeutics. Here, we report the discovery of a new sulfonamide class of small-molecule miR-122 inhibitors from a high-throughput screen using a luciferase-based reporter assay. Structure-activity relationship (SAR) studies and secondary assays led to the development of potent and selective miR-122 inhibitors. Preliminary mechanism-of-action studies suggest a role in the promoter-specific transcriptional inhibition of miR-122 expression through direct binding to the liver-enriched transcription factor hepatocyte nuclear factor 4α. Importantly, the developed inhibitors significantly reduce HCV replication in human liver cells.
Subject(s)
Hepatitis C , Liver Neoplasms , MicroRNAs , Humans , Hepacivirus/genetics , MicroRNAs/metabolism , Hepatitis C/drug therapy , Liver Neoplasms/pathology , Virus ReplicationABSTRACT
Despite intensive research of nanomaterials-based field-effect transistors (FETs) as a rapid diagnostic tool, it remains to be seen for FET sensors to be used for clinical applications due to a lack of stability, reliability, reproducibility, and scalability for mass production. Herein, we propose a remote floating-gate (RFG) FET configuration to eliminate device-to-device variations of two-dimensional reduced graphene oxide (rGO) sensing surfaces and most of the instability at the solution interface. Also, critical mechanistic factors behind the electrochemical instability of rGO such as severe drift and hysteresis were identified through extensive studies on rGO-solution interfaces varied by rGO thickness, coverage, and reduction temperature. rGO surfaces in our RFGFET structure displayed a Nernstian response of 54 mV/pH (from pH 2 to 11) with a 90% yield (9 samples out of total 10), coefficient of variation (CV) < 3%, and a low drift rate of 2%, all of which were calculated from the absolute measurement values. As proof-of-concept, we demonstrated highly reliable, reproducible, and label-free detection of spike proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a saliva-relevant media with concentrations ranging from 500 fg/mL to 5 µg/mL, with an R2 value of 0.984 and CV < 3%, and a guaranteed limit of detection at a few pg/mL. Taken together, this new platform may have an immense effect on positioning FET bioelectronics in a clinical setting for detecting SARS-CoV-2.
Subject(s)
Biosensing Techniques , COVID-19 , Graphite , COVID-19/diagnosis , Graphite/chemistry , Humans , Reproducibility of Results , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Transistors, ElectronicABSTRACT
The outbreak of 2019 coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global pandemic. Despite intensive research, the current treatment options show limited curative efficacies. Here the authors report a strategy incorporating neutralizing antibodies conjugated to the surface of a photothermal nanoparticle (NP) to capture and inactivate SARS-CoV-2. The NP is comprised of a semiconducting polymer core and a biocompatible polyethylene glycol surface decorated with high-affinity neutralizing antibodies. The multifunctional NP efficiently captures SARS-CoV-2 pseudovirions and completely blocks viral infection to host cells in vitro through the surface neutralizing antibodies. In addition to virus capture and blocking function, the NP also possesses photothermal function to generate heat following irradiation for inactivation of virus. Importantly, the NPs described herein significantly outperform neutralizing antibodies at treating authentic SARS-CoV-2 infection in vivo. This multifunctional NP provides a flexible platform that can be readily adapted to other SARS-CoV-2 antibodies and extended to novel therapeutic proteins, thus it is expected to provide a broad range of protection against original SARS-CoV-2 and its variants.
Subject(s)
Antibodies, Neutralizing/administration & dosage , Antibodies, Viral/administration & dosage , COVID-19/therapy , Immunoconjugates/administration & dosage , Nanoparticles , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/physiology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/immunology , Antigen-Antibody Reactions , COVID-19/immunology , COVID-19/virology , Drug Evaluation, Preclinical , Hot Temperature , Humans , Immunoconjugates/immunology , Immunoconjugates/therapeutic use , Light , Mice , Nanoparticles/therapeutic use , Phosphatidylethanolamines , Polyethylene Glycols , Polymers , Receptors, Virus/physiology , Semiconductors , Spike Glycoprotein, Coronavirus/immunology , Thiadiazoles , Virus InactivationABSTRACT
Although chimeric antigen receptor (CAR) T-cell therapy has transformed cancer treatment, high-quality and universal CAR-staining reagents are urgently required to manufacture CAR T cells, predict therapy response, decipher CAR biology, and engineer new CARs. Here, we developed tetrameric and dodecameric forms of a multifunctional and extensible category of high-avidity CAR-staining reagents: antigen-multimers. Antigen-multimers detected CARs against CD19, HER2, and Tn-glycoside with significantly higher specificity, sensitivity, and precision than existing reagents. In addition to accurate CAR T-cell detection by flow cytometry, antigen-multimers also enabled ≥100-fold magnetic enrichment of rare CAR T cells, selective CAR T-cell stimulation, and high-dimensional CAR T-cell profiling by single-cell multi-omics analyses. Finally, antigen-multimers accurately captured clinical anti-CD19 CAR T cells from patients' cellular infusion products, post-infusion peripheral blood, and tumor biopsies. Antigen-multimers can be readily extended to other CAR systems by switching its antigen ligand. As such, antigen-multimers have broad clinical and research applications.
ABSTRACT
High-throughput matrix-assisted laser desorption/ionization mass spectrometry (HT-MALDI-MS) has garnered considerable attention within the drug discovery industry as an information-rich alternative to assays using light-based detection methods. To date, these efforts have been primarily focused on assays using protein or peptide substrates. Methods for RNA or DNA analysis by HT-MALDI-MS have not been extensively reported due to the challenges associated with MALDI-MS of oligonucleotides, including the propensity to form multiple salt adducts, low ionization potential, and ease of fragmentation. The objective of this work was to develop a platform suitable for HT-MS analysis of RNA and DNA substrates that overcomes these hurdles by combining on-surface sample preparation with soft ionization. This has been accomplished through the selective immobilization of fluorous-tagged oligonucleotides on a fluorous-modified MS target plate, followed by on-surface enrichment, matrix addition, and direct laser desorption/ionization, a process dubbed fluorous HT-MS (F-HT-MS). The work has resulted in methods by which RNA and DNA substrates can be detected at nanomolar concentrations from a typical assay buffer system using procedures that are amenable to full automation. The protocols were applied to an miRNA biogenesis assay, demonstrating its potential for RNA processes and thereby filling a prominent gap in RNA drug discovery: the paucity of in vitro functional assays.
Subject(s)
DNA/analysis , High-Throughput Screening Assays/methods , RNA/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , DNA/chemistry , Drug Discovery , Oligonucleotides/analysis , RNA/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Structure-Activity RelationshipABSTRACT
The outbreak of 2019 coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global pandemic. Despite intensive research including several clinical trials, currently there are no completely safe or effective therapeutics to cure the disease. Here we report a strategy incorporating neutralizing antibodies conjugated on the surface of a photothermal nanoparticle to actively capture and inactivate SARS-CoV-2. The photothermal nanoparticle is comprised of a semiconducting polymer core and a biocompatible polyethylene glycol surface decorated with neutralizing antibodies. Such nanoparticles displayed efficient capture of SARS-CoV-2 pseudoviruses, excellent photothermal effect, and complete inhibition of viral entry into ACE2-expressing host cells via simultaneous blocking and inactivating of the virus. This photothermal nanoparticle is a flexible platform that can be readily adapted to other SARS-CoV-2 antibodies and extended to novel therapeutic proteins, thus providing a broad range of protection against multiple strains of SARS-CoV-2.
ABSTRACT
MicroRNAs (miRNAs) are short, non-coding RNA molecules estimated to regulate expression of a large number of protein-coding genes and are implicated in a variety of biological processes such as development, differentiation, proliferation, and cell survival. Dysregulation of miRNAs has been attributed to the onset and progression of various human diseases, including cancer. MicroRNA-21 (miR-21), one of the most established oncogenic miRNAs, is found to be upregulated in a wide range of cancers making it an attractive therapeutic target. Employment of a luciferase-based live-cell reporter assay in a high-throughput screen of >300,000 small molecules led to the discovery of a new class of ether-amide miR-21 inhibitors. Following a structure-activity relationship study, an optimized lead molecule was found to inhibit miR-21 transcription. Furthermore, the inhibitor demonstrated cytotoxicity in a cervical cancer cell line via induction of apoptosis and was capable of reducing microtumor formation in a long-term clonogenic assay. Altogether, this work reports the discovery of a new small molecule inhibitor of miR-21 and demonstrates its potential as an alternative approach in cancer therapy.
Subject(s)
MicroRNAs/metabolism , Cell Survival , Humans , Structure-Activity RelationshipABSTRACT
Chemical probes of microRNA (miRNA) function are potential tools for understanding miRNA biology that also provide new approaches for discovering therapeutics for miRNA-associated diseases. MicroRNA-21 (miR-21) is an oncogenic miRNA that is overexpressed in most cancers and has been strongly associated with driving chemoresistance in cancers such as renal cell carcinoma (RCC). Using a cell-based luciferase reporter assay to screen small molecules, we identified a novel inhibitor of miR-21 function. Following structure-activity relationship studies, an optimized lead compound demonstrated cytotoxicity in several cancer cell lines. In a chemoresistant-RCC cell line, inhibition of miR-21 via small molecule treatment rescued the expression of tumor-suppressor proteins and sensitized cells to topotecan-induced apoptosis. This resulted in a >10-fold improvement in topotecan activity in cell viability and clonogenic assays. Overall, this work reports a novel small molecule inhibitor for perturbing miR-21 function and demonstrates an approach to enhancing the potency of chemotherapeutics specifically for cancers derived from oncomir addiction.
Subject(s)
Carcinoma, Renal Cell/pathology , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Kidney Neoplasms/pathology , MicroRNAs/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Topotecan/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , HumansABSTRACT
Biological processes are naturally regulated with high spatial and temporal control, as is perhaps most evident in metazoan embryogenesis. Chemical tools have been extensively utilized in cell and developmental biology to investigate cellular processes, and conditional control methods have expanded applications of these technologies toward resolving complex biological questions. Light represents an excellent external trigger since it can be controlled with very high spatial and temporal precision. To this end, several optically regulated tools have been developed and applied to living systems. In this review we discuss recent developments of optochemical tools, including small molecules, peptides, proteins, and nucleic acids that can be irreversibly or reversibly controlled through light irradiation, with a focus on applications in cells and animals.
Subject(s)
Cell Physiological Phenomena/radiation effects , Optogenetics/methods , Photochemistry/methods , Animals , Cell Physiological Phenomena/drug effects , Drug Discovery/methods , Humans , Light , Molecular Docking Simulation , Nucleic Acids/genetics , Nucleic Acids/metabolism , Peptides/genetics , Peptides/metabolism , Photochemical Processes , Protein Multimerization/drug effects , Protein Multimerization/radiation effects , Proteins/genetics , Proteins/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacologyABSTRACT
DNA-based logic gates can be assembled into computational devices that generate a specific output signal in response to oligonucleotide input patterns. The ability to interface with biological and chemical environments makes DNA computation a promising technology for monitoring cellular systems. However, DNA logic gate circuits typically provide a single-stranded oligonucleotide output, limiting the ability to effect biology. Here, we introduce a novel DNA logic gate design capable of yielding a small molecule output signal. Employing a Staudinger reduction as a trigger for the release and activation of a small molecule fluorophore, we constructed AND and OR logic gates that respond to synthetic microRNA (miRNA) inputs. Connecting the gates in series led to more complex DNA circuits that provided a small molecule output in response to a specific pattern of three different miRNAs. Moreover, our gate design can be readily multiplexed as demonstrated by simultaneous small molecule activation from two independent DNA circuits.