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Hydrothermal deposition is emerging as a highly potential route for antimony-based solar cells, in which the Sb2(S,Se)3 is typically in situ grown on a common toxic CdS buffer layer. The narrow band gap of CdS causes a considerable absorption in the short-wavelength region and then lowers the current density of the device. Herein, TiO2 is first evaluated as an alternative Cd-free buffer layer for hydrothermally derived Sb2S3 solar cells. But it suffers from a severely inhomogeneous Sb2S3 coverage, which is effectively eliminated by inserting a Zn(O,S) layer. The surface atom of sulfur in Zn(O,S) uniquely provides a chemical bridge to enable the quasi-epitaxial deposition of Sb2S3 thin film, confirming by both morphology and binding energy analysis using DFT. Then the results of the first-principles calculations also show that Zn(O,S)/Sb2S3 has a more stable structure than TiO2/Sb2S3. The resultant perfect Zn(O,S)/Sb2S3 junction, with a suitable band alignment and excellent interface contact, delivers a remarkably enhanced JSC and VOC for Sb2S3 solar cells. The device efficiency with the TiO2/Zn(O,S) buffer layer boosts from 0.54% to 3.70% compared with the counterpart of TiO2, which has a champion efficiency of Cd-free Sb2S3 solar cells with a structure of ITO/TiO2/Zn(O,S)/Sb2S3/Carbon/Ag by in situ hydrothermal deposition. This work provides a guideline for the hydrothermal deposition of antimony-based films upon a nontoxic buffer layer.

An enantioselective synthesis of spiropyrazolone-fused cyclopenta[c]chromen-4-ones is demonstrated via a (3+2) cycloaddition reaction. The reactions of 3-homoacylcoumarins and α,β-unsaturated pyrazolones in the presence of the cinchona-alkaloid derived hydrogen-bonding catalyst provide aforementioned spiropyrazolone-chromenone adducts bearing five contiguous stereocenters, of which one is the spiro all-carbon quaternary stereocenter in high yields (up to 98%) with good to excellent stereoselectivities (>25:1 dr and up to 99% ee). This one-pot methodology could also be practically demonstrated on a gram-scale with similar efficacy.

We report an efficient method for the direct β-acylation of 2-ylideneoxindoles with acyl chlorides that is catalyzed by organophosphanes in the presence of base. A variety of functionalized 2-ylideneoxindoles were prepared in moderate to good yields under mild, metal-free conditions via a tandem phospha-Michael/O-acylation/intramolecular cyclization/rearrangement sequence. Mechanistic investigations revealed that C–O bond cleavage on a possible betaine intermediate is the key step for the installation of the keto-functionality at the β-position of 2-ylideneoxindoles in a highly stereospecific manner. The synthetic utility of this protocol could also be proven by a scale-up reaction and synthetic transformations of the products.

An efficient protocol for the construction of functionalized 3-alkenyl benzofurans is demonstrated under metal-free conditions using catalytic amount of phosphine proceeding an intramolecular Wittig reaction. This one-pot reaction initiated by the phospha-Michael addition of phosphine to O-acylated nitrostyrene, in which phosphine was in-situ-generated from the chemoselective reduction of phosphine oxide with PhSiH3, would provide the phosphorus ylide to result in the aforementioned multifunctionalized benzofuran via O-acylation/nitrous acid elimination/Wittig reaction.

[Image: see text] Proteolysis-targeting chimeras (PROTACs) degrade target proteins by engaging the ubiquitin-proteasome system. Assays detecting target–PROTAC-E3 ligase ternary complexes are critical for PROTAC development. Both time-resolved fluorescence energy transfer (TR-FRET) assays and amplified luminescent proximity homogeneous assays can characterize ternary complexes and assess PROTAC efficacy; stepwise optimization protocols for these assays are lacking. To identify assay conditions that can be applied to various targets and PROTACs, we used a stepwise approach to optimize a TR-FRET assay of BRD2(BD1)/PROTACs/CRBN ternary complexes. This assay is sensitive and specific and responds to the bivalent PROTACs dBET1, PROTAC BET Degrader-1, and PROTAC BET Degrader-2 but not to non-PROTAC ligands of BRD2(BD1) or CRBN. The activity rank order of dBET1, PROTAC BET Degrader-1, and PROTAC BET Degrader-2 in the TR-FRET assay corresponded with previously reported cell growth inhibition assays, indicating the effectiveness of our assay for predicting PROTAC cellular activity. The TR-FRET ternary complex formation assay for BRD2(BD1)/PROTAC/CRBN can be configured to characterize the binding activities of BRD2(BD1) and CRBN ligands with the same compound activity rank order as that of previously reported binary binding assays for individual targets but with the advantage of simultaneously assessing the ligand activities for both targets. Our assay is modular in nature, as BRD2(BD1) can be replaced with other BRDs and successfully detect ternary complexes without modifying other assay conditions. Therefore, the TR-FRET ternary complex assay for BRDs provides a general assay protocol for establishing assays for other targets and bivalent molecules.

A new method for the construction of functionalized furo[3,2-c]coumarins via MBH-type/acyl-transfer/Wittig reaction is reported. The current approach would open a new route for the simultaneous formation of two rings in a one-pot reaction which is accompanied by incorporation of a keto functionality on the furan ring by activating the terminal alkynoates with phosphine. Furthermore, this protocol could also be applicable to the internal alkynoates/propiolamides to generate the 2,3-disubstituted furo[3,2-c]coumarins/furo[3,2-c]quinolinones by excluding the acyl-transfer reaction.

The von Hippel-Lindau (VHL) tumor suppressor associates with transcription factors elongin-C and elongin-B to form the VHL-elongin-C-elongin-B protein complex and carry out its functions, such as degradation of hypoxia-inducible factors. VHL ligands are used not only to modulate hypoxia-signaling pathways and potentially treat chronic anemia or ischemia but also to form bivalent ligands as proteolysis-targeting chimeras to degrade proteins for potential therapeutic applications. Sensitive and selective VHL-based binding assays are critical for identifying and characterizing VHL ligands with high-throughput screening approaches. VHL ligand-binding assays, such as isothermal titration calorimetry, surface plasmon resonance, and fluorescence polarization assays, are reported but with limitations. Isothermal titration calorimetry requires higher protein concentrations with a lower throughput than fluorescence-based assays do. Surface plasmon resonance requires protein immobilization, which introduces variation and is not suitable for testing a large number of ligands. Fluorescence polarization can be sensitive with high-throughput capability but is susceptible to assay interference, and small-molecule-based fluorescent probes are not available. We developed the first small-molecule-based high-affinity VHL fluorescent probe BODIPY FL VH032 (5), with a K d of 3.01 nM, for a time-resolved fluorescence resonance energy-transfer assay. This new assay is sensitive, selective, resistant to assay interference, and capable of characterizing VHL ligands with a wide range of affinities. It is also suitable for VHL ligand identification and characterization with high-throughput screening.

Ligands for cereblon, a component of a functional E3 ligase complex that targets proteins for proteolysis, are critical for developing molecular glues and proteolysis-targeting chimeras (PROTACs), which have therapeutic implications for various diseases. However, the lack of sensitivity of previously reported assays limits characterization of cereblon ligands. To address this shortcoming, we developed BODIPY FL Thalidomide (10) as a high-affinity fluorescent probe for the human cereblon protein, with a K(d) value of 3.6 nM. We then used BODIPY FL Thalidomide (10) to develop a cereblon time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay. The IC(50) values of the cereblon ligand pomalidomide (8) were 6.4 nM in our cereblon TR-FRET binding assay, 264.8 nM in a previously reported Cy5-conjugated thalidomide (7)-mediated fluorescence polarization (FP) assay, and 1.2 μM in a previously reported Cy5-conjugated cereblon modulator (compound 7) (9)-mediated TR-FRET assay, indicating that our cereblon TR-FRET binding assay is 41- and 187-fold more sensitive than these two previously published assays. With our cereblon TR-FRET binding assay, we detected binding of cereblon ligands but not binding of bromodomain-containing protein 4 or von Hippel–Lindau ligands, thereby demonstrating its selectivity. Our cereblon TR-FRET binding assay was very stable and detected changes in phthalimide activity due to thalidomide isomerization. Therefore, the BODIPY FL Thalidomide (10)-mediated cereblon TR-FRET binding assay we designed is highly sensitive, selective, and stable and will aid the development and characterization of novel cereblon ligands.

Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded the development of MAGE-directed therapeutics. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-Aminoquinolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for developing cancer-specific therapeutics., Testis-restricted melanoma antigen (MAGE) proteins function as substrate adapters for E3 ubiquitin ligases. Biochemical and structural analyses of MAGE-A11 provide insight into the substrate binding mode of MAGE proteins and enable discovery of potent, cytotoxic inhibitors of MAGE-A11:substrate interaction.

An efficient method for the diversity-oriented synthesis of spiropentadiene pyrazolones and 1H-oxepino[2,3-c]pyrazoles is reported. The methodology attributes O-acylation of phosphorus zwitterions which were formed by a tandem phospha-1,6-addition of PBu3 to α,β,γ,δ-unsaturated pyrazolones, further generating betaine intermediates that preferentially resulted in the aforementioned cyclic products in a diversity-oriented manner. The mechanistic investigations revealed that formation of the betaines is the key step to provide the products via an intramolecular Wittig reaction or an unprecedented δ-C-acylation/cyclization/Wittig reaction.

Background: Lung cancer has a high mortality rate and is resistant to multiple chemotherapeutics. Natural Borneol (NB) is a monoterpenoid compound that facilitates the bioavailability of drugs. In this study, we investigated the effects of NB on chemosensitivity in the A549 human lung adenocarcinoma cell line and to elucidate therapeutic molecular target of NB. Methods: The chemosensitivity effects of NB in A549 cells were examined by MTT assay. The mechanism of NB action was evaluated using flow cytometry and Western blotting assays. Surface plasmon resonance (SPR) and LC-MS combined analysis (MS-SPRi) was performed to elucidate the candidate molecular target of NB. The chemosensitizing capacity of NB in vivo was assessed in nude mice bearing A549 tumors. Results: NB pretreatment sensitized A549 cells to low doxorubicin (DOX) dosage, leading to a 15.7% to 41.5% increase in apoptosis. This increase was correlated with ERK and AKT inactivation and activation of phospho-p38 MAPK, phospho-JNK, and phosphor-p53. Furthermore, this synergism depends on reactive oxygen species (ROS) generation. MS-SPRi analysis revealed that transient receptor potential melastatin-8 (TRPM8) is the candidate target of NB in potentiating DOX killing potency. Genetically, TRPM8 knock-down significantly suppresses the chemosensitizing effects of NB and inhibits ROS generation through restraining calcium mobilization. Moreover, pretreatment with NB synergistically enhances the anticancer effects of DOX to delay tumor progression in vivo. Conclusions: These results suggest that TRPM8 may be a valid therapeutic target in the potential application of NB, and show that NB is a chemosensitizer for lung cancer treatment.

The xenobiotic receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are activated by structurally diverse chemicals to regulate the expression of target genes, and they have overlapping regulation in terms of ligands and target genes. Receptor-selective agonists are, therefore, critical for studying the overlapping function of PXR and CAR. An early effort identified 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) as a selective human CAR (hCAR) agonist, and this has since been widely used to distinguish the function of hCAR from that of human PXR (hPXR). The selectivity was demonstrated in a green monkey kidney cell line, CV-1, in which CITCO displayed >100-fold selectivity for hCAR over hPXR. However, whether the selectivity observed in CV-1 cells also represented CITCO activity in liver cell models was not hitherto investigated. In this study, we showed that CITCO: 1) binds directly to hPXR; 2) activates hPXR in HepG2 cells, with activation being blocked by an hPXR-specific antagonist, SPA70; 3) does not activate mouse PXR; 4) depends on tryptophan-299 to activate hPXR; 5) recruits steroid receptor coactivator 1 to hPXR; 6) activates hPXR in HepaRG cell lines even when hCAR is knocked out; and 7) activates hPXR in primary human hepatocytes. Together, these data indicate that CITCO binds directly to the hPXR ligand-binding domain to activate hPXR. As CITCO has been widely used, its confirmation as a dual agonist for hCAR and hPXR is important for appropriately interpreting existing data and designing future experiments to understand the regulation of hPXR and hCAR. SIGNIFICANCE STATEMENT: The results of this study demonstrate that 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) is a dual agonist for human constitutive androstane receptor (hCAR) and human pregnane X receptor (hPXR). As CITCO has been widely used to activate hCAR, and hPXR and hCAR have distinct and overlapping biological functions, these results highlight the value of receptor-selective agonists and the importance of appropriately interpreting data in the context of receptor selectivity of such agonists.

Targeted protein degradation is a powerful induced-proximity tool to control cellular concentrations of native proteins using small molecules. However, the design of selectivity in protein degradation remains challenging. In the case of Bromodomain and Extra-Terminal (BET) family proteins, BRD4 has emerged as the primary therapeutic target over other family members BRD2, 3 and T, but strategies to selectively degrade BRD4 rely on the use of pan-BET inhibitors optimized for BRD4:E3 protein-ubiquitin ligase (E3) ternary complex formation. Here, we report a potent and selective inhibitor for the first bromodomain of BRD4, iBRD4-BD1 (IC50 = 12 nM, 23-6200-fold intra-BET selectivity). We further use this novel inhibitor to develop dBRD4-BD1 that induces selective degradation of BRD4 at a DC50 of 280 nM. The design of BRD4 selectivity up-front enables the study of BRD4 biology in the absence of wider BET-inhibition, simplifies design of future BRD4-selective degraders as new E3 recruiting ligands are discovered, and provides a tool to design additional heterobifunctional BRD4-selective probes.

Alzheimer’s disease (AD) is a common neurodegenerative disease presenting with progressive memory and cognitive impairments. One of the pathogenic mechanisms of AD is attributed to the aggregation of misfolded amyloid β (Aβ), which induces neurotoxicity by reducing the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TRKB) and increasing oxidative stress, caspase-1, and acetylcholinesterase (AChE) activities. Here, we have found the potential of two novel synthetic coumarin derivatives, ZN014 and ZN015, for the inhibition of Aβ and neuroprotection in SH-SY5Y neuroblastoma cell models for AD. In SH-SY5Y cells expressing the GFP-tagged Aβ-folding reporter, both ZN compounds reduced Aβ aggregation, oxidative stress, activities of caspase-1 and AChE, as well as increased neurite outgrowth. By activating TRKB-mediated extracellular signal-regulated kinase (ERK) and AKT serine/threonine kinase 1 (AKT) signaling, these two ZN compounds also upregulated the cAMP-response-element binding protein (CREB) and its downstream BDNF and anti-apoptotic B-cell lymphoma 2 (BCL2). Knockdown of TRKB attenuated the neuroprotective effects of ZN014 and ZN015. A parallel artificial membrane permeability assay showed that ZN014 and ZN015 could be characterized as blood–brain barrier permeable. Our results suggest ZN014 and ZN015 as novel therapeutic candidates for AD and demonstrate that ZN014 and ZN015 reduce Aβ neurotoxicity via pleiotropic mechanisms.

A highly diastereoselective method for the synthesis of novel spiro-tetrahydroquinoline derivatives is reported here, using a one-pot reaction method. All compounds were characterized by 1H-nuclear magnetic resonance (NMR) and mass spectroscopy, and their stereo configurations were confirmed by X-ray analysis. These activities of these derivatives were then tested in human keratocyte cells. The responses of cells to treatment with selected compounds were studied using scratch analysis, and the compounds were tested in a mouse excision wound model. Three of the derivatives demonstrated significant wound-healing activities.

An efficient synthesis of functionalized benzofurans is achieved under mild and metal-free conditions from stable para-quinone methides by treatment with phosphine, acyl chloride, and a base. This ...

The pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are ligand-activated nuclear receptors (NRs) that are notorious for their role in drug metabolism, causing unintended drug-drug interactions and decreasing drug efficacy. They control the xenobiotic detoxification system by regulating the expression of an array of drug-metabolizing enzymes and transporters that excrete exogenous chemicals and maintain homeostasis of endogenous metabolites. Much effort has been invested in recognizing potential drugs for clinical use that can activate PXR and CAR to enhance the expression of their target genes, and in identifying PXR and CAR inhibitors that can be used as co-therapeutics to prevent adverse effects. Here, we present current technologies and assays used in the quest to characterize PXR and CAR modulators, which range from biochemical to cell-based and animal models.

An efficient organocatalytic quadruple cascade reaction resulting in spiroxindole scaffolds bearing five quaternary stereocenters is reported. The complex cascade reaction is triggered by the scarcely explored vinylogous Michael addition of 3-alkylidene oxindoles to fully substituted enones and demonstrates the usefulness of the latter as efficient Michael acceptors in generating complex caged products in 26–92% yields, 14–98% ee and up to >25 : 1 d.r. values.

Tilted fiber Bragg grating, which has the advantages of both fiber Bragg grating and long-period fiber grating, has been widely studied for sensing in many fields, especially in the field of biochemistry. Calmodulin, which has a wide distribution in eukaryotes, can regulate several enzymes such as adenylate cyclase and guanylate cyclase and mediates several cellular processes such as cell proliferation and cyclic nucleotide metabolism. The abnormal levels of calmodulin in the body will result in serious effects from metabolism to nerve growth and memory. Therefore, it is important to measure the calmodulin concentration in the body. In this work, we propose and experimentally demonstrate a plasmonic tilted fiber Bragg grating-based biosensor for calmodulin detection. The biosensor was made using an 18° tilted fiber Bragg grating with a 50 nm-thick gold nanofilm coating the surface of the fiber, and transient receptor potential channels were bonded onto the surface of the gold nanofilm to serve as bio-detectors for calmodulin detection. Experimental results showed that the limit of detection using our biosensor was 0.44 nM. Furthermore, we also demonstrated that the interaction between calmodulin and transient receptor potential channels was quite weak without calcium in the solution, which agrees with the biology. Our proposed biosensor has a simple structure, is easy to manufacture, and is of small size, making it a good choice for real-time, label-free, and microliter-volume biomolecule detection.

The phytohormone auxin controls a myriad of processes in plants, at least in part through its regulation of cell expansion. The "acid growth hypothesis" has been proposed to explain auxin-stimulated cell expansion for five decades, but the mechanism underlying auxin-induced cell wall acidification is poorly characterized. Auxin induces the phosphorylation and activation of the plasma membrane (PM) H+-ATPase that pumps protons into the apoplast, yet how auxin activates its phosphorylation remains elusive. Here, we show that the transmembrane kinase (TMK) auxin signaling proteins interact with PM H+-ATPases and activate their phosphorylation to promote cell wall acidification and hypocotyl cell elongation in Arabidopsis. Auxin induced TMK's interaction with H+-ATPase on the plasma membrane within 1-2 minutes as well as TMK-dependent phosphorylation of the penultimate Thr residue. Genetic, biochemical, and molecular evidence demonstrates that TMKs are required for auxin-induced PM H+-ATPase activation, apoplastic acidification, and cell expansion. Thus, our findings reveal a crucial connection between auxin and PM H+-ATPase activation in regulating apoplastic pH changes and cell expansion via TMK-based cell surface auxin signaling.

The phytohormone auxin controls many processes in plants, at least in part through its regulation of cell expansion1. The acid growth hypothesis has been proposed to explain auxin-stimulated cell expansion for five decades, but the mechanism that underlies auxin-induced cell-wall acidification is poorly characterized. Auxin induces the phosphorylation and activation of the plasma membrane H+-ATPase that pumps protons into the apoplast2, yet how auxin activates its phosphorylation remains unclear. Here we show that the transmembrane kinase (TMK) auxin-signalling proteins interact with plasma membrane H+-ATPases, inducing their phosphorylation, and thereby promoting cell-wall acidification and hypocotyl cell elongation in Arabidopsis. Auxin induced interactions between TMKs and H+-ATPases in the plasma membrane within seconds, as well as TMK-dependent phosphorylation of the penultimate threonine residue on the H+-ATPases. Our genetic, biochemical and molecular evidence demonstrates that TMKs directly phosphorylate plasma membrane H+-ATPase and are required for auxin-induced H+-ATPase activation, apoplastic acidification and cell expansion. Thus, our findings reveal a crucial connection between auxin and plasma membrane H+-ATPase activation in regulating apoplastic pH changes and cell expansion through TMK-based cell surface auxin signalling., Auxin induces transmembrane-kinase-dependent activation of H+-ATPase in the plasma membrane through phosphorylation of its penultimate threonine residue, promoting apoplastic acidification and hypocotyl cell elongation in Arabidopsis.

An efficient protocol for the chemoselective construction of the indeno[1,2-b]pyrroles and rearranged indeno[1,2-b]pyrrole derivatives is reported via an N-acylation/cyclization/Wittig reaction. Extensive mechanistic investigations revealed that the initially formed crucial spiro-indene-1,2′-[1,3,4]oxadiazol intermediate further reacts with phosphine to generate betaine, thus predominately resulting in the aforementioned heteroarenes proceeding by a Wittig reaction.

Pregnane X receptor (PXR) plays roles in detoxification and other physiologic processes. PXR activation may enhance drug metabolism (leading to adverse drug reactions) or inhibit inflammation. Therefore, PXR agonists, antagonists, and inverse agonists may serve as research tools and drug candidates. However, a specific PXR modulator with an associated structure–activity relationship (SAR) is lacking. Based on the scaffold of specific human PXR (hPXR) antagonist SPA70 (10), we developed 81 SPA70 analogs and evaluated their receptor binding and cellular activities. Interestingly, analogs with subtle structural differences displayed divergent cellular activities, including agonistic, dual inverse agonistic and antagonistic, antagonistic, and partial agonistic/partial antagonistic activities (as in compound 111, 10, 97, and 42, respectively). We generated a pharmacophore model that represents 81 SPA70 analogs, and docking models that correlate strong interactions between the compounds and residues in the AF-2 helix with agonistic activity. These compounds are novel chemical tools for studying hPXR.

Abnormal accumulations of misfolded Aβ and tau proteins are major components of the hallmark plaques and neurofibrillary tangles in the brains of Alzheimer’s disease (AD) patients. These abnormal protein deposits cause neurodegeneration through a number of proposed mechanisms, including downregulation of the cAMP-response-element (CRE) binding protein 1 (CREB) signaling pathway. Using CRE-GFP reporter cells, we investigated the effects of three coumarin-chalcone derivatives synthesized in our lab on CREB-mediated gene expression. Aβ-GFP- and ΔK280 tauRD-DsRed-expressing SH-SY5Y cells were used to evaluate these agents for possible antiaggregative, antioxidative, and neuroprotective effects. Blood-brain barrier (BBB) penetration was assessed by pharmacokinetic studies in mice. Of the three tested compounds, (E)-3-(3-(4-(dimethylamino)phenyl)acryloyl)-4-hydroxy-2H-chromen-2-one (LM-021) was observed to increase CREB-mediated gene expression through protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and extracellular signal-regulated kinase (ERK) in CRE-GFP reporter cells. LM-021 exhibited antiaggregative, antioxidative, and neuroprotective effects mediated by the upregulation of CREB phosphorylation and its downstream brain-derived neurotrophic factor and BCL2 apoptosis regulator genes in Aβ-GFP- and ΔK280 tauRD-DsRed-expressing SH-SY5Y cells. Blockage of the PKA, CaMKII, or ERK pathway counteracted the beneficial effects of LM-021. LM-021 also exhibited good BBB penetration ability, with brain to plasma ratio of 5.3%, in in vivo pharmacokinetic assessment. Our results indicate that LM-021 works as a CREB enhancer to reduce Aβ and tau aggregation and provide neuroprotection. These findings suggest the therapeutic potential of LM-021 in treating AD.

Summary Histone lysine demethylases (KDMs) play critical roles in oncogenesis and therefore may be effective targets for anticancer therapy. Using a time-resolved fluorescence resonance energy transfer demethylation screen assay, in combination with multiple orthogonal validation approaches, we identified geldanamycin and its analog 17-DMAG as KDM inhibitors. In addition, we found that these Hsp90 inhibitors increase degradation of the alveolar rhabdomyosarcoma (aRMS) driver oncoprotein PAX3-FOXO1 and induce the repressive epigenetic mark H3K9me3 and H3K36me3 at genomic loci of PAX3-FOXO1 targets. We found that as monotherapy 17-DMAG significantly inhibits expression of PAX3-FOXO1 target genes and multiple oncogenic pathways, induces a muscle differentiation signature, delays tumor growth and extends survival in aRMS xenograft mouse models. The combination of 17-DMAG with conventional chemotherapy significantly enhances therapeutic efficacy, indicating that targeting KDM in combination with chemotherapy may serve as a therapeutic approach to PAX3-FOXO1-positive aRMS., Graphical Abstract, Highlights • Identification of geldanamycin/17-DMAG as histone lysine demethylase inhibitors • Geldanamycin/17-DMAG causes degradation of PAX3-FOXO1, an Hsp90 client • Geldanamycin/17-DMAG induces epigenetic changes and targets PAX3-FOXO1 pathway • 17-DMAG alone or combined with chemotherapy show potency to PAX3-FOXO1 xenografts, Molecular Biology; Cancer; Omics

An efficient method for the direct β-acylation of arylidene pyrazolones and thiazolones with acyl chlorides in the presence of a base catalyzed by organophosphanes is reported. A variety of functionalized 4-arylidene pyrazolone and 5-arylidene thiazolone derivatives were prepared under metal-free and mild conditions via a tandem phospha-Michael addition/O-acylation/intramolecular cyclization/rearrangement sequence. Our mechanistic investigations revealed that the reaction is highly stereospecific to provide exclusively cis-isomers, and the methodology can also be scaled up with similar efficacy.

Cell polarity is fundamental to the development of both eukaryotes and prokaryotes, yet the mechanisms behind its formation are not well understood. Here we found that, phytohormone auxin-induced, sterol-dependent nanoclustering of cell surface transmembrane receptor kinase 1 (TMK1) is critical for the formation of polarized domains at the plasma membrane (PM) during the morphogenesis of cotyledon pavement cells (PC) in Arabidopsis. Auxin-induced TMK1 nanoclustering stabilizes flotillin1-associated ordered nanodomains, which in turn promote the nanoclustering of ROP6 GTPase that acts downstream of TMK1 to regulate cortical microtubule organization. In turn, cortical microtubules further stabilize TMK1- and flotillin1-containing nanoclusters at the PM. Hence, we propose a new paradigm for polarity formation: A diffusive signal triggers cell polarization by promoting cell surface receptor-mediated nanoclustering of signaling components and cytoskeleton-mediated positive feedback that reinforces these nanodomains into polarized domains., The significance of protein nanoclustering in cell polarization is unclear. Here Pan et al. show that auxin-induced TMK1/sterol nanoclustering as well as microtubule-based positive feedback regulation of the TMK1/sterol nanoclusters is critical for cell polarity formation in Arabidopsis.

Spatial partitioning is a propensity of biological systems orchestrating cell activities in space and time. The dynamic regulation of plasma membrane nano-environments has recently emerged as a key fundamental aspect of plant signaling, but the molecular components governing it are still mostly unclear. The receptor kinase FERONIA (FER) controls complex formation of the immune receptor kinase FLAGELLIN SENSING 2 (FLS2) with its co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), and this function is inhibited by the FER ligand RAPID ALKALANIZATION FACTOR 23 (RALF23). Here, we show that FER regulates the plasma membrane nanoscale organization of FLS2 and BAK1. Our study demonstrates that akin to FER, leucine-rich repeat (LRR) extensin (LRXs) proteins contribute to RALF23 responsiveness, regulate BAK1 nanoscale organization and immune signaling. Furthermore, RALF23 perception leads to rapid modulation of FLS2 and BAK1 nanoscale organization and its inhibitory activity on immune signaling relies on FER kinase activity. Our results suggest that perception of RALF peptides by FER and LRXs actively modulates the plasma membrane nanoscale landscape to regulate cell surface signaling by other receptor kinases.

Microtubule‐associated protein Tau, abundant in the central nervous system (CNS), plays crucial roles in microtubule assembly and stabilization. Abnormal Tau phosphorylation and aggregation are a common pathogenic hallmark in Alzheimer's disease (AD). Hyperphosphorylation of Tau could change its conformation and result in self‐aggregation, increased oxidative stress, and neuronal death. In this study, we examined the potential of licochalcone A (a natural chalcone) and five synthetic derivatives (LM compounds) for inhibiting Tau misfolding, scavenging reactive oxygen species (ROS) and providing neuroprotection in human cells expressing proaggregant ΔK280 TauRD‐DsRed. All test compounds were soluble up to 100 μM in cell culture media and predicted to be orally bioavailable and CNS‐active. Among them, licochalcone A and LM‐031 markedly reduced Tau misfolding and associated ROS, promoted neurite outgrowth, and inhibited caspase 3 activity in ΔK280 TauRD‐DsRed 293 and SH‐SY5Y cells. Mechanistic studies showed that LM‐031 upregulates HSPB1 chaperone, NRF2/NQO1/GCLC pathway, and CREB‐dependent BDNF/AKT/ERK/BCL2 pathway in ΔK280 TauRD‐DsRed SH‐SY5Y cells. Decreased neurite outgrowth upon induction of ΔK280 TauRD‐DsRed was rescued by LM‐031, which was counteracted by knockdown of NRF2 or CREB. LM‐031 further rescued the downregulated NRF2 and pCREB, reduced Aβ and Tau levels in hippocampus and cortex, and ameliorated cognitive deficits in streptozocin‐induced hyperglycemic 3 × Tg‐AD mice. Our findings strongly indicate the potential of LM‐031 for modifying AD progression by targeting HSPB1 to reduce Tau misfolding and activating NRF2 and CREB pathways to suppress apoptosis and promote neuron survival, thereby offering a new drug development avenue for AD treatment., Through upregulating HSPB1 chaperone to reduce Tau misfolding, and enhancing NRF2 and CREB pathways to reduce ROS and apoptosis in ΔK280 TauRD‐DsRed SH‐SY5Y cells, as well as promoting neuron survival and cognitive function in hyperglycemic 3×Tg‐AD mice, LM‐031 (3‐benzoyl‐5‐hydroxychromen‐2‐one) displays potential for Alzheimer’s disease treatment.

Background Lung cancer possesses high mortality rate and tolerances to multiple chemotherapeutics. Natural Borneol (NB) is a monoterpenoid compound that found to facilitate the bioavailability of drugs. In this study, we attempted to investigate effects of NB on the chemosensitivity in A549 cells and try to elucidate its therapeutic target. Methods The effects of NB on chemosensitivity in A549 cells was examined by MTT assay. The mechanism studies were evaluated by flow cytometry and western blotting assay. Surface plasmon resonance (SPR) and LC-MS combined analysis (MS-SPRi) was performed to elucidate the candidate target of NB contributes to this synergism. The chemosensitizing capacity of NB in vivo was conducted in nude mice bearing A549 tumors. Results NB pretreatment sensitizes A549 cells to low dosage of DOX, leading to a 15.7% to 41.5% increase in apoptosis, which is corelated with ERK and AKT inactivation but activation of phosphor-p38MAPK, -JNK and p53. Furthermore, this synergism depends on reactive oxygen species (ROS) generation. The MS-SPRi analysis reveals that the transient receptor potential melastatin-8 (TRPM8) is the interaction target of NB in potentiating DOX killing potency. Genetically knock down of TRPM8 significantly suppress the chemosensitizing effects of NB with the involvement of inhibiting ROS generation through restraining calcium mobilization. Moreover, pretreatment of NB synergistically enhanced the anticancer effects of DOX to delay tumor progression in vivo. Conclusions These results suggest that TRPM8 may be a valid therapeutic target in the potential application of NB serves as a chemosensitizer for lung cancer treatment.

Spinocerebellar ataxia type 17 (SCA17) is caused by a CAG/CAA expansion mutation encoding an expanded polyglutamine (polyQ) tract in TATA-box binding protein (TBP), a general transcription initiation factor. Suppression of cAMP-responsive element binding protein- (CREB-) dependent transcription, impaired nuclear factor erythroid 2-related factor 2 (NRF2) signaling, and interaction of AMP-activated protein kinase (AMPK) with increased oxidative stress have been implicated to be involved in pathogenic mechanisms of polyQ-mediated diseases. In this study, we demonstrated decreased pCREB and NRF2 and activated AMPK contributing to neurotoxicity in SCA17 SH-SY5Y cells. We also showed that licochalcone A and the related in-house derivative compound 3-benzoyl-5-hydroxy-2H-chromen-2-one (LM-031) exhibited antiaggregation, antioxidative, antiapoptosis, and neuroprotective effects in TBP/Q79-GFP-expressing cell models. LM-031 and licochalcone A exerted neuroprotective effects by upregulating pCREB and its downstream genes, BCL2 and GADD45B, and enhancing NRF2. Furthermore, LM-031, but not licochalcone A, reduced activated AMPKα. Knockdown of CREB and NRF2 and treatment of AICAR (5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside), an AMPK activator, attenuated the aggregation-inhibiting and neurite outgrowth promoting effects of LM-031 on TBP/Q79 SH-SY5Y cells. The study results suggest the LM-031 as potential therapeutics for SCA17 and probable other polyQ diseases.

Histone lysine demethylases (KDMs) are emerging as therapeutic targets in cancer. Development of potent KDM inhibitors may provide additional options for epigenomics-oriented therapies. Using a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) functional demethylation assay, in combination with a high-content immunofluorescence imaging phenotypic screen, Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Ion Cyclotron Resonance mass spectrometry (MALDI-FTICR MS) and Amplified Luminescent Proximity Homogeneous Assay (ALPHA), we identified geldanamycin, an inhibitor of heat shock protein 90 (Hsp90), as a novel inhibitor of JmjC-domain containing demethylases such as KDM4B. We further found that geldanamycin can destabilize the PAX3-FOXO1 fusion oncoprotein, an Hsp90 client, which is a driver of clinically unfavorable alveolar rhabdomyosarcoma (aRMS). We then hypothesized that dual inhibition of PAX3-FOXO1 and epigenetic modifiers of aRMS would have synergistic antitumor activity. We repurposed the geldanamycin analog 17-DMAG to target aRMS and found that 17-DMAG significantly delays tumor growth, extends survival in xenograft mouse models, and inhibits expression of PAX3-FOXO1 targets and multiple oncogenic pathways including MYC, E2F and NOTCH. In addition, the combination of 17-DMAG with conventional chemotherapy or the bromodomain inhibitor JQ1 significantly enhances therapeutic efficacy. In summary, we have identified geldanamycin and 17-DMAG as dual KDM/Hsp90 inhibitors and 17-DMAG is efficacious against PAX3-FOXO1-driven rhabdomyosarcoma.

Pregnane X receptor (PXR) is activated by chemicals to transcriptionally regulate drug disposition and possibly decrease drug efficacy and increase resistance, suggesting therapeutic value for PXR antagonists. We previously reported the antagonist SPA70 and its analog SJB7, which unexpectedly is an agonist. Here, we describe another unexpected observation: mutating a single residue (W299A) within the PXR ligand-binding domain converts SPA70 to an agonist. After characterizing wild-type and W299A PXR activity profiles, we used molecular dynamics simulations to reveal that in wild-type PXR, agonists stabilize the activation function 2 (AF-2) helix in an "inward" position, but SPA70 displaces the AF-2. In W299A, however, SPA70 stabilizes the AF-2 "inward", like agonists. We validated our model by predicting the antagonist SJC2 to be a W299A agonist, which was confirmed experimentally. Our work correlates previously unobserved ligand-induced conformational changes to PXR cellular activity and, for the first time, reveals how PXR antagonists work.

Polyglutamine (polyQ)-mediated spinocerebellar ataxias (SCA) are caused by mutant genes with expanded CAG repeats encoding polyQ tracts. The misfolding and aggregation of polyQ proteins result in increased reactive oxygen species (ROS) and cellular toxicity. Inflammation is a common manifestation of oxidative stress and inflammatory process further reduces cellular antioxidant capacity. Increase of activated microglia in the pons of SCA type 3 (SCA3) patients suggests the involvement of neuroinflammation in the disease pathogenesis. In this study, we evaluated the anti-inflammatory potentials of indole compound NC009-1, 4-aminophenol-arachidonic acid derivative AM404, quinoline compound VB-037 and chalcone-coumarin derivative LM-031 using human HMC3 microglia and SCA3 ATXN3/Q75-GFP SH-SY5Y cells. The four tested compounds displayed anti-inflammatory activity by suppressing NO, IL-1β, TNF-α and IL-6 production and CD68 expression of IFN-γ-activated HMC3 microglia. In retinoic acid-differentiated ATXN3/Q75-GFP SH-SY5Y cells inflamed with IFN-γ-primed HMC3 conditioned medium, treatment with the tested compounds mitigated the increased caspase 1 activity and lactate dehydrogenase release, reduced polyQ aggregation and ROS and/or promoted neurite outgrowth. Examination of IL-1β- and TNF-α-mediated signaling pathways revealed that the tested compounds decreased IκBα/P65, JNK/JUN and/or P38/STAT1 signaling. The study results suggest the potential of NC009-1, AM404, VB-037 and LM-031 in treating SCA3 and probable other polyQ diseases.

In this account, our group’s efforts towards exploring new substrates as precursors for the Wittig reaction have been discussed. Several new strategies developed by our group for the generation of requisite ylides for the Wittig reaction are highlighted. The idea behind the development of some chemoselective and diversity-oriented strategies are discussed in detail in a progressive manner. These strategies encompass a wide range of substrates that are employed for the synthesis of an array of heterocycles and multifunctional olefins and present a huge scope for their application on an industrial level.1 Introduction2 Development of New Methods to Effect Intramolecular Wittig Reaction3 Development of a Catalytic Wittig Reaction4 New Synthesis of Bis-Heteroarenes5 Direct β-Acylation of 2-Arylidene-1,3-indandiones6 Doubly Chemoselective Protocol for the Diversity-Oriented Synthesis of Coumarin Derivatives7 Conclusion

A highly efficient and chemoselective one-pot protocol for the diversity-oriented synthesis of two types of coumarin-based formal cross-coupling adducts, furo[3,2-c]coumarins and 3-benzofuranyl chromenones, is described. Key attributes of the methodology are an initial chemoselective acylation of functionalized phosphorus zwitterions and a subsequent chemoselective intramolecular Wittig reaction that preferentially resulted in one of the two coumarin derivatives in high yield, depending on relative reactivities and the addition sequence of the acylating agents.

Phosphines constitute key organophosphorus reagents that are being widely used in industry as well as in research laboratories since over a century. Specifically, in last two decades, there have been numerous reports on phosphine-mediated and phosphine-catalyzed reactions. In terms of industrial application, phosphine-mediated reactions have received much attention when compared to the catalytic versions for the synthesis of a variety of structurally diverse organic frameworks. In this digest review, we wish to summarize the recent advances (2014–2017) in phosphine-mediated reactions that are primarily based on stoichiometric application of phosphine.

An organobase-directed, regiodivergent 1,3-dipolar cycloaddition of azomethine ylides and 2-hydoxybenzylidene indandiones is reported. The scarcely explored reversal of the nucleophilic site in azomethine ylides has been exploited for their regiodivergent (3+2) cycloaddition, which subsequently resulted in two different cascade processes to generate functionally distinct chromenopyrrolidines in a diversity oriented manner.

A new type of all-carbon 1,3-dipole precursor, 3-homoacylcoumarin, was employed for the stereoselective (3+2) cycloaddition with indandione alkylidenes to generate a series of coumarin/indandione-fused spirocyclopentanes bearing four contiguous stereogenic centers. While two reaction pathways progressed simultaneously, detailed mechanistic investigation revealed that the highly efficient stereoselective concerted route dominated the extremely slow stepwise pathway.

Human pregnane X receptor (hPXR) is a nuclear receptor that regulates the expression of phase I and phase II drug-metabolism enzymes, as well as that of drug transporters. hPXR is a “xenobiotics sensor” and can be activated by structurally diverse compounds. The activation of hPXR by its agonists increases the clearance of xenobiotics by increasing the expression of drug-metabolism enzymes and drug transporters, possibly leading to drug toxicity, drug resistance, and other adverse drug reactions. Therefore, hPXR antagonists might attenuate agonist-mediated activation of hPXR and reduce the risk of adverse drug reactions. Several hPXR antagonists have been reported, but none of them is specific for hPXR. In this study, we present the first large-scale, unbiased, cell-based high-throughput screen to identify specific hPXR antagonists. Among the 132,975 compounds screened, we identified the 1,4,5-substituted 1,2,3-triazole analogs as potent and specific hPXR antagonists by sequentially performing primary screening, retesting, and dose–response analysis using cell-based hPXR gene reporter and receptor binding assays, as well as receptor and promoter specificity assays. The compound SJ000076745-1 is the most potent and specific hPXR antagonist in the 1,4,5-substituted 1,2,3-triazole chemical class, having a cell-based hPXR antagonist 50% inhibitory concentration (IC50) value of 377 ± 16 nM and an hPXR binding inhibitory IC50 value of 563 ± 40 nM.

The earth-abundant material CuSbS2 (CAS) exhibits excellent optical and electrical properties as a potential absorber material for thin-film solar cells. However, the preparation of phase-pure CAS is still challenging due to the multi-phase characters of Cu-Sb-S system and antimony (Sb) loss during the annealing. In this work, an oxides nanoparticles-derived route is first investigated to synthesize CAS thin film. The Sb loss can be alleviated slightly by increasing sulfurization pressure. To control the film composition and phase purity, the effect of Cu/Sb ratios on the phase evolution is carefully investigated, and phase formation mechanism is then well established. Finally, the phase-pure CAS thin film with large grains is prepared with the sufficient Sb provided as a sacrificial material. An obvious photoelectric response of CAS thin film and its related solar cell are observed, suggesting a potential strategy for the fabrication of CAS solar cells from oxides nanoparticles ink.

Summary Identification of diverse chemotypes of selective KDM4 inhibitors is important for exploring and validating the roles of KDM4s in the pathogenesis of human disease and for developing therapies. Here, we report a protocol for high-throughput screening of KDM4 inhibitors using TR-FRET demethylation functional assay. We describe this protocol for screen of KDM4B inhibitors, which can be modified to screen inhibitors of other JmjC-domain-containing KDMs. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021)., Graphical abstract, Highlights • Describes protein expression and purification of KDM4B catalytic domain • Describes preparation and optimization of KDM4B TR-FRET reagents and conditions • Describes high-throughput KDM4B TR-FRET screening procedure, Identification of diverse chemotypes of selective KDM4 inhibitors is important for exploring and validating the roles of KDM4s in the pathogenesis of human disease and for developing therapies. Here, we report a protocol for high-throughput screening of KDM4 inhibitors using TR-FRET demethylation functional assay. We describe this protocol for screen of KDM4B inhibitors, which can be modified to screen inhibitors of other JmjC-domain-containing KDMs.