Mary M. Mader

The promise and peril of chemical probes

Chemical probes are powerful reagents with increasing impacts on biomedical research. However, probes of poor quality or that are used incorrectly generate misleading results. To help address these shortcomings, we will create a community-driven wiki resource to improve quality and convey current best practice.

Complete Inhibition of Anisomycin and UV Radiation but Not Cytokine Induced JNK and p38 Activation by an Aryl-substituted Dihydropyrrolopyrazole Quinoline and Mixed Lineage Kinase 7 Small Interfering RNA

Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC50 of 70 nm for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nm or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1β or tumor necrosis factor-α-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stress-activated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.

Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G2/M transition. We find that activation of Aurora A first occurs at centrosomes at late G2 and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.

LLY-507, a Cell-active, Potent, and Selective Inhibitor of Protein-lysine Methyltransferase SMYD2

Background: SMYD2 is a methyltransferase whose role in cancer is poorly understood and is lacking cell-active chemical tools. Results: We describe LLY-507, a small molecule inhibitor of SMYD2. Conclusion: LLY-507 is potent, selective, cell-active, and binds SMYD2 in a high resolution co-crystal. Significance: LLY-507 is a first-in-class cell-potent chemical probe that will be valuable in dissecting SMYD2 biology. SMYD2 is a lysine methyltransferase that catalyzes the monomethylation of several protein substrates including p53. SMYD2 is overexpressed in a significant percentage of esophageal squamous primary carcinomas, and that overexpression correlates with poor patient survival. However, the mechanism(s) by which SMYD2 promotes oncogenesis is not understood. A small molecule probe for SMYD2 would allow for the pharmacological dissection of this biology. In this report, we disclose LLY-507, a cell-active, potent small molecule inhibitor of SMYD2. LLY-507 is >100-fold selective for SMYD2 over a broad range of methyltransferase and non-methyltransferase targets. A 1.63-Å resolution crystal structure of SMYD2 in complex with LLY-507 shows the inhibitor binding in the substrate peptide binding pocket. LLY-507 is active in cells as measured by reduction of SMYD2-induced monomethylation of p53 Lys370 at submicromolar concentrations. We used LLY-507 to further test other potential roles of SMYD2. Mass spectrometry-based proteomics showed that cellular global histone methylation levels were not significantly affected by SMYD2 inhibition with LLY-507, and subcellular fractionation studies indicate that SMYD2 is primarily cytoplasmic, suggesting that SMYD2 targets a very small subset of histones at specific chromatin loci and/or non-histone substrates. Breast and liver cancers were identified through in silico data mining as tumor types that display amplification and/or overexpression of SMYD2. LLY-507 inhibited the proliferation of several esophageal, liver, and breast cancer cell lines in a dose-dependent manner. These findings suggest that LLY-507 serves as a valuable chemical probe to aid in the dissection of SMYD2 function in cancer and other biological processes.

Characterization of LY2228820 Dimesylate, a Potent and Selective Inhibitor of p38 MAPK with Antitumor Activity

p38α mitogen-activated protein kinase (MAPK) is activated in cancer cells in response to environmental factors, oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production of cytokines in the tumor microenvironment, such as TNF-α, interleukin-1β (IL-1β), IL-6, and CXCL8 (IL-8). p38α MAPK is highly expressed in human cancers and may play a role in tumor growth, invasion, metastasis, and drug resistance. LY2228820 dimesylate (hereafter LY2228820), a trisubstituted imidazole derivative, is a potent and selective, ATP-competitive inhibitor of the α- and β-isoforms of p38 MAPK in vitro (IC50 = 5.3 and 3.2 nmol/L, respectively). In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC50 = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 μmol/L. LY2228820 also reduced TNF-α secretion by lipopolysaccharide/IFN-γ–stimulated macrophages (IC50 = 6.3 nmol/L). In mice transplanted with B16-F10 melanoma, tumor phospho-MK2 (p-MK2) was inhibited by LY2228820 in a dose-dependent manner [threshold effective dose (TED)70 = 11.2 mg/kg]. Significant target inhibition (>40% reduction in p-MK2) was maintained for 4 to 8 hours following a single 10 mg/kg oral dose. LY2228820 produced significant tumor growth delay in multiple in vivo cancer models (melanoma, non–small cell lung cancer, ovarian, glioma, myeloma, breast). In summary, LY2228820 is a p38 MAPK inhibitor, which has been optimized for potency, selectivity, drug-like properties (such as oral bioavailability), and efficacy in animal models of human cancer. Mol Cancer Ther; 13(2); 364–74. ©2013 AACR.

Structure-guided expansion of kinase fragment libraries driven by support vector machine models

This work outlines a new de novo design process for the creation of novel kinase inhibitor libraries. It relies on a profiling paradigm that generates a substantial amount of kinase inhibitor data from which highly predictive QSAR models can be constructed. In addition, a broad diversity of X-ray structure information is needed for binding mode prediction. This is important for scaffold and substituent site selection. Borrowing from FBDD, the process involves fragmentation of known actives, proposition of binding mode hypotheses for the fragments, and model-driven recombination using a pharmacophore derived from known kinase inhibitor structures. The support vector machine method, using Merck atom pair derived fingerprint descriptors, was used to build models from activity from 6 kinase assays. These models were qualified prospectively by selecting and testing compounds from the internal compound collection. Overall hit and enrichment rates of 82% and 2.5%, respectively, qualified the models for use in library design. Using the process, 7 novel libraries were designed, synthesized and tested against these same 6 kinases. The results showed excellent results, yielding a 92% hit rate for the 179 compounds that made up the 7 libraries. The results of one library designed to include known literature compounds, as well as an analysis of overall substituent frequency, are discussed.

Characterization of LY3023414, a Novel PI3K/mTOR Dual Inhibitor Eliciting Transient Target Modulation to Impede Tumor Growth

The PI3K/AKT/mTOR pathway is among the most frequently altered pathways in cancer cell growth and survival. LY3023414 is a complex fused imidazoquinolinone with high solubility across a wide pH range designed to inhibit class I PI3K isoforms and mTOR kinase. Here, we describe the in vitro and in vivo activity of LY3023414. LY3023414 was highly soluble at pH 2–7. In biochemical testing against approximately 266 kinases, LY3023414 potently and selectively inhibited class I PI3K isoforms, mTORC1/2, and DNA-PK at low nanomolar concentrations. In vitro, inhibition of PI3K/AKT/mTOR signaling by LY3023414 caused G1 cell-cycle arrest and resulted in broad antiproliferative activity in cancer cell panel screens. In vivo, LY3023414 demonstrated high bioavailability and dose-dependent dephosphorylation of PI3K/AKT/mTOR pathway downstream substrates such as AKT, S6K, S6RP, and 4E-BP1 for 4 to 6 hours, reflecting the drugs half-life of 2 hours. Of note, equivalent total daily doses of LY3023414 given either once daily or twice daily inhibited tumor growth to similar extents in multiple xenograft models, indicating that intermittent target inhibition is sufficient for antitumor activity. In combination with standard-of-care drugs, LY3023414 demonstrated additive antitumor activity. The novel, orally bioavailable PI3K/mTOR inhibitor LY3023414 is highly soluble and exhibits potent in vivo efficacy via intermittent target inhibition. It is currently being evaluated in phase I and II trials for the treatment of human malignancies. Mol Cancer Ther; 15(10); 2344–56. ©2016 AACR.

A new access to 3,5-disubstituted piperazinones via Pd(0)-catalyzed amination

Abstract An original synthetic route toward 3,5-disubstituted piperazinones has been developed. The method relies upon a 6- exo intramolecular process between a sulfonylated nitrogen atom of amino acid derivation and an η 3 -allyl-palladium moiety. This cyclization process generates the two possible ( cis and trans ) diastereoisomers whose ratio depends on the amino acid employed. The bulkier the amino acid residue, the higher the observed cis : trans ratio. Convincing evidence for reversible intramolecular addition of the nitrogen nucleophile to the η 3 -allyl-palladium complex is put forward.

Computational Investigation of the Role of Fluoride in Tamao Oxidations

The Tamao oxidation of alkoxysilanes was investigated computationally to determine the role of fluoride, a key additive, in this reaction. A sequence of fluoride equilibria as well as possible transition states, mediated by basic and neutral peroxide, respectively, were examined, and a potential energy surface was calculated which was consistent with the typical synthetic methods required for the conversion of alkoxysilanes to alcohols.

A mild method for protodesilylation of α-dimethylphenylsilyl ester substrates

Mild conditions (1.2 eq. Hg(OAc)2, 1.2 eq. TBAF in 1:1 MeOH/THF; 35 min at 0 °C) have been developed for the protodesilylation of α-dimethylphenylsilyl esters. An enolate-dependent mechanism for the reaction was supported through studies indicating the clean incorporation of deuterium. To further investigate the mechanism, the optimal conditions as well as the kinetics of the reaction were explored.