Longqin Hu

Small Molecule Modulators of Keap1-Nrf2-ARE Pathway as Potential Preventive and Therapeutic Agents

Kelch‐like ECH‐associated protein 1 (Keap1)‐nuclear factor erythroid 2‐related factor 2 (Nrf2)‐antioxidant response elements (ARE) pathway represents one of the most important cellular defense mechanisms against oxidative stress and xenobiotic damage. Activation of Nrf2 signaling induces the transcriptional regulation of ARE‐dependent expression of various detoxifying and antioxidant defense enzymes and proteins. Keap1‐Nrf2‐ARE signaling has become an attractive target for the prevention and treatment of oxidative stress‐related diseases and conditions including cancer, neurodegenerative, cardiovascular, metabolic, and inflammatory diseases. Over the last few decades, numerous Nrf2 inducers have been developed and some of them are currently undergoing clinical trials. Recently, overactivation of Nrf2 has been implicated in cancer progression as well as in drug resistance to cancer chemotherapy. Thus, Nrf2 inhibitors could potentially be used to improve the effectiveness of cancer therapy. Herein, we review the signaling mechanism of Keap1‐Nrf2‐ARE pathway, its disease relevance, and currently known classes of small molecule modulators. We also discuss several aspects of Keap1–Nrf2 interaction, Nrf2‐based peptide inhibitor design, and the screening assays currently used for the discovery of direct inhibitors of Keap1‐Nrf2 interaction.

Design of anticancer prodrugs for reductive activation.

Anticancer prodrugs designed to target specifically tumor cells should increase therapeutic effectiveness and decrease systemic side effects in the treatment of cancer. Over the last 20 years, significant advances have been made in the development of anticancer prodrugs through the incorporation of triggers for reductive activation. Reductively activated prodrugs have been designed to target hypoxic tumor tissues, which are known to overexpress several endogenous reductive enzymes. In addition, exogenous reductive enzymes can be delivered to tumor cells through fusion with tumor‐specific antibodies or overexpressed in tumor cells through gene delivery approaches. Many anticancer prodrugs have been designed to use both the endogenous and exogenous reductive enzymes for target‐specific activation and these prodrugs often contain functional groups such as quinones, nitroaromatics, N‐oxides, and metal complexes. Although no new agents have been approved for clinical use, several reductively activated prodrugs are in various stages of clinical trial. This review mainly focuses on the medicinal chemistry aspects of various classes of reductively activated prodrugs including design principles, structure‐activity relationships, and mechanisms of activation and release of active drug molecules.

Discovery of a small-molecule inhibitor and cellular probe of Keap1-Nrf2 protein-protein interaction.

A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization assay identified a small molecule as a first-in-class direct inhibitor of Keap1-Nrf2 protein-protein interaction. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated as ML334 (LH601A), which is at least 100× more potent than the other stereoisomers. The stereochemistry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translocation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary but tractable structure-activity relationships support its use as a lead for our ongoing optimization.

NITROREDUCTASE: A PRODRUG‐ACTIVATING ENZYME FOR CANCER GENE THERAPY

1. The prodrug CB1954 (5‐(aziridin‐1‐yl)‐2,4‐dinitrobenzamide) is activated by Escherichia coli nitroreductase (NTR) to a potent DNA‐crosslinking agent.

Nitrobenzocyclophosphamides as potential prodrugs for bioreductive activation: Synthesis, stability, enzymatic reduction, and antiproliferative activity in cell culture

In efforts to obtain potential anticancer prodrugs for gene-directed enzyme prodrug therapy using Eschericia coli nitroreductase, a series of four benzocyclophosphamide analogues were designed and synthesized incorporating a strategically placed nitro group in a position para to the benzylic carbon for reductive activation. All four analogues were found to be stable in phosphate buffer at pH 7.4 and 37 degrees C and were good substrates of E. coli nitroreductase with half lives between 7 and 24 min at pH 7.0 and 37 degrees C. However, only two analogues 6a and 6c, both with a benzylic oxygen in the phosphorinane ring para to the nitro group, showed a modest 33-36-fold enhanced cytotoxicity in E. coli nitroreductase-expressing cells. These results suggest that good substrate activity and the para benzylic oxygen are required for activation by E. coli nitroreductase. Compounds 6a and 6c represent a new structure type for reductive activation and a lead for further modification in the development of better analogues with improved selective toxicity to be used in gene-directed enzyme prodrug therapy.

Structural Influence of Isothiocyanates on the Antioxidant Response Element (ARE)-Mediated Heme Oxygenase-1 (HO-1) Expression

PurposeIsothiocyanates (ITCs), existing abundantly in cruciferous vegetables, is one class of promising dietary cancer chemopreventive agents that possess strong cancer protective effects by modulation of phase II detoxifying/antioxidant enzyme activities. However, limited studies regarding to the structure-activity relationship (SAR) of ITCs on the induction of phase II detoxifying/antioxidant enzymes are reported. In this study, the effects of ten structurally related isothiocyanates on the antioxidant response element (ARE)-mediated antioxidant enzyme heme oxygenase-1 (HO-1) induction in human hepatoma HepG2-C8 cells were evaluated.Materials and MethodsAfter exposure of HepG2-C8 cells to ITCs, cell viability, luciferase reporter assay, Western blot analysis and quantitative real-time PCR were conducted.ResultsTreatments with most ITCs significantly activated ARE-mediated luciferase activity with different maximal degree of ARE induction. In addition, ITCs caused a substantial induction of HO-1 protein, which was closely correlated with inductive level of Nrf2 protein. Real-time PCR revealed that the expression of HO-1 mRNA and protein was significantly increased after treatments with ITCs, although not directly correlated. HO-1 induction by ITCs was attenuated in HepG2-C8 cells transiently transfected with a dominant negative mutant of Nrf2 (Nrf2-M4), whereas it was totally absent in Nrf2−/− mouse embryonic fibroblasts. In addition, ARE activation by ITCs was associated with the depletion of intracellular glutathione.ConclusionCollectively, our results demonstrate that the ITC class of compounds activates ARE-mediated HO-1 gene transcription through Nrf2/ARE signaling pathway, however, their inductive effects are quite specific, depending on the chemical structure. These results suggest the possibility that some synthetic ITCs might have superior chemopreventive activity than natural ITCs.

Analysis of Multiple Metabolites of Tocopherols and Tocotrienols in Mice and Humans

Tocopherols and tocotrienols, collectively known as vitamin E, are essential antioxidant nutrients. The biological fates and metabolite profiles of the different forms are not clearly understood. The objective of this study is to simultaneously analyze the metabolites of different tocopherols and tocotrienols in mouse and human samples. Using HPLC/electrochemical detection and mass spectrometry, 18 tocopherol-derived and 24 tocotrienol-derived side-chain degradation metabolites were identified in fecal samples. Short-chain degradation metabolites, in particular gamma- and delta-carboxyethyl hydroxychromans (CEHCs) and carboxymethylbutyl hydroxychromans (CMBHCs) were detected in urine, serum, and liver samples, with tocopherols additionally detected in serum and liver samples. The metabolite profiles of tocotrienols and tocopherols were similar, but new tocotrienol metabolites with double bonds were identified. This is the first comprehensive report describing simultaneous analysis of different side-chain metabolites of tocopherols and tocotrienols in mice and humans. Urinary metabolites may serve as useful biomarkers for the nutritional assessment of vitamin E.

Anti-NF-κB and Anti-inflammatory Activities of Synthetic Isothiocyanates: effect of chemical structures and cellular signaling

Many cancer chemopreventive agents have been associated with lower cancer risk by suppressing nuclear factor-kappaB (NF-kappaB) signaling pathways, which subsequently leads to attenuated pro-inflammatory mediators and activities. Of the natural compounds, the isothiocyanates (ITCs) found in cruciferous vegetables have received particular attention because of their potential anti-cancer effects. However, limited studies regarding the influence of ITCs structure on NF-kappaB transactivation and anti-inflammatory action are reported. In the present study, the anti-inflammatory potential of ten structurally divergent synthetic ITCs were evaluated in HT-29-N9 human colon cancer cells and RAW 264.7 murine macrophages. The effect of ITCs on the basal transcriptional activation of NF-kappaB and the inflammatory response to bacterial lipopolysaccharide (LPS) were assessed. The synthetic ITC analogs suppressed NF-kappaB-mediated pro-inflammatory gene transcription. Among the ITC analogs, tetrahydrofurfuryl isothiocyanate, methyl-3-isothiocyanatopropionate, 3-morpholinopropyl isothiocyanate and 3,4-methyelendioxybenzyl isothiocyanate showed stronger NF-kappaB inhibition as compared to the parent compound, phenylethyl isothiocyanate (PEITC). Molecular analysis revealed that several of the pro-inflammatory mediators and cytokines (iNOS, COX-2, IL-1beta, IL-6 and TNF-alpha) were reduced by ITCs, and correlated with the downregulation of NF-kappaB signaling pathways. Immunoblotting showed that ITCs suppressed LPS-induced phosphorylation and degradation of IkappaB alpha and decreased nuclear translocation of p65. In parallel, ITCs suppressed the phosphorylation of IkappaB kinase alpha/beta (IKKalpha/beta). Taken together, our findings provide the possibility that synthetic ITC analogs might have promising cancer chemopreventive potential, based on their stronger anti-NF-kappaB and anti-inflammatory activities, than the natural ITCs.

Exploiting the Drug-Activating Properties of a Novel Trypanosomal Nitroreductase

ABSTRACT Nitroheterocyclic prodrugs have been used to treat trypanosomal diseases for more than 40 years. Recently, the key step involved in the activation of these compounds has been elucidated and shown to be catalyzed by a type I nitroreductase (NTR). This class of enzyme is normally associated with bacteria and is absent from most eukaryotes, with trypanosomes being a major exception. Here we exploit this difference by evaluating the trypanocidal activity of a library of nitrobenzylphosphoramide mustards against bloodstream-form Trypanosoma brucei parasites. Biochemical screening against the purified enzyme revealed that a subset of halogenated nitroaromatic compounds were effective substrates for T. brucei NTR (TbNTR), having apparent Kcat/Km values approximately 100 times greater than nifurtimox. When tested against T. brucei, cytotoxicity mirrored enzyme activity, with 50% inhibitory concentrations of the most potent substrates being less than 10 nM. T. brucei NTR plays a key role in parasite killing: heterozygous lines displayed resistance to the compounds, while parasites overexpressing the enzyme showed hypersensitivity. We also evaluated the cytotoxicities of substrates with the highest trypanocidal activities by using mammalian THP-1 cells. The relative toxicities of these newly identified compounds were much lower than that of nifurtimox. We conclude that halogenated nitrobenzylphosphoramide mustards represent a novel class of antitrypanosomal agents, and their efficacy validates the strategy of specifically targeting NTR activity to develop new therapeutics.

Optimization of fluorescently labeled Nrf2 peptide probes and the development of a fluorescence polarization assay for the discovery of inhibitors of Keap1-Nrf2 interaction

Activation of the antioxidant response element (ARE) upregulates enzymes involved in detoxification of electrophiles and reactive oxygen species. The induction of ARE genes is regulated by the interaction between redox sensor protein Keap1 and the transcription factor Nrf2. Fluorescently labeled Nrf2 peptides containing the ETGE motif were synthesized and optimized as tracers in the development of a fluorescence polarization (FP) assay to identify small-molecule inhibitors of the Keap1-Nrf2 interaction. The tracers were optimized to increase the dynamic range of the assay and their binding affinities to the Keap1 Kelch domain. The binding affinities of Nrf2 peptide inhibitors obtained in our FP assay using FITC-9mer Nrf2 peptide amide as the probe were in good agreement with those obtained previously by a surface plasmon resonance assay. The FP assay exhibits considerable tolerance toward DMSO and produced a Z′ factor greater than 0.6 in a 384-well format. Further optimization of the probe led to cyanine-labeled 9mer Nrf2 peptide amide, which can be used along with the FITC-9mer Nrf2 peptide amide in a high-throughput screening assay to discover small-molecule inhibitors of Keap1-Nrf2 interaction.