Fluorescence Energy Transfer Research Articles

Novel isocyanobenzene carbazole-based thermally activated delayed fluorophors towards solution-processed blue emitting OLED devices with high efficiency

Achieving efficient blue emission is an exigent challenge for the application of thermally activated delayed fluorescent organic light-emitting diodes (TADF-OLEDs). Carbazole benzonitrile-based TADF molecules exhibit promising performance in achieving the goal. Here, by replacing the cyano group in the sensitizer with the isocyano group, a 9 nm hypochromatic shift in photoluminescence spectra of the sensitizer and an enhanced efficiency of the Förster energy transfer (FET) in TADF-sensitized fluorescence (TSF) is found. By using the isocyano-based compounds as sensitizers, a maximum external quantum efficiency of 24.5% is obtained in solution-processed OLEDs, surpassing the cyano-based counterparts with similar structures. Moreover, the full width at half maximum (FWHM) of the emitter in the electroluminescence spectrum is also decreased from 47 nm to 38 nm, indicating a more complete energy transfer.

Luminescent Terbium Probe for Time‐Resolved FRET and NSET Binding Assays with Quantum Dots and Gold Nanoparticles

Time‐gated or time‐resolved FRET (TR‐FRET) assays are important tools in biosensing, bioimaging, drug screening, and molecular diagnostics. Efficient TR‐FRET assays require stable lanthanide complexes with high absorption cross sections, high quantum yields, and long photoluminescence lifetimes. Owing to their challenging synthesis, such complexes are relatively rare and new components are of potential interest when developing TR‐FRET probes. Here, we evaluate the recently developed Tb complex CoraFluor‐1 concerning its analytical performance in terbium‐to‐quantum dot FRET and terbium‐to‐gold nanoparticle NSET assays using the prototypical biological recognition system of biotin and streptavidin. Biological binding was quantifiable at sub‐picomolar concentrations in small sample volumes, with broad applicability demonstrated across three commercial fluorescence plate readers used for time‐resolved, spectrally‐resolved, and clinical bioanalysis. Overall, CoraFluor‐1 provided excellent analytical performance as both FRET and NSET donor, validating its potential for developing new TR‐FRET probes for biosensing and bioimaging.

A narrow ratio of nucleic acid to SARS-CoV-2 N-protein enables phase separation

SARS-CoV-2 Nucleocapsid protein (N) is a viral structural protein that packages the 30kb genomic RNA inside virions and forms condensates within infected cells through liquid-liquid phase separation (LLPS). In both soluble and condensed forms, N has accessory roles in the viral life cycle including genome replication and immunosuppression. The ability to perform these tasks depends on phase separation and its reversibility. The conditions that stabilize and destabilize N condensates and the role of N-N interactions are poorly understood. We have investigated LLPS formation and dissolution in a minimalist system comprised of N protein and an ssDNA oligomer just long enough to support assembly. The short oligo allows us to focus on the role of N-N interaction. We have developed a sensitive FRET assay to interrogate LLPS assembly reactions from the perspective of the oligonucleotide. We find that N alone can form oligomers but that oligonucleotide enables their assembly into a three-dimensional phase. At a ∼1:1 ratio of N to oligonucleotide, LLPS formation is maximal. We find that a modest excess of N or of nucleic acid causes the LLPS to break down catastrophically. Under the conditions examined here, assembly has a critical concentration of about 1 μM. The responsiveness of N condensates to their environment may have biological consequences. A better understanding of how nucleic acid modulates N-N association will shed light on condensate activity and could inform antiviral strategies targeting LLPS.

Anti-coronavirus and anti-pulmonary inflammation effects of iridoids, the common component from Chinese herbal medicines for the treatment of COVID-19.

The practice of Chinese herbal medicines for the treatment of COVID-19 in China played an essential role for the control of mortality rate and reduction of recovery time. The iridoids is one of the main constituents of many heat-clearing and detoxifying Chinese medicines that were largely planted and frequently used in clinical practice. Twenty-three representative high content iridoids from several staple Chinese medicines were obtained and tested by a SARS-CoV-2 pseudo-virus entry-inhibition assay on HEK-293T/ACE2 cells, a live HCoV-OC43 virus infection assay on HRT-18 cells, and a SARS-CoV-2 3CL protease inhibitory FRET assay followed by molecular docking simulation. The anti-pulmonary inflammation activities were further evaluated on a TNF-α induced inflammation model in A549 cells and preliminary SARs were concluded. The results showed that specnuezhenide (7), cornuside (12), neonuezhenide (15), and picroside III (21) exhibited promising antiviral activities, and neonuezhenide (15) could inhibit 3CL protease with an IC50 of 14.3μM. Docking computation showed that compound 15 could bind to 3CL protease through a variety of hydrogen bonding and hydrophobic interactions. In the anti-pulmonary inflammation test, cornuside (12), aucubin (16), monotropein (17), and shanzhiside methyl ester (18) could strongly decrease the content of IL-1β and IL-8 at 10μM. Compound 17 could also upregulate the expression of the anti-inflammatory cytokine IL-10 significantly. The iridoids exhibited both anti-coronavirus and anti-pulmonary inflammation activities for their significance of existence in Chinese herbal medicines, which also provided a theoretical basis for their potential utilization in the pharmaceutical and food industries.

Substrate-Based Ligand Design for Phenazine Biosynthesis Enzyme PhzF.

The phenazine pyocyanin is an important virulence factor of the pathogen Pseudomonas aeruginosa, which is on the WHO list of antibiotic resistant "priority pathogens". In this study the isomerase PhzF, a key bacterial enzyme of the pyocyanin biosynthetic pathway, was investigated as a pathoblocker target. The aim of the pathoblocker strategy is to reduce the virulence of the pathogen without killing it, thus preventing the rapid development of resistance. Based on crystal structures of PhzF, derivatives of the inhibitor 3-hydroxyanthranilic acid were designed. Co-crystal structures of the synthesized derivatives with PhzF revealed spacial limitations of the binding pocket of PhzF in the closed conformation. In contrast, ligands aligned to the open conformation of PhzF provided more room for structural modifications. The intrinsic fluorescence of small 3-hydroxyanthranilic acid derivatives enabled direct affinity determinations using FRET assays. The analysis of structure-activity relationships showed that the carboxylic acid moiety is essential for binding to the target enzyme. The results of this study provide fundamental structural insights that will be useful for the design of PhzF-inhibitors.

Single-molecule analysis reveals the phosphorylation of FLS2 governs its spatiotemporal dynamics and immunity

The Arabidopsis thaliana FLAGELLIN-SENSITIVE2 (FLS2), a typical receptor kinase, recognizes the conserved 22 amino acid sequence in the N-terminal region of flagellin (flg22) to initiate plant defense pathways, which was intensively studied in the past decades. However, the dynamic regulation of FLS2 phosphorylation at the plasma membrane after flg22 recognition needs further elucidation. Through single-particle tracking, we demonstrated that upon flg22 treatment the phosphorylation of Ser-938 in FLS2 impacts its spatiotemporal dynamics and lifetime. Following Förster resonance energy transfer-fluorescence lifetime imaging microscopy and protein proximity indexes assays revealed that flg22 treatment increased the co-localization of GFP-tagged FLS2/FLS2S938D but not FLS2S938A with AtRem1.3-mCherry, a sterol-rich lipid marker, indicating that the phosphorylation of FLS2S938 affects FLS2 sorting efficiency to AtRem1.3-associated nanodomains. Importantly, we found that the phosphorylation of Ser-938 enhanced flg22-induced FLS2 internalization and immune responses, demonstrating that the phosphorylation may activate flg22-triggered immunity through partitioning FLS2 into functional AtRem1.3-associated nanodomains, which fills the gap between the FLS2S938 phosphorylation and FLS2-mediated immunity.

Single-molecule analysis reveals the phosphorylation of FLS2 governs its spatiotemporal dynamics and immunity.

The Arabidopsis thaliana FLAGELLIN-SENSITIVE2 (FLS2), a typical receptor kinase, recognizes the conserved 22 amino acid sequence in the N-terminal region of flagellin (flg22) to initiate plant defense pathways, which was intensively studied in the past decades. However, the dynamic regulation of FLS2 phosphorylation at the plasma membrane after flg22 recognition needs further elucidation. Through single-particle tracking, we demonstrated that upon flg22 treatment the phosphorylation of Ser-938 in FLS2 impacts its spatiotemporal dynamics and lifetime. Following Förster resonance energy transfer-fluorescence lifetime imaging microscopy and protein proximity indexes assays revealed that flg22 treatment increased the co-localization of GFP-tagged FLS2/FLS2S938D but not FLS2S938A with AtRem1.3-mCherry, a sterol-rich lipid marker, indicating that the phosphorylation of FLS2S938 affects FLS2 sorting efficiency to AtRem1.3-associated nanodomains. Importantly, we found that the phosphorylation of Ser-938 enhanced flg22-induced FLS2 internalization and immune responses, demonstrating that the phosphorylation may activate flg22-triggered immunity through partitioning FLS2 into functional AtRem1.3-associated nanodomains, which fills the gap between the FLS2S938 phosphorylation and FLS2-mediated immunity.

Design and Discovery of New Collagen V-Derived FGF2-Blocking Natural Peptides Inhibiting Lung Squamous Cell Carcinoma In Vitro and In Vivo.

Aberrant FGF2/FGFR signaling is implicated in lung squamous cell carcinoma (LSCC), posing treatment challenges due to the lack of targeted therapeutic options. Designing drugs that block FGF2 signaling presents a promising strategy different from traditional kinase inhibitors. We previously reported a ColVα1-derived fragment, HEPV (127AA), that inhibits FGF2-induced angiogenesis. However, its large size may limit therapeutic application. This study combines rational peptide design, molecular dynamics simulations, knowledge-based prediction, and GUV and FRET assays to identify smaller peptides with FGF2-blocking properties. We synthesized two novel peptides, HBS-P1 (45AA) and HBS-P2 (66AA), that retained the heparin-binding site. Both peptides demonstrated anti-LSCC and antiangiogenesis properties in cell viability and microvessel network induction assays. In two LSCC subcutaneous models, HBS-P1, with its affinity for FGF2 and enhanced penetration ability, demonstrated substantial therapeutic potential without apparent toxicities. Our study provides the first evidence supporting the development of collagen V-derived natural peptides as FGF2-blocking agents for LSCC treatment.

Direct measurements of neurosteroid binding to specific sites on GABAA receptors.

Neurosteroids are allosteric modulators of GABAA currents, acting through several functional binding sites although their affinity and specificity for each site are unknown. The goal of this study was to measure steady-state binding affinities of various neurosteroids for specific sites on the GABAA receptor. Two methods were developed to measure neurosteroid binding affinity: (1) quenching of specific tryptophan residues in neurosteroid binding sites by the neurosteroid 17-methylketone group, and (2) FRET between MQ290 (an intrinsically fluorescent neurosteroid) and tryptophan residues in the binding sites. The assays were developed using ELIC-α1GABAAR, a chimeric receptor containing transmembrane domains of the α1-GABAA receptor. Tryptophan mutagenesis was used to identify specific interactions. Allopregnanolone (3α-OH neurosteroid) was shown to bind at intersubunit and intrasubunit sites with equal affinity, whereas epi-allopregnanolone (3β-OH neurosteroid) binds at the intrasubunit site. MQ290 formed a strong FRET pair with W246, acting as a site-specific probe for the intersubunit site. The affinity and site-specificity of several neurosteroid agonists and inverse agonists was measured using the MQ290 binding assay. The FRET assay distinguishes between competitive and allosteric inhibition of MQ290 binding and demonstrated an allosteric interaction between the two neurosteroid binding sites. The affinity and specificity of neurosteroid binding to two sites in the ELIC-α1GABAAR were directly measured and an allosteric interaction between the sites was revealed. Adaptation of the MQ290 FRET assay to a plate-reader format will enable screening for high affinity agonists and antagonists for neurosteroid binding sites.

Human cytochrome C natural variants: Studying the membrane binding properties of G41S and Y48H by fluorescence energy transfer and molecular dynamics

Cytochrome C (cyt C), the protein involved in oxidative phosphorylation, plays several other crucial roles necessary for both cell life and death. Studying natural variants of cyt C offers the possibility to better characterize the structure-to-function relationship that modulates the different activities of this protein. Naturally mutations in human cyt C (G41S and Y48H) occur in the protein central Ω-loop and cause thrombocytopenia 4. In this study, we have investigated the binding of such variants and of wild type (wt) cyt C to synthetic cardiolipin-containing vesicles. The mutants have a lower propensity in membrane binding, displaying higher dissociation constants with respect to the wt protein. Compressibility measurements reveal that both variants are more flexible than the wt, suggesting that the native central Ω-loop is important for the interaction with membranes. Such hypothesis is supported by molecular dynamics simulations. A minimal distance analysis indicates that in the presence of cardiolipin the central Ω-loop of the mutants is no more in contact with the membrane, as it happens instead in the case of wt cyt C. Such finding might provide a hint for the reduced membrane binding capacity of the variants and their enhanced peroxidase activity in vivo.

Replication protein A dynamically re-organizes on primer/template junctions to permit DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis.

DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, PCNA, carry out DNA synthesis during lagging strand replication, initiation of leading strand replication, and the major DNA damage repair and tolerance pathways. Pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process involving the major single strand DNA (ssDNA)-binding protein complex, RPA, the processivity sliding clamp loader, RFC, PCNAand pol δ. During this process, the interactions of RPA, RFCand pol δ with a P/T junction all significantly overlap. A burning issue that has yet to be resolved is how these overlapping interactions are accommodated during this process. To address this, we design and utilize novel, ensemble FRET assays that continuously monitor the interactions of RPA, RFC, PCNAand pol δ with DNA as pol δ holoenzymes are assembled and initiate DNA synthesis. Results from the present study reveal that RPA remains engaged with P/T junctions throughout this process and the RPA•DNA complexes dynamically re-organize to allow successive binding of RFC and pol δ. These results have broad implications as they highlight and distinguish the functional consequences of dynamic RPA•DNA interactions in RPA-dependent DNA metabolic processes.

Identification of Cinnamein, a Component of Balsam of Tolu/Peru, as a New Ligand of PPARα for Plaque Reduction and Memory Protection in a Mouse Model of Alzheimer's Disease.

Despite intense investigations, no effective treatment is yet available to reduce plaques and protect memory and learning in patients with Alzheimer's disease (AD), the most common neurodegenerative disorder. Therefore, it is important to identify a non-toxic, but effective, treatment option for AD. Cinnamein, a nontoxic compound, is naturally available in Balsam of Peru and Tolu Balsam. We examined whether cinnamein treatment could decrease plaques and improve cognitive functions in 5XFAD mouse model of AD. We employed in silico analysis, time-resolved fluorescence energy transfer assay, thermal shift assay, primary neuron isolation, western blot, immunostaining, immunohistochemistry, Barnes maze, T maze, and open field behavior. Oral administration of cinnamein led to significant reduction in amyloid-β plaque deposits in the brain and protection of spatial learning and memory in 5XFAD mice. Peroxisome proliferator-activated receptor alpha (PPARα), a nuclear hormone receptor, is involved in plaque lowering and increase in hippocampal plasticity. While investigating underlying mechanisms, we found that cinnamein served as a ligand of PPARα. Accordingly, oral cinnamein upregulated the level of PPARα, but not PPARβ, in the hippocampus, and remained unable to decrease plaques from the hippocampus and improve memory and learning in 5XFAD mice lacking PPARα. While A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is one of the drivers of nonamyloidogenic pathway, transcription factor EB (TFEB) is considered as the master regulator of autophagy. Cinnamein treatment was found to upregulate both ADAM10 and TFEB in the brain of 5XFAD mice via PPARα. Our results suggest that this balsam component may have therapeutic importance in AD.

Fluorescence-Selective Absorption and Circularly Polarized Fluorescence Energy Transfer Assist the Generation of Multicolor Circularly Polarized Luminescence in Chiral Helical Polyacetylene-Based Janus Nanofibers.

Chiroptical nanomaterials with circularly polarized luminescence (CPL) performance have aroused increasing attention. Herein, multicolor CPL-active Janus nanofibers are prepared through a simple parallel electrospinning method using chiral helical polyacetylenes as the chiral source and achiral fluorophores as the fluorescent source. Interestingly, despite a direct spatial isolation between the chiral component and the fluorescent component, blue and green CPL emissions can still be obtained due to the fluorescence-selective absorption behavior of chiral helical polyacetylenes, with a satisfactory dissymmetric factor (glum) of 2 × 10-2 and 2.5 × 10-3, respectively. Moreover, by taking advantage of the circular polarization fluorescence energy transfer process, red CPL emission is further achieved using the obtained blue and green CPL as energy donors and the achiral red fluorophore as an energy acceptor. The present work offers a facile approach to prepare multilevel-structured chiroptical materials with promising application potentials in a flexible photoelectric device.

Acylhydrazones derived from isonicotinic acid: Synthesis, characterization, and evaluation against Alzheimer's disease biomarkers

Acylhydrazones have garnered attention as a promising structural motif due to their capacity to establish hydrogen bonds and interact effectively with biomolecular targets. In this study, we synthesized ten distinct acylhydrazones using the carbohydrazide of isonicotinic acid (INH) in combination with various substituted aromatic aldehydes. The compounds were obtained with moderate chemical yields, in the range of 28 to 82 %. Following thorough spectroscopic characterization, we proceeded to investigate the impact of the synthesized compounds on key biochemical indicators associated with Alzheimer's disease. We examined the inhibition of acetylcholinesterase (AChE) and β-secretase (BACE-1) through Ellman's colorimetric assay and FRET assay, respectively. The formation of Aβ fibrils was assessed using Thioflavin T fluorescence emission, while the compounds' ability to scavenge free radicals was evaluated using the DPPH method. Notably, the most of compounds demonstrated inhibitory effects on AChE, with the acylhydrazone derived from 3,5-difluorobenzaldehyde and INH displaying the highest potency. Among the array of tested compounds, the derivative sourced from 4-trifluoromethylbenzaldehyde exhibited AChE and BACE-1 inhibitory activity, with IC50 values of 2.98 and 63.9 µM, respectively. This compound also has an effect on inhibiting the formation of Aβ fibrils (IC50 = 17.1 µM) and DPPH radical scavenging activity in 88 %. Physicochemical and pharmacokinetic properties were in silico calculated using SwissADME software and all compounds were predicted to have good oral bioavailability.

Rapid Colorimetric Quantita2tive Portable Platform for Detection of Brucella melitensis Based on a Fluorescence Resonance Energy Transfer Assay and Nanomagnetic Particles.

Brucellosis is a bacterial zoonotic disease that requires major attention for both health and financial facilities in many parts of the world including the Mediterranean and the Middle East. The existing gold standard diagnosis relies on the culturing technique, which is costly and time-consuming with a duration of up to 45 days. The Brucella protease biosensor represents a new detection approach that will lead to low-cost point-of-care devices for sensitive Brucella detection. In addition, the described diagnostic device is portable and simple to operate by a nurse or non-skilled clinician making it appropriate for the low-resource setting. In this study, we rely on the total extracellular protease proteolytic activity on specific peptide sequences identified using the FRET assay by high-throughput screening from the library of peptide (96 short peptides such as dipeptides and tripeptides) substrates for Brucella melitensis (B. melitensis). The B. melitensis-specific protease substrate was utilized in the development of the paper-based colorimetric assay. Two specific and highly active dipeptide substrates were identified (FITC-Ahx-K-r-K-Ahx-DABCYL and FITC-Ahx-R-r-K-Ahx-DABCYL). The peptide-magnetic bead nanoprobe sensors developed based on these substrates were able to detect the Brucella with LOD as low as 1.5 × 102 and 1.5 × 103 CFU/mL using K-r dipeptide and R-r dipeptide substrates, respectively, as the recognition element. The samples were tested using this sensor in few minutes. Cross-reactivity studies confirmed that the other proteases extracted from closely related pathogens have no significant effect on the sensor. To the best of our knowledge, this assay is the first assay that can be used with low-cost, rapid, direct, and visual detection of B. melitensis.

Luminescence Dynamics of BaAl2O4:Eu2+ Phosphor.

We studied steady-state and time-resolved photoluminescence of Eu doped BaAl2O4 phosphor. The undoped BaAl2O4 sample shows a dominant blue emission band at ~ 428nm and two secondary maxima at ~ 405 and 456nm due to F-centre and aggregate defects such as F2 -centre. The samples after doping of Eu at 1-5% show additional emission bands at ~ 485 and 518 nm due to Eu2+ centre and a red emission band at ~ 657nm is attributed to Eu3+ centre. The sample doped with 2% of Eu shows anomalous emission having the dominant peak at ~ 494nm. The average luminescence lifetime of the emission band at ~ 428nm in the undoped sample was estimated to be (3.29 ± 0.91) ns. The average luminescence lifetime of this emission band after doping of Eu was found to increase by 102 orders of magnitude. The intensity of the 428nm blue emission band was found to quench after doping of Eu beyond 3%. The concentration quenching effect was attributed to dipole-quadrupole interaction. Further, a non-radiative fluorescence energy transfer mechanism from an extrinsic Eu2+ centre to an intrinsic F-centre is proposed to describe the luminescence dynamics of the samples.

BRD2-specific inhibitor, BBC0403, inhibits the progression of osteoarthritis pathogenesis in osteoarthritis-induced C57BL/6 male mice.

The discovery of new bromo- and extra-terminal inhibitors presents new drugs to treat osteoarthritis (OA). The new drug, BBC0403, was identified in the DNA-encoded library screening system by searching for compounds that target BRD (bromodomain-containing) proteins. The binding force with BRD proteins was evaluated using time-resolved fluorescence energy transfer (TR-FRET) and binding kinetics assays. Subsequently, in vitro and ex vivo analyses demonstrated the effects of the BRD2 inhibitor, BBC0403, on OA. For animal experiments, medial meniscus destabilization was performed to create a 12-week-old male C57BL/6 mouse model, and intra-articular (i.a.) injections were administered. Histological and immunohistochemical analyses were then performed. The underlying mechanism was confirmed by gene set enrichment analysis (GSEA) using RNA-seq. TR-FRET and binding kinetics assays revealed that BBC0403 exhibited higher binding specificity for BRD2 compared to BRD3 and BRD4. The anti-OA effects of BBC0403 were tested at concentrations of 5, 10 and 20 μM (no cell toxicity in the range tested). The expression of catabolic factors, prostaglandin E2 (PGE2) production and extracellular matrix (ECM) degradation was reduced. Additionally, the i.a. injection of BBC0403 prevented OA cartilage degradation in mice. Finally, BBC0403 was demonstrated to suppress NF-κB and MAPK signalling pathways. This study demonstrated that BBC0403 is a novel BRD2-specific inhibitor and a potential i.a.-injectable therapeutic agent to treat OA.

Reversible Photochromism for Dynamically Tuning Full‐Color Circularly Polarized Luminescence Toward Multi‐Level Anti‐Counterfeiting Application

AbstractPhotochromic circularly polarized luminescence (CPL) demonstrates broad application prospects in the field of advanced functional materials. However, the current photochromic CPL focuses predominantly on single‐band CPL adjustment, and how to achieve dynamically tunable CPL in multi‐band is still highly challenging. This work reports the success in utilizing reversible photochromism for realizing full‐color CPL emission. Photochromic flexible films are created consisting of triple components: spiropyran (SP) working as photochromic matter, polyurethane (PU) as matrix, and chiral helical polyacetylene as chiral component. Further introducing blue fluorophore 9‐anthracene carboxylic acid (ACA) in the film, blue CPL and red CPL are emitted respectively through selective absorption/transmission and circularly polarized fluorescence energy transfer (CPF‐ET). Doping the photochromic film with diverse fluorophores provides full‐color CPL emission. Adding Zn2+ further intensifies fluorescence and CPL via forming merocyanine (MC)‐Zn2+ complex. Taking advantage of the diverse fluorescence and CPL emission, anti‐counterfeiting, and secondary encryption application of the resulting photochromic CPL materials are demonstrated.

Abstract 4535: JAB-26766: A small-molecule, orally bioavailable PARP7 inhibitor with high potency and selectivity

Abstract Background: PARP7 (also referred to as TIPARP or ARTD14) is a mono-ADP-ribosyl transferase that inhibits type I interferon (IFN) signaling. Targeting PARP7 to restore antitumor immunity represents a promising treatment strategy. We have developed JAB-26766, an orally bioavailable, highly potent, and selective PARP7 inhibitor. Methods: A time-resolved fluorescence energy transfer (TR-FRET) assay was applied to determine the binding and inhibition of PARP7 by JAB-26766. IFN-β secretion assay, STAT1 phosphorylation assay, and ISG (interferon stimulated gene) mRNA assay were performed to evaluate the inhibitory activities of JAB-26766 on the downstream signaling of PARP7. CellTiter-Glo assay was performed to evaluate cell viability upon treatment of JAB-26766. In vivo PK-PD study was conducted to evaluate the relationship between JAB-26766 concentration and the level of tumor STAT1 phosphorylation. The antitumor activity of JAB-26766 either as a single agent or in different drug combinations was evaluated in multiple mouse models. Results: High potency of JAB-26766 on PARP7 inhibition as well as >1800-fold selectivity over PARP2 was observed through biochemical assays. At the cellular level, JAB-26766 induced IFN-β secretion, up-regulated ISG mRNA levels, increased phosphorylation of STAT1, and inhibited cell viability of multiple tumor cell lines (with most IC50 values below 50 nM). JAB-26766 combined with a STING agonist resulted in synergistic activation of STING pathway, as evidenced by increased CXCL10 secretion, and inhibited tumor cell proliferation. In vivo PK-PD study showed that JAB-26766 exposure in plasma and tumor has good correlation with induction of tumor STAT1 phosphorylation. Furthermore, JAB-26766 showed potent antitumor activity and good tolerability either as monotherapy or combined with STING agonist or anti-PD-1 antibody in mouse models of non-small cell lung cancer, breast cancer and colorectal cancer. Conclusions: JAB-26766 is an orally bioavailable, highly potent, and selective PARP7 inhibitor that shows early promising anti-tumor activities. Citation Format: Di Kang, Yanping Wang, Xin Sun, Man Yan, Haijun Li, Mingming Chen, Yiwei Lin, Wei Long. JAB-26766: A small-molecule, orally bioavailable PARP7 inhibitor with high potency and selectivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4535.

Diagnostic accuracy of serum matrix metalloproteinase-7 as a biomarker of biliary atresia in a large North American cohort.

High levels of serum matrix metalloproteinase-7 (MMP-7) have been linked to biliary atresia (BA), with wide variation in concentration cutoffs. We investigated the accuracy of serum MMP-7 as a diagnostic biomarker in a large North American cohort. MMP-7 was measured in serum samples of 399 infants with cholestasis in the Prospective Database of Infants with Cholestasis study of the Childhood Liver Disease Research Network, 201 infants with BA and 198 with non-BA cholestasis (age median: 64 and 59 days, p = 0.94). MMP-7 was assayed on antibody-bead fluorescence (single-plex) and time resolved fluorescence energy transfer assays. The discriminative performance of MMP-7 was compared with other clinical markers. On the single-plex assay, MMP-7 generated an AUROC of 0.90 (CI: 0.87-0.94). At cutoff 52.8ng/mL, it produced sensitivity = 94.03%, specificity = 77.78%, positive predictive value = 64.46%, and negative predictive value = 96.82% for BA. AUROC for gamma-glutamyl transferase = 0.81 (CI: 0.77-0.86), stool color = 0.68 (CI: 0.63-0.73), and pathology = 0.84 (CI: 0.76-0.91). Logistic regression models of MMP-7 with other clinical variables individually or combined showed an increase for MMP-7+gamma-glutamyl transferase AUROC to 0.91 (CI: 0.88-0.95). Serum concentrations produced by time resolved fluorescence energy transfer differed from single-plex, with an optimal cutoff of 18.2ng/mL. Results were consistent within each assay technology and generated similar AUROCs. Serum MMP-7 has high discriminative properties to differentiate BA from other forms of neonatal cholestasis. MMP-7 cutoff values vary according to assay technology. Using MMP-7 in the evaluation of infants with cholestasis may simplify diagnostic algorithms and shorten the time to hepatoportoenterostomy.