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Membrane proteins have key functions in signal transduction, transport, and metabolism. Therefore, deciphering the interactions between membrane proteins provides crucial information on signal transduction and the spatiotemporal organization of protein complexes. However, detecting the interactions and behaviors of membrane proteins in their native environments remains difficult. Förster resonance energy transfer (FRET) is a powerful tool for quantifying the dynamic interactions and assembly of membrane proteins without disrupting their local environment, supplying nanometer-scale spatial information and nanosecond-scale temporal information. In this review, we briefly introduce the basic principles of FRET and assess the current state of progress in the development of new FRET techniques (such as FRET-FLIM, homo-FRET, and smFRET) for the analysis of plant membrane proteins. We also describe the various FRET-based biosensors used to quantify the homeostasis of signaling molecules and the active state of kinases. Furthermore, we summarize recent applications of these advanced FRET sensors in probing membrane protein interactions, stoichiometry, and protein clustering, which have shed light on the complex biological functions of membrane proteins in living plant cells.

Being sessile in soil, plant cells rely on cell-surface receptors to sense and transduce environmental stimulus signals into intracellular responses. FERONIA (FER), a Catharanthus roseus receptor-like kinase 1-like protein, has emerged as a versatile regulator of plant growth, development, and stress responses. In recent years, accumulating studies have witnessed rapid advances in dissecting the mechanisms underlying the interaction between FER and its partners in response to pathogen invasion, particularly regulation of immune complex formation and signalling. Moreover, hormonal signalling, rhizosphere microbiota and other constituents are also extensively involved in these processes.

A series of osimertinib derivatives without acrylamide groups were synthesized and their inhibitory rates against L858R/T790M/C797S mutated EGFR kinase and antiproliferation activities against non-small cell lung cancer cell lines (A549, H1975) were evaluated. The preferred compounds were selected and their in vitro inhibitory activities against various EGFR kinases (wild-type, L858R/T790M, L858R/T790M/C797S) and c-Met kinase were tested. Compound 9h showed remarkable inhibitory activity against the wild type (IC

Based on its inhibition by antagonists, the A

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. DNA repair genes play a vital role in cancer development. However, there has been very little research about DNA repair genes in UM. This study aimed to evaluate the importance of DNA repair genes and established a signature for predicting prognosis and immune features of UM. In this study, we mined TCGA database through bioinformatics analysis, and the intersect was taken between DNA repair genes and prognosis related genes and yielded 52 genes. We divided 80 UM patients into C1 and C2 subtypes. GSEA results indicated that abundant cancer-promoting functions and signaling pathways were activated in C2 subtype and the proportion of SNVs was higher in C2 than in C1 which suggested a worse prognosis. We built a six DNA repair genes model including ITPA, CETN2, CCNO, POLR2J, POLD1, and POLA1 by LASSO regression to predict prognosis of UM patients and utilized the median value of risk scores as the cutoff point to differentiate high risk and low risk group. The survival analyses and the receiver operating characteristic (ROC) curves in the validation group and entire data set confirmed the accuracy of this model. We also constructed a nomogram based on age and risk scores to evaluate the relationship between risk scores and clinical outcome. The calibration curve of the overall survival (OS) indicated that the performance of this model is steady and robust. Finally, the enrichment analysis showed that there were complex regulatory mechanisms in UM patients. The immune infiltration analysis indicated that the immune infiltration in C2 in the high risk group was different from that in the low risk group. Our findings indicated that the DNA repair genes may be related to UM prognosis and provide new insight into the underlying mechanisms.

Isoform 2 of claudin 18 (CLDN18.2) is overexpressed in gastric cancer and may be a promising imaging target. In this study, we constructed three anti-CLDN18.2 antibodies and compared them in preclinical experiments.Screening from anti-CLDN18.2 nanobody library, we constructed three antibodies, anti-CLDN18.2 VHH (recombinant single-chain antibody fused with poly-histidine-tag), anti-CLDN18.2 VHH-ABD (recombinant single-chain antibody fused fused with albumin binding domain), and anti-CLDN18.2 VHH-Fc (recombinant single-chain antibody fused with IgG1-Fc) and radiolabeled withThe EC50 were 12.21 nM, 2.48 nM, and 0.14 nM for anti-CLDN18.2 VHH, anti-CLDN18.2 VHH-ABD, and anti-CLDN18.2 VHH-Fc, respectively.Both anti-CLDN18.2 VHH-ABD and anti-CLDN18.2 VHH-Fc can be efficiently and stably radiolabeled with

On the basis of N-(3-amino-4-methoxyphenyl)acrylamide scaffold, a series of novel compounds containing 3-substitutional-1-methyl-1H-indole, 2-substitutional pyrrole or thiophene moieties were synthesized and their in vitro antiproliferation activities against A549 and H1975 cell lines were evaluated. The results indicated that most of the compounds showed moderate to excellent antitumor activities. Especially, compounds 9a (A549 IC

Efficient and convenient one-pot, three-component reactions of pyrrolidone, aromatic aldehydes and 1-phenyl-3-trifluoromethyl-5-pyrazolone afforded highly functionalized trifluoromethylated pyrazol-4-ylpyrrole-2,5-dione derivatives in good yields. The effect of solvents on the reaction efficiency and yield was briefly investigated. The structures of products were determined by spectral methods and X-ray diffraction analysis. The latter showed that the products formed have a strong intramolecular hydrogen bond, which made them particularly stable and the corresponding annulated products were not obtained by treatment with dehydrating reagents.

Claudin 18.2 (CLDN18.2) is aberrantly overexpressed in gastric cancer, pancreatic adenocarcinomas, and a fraction of non-small cell lung cancer. Its tumor specificity allows CLDN18.2 to be an attractive target for developing biotherapeutics for the treatment of gastric and pancreatic cancers. DR30303 is a humanized ant-CLDN18.2 heavy chain antibody Fc fusion protein (VHH-Fc) generated via Doer Biologics’ proprietary SMART-VHHBody platform. DR30303 exhibits strong and highly selective binding to CLDN18.2, but not its close splicing variant CLDN18.1. In addition, it exhibits enhanced ADCC activity as a result of Fc engineering and low immunogenicity risk validated via in-silico T-cell epitope predictor. Its smaller size (78 kDa vs 150 kDa for conventional full-length IgG) allows DR30303 to penetrate solid tumors faster and deeper, resulting in a strong competitive advantage and differentiation compared with other anti-CLDN18.2 antibodies in the market. Single-agent therapeutic potency of DR30303 was demonstrated in various gastric and pancreatic cell-derived xenograft (CDX) mouse models with a minimum effective dose of 0.3 mg/kg dose of DR30303 led to TGI≥90%, which is significantly more potent (p Citation Format: Yongliang Fang, Gaofeng Yao, Weixiang Zhong, Wenwen Duan, Zhenxing Zhou, Xiaofang Wen, Yonglu Chen, Jingjin Fang, Yongwei Wang, Weiqin Jiang, Junfang Xu, Yanshan Huang. DR30303, a SMART-VHHBody powered anti-CLDN18.2 VHH-Fc with enhanced ADCC activity for the treatment of gastric and pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2857.

On the basis of N-(3-amino-4-methoxyphenyl)acrylamide scaffold, a series of novel compounds containing 3-substitutional-1-methyl-1H-indole, 2-substitutional pyrrole or thiophene moieties were synthesized and their in vitro antiproliferation activities against A549 and H1975 cell lines were evaluated. The results indicated that most of the compounds showed moderate to excellent antitumor activities. Especially, compounds 9a (A549 IC50 = 1.96 μM, H1975 IC50 = 0.095 μM), 17i (A549 IC50 = 4.17 μM, H1975 IC50 = 0.052 μM), 17j (A549 IC50 = 1.67 μM, H1975 IC50 = 0.061 μM) exhibited comparable antitumor activities and selectivity ratios compared to the positive control osimertinib (A549 IC50 = 2.91 μM, H1975 IC50 = 0.064 μM). In vitro inhibitory activities against EGFR kinases containing different mutations were also tested. Compound 17i showed remarkable inhibitory activity (with IC50 value of 1.7 nM) to EGFRL858R/T790M kinase and selectivity (22-folds compared to EGFRWT kinase). Furthermore, acridine orange/ethidium bromide (AO/EB) staining assay, cell apoptosis assay, cell cycle distribution assay and wound-healing assay of the compounds 9a and 17i were performed on H1975 cell line. The results showed dose-dependent activities of the induction of apoptosis, G0/G1-phase arrestation and inhibition of migration, which were similar to the positive control osimertinib. Additionally, molecular docking analysis was performed to seek the possible binding mode between the selected compounds (9a, 17i-17j) and EGFRL858R/T790M kinase. The results demonstrated that compound 17i is a promising candidate and worth further study.

One-pot, four-component reactions of 1,2-diphenylpyrazolidine-3,5-dione, ethyl 4,4,4-trifluoroacetoacetate, aromatic aldehyde and ammonium acetate afforded highly functionalized ethyl 8-hydroxy-1,4-dioxo-2,3,6,10-tetraphenyl-8-(trifluoromethyl)-2,3,7-triazaspiro[4.5]decane-9-carboxylate derivatives 5 in good yields. The most advantage of this reaction was the high diastereoselectivity because only one diastereoisomer 5 was produced. The effect of NH4OAc and solvents on the reaction efficiency and yield was briefly investigated. Meanwhile, the further transformation of hemi-aminal moieties to the corresponding dehydrated product was also achieved under the mild reaction conditions. And a plausible reaction mechanism for the formation of products 5 was illustrated based on the control experiments.

Cannabinoids are widely studied as therapeutic agents for the treatment of various diseases. Among them, THC and CBD are two important phytocannabinoids which have served as structural templates for the design of synthetic analogs. In this study, we designed and synthesized a variety of novel cannabinoids based on the structural backbones of THC and CBD using the carbon-silicon switch strategy. A dimethyl silyl group was introduced as the tail group and two series of novel compounds were designed and synthesized, which showed a wide range of binding affinity for CB1 and CB2 receptors. Among them, compound 15b was identified as a non-selective CB1 and CB2 agonist and 38b as a selective agonist for the CB2 receptor. Preliminary screening showed that both compounds have improved metabolic stability than their carbon analogs and good in vivo pharmacokinetic profiles. Furthermore, both 15b and 38b significantly alleviated the phenotype of experimental autoimmune encephalomyelitis (EAE) in mice.

A series of poly-heterocycles containing hemi-aminal moieties: 6’-hydroxy trifluoromethylated spiro [indoline-3,4’-pyrazolo [3,4-b]pyridine] derivatives were efficiently synthesized in ethanol catalyzed by NH4OAc via one-pot, three-component reactions of 3-aminopyrazole, ethyl 4,4,4-trifluoroacetoacetate and isatin. Treatment of this hemi-aminal containing products with the dehydrating reagent SOCl2/Pyridine in acetonitrile afforded the corresponding dehydrating trifluoromethylated polyheterocyclic spiro [indoline-3,4’-pyrazolo [3,4-b]pyridine] derivatives. Furthermore, a more efficient, straightforward approach to formation of their dehydrating products has been developed via one-pot, three-component reactions catalyzed by hydrochloric acid. Meanwhile, the structures of products were fully confirmed by spectroscopic data, two representative products were further confirmed by XRD analysis, respectively.

According to the binding mode of ABBV-744 with bromodomains and the cape space of HDAC, the novel selective HDAC/BRD4 dual inhibitors were designed and synthesized by the pharmacophore fusion strategy. Evaluating the biomolecular activities through SARs exploration identified three kinds of selective dual inhibitors 41c (HDAC1/BRD4), 43a (pan-HDAC/BRD4) and 43d (HDAC6/BRD4(BD2)), whose target-related cellular activities in MV-4-11 cells were also confirmed. Significantly, the selective dual inhibitor 41c (HDAC1/BRD4) exhibited synergistic effects against MV-4-11 cells, which strongly induced G0/G1 cell cycle arrest and apoptosis, and the first HDAC6/BRD4(BD2) dual inhibitor was found. This study provides support for selective HDAC/BRD4 dual inhibitors as epigenetic probes based on pyrrolopyridone core for the future biological evaluation in different cancer cell lines.

A series of non-annulated products: trifluoromethylpyrazolone-tethered trisubstituted methane derivatives were synthesized in high yields in acetonitrile solvent catalyzed by NH4OAc via one-pot, three-component reactions of 2-hydroxy-1,4-naphthoquinone, aromatic aldehydes and 1-aryl-3-trifluoromethyl-5-pyrazolone. Treatment of trifluoromethylpyrazolone-tethered trisubstituted methane derivatives with the dehydrating agent SOCl2/Pyridine in acetonitrile afforded the corresponding annulated fused polyheterocyclic products trifluoromethylated benzo[6,7]chromeno[2,3-c]pyrazole-5,10-dione derivatives. Furthermore, an efficient, straightforward protocol has been developed for the synthesis of trifluoromethylated benzo[6,7]chromeno[2,3-c]pyrazole-5,10-dione derivatives at medium yields via one-pot, two-step, three-component reactions. Meanwhile, the structures of typical products were further confirmed by XRD-analysis.

Simultaneous blockade of more than one pathway is considered to be a promising approach to overcome the low efficacy and acquired resistance of cancer therapies. Thus, a novel series of c-Met/HDAC bifunctional inhibitors was designed and synthesized by merging pharmacophores of c-Met and HDAC inhibitors. The most potent compound, 2m, inhibited c-Met kinase and HDAC1, with IC50 values of 0.71 and 38 nM, respectively, and showed efficient antiproliferative activities against both EBC-1 and HCT-116 cells with greater potency than the reference drug Chidamide. Western blot analysis revealed that compound 2m inhibited phosphorylation of c-Met and c-Met downstream signaling proteins and increased expression of Ac-H3 and p21 in EBC-1 cells in a dose-dependent manner. Our study presents novel compounds for the further exploration of dual c-Met/HDAC pathway inhibition achieved with a single molecule.

Proteins are frequently processed by proteases in cell signaling pathways to perform their biological functions in response to environmental stimuli. Identification of the exact cleavage sites provides necessary information for the study of their biological functions. Although proteomic approaches for profiling of protein N-termini have been developed extensively in the past few years, the N-terminal profiling strategy has its inherent disadvantages. Therefore, C-terminal profiling approaches might be a complementary approach for the identification of protein cleavages although it has similar shortcomings as N-terminal profiling methods. In this protocol, we describe an approach, termed ProC-TEL: Profiling of Protein C-Termini by Enzymatic Labeling, for affinity labeling of protein C-termini for a protein or proteome. This method uses the transpeptidase activity of carboxypeptidase Y to label protein C-termini with an affinity biotin tag for subsequent isolation with avidin beads and identification by mass spectrometer. It is complementary to the N-terminal profiling approaches and can be used to identify proteolytic cleavages for a specific protease or in cell signaling events, such as apoptosis.

Proteins are frequently processed by proteases in cell signaling pathways to perform their biological functions in response to environmental stimuli. Identification of the exact cleavage sites provides necessary information for the study of their biological functions. Although proteomic approaches for profiling of protein N-termini have been developed extensively in the past few years, the N-terminal profiling strategy has its inherent disadvantages. Therefore, C-terminal profiling approaches might be a complementary approach for the identification of protein cleavages although it has similar shortcomings as N-terminal profiling methods. In this protocol, we describe an approach, termed ProC-TEL: Profiling of Protein C-Termini by Enzymatic Labeling, for affinity labeling of protein C-termini for a protein or proteome. This method uses the transpeptidase activity of carboxypeptidase Y to label protein C-termini with an affinity biotin tag for subsequent isolation with avidin beads and identification by mass spectrometer. It is complementary to the N-terminal profiling approaches and can be used to identify proteolytic cleavages for a specific protease or in cell signaling events, such as apoptosis.

Proteolytic cleavage is one of the post-translational modifications and plays important roles in many biological processes, such as apoptosis and tumor cell metastasis. The identification of the cleavage events can improve our understanding of their biological functions in these processes. Although proteomic approaches using N-terminal labeling have resulted in the discovery of many proteolytic cleavages, this strategy has its own inherent drawbacks. Labeling of protein C-termini is an alternative approach. Here, we optimized the labeling procedure in the profiling protein C-termini by enzymatic labeling (ProC-TEL) and improved the labeling efficiency for the positive isolation of protein C-terminal peptides and mass spectrometric identification. We applied this approach to a complex protein mixture from Escherichia coli and identified many C-terminal peptides and internal cleaved peptides from more than 120 proteins. From the identified cleavages, we found several previously known internal proteolytic cleavage sites and many novel ones which may play roles in regulating normal biological processes. This work provides a potential new way, complementary to the N-terminomics, for the identification of proteolytic cleavages in complex biological systems.

Caspase activation and proteolytic cleavages are the major events in the early stage of apoptosis. Identification of protein substrates cleaved by caspases will reveal the occurrence of the early events in the apoptotic process and may provide potential drug targets for cancer therapy. Although several N-terminal MS-based proteomic approaches have been developed to identify proteolytic cleavages, these methods have their inherent drawbacks. Here we apply a previously developed proteomic approach, protein C-terminal enzymatic labeling (ProC-TEL), to identify caspase cleavage events occurring in the early stage of the apoptosis of a myeloma cell line induced by kinase inhibition. Both previously identified and novel caspase cleavage sites are detected and the reduction of the expression level of several proteins is confirmed biochemically upon kinase inhibition although the current ProC-TEL procedure is not fully optimized to provide peptide identifications comparable to N-terminal labeling approaches. The identified cleaved proteins form a complex interaction network with central hubs determining morphological changes during the apoptosis. Sequence analyses show that some ProC-TEL identified caspase cleavage events are unidentifiable when traditional N-terminomic approaches are utilized. This work demonstrates that ProC-TEL is a complementary approach to the N-terminomics for the identification of proteolytic cleavage events such as caspase cleavages in signaling pathways.

Background Schistosomiasis is a major health problem in tropical and sub-tropical areas caused by species of trematode belonging to the genus Schistosoma. The treatment and control of this disease has been relying on the use of a single drug praziquantel. However, the drug resistance concern urged the development of new drugs against schistosoma. Here, we report our systematic biological evaluation of DW-3-15, a new lead compound developed based on our conjugation design rationale as an effective anti-schistosomal agent. Methodology/Principal Findings The antischistosomal activity of DW-3-15 was systematically evaluated in S. japonicum infected mouse model for its stage-sensitivity and dose response. The results revealed that DW-3-15 exhibited 60–85% worm reduction rate against different development stage of worm. Scanning electron microscopy (SEM) observation indicated that DW-3-15 may damage to the tegument of male schistosomes. Conclusions/Significance Our results demonstrated that DW-3-15 showed potent anti-schistosomal activities in vivo. The results strongly support our conjugation design strategy of artemisinin analogs and further development of DW-3-15 as a new lead compound as anti-schistosomal agent.