Stereoselective [4+3]-Cycloaddition of 2-Amino-ß-nitrostyrenes with Azaoxyallyl Cations to Access Functionalized 1,4-Benzodiazepin-3-ones.

The 1,4-benzodiazepine structural framework is a fascinating element commonly found in biologically active and pharmaceutically relevant compounds. A highly efficient method for synthesizing 1,4-benzodiazepin-3-ones is described, involving a [4+3]-cycloaddition reaction between 2-amino-ß-nitrostyrenes and α-bromohydroxamate, with Cs2CO3 used as a base. This process yielded the desired 1,4-benzodiazepines in good yields. Furthermore, an organocatalytic asymmetric [4+3]-cycloaddition was successfully accomplished using a bifunctional squaramide-based catalyst. This approach enabled the enantioselective synthesis of chiral 1,4-benzodiazepines with commendable yields and moderate enantioselectivities, reaching up to 80% yield and 72% ee.

Organocatalytic Enantioselective Synthesis of Polycyclic Benzosultams from 2-Amino-ß-nitrostyrenes with Cyclic N-Sulfonyl Ketimines.

A highly efficient enantioselective [4 + 2] cycloaddition of 2-amino-ß-nitrostyrenes with cyclic N-sulfonyl ketimines has been developed. This reaction utilizes an organocatalytic approach, employing a multiple-hydrogen-bonding bifunctional squaramide-based catalyst. The process allows for the precise synthesis of chiral polycyclic benzosultams, showcasing intricate structures that incorporate chiral quaternary centers. Noteworthy outcomes of this method include high yields with excellent enantioselectivities and diastereoselectivities (up to 97% yield, 96% ee, and >20:1 dr).

[4+2]-Cycloaddition of 2-Aminophenyl Enones with Cyclic N-Sulfimines to Access Enantioenriched Ring-Fused Aminals.

Ring-fused aminal is an interesting structural skeleton in biologically active and pharmaceutically relevant compounds. A novel and efficient method for synthesizing benzosulfamidate-fused tetrahydroquinazolines is described. By employing the [4+2]-cycloaddition of 2-aminophenyl enones with cyclic N-sulfimines in the presence of DMAP as a base, the desired benzosulfamidate-fused tetrahydroquinazolines were obtained in good yields with high diastereoselectivities. Furthermore, an organocatalytic asymmetric [4+2]-cycloaddition was successfully achieved using a squaramide-based catalyst, enabling the enantioselective synthesis of chiral ring-fused tetrahydroquinazolines with high yields and enantio- as well as diastereoselectivities (up to 89 % yield, 94 % ee, and >30 : 1 dr).

Organocatalytic Asymmetric [4 + 2]-Cycloadditions of 2-Aminophenyl Enones with Isatin-Derived Ketimines: Diastereo- and Enantioselective Synthesis of Spirooxindole-Tetrahydroquinazolines.

A novel method for the enantioselective synthesis of spiro N,N-heterocyclic oxindoles has been developed, employing asymmetric [4 + 2]-cycloadditions of 2-aminophenyl enones with isatin-derived ketimines. This method employs an organocatalytic approach, utilizing a bifunctional squaramide-based catalyst. It enables the precise synthesis of chiral spirooxindole-tetrahydroquinazolines with intricate structures, featuring chiral quaternary centers. This process achieves remarkable results, including high yields and exceptional levels of enantioselectivity and diastereoselectivity (up to 96% yield, 95% ee, and >20:1 dr).

Organocatalytic Asymmetric [3 + 2]-Annulations of γ-Sulfonamido/γ-Hydroxy-α,ß-Unsaturated Ketones with Cyclic N-Sulfimines: Synthesis of Chiral Polyheterotricyclic Imidazolidines and Oxazolidines.

The first organocatalytic asymmetric [3 + 2]-annulation of γ-sulfonamido-α,ß-unsaturated ketones with cyclic N-sulfimines has been developed, and enantioenriched functionalized polyheterotricyclic imidazolidines were obtained in good yields and with excellent enantioselectivities. This approach was also extended to the asymmetric [3 + 2]-annulation of γ-hydroxy-α,ß-unsaturated ketones, affording enantioenriched polyheterotricyclic oxazolidines. In addition, base-catalyzed [3 + 2]-annulations of γ-sulfonamido/γ-hydroxy-α,ß-unsaturated ketones with cyclic N-sulfimines were re-investigated under mild reaction conditions for the synthesis of racemic polyheterotricyclic imidazolidines and oxazolidines with excellent diastereoselectivities.

Proteome Multimarker Panel for the Early Detection of Hepatocellular Carcinoma: Multicenter Derivation, Validation, and Comparison.

Conventional methods for the surveillance of hepatocellular carcinoma (HCC) by imaging, with and without serum tumor markers, are suboptimal with regard to accuracy. We aimed to develop and validate a reliable serum biomarker panel for the early detection of HCC using a proteomic technique. This multicenter case-control study comprised 727 patients with HCC and patients with risk factors but no HCC. We developed a multiple reaction monitoring-mass spectrometry (MRM-MS) multimarker panel using 17 proteins from the sera of 398 patients. Area under the receiver operating characteristics curve (AUROC) values of this MRM-MS panel with and without α-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist-II (PIVKA-II) were compared. The combination and standalone MRM-MS panels had higher AUROC values than AFP in the training (0.940 and 0.929 vs 0.775, both P < 0.05), test (0.894 and 0.893 vs 0.593, both P < 0.05), and confirmation sets (0.961 and 0.937 vs 0.806, both P < 0.05) in detecting small single HCC. The combination and standalone MRM-MS panels had significantly higher AUROC values than the GALAD score (0.945 and 0.931 vs 0.829, both P < 0.05). Our proteome 17-protein multimarker panel distinguished HCC patients from high-risk controls and had high accuracy in the early detection of HCC.

Comparison of Fucose-Specific Lectins to Improve Quantitative AFP-L3 Assay for Diagnosing Hepatocellular Carcinoma Using Mass Spectrometry.

Glycoproteins have many important biological functions. In particular, aberrant glycosylation has been observed in various cancers, such as liver cancer. A well-known glycoprotein biomarker is α-fetoprotein (AFP), a surveillance biomarker for hepatocellular carcinoma (HCC) that contains a glycosylation site at asparagine 251. The low diagnostic sensitivity of AFP led researchers to focus on AFP-L3, which has the same sequence as conventional AFP but contains a fucosylated glycan. AFP-L3 has high affinity for Lens culinaris agglutinin (LCA) lectin, prompting many groups to use it for detecting AFP-L3. However, a few studies have identified more effective lectins for fractionating AFP-L3. In this study, we compared the amounts of enriched AFP-L3 with five fucose-specific lectins─LCA, Lotus tetragonolobus lectin (LTL), Ulex europaeus agglutinin I (UEA I), Aleuria aurantia lectin (AAL), and Aspergillus oryzae lectin (AOL)─to identify better lectins and improve HCC diagnostic assays using mass spectrometry (MS). Our results indicate that LTL was the most effective lectin for capturing AFP-L3 species, yielding approximately 3-fold more AFP-L3 than LCA from the same pool of HCC serum samples. Thus, we recommend the use of LTL for AFP-L3 assays, given its potential to improve the diagnostic sensitivity in patients having limited results by conventional LCA assay. The MS data have been deposited to the PeptideAtlas (PASS01752).

A Clinically Applicable 24-Protein Model for Classifying Risk Subgroups in Pancreatic Ductal Adenocarcinomas using Multiple Reaction Monitoring-Mass Spectrometry.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) subtypes have been identified using various methodologies. However, it is a challenge to develop classification system applicable to routine clinical evaluation. We aimed to identify risk subgroups based on molecular features and develop a classification model that was more suited for clinical applications. EXPERIMENTAL DESIGN: We collected whole dissected specimens from 225 patients who underwent surgery at Seoul National University Hospital [Seoul, Republic of Korea (South)], between October 2009 and February 2018. Target proteins with potential relevance to tumor progression or prognosis were quantified with robust quality controls. We used hierarchical clustering analysis to identify risk subgroups. A random forest classification model was developed to predict the identified risk subgroups, and the model was validated using transcriptomic datasets from external cohorts (N = 700), with survival analysis. RESULTS: We identified 24 protein features that could classify the four risk subgroups associated with patient outcomes: stable, exocrine-like; activated, and extracellular matrix (ECM) remodeling. The "stable" risk subgroup was characterized by proteins that were associated with differentiation and tumor suppressors. "Exocrine-like" tumors highly expressed pancreatic enzymes. Two high-risk subgroups, "activated" and "ECM remodeling," were enriched in terms such as cell cycle, angiogenesis, immunocompetence, tumor invasion metastasis, and metabolic reprogramming. The classification model that included these features made prognoses with relative accuracy and precision in multiple cohorts. CONCLUSIONS: We proposed PDAC risk subgroups and developed a classification model that may potentially be useful for routine clinical implementations, at the individual level. This clinical system may improve the accuracy of risk prediction and treatment guidelines.See related commentary by Thakur and Singh, p. 3272.

Development and Multiple Validation of the Protein Multi-marker Panel for Diagnosis of Pancreatic Cancer.

PURPOSE: To develop and validate a protein-based, multi-marker panel that provides superior pancreatic ductal adenocarcinoma (PDAC) detection abilities with sufficient diagnostic performance. EXPERIMENTAL DESIGN: A total of 959 plasma samples from patients at multiple medical centers were used. To construct an optimal, diagnostic, multi-marker panel, we applied data preprocessing procedure to biomarker candidates. The multi-marker panel was developed using a training set comprised of 261 PDAC cases and 290 controls. Subsequent evaluations were performed in a validation set comprised of 65 PDAC cases and 72 controls. Further validation was performed in an independent set comprised of 75 PDAC cases and 47 controls. RESULTS: A multi-marker panel containing 14 proteins was developed. The multi-marker panel achieved AUCs of 0.977 and 0.953 for the training set and validation set, respectively. In an independent validation set, the multi-marker panel yielded an AUC of 0.928. The diagnostic performance of the multi-marker panel showed significant improvements compared with carbohydrate antigen (CA) 19-9 alone (training set AUC = 0.977 vs. 0.872, P < 0.001; validation set AUC = 0.953 vs. 0.832, P < 0.01; independent validation set AUC = 0.928 vs. 0.771, P < 0.001). When the multi-marker panel and CA 19-9 were combined, the diagnostic performance of the combined panel was improved for all sets. CONCLUSIONS: This multi-marker panel and the combined panel showed statistically significant improvements in diagnostic performance compared with CA 19-9 alone and has the potential to complement CA 19-9 as a diagnostic marker in clinical practice.

Web portal for analytical validation of MRM-MS assay abided with integrative multinational guidelines.

Multiple reaction monitoring-mass spectrometry became a mainstream method for quantitative proteomics, which made the validation of a method and the analyzed data important. In this portal for validation of the MRM-MS assay, we developed a website that automatically evaluates uploaded MRM-MS data, based on biomarker assay guidelines from the European Medicines Agency, the US Food & Drug Administration, and the Korea Food & Drug Administration. The portal reads a Skyline output file and produces the following results-calibration curve, specificity, sensitivity, carryover, precision, recovery, matrix effect, recovery, dilution integrity, stability, and QC-according to the standards of each independent agency. The final tables and figures that pertain to the 11 evaluation categories are displayed in an individual page. Spring boot was used as a framework for development of the webpage, which follows MVC Pattern. JSP, HTML, XML, and Java Script were used to develop the webpage. A server was composed of Apache Tomcat, MySQL. Input files were skyline-derived output files (csv file), and each files were organized by specific columns in order. SQL, JAVA were interworked to evaluate all the categories and show the results. Method Validation Portal can be accessed via any kind of explorer from https://pnbvalid.snu.ac.kr.

Canine non-B, non-T NK lymphocytes have a potential antibody-dependent cellular cytotoxicity function against antibody-coated tumor cells.

BACKGROUND: The antibody-dependent cellular cytotoxicity (ADCC) is a cell-mediated immune defense mechanism in which effector immune cells actively lyse antibody-coated target cells. The ADCC of tumor cells is employed in the treatment of various cancers overexpressing unique antigens, and only natural killer (NK) cells are known to be major effectors of antibody mediated ADCC activity. Canine NK cells are still defined as non-B, non-T large granular lymphocytes because of the lack of information regarding the NK cell-restricted specific marker in dogs, and it has never been demonstrated that canine NK cells have ADCC ability against tumor cells. In the present study, we investigated whether canine non-B, non-T NK cells have ADCC ability against target antibody-coated tumor cells, using cetuximab and trastuzumab, the only human antibodies reported binding to canine cancer cells. RESULTS: Activated canine non-B, non-T NK cells (CD3-CD21-CD5-TCRαß-TCRγδ-) for 13~17 days ex vivo showed ADCC ability against trastuzumab- or cetuximab-coated target tumor cells expressing various levels of human epidermal growth factor receptor 2 (HER-2) and epidermal growth factor receptor (EGFR). Trastuzumab and cetuximab induced significant ADCC responses of canine NK cells even in CMT-U334 and CF41.Mg cells expressing low levels of HER-2 and/or EGFR, as well as in SKBR3 and DU145 cells overexpressing HER-2 and/or EGFR. The trastuzumab-mediated ADCC activity of NK cells was significantly enhanced by treatment with rcIL-21. CONCLUSIONS: The results of this study suggest that canine non-B, non-T NK lymphocytes have a potential ADCC function and that combinational strategies of monoclonal antibodies with either cytokines, which activate NK cells in vivo, or adoptive transfer of NK cells may be a feasible method for amplifying the efficacy of immunotherapy against malignant cancers even with very low expression of target molecules in dogs.

Clinically Applicable Deep Learning Algorithm Using Quantitative Proteomic Data.

Deep learning (DL), a type of machine learning approach, is a powerful tool for analyzing large sets of data that are derived from biomedical sciences. However, it remains unknown whether DL is suitable for identifying contributing factors, such as biomarkers, in quantitative proteomics data. In this study, we describe an optimized DL-based analytical approach using a data set that was generated by selected reaction monitoring-mass spectrometry (SRM-MS), comprising SRM-MS data from 1008 samples for the diagnosis of pancreatic cancer, to test its classification power. Its performance was compared with that of 5 conventional multivariate and machine learning methods: random forest (RF), support vector machine (SVM), logistic regression (LR), k-nearest neighbors (k-NN), and naïve Bayes (NB). The DL method yielded the best classification (AUC 0.9472 for the test data set) of all approaches. We also optimized the parameters of DL individually to determine which factors were the most significant. In summary, the DL method has advantages in classifying the quantitative proteomics data of pancreatic cancer patients, and our results suggest that its implementation can improve the performance of diagnostic assays in clinical settings.

Comparison of Phenotypic and Functional Characteristics Between Canine Non-B, Non-T Natural Killer Lymphocytes and CD3+CD5dimCD21- Cytotoxic Large Granular Lymphocytes.

Natural killer (NK) cells play a pivotal role in the immune response against infections and malignant transformation, and adopted transfer of NK cells is thought to be a promising therapeutic approach for cancer patients. Previous reports describing the phenotypic features of canine NK cells have produced inconsistent results. Canine NK cells are still defined as non-B and non-T (CD3-CD21-) large granular lymphocytes. However, a few reports have demonstrated that canine NK cells share the phenotypic characteristics of T lymphocytes, and that CD3+CD5dimCD21- lymphocytes are putative canine NK cells. Based on our previous reports, we hypothesized that phenotypic modulation could occur between these two populations during activation. In this study, we investigated the phenotypic and functional differences between CD3+CD5dimCD21- (cytotoxic large granular lymphocytes) and CD3-CD5-CD21- NK lymphocytes before and after culture of peripheral blood mononuclear cells isolated from normal dogs. The results of this study show that CD3+CD5dimCD21- lymphocytes can be differentiated into non-B, non-T NK (CD3-CD5-CD21-TCRαß-TCRγδ-GranzymeB+) lymphocytes through phenotypic modulation in response to cytokine stimulation. In vitro studies of purified CD3+CD5dimCD21- cells showed that CD3-CD5-CD21- cells are derived from CD3+CD5dimCD21- cells through phenotypic modulation. CD3+CD5dimCD21- cells share more NK cell functional characteristics compared with CD3-CD5-CD21- cells, including the expression of T-box transcription factors (Eomes, T-bet), the production of granzyme B and interferon-γ, and the expression of NK cell-related molecular receptors such as NKG2D and NKp30. In conclusion, the results of this study suggest that CD3+CD5dimCD21- and CD3-CD5-CD21- cells both contain a subset of putative NK cells, and the difference between the two populations may be due to the degree of maturation.

Effect of shell-crosslinking of micelles on endocytosis and exocytosis: acceleration of exocytosis by crosslinking.

Uptake of drug-loaded micelles by tumour cell lines can be the crucial step in the creation of an efficient drug delivery system. Crosslinking of micelles has increasingly been proposed as a pathway to create stable nanoparticles. So far, little is known how crosslinking can affect the interaction of these nanocarriers with cells. The aim of this study is therefore to investigate the effect of crosslinking on exo- and endocytosis. RAFT (reversible addition fragmentation chain transfer) polymerization has been used to synthesize the block copolymer poly(methyl methacrylate)-block-poly(polyethylene glycol methyl ether methacrylate) PMMA-b-P(PEGMEMA), which was subsequently self-assembled into micelles of 20 nm. For comparison, the micelles were shell-crosslinked by incorporating methacrylic acid into the shell, which was used as a reactive group for crosslinking with 1,8-diaminooctane. The hydrodynamic diameter of the shell-crosslinked micelle was with 25 nm similar to that of the non-crosslinked one. Endocytosis of both micelles was significantly reduced by the presence of NaN3 or at 4 °C suggesting an energy dependent process. The internalization pathways of the block copolymer micelles in OVCAR-3 cells were elucidated using endocytosis inhibitors. Both nanoparticles, micelles and shell-crosslinked micelles, were internalized by caveolae mediated endocytosis while clathrin mediated endocytosis did not play a noticeable role. Shell-crosslinking therefore did not have an effect on endocytosis. However, a considerable difference was found in the exocytosis of both particles. While the micelle was lodged inside the cell for an extended period of time with less than 3% released in two hours, the shell-crosslinked micelle quickly exited the OVCAR-3 cells (25% in two hours). For comparison, a small molecule (Lucifer yellow) was found to be only marginally faster than the crosslinked micelles (40% in 2 h). These results could have implications on the use polymer-drug conjugates or drug carriers where the drug needs to be released before the polymer undergoes exocytosis.

Zwitterionic guanidine-based oligomers mimicking cell-penetrating peptides as a nontoxic alternative to cationic polymers to enhance the cellular uptake of micelles.

The aim of this work is to generate polymer micelles decorated with a synthetic version of cell-penetrating peptides, which are often rich in arginine with its positively charged guanidine group. A methacrylate-based monomer with guanidinium as functional groups was prepared using arginine (M-Arg) as a building block, resulting in a zwitterionic monomer. RAFT (reversible addition-fragmentation chain transfer) polymerization was employed to generate triblock copolymers with poly(methyl methacrylate)-block-poly(polyethylene glycol methyl ether methacrylate) as the first two blocks, which were subsequently chain extended with the guanidine-based monomer to generate micelles with guanidinium functional groups on the surface. To simulate the actual oligoarginine peptide, which only carries cationic charges, the carboxylate group of P(M-Arg) was methylated to convert the zwitterionic polymer into a cationic polymer P(Me-M-Arg). For comparison, micelles based on triblock copolymers with a third block with permanently cationic charges, poly(2-methacryolyloxy ethyl) trimethyl ammonium chloride (PTMA), was prepared. The hydrodynamic diameters of the micelles were approximately 30-40 nm based on DLS and TEM. A direct correlation between surface charge (zeta potential ζ) and cytotoxicity was observed. The micelles based on the zwitterionic P(M-Arg) were nontoxic (ζ = -10 mV at pH = 7), while the methylated version P(Me-M-Arg) with a high cationic charge (ζ = +35 mV at pH = 7) were observed to be toxic. The cellular uptake of the block copolymers by OVCAR-3 ovarian cancer cell lines was found to be relatively fast (about 35% in 3 min) reaching an equilibrium after approximately 30 min. Both micelles, with either P(M-Arg) or P(Me-M-Arg) on the surface, showed an enhanced uptake compared to micelles with P(PEGMEMA) as shell only. In fact, the percentage of uptake was similar, with the difference that cells incubated with micelles with P(M-Arg) (zwitterionic) stayed alive, while P(Me-M-Arg) (cationic) led to significant cell death.

Shell cross-linking of cyclodextrin-based micelles via supramolecular chemistry for the delivery of drugs.

A block copolymer based on poly(N-isopropyl acrylamide) (PNIPAAm) and a block with a statistical distribution of poly(2-hydroxyethyl acrylate) (PHEA) and repeating unit with carrying ß-cyclodextrin was prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization and click reaction. Addition of poly(2-hydroxyethyl acrylate-s-adamantylmethyl acrylate) P(HEA(17) -s-AdMA(7) ) above the LCST of the block copolymer led to capture of the micelle structure of 36 nm against disassembly. The drug- (albendazole) loaded supramolecular assembly, which was fixed via host-guest complexation between ß-cyclodextrin and adamantane, was then tested as a drug carrier. Cell viability studies using human ovarian carcinoma cell line (OVCAR-3) cell lines show a higher toxicity of the shell cross-linked micelle compared with the free block copolymer.

Effect of cross-linking on the performance of micelles as drug delivery carriers: a cell uptake study.

Poly(polyethylene glycol methyl ether methacrylate-co-methacrylic acid)-block-poly(methyl methacrylate) P(PEGMEMA-co-MAA)-b-PMMA block copolymer were prepared via RAFT (reversible addition-fragmentation chain transfer) polymerization and subsequently self-assembled into micelles as a drug delivery carrier for albendazole (ABZ). For comparison, the micelles were additionally cross-linked to study the effect of shell-cross-linking on the biological activity. The hydrodynamic diameter of cross-linked and un-cross-linked micelles was approximately 40 nm in both cases. While the cross-linked micelle was stable even in good solvents for both blocks, the un-cross-linked micelle was found to lose its integrity in cell growth media. Crosslinking had a major effect on the rate of drug release reducing it dramatically from 50% (uncrosslinked) to around 20% (crosslinked) over a 30 h incubation period. Both drug delivery systems were tested on human prostate cancer cells (PC-3, DU-145) and human ovarian cancer cells (OVCAR-3, A-2780). No toxic effects were measured with the unloaded micelle while the ABZ loaded un-cross-linked micelle lead to IC(50) values between 0.2 and 0.9 µM depending on the cell line. The IC(50) dropped to values between 0.006 and 0.06 µM, depending on cell line, once the micelles were stabilized by cross-linking. Three treatment cycles with ABZ for one day, followed by two days incubation in media using ABZ-loaded drug carriers led to complete cell death even at low concentrations in the case of the cross-linked micelle only. Cellular uptake has been studied using fluorescently labeled micelles and Nile red as model drug, showing cell uptake above the CMC but no micelle uptake below the CMC. Additional biological studies, such as colony formation assay and tubulin disorganization tests, were also performed to gain more insight into the effect of cross-linking of the shell of the micelle. In conclusion, shell-cross-linking is highly recommended, even for glassy micelles, for an efficient cellular uptake at low concentrations.

An optimized RGD-decorated micellar drug delivery system for albendazole for the treatment of ovarian cancer: from RAFT polymer synthesis to cellular uptake.

Block copolymers were prepared via RAFT polymerization with P(PEGMEMA) as the hydrophilic block to form micelles for the controlled delivery of ABZ. The group contribution method was used to estimate the partial solubility parameters for ABZ and various polymers as potential core-forming block to achieve optimum compatibility. Different ratios between MMA and LMA, a non-compatible monomer, were prepared. Cytotoxicity tests revealed a high toxicity of the ABZ-loaded micelle resulting in 80% cell deaths at a micelle concentration of 10 µg · mL(-1) . Cellular uptake of micelles has been studied using fluorescently labeled micelles, showing that a large fraction of micelles is readily taken up by OVCAR-3 cells. RGD-conjugated micelles were prepared and showed an increased cellular uptake.