Gunda I. Georg

Palladium(II)-Catalyzed Direct Arylation of Enaminones Using Organotrifluoroborates

A Pd(II)-catalyzed reaction for the direct arylation of cyclic enaminones is reported. The reactivity of electron-rich, electron-poor, and sterically encumbered organotrifluoroborates was investigated. This reaction represents a unique use for organotrifluoroborates as coupling partners and discloses the utility of enaminones for direct-functionalization reactions. It provides immediate access to arylpiperidine, indolizidine, and quinolizidine scaffolds from the corresponding mono- and bicyclic, unattenuated enaminones.

A Preclinical Evaluation of Minnelide as a Therapeutic Agent Against Pancreatic Cancer

Minnelide prevents tumor formation, causes tumor regression, and increases survival in multiple models of pancreatic cancer. Vegetation Is Good for You Your mom always told you to eat your vegetables, but what she probably didn’t tell you is that other plants can be good for you as well. Tripterygium wilfordii, sometimes known as the Thunder God vine, has various uses in traditional Chinese medicine. To better understand and improve upon the healing properties of this vine, the active ingredients have been isolated and characterized. One component of T. wilfordii, triptolide, has shown promising effects against pancreatic cancer cells. New therapies for pancreatic cancer—which is one of the most lethal human malignancies—are desperately needed, but triptolide is poorly soluble in water and thus has limited clinical use. Now, Chugh et al. synthesize a water-soluble form of triptolide, Minnelide, and demonstrate efficacy against pancreatic cancer in multiple animal models. The authors tested Minnelide both in vitro and in multiple preclinical models of pancreatic cancer. Each model has distinct advantages and limitations: Well-studied cancer cell lines and translationally relevant patient tumors were transplanted into mice that lack immune systems, whereas a spontaneous model in immunosufficient mice was, by necessity, a mouse tumor. By combining these approaches, the authors addressed many caveats that frequently plague preclinical studies. Indeed, Minnelide was highly effective in treating pancreatic cancer in all of these complementary models. The next step is to take Minnelide into early clinical trials to see if these results can be reproduced in human patients with pancreatic cancer. Pancreatic cancer is one of the most lethal human malignancies with an all-stage 5-year survival frequency of <5%, which highlights the urgent need for more effective therapeutic strategies. We have previously shown that triptolide, a diterpenoid, is effective against pancreatic cancer cells in vitro as well as in vivo. However, triptolide is poorly soluble in water, limiting its clinical use. We therefore synthesized a water-soluble analog of triptolide, named Minnelide. The efficacy of Minnelide was tested both in vitro and in multiple independent yet complementary in vivo models of pancreatic cancer: an orthotopic model of pancreatic cancer using human pancreatic cancer cell lines in athymic nude mice, a xenograft model where human pancreatic tumors were transplanted into severe combined immunodeficient mice, and a spontaneous pancreatic cancer mouse model (KRasG12D; Trp53R172H; Pdx-1Cre). In these multiple complementary models of pancreatic cancer, Minnelide was highly effective in reducing pancreatic tumor growth and spread, and improving survival. Together, our results suggest that Minnelide shows promise as a potent chemotherapeutic agent against pancreatic cancer, and support the evaluation of Minnelide in clinical trials against this deadly disease.

Acetyl-lysine Binding Site of Bromodomain-Containing Protein 4 (BRD4) Interacts with Diverse Kinase Inhibitors.

Members of the bromodomain and extra terminal (BET) family of proteins are essential for the recognition of acetylated lysine (KAc) residues in histones and have emerged as promising drug targets in cancer, inflammation, and contraception research. In co-crystallization screening campaigns using the first bromodomain of BRD4 (BRD4-1) against kinase inhibitor libraries, we identified and characterized 14 kinase inhibitors (10 distinct chemical scaffolds) as ligands of the KAc binding site. Among these, the PLK1 inhibitor BI2536 and the JAK2 inhibitor TG101209 displayed strongest inhibitory potential against BRD4 (IC50 = 25 nM and 130 nM, respectively) and high selectivity for BET bromodomains. Comparative structural analysis revealed markedly different binding modes of kinase hinge-binding scaffolds in the KAc binding site, suggesting that BET proteins are potential off-targets of diverse kinase inhibitors. Combined, these findings provide a new structural framework for the rational design of next-generation BET-selective and dual-activity BET-kinase inhibitors.

Palladium(II)-catalyzed dehydrogenative alkenylation of cyclic enaminones via the Fujiwara-Moritani reaction

A new Pd(II)-catalyzed dehydrogenative alkenylation reaction involving two alkenes was developed. A variety of nonaromatic, cyclic enaminones were successfully coupled to primary and secondary alkenes yielding a series of unique 1,3-dienes. The generality of this transformation presents a useful strategy for directly cross-coupling alkenes and offers an attractive new approach to functionalize enaminones.

β-Amyloid-Induced Neurodegeneration and Protection by Structurally Diverse Microtubule-Stabilizing Agents

Deposition of β-amyloid peptide (Aβ) and hyperphosphorylation of the τ protein are associated with neuronal dysfunction and cell death in Alzheimers disease. Although the relationship between these two processes is not yet understood, studies have shown that both in vitro and in vivo exposure of neurons to Aβ leads to τ hyperphosphorylation and neuronal dystrophy. We previously reported that the microtubule-stabilizing drug paclitaxel (Taxol) protects primary neurons against toxicity induced by the Aβ25-35 peptide. The studies in this report were undertaken to characterize the actions of paclitaxel more fully, to assess the effectiveness of structurally diverse microtubulestabilizing agents in protecting neurons, and to determine the time course of the protective effects of the drugs. Primary neurons were exposed to Aβ in the presence or absence of several agents shown to interact with microtubules, and neuronal survival was monitored. Paclitaxel protected neurons against Aβ1-42 toxicity, and paclitaxel-treated cultures exposed to Aβ showed enhanced survival over Aβ-only cultures for several days. Neuronal apoptosis induced by Aβ was blocked by paclitaxel. Other taxanes and three structurally diverse microtubule-stabilizing compounds also significantly increased survival of Aβ-treated cultures. At concentrations below 100 nM, the drugs that protected the neurons did not produce detectable toxicity when added to the cultures alone. Although multiple mechanisms are likely to contribute to the neuronal cell death induced by oligomeric or fibrillar forms of Aβ, low concentrations of drugs that preserve the integrity of the cytoskeletal network may help neurons survive the toxic cascades initiated by these peptides.

Understanding tubulin–Taxol interactions: Mutations that impart Taxol binding to yeast tubulin

We have successfully used mutagenesis to engineer Taxol (paclitaxel) binding activity in Saccharomyces cerevisiae tubulin. Taxol, a successful antitumor agent, acts by promoting tubulin assembly and stabilizing microtubules. Several structurally diverse antimitotic compounds, including the epothilones, compete with Taxol for binding to mammalian microtubules, suggesting that Taxol and these compounds share an overlapping binding site. However, Taxol has no effect on tubulin or microtubules from S. cerevisiae, whereas epothilone does. After considering data on Taxol binding to mammalian tubulin and recent modeling studies, we have hypothesized that differences in five key amino acids are responsible for the lack of Taxol binding to yeast tubulin. After changing these amino acids to those found in mammalian brain tubulin, we observed Taxol-related activity in yeast tubulin comparable to that in mammalian tubulin. Importantly, this experimental system can be used to reveal tubulin interactions with Taxol, the epothilones, and other Taxol-like compounds.

Discovery of a Potential Allosteric Ligand Binding Site in CDK2.

Cyclin-dependent kinases (CDKs) are key regulatory enzymes in cell cycle progression and transcription. Aberrant activity of CDKs has been implicated in a number of medical conditions, and numerous small molecule CDK inhibitors have been reported as potential drug leads. However, these inhibitors exclusively bind to the ATP site, which is largely conserved among protein kinases, and clinical trials have not resulted in viable drug candidates, attributed in part to the lack of target selectivity. CDKs are unique among protein kinases, as their functionality strictly depends on association with their partner proteins, the cyclins. In an effort to identify potential target sites for disruption of the CDK-cyclin interaction, we probed the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS) with human CDK2 and cyclin A using fluorescence spectroscopy and protein crystallography. ANS interacts with free CDK2 in a saturation-dependent manner with an apparent K(d) of 37 μM, and cyclin A displaced ANS from CDK2 with an EC(50) value of 0.6 μM. Co-crystal structures with ANS alone and in ternary complex with ATP site-directed inhibitors revealed two ANS molecules bound adjacent to one another, away from the ATP site, in a large pocket that extends from the DFG region above the C-helix. Binding of ANS is accompanied by substantial structural changes in CDK2, resulting in a C-helix conformation that is incompatible for cyclin A association. These findings indicate the potential of the ANS binding pocket as a new target site for allosteric inhibitors disrupting the interaction of CDKs and cyclins.

Protection Against β‐Amyloid Toxicity in Primary Neurons by Paclitaxel (Taxol)

Abstract: Neurofibrillary tangles in Alzheimers disease contain aggregates of abnormally phosphorylated microtubule‐associated protein τ, indicating that microtubule breakdown is a primary event in the neurodegenerative cascade. Recent studies have shown that addition to neuronal cultures of amyloid peptides found in Alzheimers leads to abnormal phosphorylation of τ and neurofibrillary pathology. We tested the possibility that the microtubule‐stabilizing drug paclitaxel (Taxol) might protect primary neurons against amyloid‐induced toxicity. Neurons exposed to aggregated amyloid peptides 25–35 and 1–42 became pyknotic with degenerating neurites within 24 h. Treatment of cultures with paclitaxel either 2 h before or 2 h after addition of the peptide prevented these morphological alterations. When numbers of viable cells were determined in cultures exposed to amyloid peptide with or without paclitaxel for 24 or 96 h, the percentage of surviving cells was significantly higher in paclitaxel‐treated cultures, and activation of the apoptosis‐associated protease CPP32 was significantly reduced. These observations indicate that microtubule‐stabilizing drugs may help slow development of the neurofibrillary pathology that leads to the loss of neuronal integrity in Alzheimers disease.

Stabilization of the cyclin‐dependent kinase 5 activator, p35, by paclitaxel decreases β‐amyloid toxicity in cortical neurons

One hallmark of Alzheimers disease (AD) is the formation of neurofibrillary tangles, aggregated paired helical filaments composed of hyperphosphorylated tau. Amyloid‐β (Aβ) induces tau hyperphosphorylation, decreases microtubule (MT) stability and induces neuronal death. MT stabilizing agents have been proposed as potential therapeutics that may minimize Aβ toxicity and here we report that paclitaxel (taxol) prevents cell death induced by Aβ peptides, inhibits Aβ‐induced activation of cyclin‐dependent kinase 5 (cdk5) and decreases tau hyperphosphorylation. Taxol did not inhibit cdk5 directly but significantly blocked Aβ‐induced calpain activation and decreased formation of the cdk5 activator, p25, from p35. Taxol specifically inhibited the Aβ‐induced activation of the cytosolic cdk5‐p25 complex, but not the membrane‐associated cdk5‐p35 complex. MT‐stabilization was necessary for neuroprotection and inhibition of cdk5 but was not sufficient to prevent cell death induced by overexpression of p25. As taxol is not permeable to the blood–brain barrier, we assessed the potential of taxanes to attenuate Aβ toxicity in adult animals using a succinylated taxol analog (TX67) permeable to the blood–brain barrier. TX67, but not taxol, attenuated the magnitude of both basal and Aβ‐induced cdk5 activation in acutely dissociated cortical cultures prepared from drug treated adult mice. These results suggest that MT‐stabilizing agents may provide a therapeutic approach to decrease Aβ toxicity and neurofibrillary pathology in AD and other tauopathies.

Polymer-bound triphenylphosphine as traceless reagent for mitsunobu reactions in combinatorial chemistry: Synthesis of aryl ethers from phenols and alcohols

Abstract The synthesis of aryl ethers from phenols and alcohols using polymer-bound triphenylphosphine and diethyl azodicar☐ylate (DEAD) is described. The polymer-bound triphenylphosphines are easily removed by filtration from the reaction products. This method is operationally simple and provides the products with high purity and in good yields.