Marie A. Colucci

Potent activity of indolequinones against human pancreatic cancer: identification of thioredoxin reductase as a potential target.

The indolequinone ES936 {5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione} was previously developed in our lab as an antitumor agent against pancreatic cancer. The objective of this study was to identify indolequinones with improved potency against pancreatic cancer and to define their mechanisms of action. Pancreatic cancer cell lines PANC-1, MIA PaCa-2, and BxPC-3 were used in in vitro assays [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) and clonogenic assays]; indolequinones displayed potent cytotoxicity against all three cell lines, and two specific classes of indolequinone were particularly potent agents. These indolequinones induced caspase-dependent apoptosis but no redox cycling or oxidative stress in MIA PaCa-2 and BxPC-3 cells. Selected indolequinones were also screened against the NCI-60 cell line panel and were found to be particularly effective against colon, renal, and melanoma cancer cells. A potential target of these indolequinones was identified as thioredoxin reductase. Indolequinones were found to be potent inhibitors of thioredoxin reductase activity both in pancreatic cancer cells and in cell-free systems. The mechanism of action of the indolequinones was shown to involve metabolic reduction, loss of a leaving group to generate a reactive electrophile resulting in alkylation of the selenocysteine residue in the active site of thioredoxin reductase. In vivo efficacy of the indolequinones was also tested in the MIA PaCa-2 pancreatic tumor xenograft in nude mice, and lead indolequinones demonstrated high efficacy and low toxicity. Inhibition of thioredoxin reductase represents a potential novel target in pancreatic cancer and may provide a biomarker of effect of lead indolequinones in this type of cancer.

Benzimidazole- and benzothiazole-quinones: excellent substrates for NAD(P)H:quinone oxidoreductase 1

A series of benzimidazole- and benzothiazole-quinones has been synthesized. The ability of these heterocyclic quinones to act as substrates for recombinant human NAD(P)H:quinone oxidoreductase (NQO1), a two-electron reductase upregulated in tumour cells, was determined. Overall, the quinones were excellent substrates for NQO1.

Discovery of Narrow Spectrum Kinase Inhibitors: New Therapeutic Agents for the Treatment of COPD and Steroid-Resistant Asthma

The discovery of a novel series of therapeutic agents that has been designed and optimized for treating chronic obstructive pulmonary disease is reported. The pharmacological strategy was based on the identification of compounds that inhibit a defined subset of kinase enzymes modulating inflammatory processes that would be effective against steroid refractory disease and exhibit a sustained duration of action after inhaled delivery.

Mechanism‐Based Inhibition of Quinone Reductase 2 (NQO2): Selectivity for NQO2 over NQO1 and Structural Basis for Flavoprotein Inhibition

A role for the flavoprotein NRH:quinone oxidoreductase 2 (NQO2, QR2) in human diseases such as malaria, leukemia and neurodegeneration has been proposed. In order to explore the potential of NQO2 as a therapeutic target, we have developed potent and selective mechanism‐based inhibitors centered on the indolequinone pharmacophore. The compounds show remarkable selectivity for NQO2 over the closely related flavoprotein NQO1, with small structural changes defining selectivity. Biochemical studies confirmed the mechanism‐based inhibition, whereas X‐ray crystallography and mass spectrometry revealed the nature of the inhibitor interaction with the protein. These indolequinones represent the first mechanism‐based inhibitors of NQO2, and their novel mode of action involving alkylation of the flavin cofactor, provides significant advantages over existing competitive inhibitors in terms of potency and irreversibility, and will open new opportunities to define the role of NQO2 in disease.

Abstract 4544: Novel indolequinone inhibitors of thioredoxin reductase are active against human pancreatic cancer

A series of indolequinones was previously developed in our lab as potential antitumor agents against pancreatic cancer. Three general classes of indolequinone consisting of 2-methyl, 2-hydroxymethyl and 2-unsubstituted derivatives with varying leaving groups at the indole 3- position exhibited marked activity against human pancreatic cancer both in vitro and in vivo. Pancreatic cancer cell lines PANC-1, MIA PaCa-2, and BxPC-3 were used to ensure effects were not cell line specific and growth inhibitory effects were measured using both the MTT and clonogenic assays. Indolequinones displayed growth inhibitory activity against pancreatic tumor cells in vitro and two specific classes of indolequinone, the 2-unsubstituted and 2-hydroxymethyl classes, were particularly active agents with IC 50 values in the 20-200nM range. Indolequinones were also found to be efficient inducers of apoptosis in pancreatic cancer cell lines at concentrations which induced growth inhibition. Selected indolequinones were also screened against the NCI-60 cell line panel to reveal the pattern of activity across various tumor types and were particularly effective against colon, renal, and melanoma cancer cells. The spectrum of activity of indolequinones in the NCI-60 was similar to established inhibitors of thioredoxin reductase. Indolequinones of both the 2-hydroxymethyl and 2-unsubstituted classes were found to inhibit thioredoxin reductase in MIA PaCa-2 cellspancreatic cancer at concentrations equivalent to those inducing growth inhibitory effects. The mechanism of inhibition of thioredoxin reductase by the indolequinones was studied in detail in cell-free systems using purified enzyme and the C-terminal selenocysteine of thioredoxin reductase was found to be the primary adduction site of the indolequinones using LC-MS analysis. Inhibition of thioredoxin reductase by indolequinones in cells resulted in a change of thioredoxin redox state and activation of the P38/JNK signaling pathway which may result in downstream apoptosis. Our results demonstrate that thioredoxin reductase is a potential target of the indolequinones in pancreatic cancer cells. (Supported by CA11441). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4544.