Biological Evaluation of Subglutinol A As a Novel Immunosuppressive Agent for Inflammation Intervention

Subglutinol A (<b>1</b>) is an immunosuppressive natural product isolated from <i>Fusarium subglutinans</i>, an endophytic fungus from the vine <i>Tripterygium wilfordii</i>. We show that <b>1</b> exerts multimodal immune-suppressive effects on activated T cells in vitro: subglutinol A (<b>1</b>) effectively blocks T cell proliferation and survival while profoundly inhibiting pro-inflammatory IFNγ and IL-17 production by fully differentiated effector Th1 and Th17 cells. Our data further reveal that <b>1</b> may exert its anti-inflammatory effects by exacerbating mitochondrial damage in T cells. Additionally, we demonstrate that <b>1</b> significantly reduces lymphocytic infiltration into the footpad and ameliorates footpad swelling in the mouse model of Th1-driven delayed-type hypersensitivity. These results suggest the potential of <b>1</b> as a novel therapeutic for inflammatory diseases

Synthesis of α,α′-<i>trans</i>-Oxepanes through an Organocatalytic Oxa-conjugate Addition Reaction

Oxepanes are found in a wide range of natural products; however, they are challenging synthetic targets due to enthalpic and entropic barriers. Organocatalytic oxa-conjugate addition reactions promoted by the <i>gem</i>-disubstituent (Thorpe–Ingold) effect stereoselectively provided α,α′-<i>trans</i>-oxepanes. In addition, the potential of an organocatalytic tandem oxa-conjugate addition/α-oxidation was demonstrated in a rapid generation of molecular complexity. These organocatalytic oxa-conjugate addition reactions would provide powerful tools for the synthesis of natural products that contain highly functionalized oxepanes

Modulation of Activity Profiles for Largazole-Based HDAC Inhibitors through Alteration of Prodrug Properties

Largazole is a potent and class I-selective histone deacetylase (HDAC) inhibitor purified from marine cyanobacteria and was demonstrated to possess antitumor activity. Largazole employs a unique prodrug strategy, via a thioester moiety, to liberate the bioactive species largazole thiol. Here we report alternate prodrug strategies to modulate the pharmacokinetic and pharmacodynamics profiles of new largazole-based compounds. The in vitro effects of largazole analogues on cancer cell proliferation and enzymatic activities of purified HDACs were comparable to the natural product. However, in vitro and in vivo histone hyperacetylation in HCT116 cells and implanted tumors, respectively, showed differences, particularly in the onset of action and oral bioavailability. These results indicate that, by employing a different approach to disguise the “warhead” moiety, the functional consequence of these prodrugs can be significantly modulated. Our data corroborate the role of the pharmacokinetic properties of this class of compounds to elicit the desired and timely functional response

Discovery of Manassantin A Protein Targets Using Large-Scale Protein Folding and Stability Measurements

Manassantin A is a natural product that has been shown to have anticancer activity in cell-based assays, but has a largely unknown mode-of-action. Described here is the use of two different energetics-based approaches to identify protein targets of manassantin A. Using the stability of proteins from rates of oxidation technique with an isobaric mass tagging strategy (iTRAQ-SPROX) and the pulse proteolysis technique with a stable isotope labeling with amino acids in cell culture strategy (SILAC-PP), over 1000 proteins in a MDA-MB-231 cell lysate grown under hypoxic conditions were assayed for manassantin A interactions (both direct and indirect). A total of 28 protein hits were identified with manassantin A-induced thermodynamic stability changes. Two of the protein hits (filamin A and elongation factor 1α) were identified using both experimental approaches. The remaining 26 hit proteins were only assayed in either the iTRAQ-SPROX or the SILAC-PP experiment. The 28 potential protein targets of manassantin A identified here provide new experimental avenues along which to explore the molecular basis of manassantin A’s mode of action. The current work also represents the first application iTRAQ-SPROX and SILAC-PP to the large-scale analysis of protein–ligand binding interactions involving a potential anticancer drug with an unknown mode-of-action

Manipulating JNK Signaling with (−)-Zuonin A

Recently, in a virtual screening strategy to identify new compounds targeting the D-recruitment site (DRS) of the c-Jun N-terminal kinases (JNKs), we identified the natural product (−)-zuonin A. Here we report the asymmetric synthesis of (−)-zuonin A and its enantiomer (+)-zuonin A. A kinetic analysis for the inhibition of c-Jun phosphorylation by (−)-zuonin A revealed a mechanism of partial competitive inhibition. Its binding is proposed to weaken the interaction of c-Jun to JNK by approximately 5-fold, without affecting the efficiency of phosphorylation within the complex. (−)-Zuonin A inhibits the ability of both MKK4 and MKK7 to phosphorylate and activate JNK. The binding site of (−)-zuonin A is predicted by docking and molecular dynamics simulation to be located in the DRS of JNK. (+)-Zuonin A also binds JNK but barely impedes the binding of c-Jun. (−)-Zuonin A inhibits the activation of JNK, as well as the phosphorylation of c-Jun in anisomycin-treated HEK293 cells, with the inhibition of JNK activation being more pronounced. (−)-Zuonin A also inhibits events associated with constitutive JNK2 activity, including c-Jun phosphorylation, basal Akt activation, and MDA-MB-231 cell migration. Mutations in the predicted binding site for (−)-zuonin A can render it significantly more or less sensitive to inhibition than wild type JNK2, allowing for the design of potential chemical genetic experiments. These studies suggest that the biological activity reported for other lignans, such as saucerneol F and zuonin B, may be the result of their ability to impede protein–protein interactions within MAPK cascades

Manipulating JNK Signaling with (−)-Zuonin A

Recently, in a virtual screening strategy to identify new compounds targeting the D-recruitment site (DRS) of the c-Jun N-terminal kinases (JNKs), we identified the natural product (−)-zuonin A. Here we report the asymmetric synthesis of (−)-zuonin A and its enantiomer (+)-zuonin A. A kinetic analysis for the inhibition of c-Jun phosphorylation by (−)-zuonin A revealed a mechanism of partial competitive inhibition. Its binding is proposed to weaken the interaction of c-Jun to JNK by approximately 5-fold, without affecting the efficiency of phosphorylation within the complex. (−)-Zuonin A inhibits the ability of both MKK4 and MKK7 to phosphorylate and activate JNK. The binding site of (−)-zuonin A is predicted by docking and molecular dynamics simulation to be located in the DRS of JNK. (+)-Zuonin A also binds JNK but barely impedes the binding of c-Jun. (−)-Zuonin A inhibits the activation of JNK, as well as the phosphorylation of c-Jun in anisomycin-treated HEK293 cells, with the inhibition of JNK activation being more pronounced. (−)-Zuonin A also inhibits events associated with constitutive JNK2 activity, including c-Jun phosphorylation, basal Akt activation, and MDA-MB-231 cell migration. Mutations in the predicted binding site for (−)-zuonin A can render it significantly more or less sensitive to inhibition than wild type JNK2, allowing for the design of potential chemical genetic experiments. These studies suggest that the biological activity reported for other lignans, such as saucerneol F and zuonin B, may be the result of their ability to impede protein–protein interactions within MAPK cascades

From in Silico Discovery to Intracellular Activity: Targeting JNK–Protein Interactions with Small Molecules

The JNK–JIP1 interaction represents an attractive target for the selective inhibition of JNK-mediated signaling. We report a virtual screening (VS) workflow, based on a combination of three-dimensional shape and electrostatic similarity, to discover novel scaffolds for the development of non-ATP competitive inhibitors of JNK targeting the JNK–JIP interaction. Of 352 (0.13%) compounds selected from the NCI Diversity Set, more than 22% registered as hits in a biochemical kinase assay. Several compounds discovered to inhibit JNK activity under standard kinase assay conditions also impeded JNK activity in HEK293 cells. These studies led to the discovery that the lignan (−)-zuonin A inhibits JNK–protein interactions with a selectivity of 100-fold over ERK2 and p38 MAPKα. These results demonstrate the utility of a virtual screening protocol to identify novel scaffolds for highly selective, cell-permeable inhibitors of JNK–protein interactions

Synthesis and Biological Evaluation of Manassantin Analogues for Hypoxia-Inducible Factor 1α Inhibition

To cope with hypoxia, tumor cells have developed a number of adaptive mechanisms mediated by hypoxia-inducible factor 1 (HIF-1) to promote angiogenesis and cell survival. Due to significant roles of HIF-1 in the initiation, progression, metastasis, and resistance to treatment of most solid tumors, a considerable amount of effort has been made to identify HIF-1 inhibitors for treatment of cancer. Isolated from <i>Saururus cernuus</i>, manassantins A (<b>1</b>) and B (<b>2</b>) are potent inhibitors of HIF-1 activity. To define the structural requirements of manassantins for HIF-1 inhibition, we prepared and evaluated a series of manassantin analogues. Our SAR studies examined key regions of manassantin’s structure in order to understand the impact of these regions on biological activity and to define modifications that can lead to improved performance and drug-like properties. Our efforts identified several manassantin analogues with reduced structural complexity as potential lead compounds for further development. Analogues <b>MA04</b>, <b>MA07</b>, and <b>MA11</b> down-regulated hypoxia-induced expression of the HIF-1α protein and reduced the levels of HIF-1 target genes, including cyclin-dependent kinase 6 (Cdk6) and vascular endothelial growth factor (VEGF). These findings provide an important framework to design potent and selective HIF-1α inhibitors, which is necessary to aid translation of manassantin-derived natural products to the clinic as novel therapeutics for cancers

Synthesis and Biological Evaluation of Manassantin Analogues for Hypoxia-Inducible Factor 1α Inhibition

To cope with hypoxia, tumor cells have developed a number of adaptive mechanisms mediated by hypoxia-inducible factor 1 (HIF-1) to promote angiogenesis and cell survival. Due to significant roles of HIF-1 in the initiation, progression, metastasis, and resistance to treatment of most solid tumors, a considerable amount of effort has been made to identify HIF-1 inhibitors for treatment of cancer. Isolated from <i>Saururus cernuus</i>, manassantins A (<b>1</b>) and B (<b>2</b>) are potent inhibitors of HIF-1 activity. To define the structural requirements of manassantins for HIF-1 inhibition, we prepared and evaluated a series of manassantin analogues. Our SAR studies examined key regions of manassantin’s structure in order to understand the impact of these regions on biological activity and to define modifications that can lead to improved performance and drug-like properties. Our efforts identified several manassantin analogues with reduced structural complexity as potential lead compounds for further development. Analogues <b>MA04</b>, <b>MA07</b>, and <b>MA11</b> down-regulated hypoxia-induced expression of the HIF-1α protein and reduced the levels of HIF-1 target genes, including cyclin-dependent kinase 6 (Cdk6) and vascular endothelial growth factor (VEGF). These findings provide an important framework to design potent and selective HIF-1α inhibitors, which is necessary to aid translation of manassantin-derived natural products to the clinic as novel therapeutics for cancers