Amphidinolide B: Total Synthesis, Structural Investigation, and Biological Evaluation

The total syntheses of amphidinolide B₁ and the proposed structure of amphidinolide B₂ have been accomplished. Key aspects of this work include the development of a practical, non-transition-metal-mediated method for the construction of the C₁₃–C₁₅ diene, the identification of α-chelation and dipole minimization models for diastereoselective methyl ketone aldol reactions, the discovery of a spontaneous Horner–Wadsworth–Emmons macrocyclization strategy, and the development of a novel late stage method for construction of an allylic epoxide moiety. The originally proposed structure for amphidinolide B₂ and diastereomers thereof display potent antitumor activities with IC₅₀ values ranging from 3.3 to 94.5 nM against human solid and blood tumor cells. Of the different stereoisomers, the proposed structure of amphidinolide B₂ is over 12-fold more potent than the C_8,9-epimer and C₁₈-epimer in human DU145 prostate cancer cells. These data suggest that the epoxide stereochemistry is a significant factor for anticancer activity

Amphidinolide B: Total Synthesis, Structural Investigation, and Biological Evaluation

The total syntheses of amphidinolide B₁ and the proposed structure of amphidinolide B₂ have been accomplished. Key aspects of this work include the development of a practical, non-transition-metal-mediated method for the construction of the C₁₃–C₁₅ diene, the identification of α-chelation and dipole minimization models for diastereoselective methyl ketone aldol reactions, the discovery of a spontaneous Horner–Wadsworth–Emmons macrocyclization strategy, and the development of a novel late stage method for construction of an allylic epoxide moiety. The originally proposed structure for amphidinolide B₂ and diastereomers thereof display potent antitumor activities with IC₅₀ values ranging from 3.3 to 94.5 nM against human solid and blood tumor cells. Of the different stereoisomers, the proposed structure of amphidinolide B₂ is over 12-fold more potent than the C_8,9-epimer and C₁₈-epimer in human DU145 prostate cancer cells. These data suggest that the epoxide stereochemistry is a significant factor for anticancer activity

Total Synthesis and Cytotoxicity Evaluation of Pareitropone and Analogues

A concise synthesis of pareitropone by oxidative cyclization of a phenolic nitronate is delineated. The use of TMSOTf as an additive to promote the facile formation of a strained norcaradiene intermediate provides convenient access to highly condensed multicyclic tropones in high yields. This synthesis is modular, efficient, and scalable, highlighting the synthetic utility of radical anion coupling reactions in annulation reactions. This work is discussed in the context of total syntheses of the tropoloisoquinoline alkaloids. Also included are the preparation of several congeners and a brief description of their biological activities

Neurospheres cultured from four GBM patients, PBT003, PBT008, PBT022 and PBT030 exhibited morphological characteristics of neural stem cells.

<p>(<b>A</b>) Morphology of PBT003, PBT008, PBT022 and PBT030 neurospheres in stem cell culture medium. (<b>B</b>) GBM neurospheres expressed specific biomarkers for normal neural stem cells. (<b>C</b>) GBM neurospheres exhibit the ability to differentiate.</p

Natural-Based Indirubins Display Potent Cytotoxicity toward Wild-Type and T315I-Resistant Leukemia Cell Lines

Drug resistance in chronic myelogenous leukemia (CML) requires the development of new CML chemotherapeutic drugs. Indirubin, a well-known mutikinase inhibitor, is the major active component of “Danggui Longhui Wan”, a Chinese traditional medicine used for the treatment of CML symptoms. An in-house collection of indirubin derivatives was screened at 1 μM on wild-type and imatinib-resistant T315I mutant CML cells. Herein are reported that only 15 analogues of the natural 6-bromoindirubin displayed potent cytotoxicity in the submicromolar range. Kinase assays in vitro show that eight out of the 15 active molecules strongly inhibited both <i>c</i>-Src and Abl oncogenic kinases in the nanomolar range. Most importantly, these eight molecules blocked the activity of T315I mutant Abl kinase at the submicromolar level and with analogue <b>22</b> exhibiting inhibitory activity at the low nanomolar range. Docking calculations suggested that active indirubins might inhibit T315I Abl kinase through an unprecedented binding to both active and Src-like inactive conformations. Analogue <b>22</b> is the first derivative of a natural product identified as an inhibitor of wild-type and imatinib-resistant T315I mutant Abl kinases

The effect of miR-4284 and miR27a inhibitors on the cytotoxic activity of BBMD3 in PBT003 and PBT030 neurospheres.

<p>(<b>A</b>) Cells from PBT003 and PBT030 neurospheres were transfected with 60 nM of anti-sense inhibitors against miR-4284, miR-27a and an unrelated control miRNA, and the inhibitor was allowed to interact with the miRNA for 48 hours prior to determining the viability of the cells. (<b>B</b>) After cells from PBT003 and PBT030 neurospheres were transfected with 60 nM anti-sense nucleotide inhibitors against miR-4284, miR-27a and an unrelated control anti-sense miRNA, and the inhibitors were allowed to interact with the miRNA for 24 hours; 5 µM of BBMD3 was added to the culture media for another 24 hour. Cell viability was determined at the end of the 48 hour period. Each experiment was performed in triplicate. The top of each bar graph represents the mean of 3 experiments, and the <i>error bars</i> represent ± the standard deviation from the mean SD. *, p<0.01; **, p<0.001.</p

The effects of BBMD3 on non-stem-like GBM tumor cells and normal neurospheres.

<p>(<b>A</b>) <b><i>Left panel</i></b>, U87 cells were treated with 0, 1, 3, 5, 8, or 10 µM BBMD3 for 24 and 48 hours, and viability was determined using the MTS assay, as described in the Methods. <b><i>Right panel</i></b>, U87 cells were treated with 0, 1, 3, 5, or 10 µM BBMD3 for 48 hours and apoptotic cells were analyzed for Annexin V-FITC reactivity and PI staining by flow cytometry. (<b>B</b>) <b><i>Left panel</i></b> shows the morphology of a normal human neurosphere. <b><i>Right panel</i></b> shows the viability of normal fetal brain derived and tumor derived neurospheres following 24 hours of treatment with 1, 5, or 10 µM BBMD3. Each experiment was performed in triplicate, and replicated as an independent experiment at least twice. The top of each bar graph represents the mean of the observed values from these replicates, and the <i>error bars</i> denote ± the standard deviation from the mean (SD).</p

Western blotting analysis of BBMD3 induced phosphorylation of JNK 1 and 2, c-Jun and total c-Fos protein expression in cells derived from human GBM neurospheres.

<p>(<b>A</b>) The effect of BBMD3 on the expression of total and phosphorylated STAT3 in cells derived from PBT003, PBT008, PBT022 and PBT030 neurospheres following a 24 hour treatment with BBMD3. (<b>B</b>) Western Blotting analysis of cell lysates probing for phosphorylated JNK expression following a 24 hour treatment of cells derived from GBM neurospheres with BBMD3. (<b>C</b>) Phosphorylation of c-Jun and quantification of c-Fos total protein expression following BBMD3 treatment.</p

Template-Catalyzed, Disulfide Conjugation of Monoclonal Antibodies Using a Natural Amino Acid Tag

The high specificity and favorable pharmacological properties of monoclonal antibodies (mAbs) have prompted significant interest in re-engineering this class of molecules to add novel functionalities for enhanced therapeutic and diagnostic potential. Here, we used the high affinity, meditope-Fab interaction to template and drive the rapid, efficient, and stable site-specific formation of a disulfide bond. We demonstrate that this template-catalyzed strategy provides a consistent and reproducible means to conjugate fluorescent dyes, cytotoxins, or “click” chemistry handles to <u>m</u>editope-<u>e</u>nabled mAbs (memAbs) and memFabs. More importantly, we demonstrate this covalent functionalization is achievable using natural amino acids only, opening up the opportunity to genetically encode cysteine meditope “tags” to biologics. As proof of principle, genetically encoded, cysteine meditope tags were added to the N- and/or C-termini of fluorescent proteins, nanobodies, and affibodies, each expressed in bacteria, purified to homogeneity, and efficiently conjugated to different memAbs and meFabs. We further show that multiple T-cell and Her2-targeting bispecific molecules using this strategy potently activate T-cell signaling pathways <i>in vitro</i>. Finally, the resulting products are highly stable as evidenced by serum stability assays (>14 d at 37 °C) and <i>in vivo</i> imaging of tumor xenographs. Collectively, the platform offers the opportunity to build and exchange an array of functional moieties, including protein biologics, among any cysteine memAb or Fab to rapidly create, test, and optimize stable, multifunctional biologics