Yan Xia

Recent advances in hypoxia-inducible factor (HIF)-1 inhibitors.

Tumor hypoxia has been recognized as a common feature of solid tumors and a negative prognostic factor for response to treatment and survival of cancer patients. The discovery of hypoxia-inducible factor-1 (HIF-1), a molecular determinant of responses to hypoxia in mammalian cells, has renewed enthusiasm for discovery and development of targeted therapies exploiting the hypoxic tumor microenvironment. HIF-1 activity in tumors depends on availability of the HIF-1α subunit, the levels of which increase under hypoxic conditions and through activation of oncogenes and/or inactivation of tumor suppressor genes. Increased HIF-1 has been correlated with increased angiogenesis, aggressive tumor growth, and poor patient prognosis, leading to current interest in HIF-1 as promising anticancer drug target. In spite of an ever increasing number of putative small molecule inhibitors of HIF-1, only a few are progressing through preclinical and early clinical development. In this review, we will discuss recent advances in discovery and development of small molecule inhibitors that target the HIF-1 pathway as potential anticancer agents.

Identification of malate dehydrogenase 2 as a target protein of the HIF-1 inhibitor LW6 using chemical probes

Hypoxia-inducible factor (HIF) regulates tumor angiogenesis and metastasis in response to low oxygen tension. In the presence of oxygen, HIF-1a is rapidly degraded through the ubiquitin–proteasome pathway. In hypoxic conditions, stabilized HIF-1a dimerizes with HIF-1b. The HIF-1a/b heterodimer binds to hypoxia response elements (HRE) in gene promoters and induces the expression of target genes involved in angiogenesis, metastasis, glycolysis, cell proliferation, and resistance to apoptosis. Increased expression of HIF-1a in many solid tumors correlates with aggressive tumor growth, therapeutic resistance, and a poor clinical outcome. HIF-1a shifts the metabolism from oxidative phosphorylation to anaerobic glycolysis. Therefore, HIF-1a is an important therapeutic target for cancer. We previously synthesized and evaluated aryloxyacetylamino benzoic acid analogues. LW6 (1 in Figure 1A) potently inhibited HIF-1a accumulation by degrading HIF1a without affecting the HIF-1a mRNA levels during hypoxia. LW6, which is commercially available, has been used in various studies as an HIF-1a inhibitor. However, the molecular target of LW6 remains unknown. To identify a drug target, chemical biological methods such as activity-based probes (ABPs), photoaffinity labeling, biotinylation, and click conjugation have been used. Herein, we identify the molecular target of 1 using chemical probes. Cellular images and direct protein interactions of 1 were examined in living cells with a series of chemical probes (2–6), which were designed using the structure– activity relationship (SAR) of 1. Synthesis and characterization data for these probes are available in the Supporting Information. The distribution of drug molecules within subcellular compartments can provide information about the mechanism of drug action. The intracellular localization of LW6 was visualized through click chemistry with probe 2, containing an acetylene group, in colon cancer HCT116 cells (Figure 1A). Both 1 and 2 suppressed HIF-1a accumulation (Figure 1B) and HRE-luciferase activity (Figure 1A; Supporting Information, Figure S12). Subsequently, the cellular localization of probe 2 was determined by a click reaction with an azidelinked Alexa Fluor 488 molecule. Notably, copper-catalyzed azide–alkyne cycloadditions (click reactions) are highly specific and efficient bio-orthogonal reactions to visualize intracellular probe distribution. We found that compound 2 was localized primarily in the cytoplasm (Figure 1C). The colocalization of compound 2 (3 mm) with the mitochondriaselective probe, MitoTracker (500 nm), indicated that 2 is specifically localized in the mitochondria, whereas the localization of an adamantyl-free probe 4 was not observed (Figure S13). The mitochondrial localization of probe 2 was Figure 1. Biological activities and cellular localization of a chemical probe for LW6. A) Formula of 1 and its clickable probe 2. B) Inhibitory effects of 1 and probe 2 on HIF-1a accumulation were determined by immunoblot analysis. C) Localization of probe 2 (3 mm, green) was detected through a click reaction using azide-linked Alexa Fluor 488 in HCT116 cells. Mitochondria were selectively stained with the MitoTracker probe (red). Nuclei (blue) were stained with 4,6-diamidino-2phenylindole (DAPI). D) Competitive binding of probe 2 (3 mm) to its target molecules in the presence or absence of 1 (10 mm). Scale bars = 20 mm.

A Novel Small Molecule Facilitates the Reprogramming of Human Somatic Cells into a Pluripotent State and Supports the Maintenance of an Undifferentiated State of Human Pluripotent Stem Cells

Booster of pluripotency: RSC133, a new synthetic derivative of indoleacrylic acid/indolepropionic acid, exhibits dual activity by inhibiting histone deacetylase and DNA methyltransferase. Furthermore it potently improves the reprogramming of human somatic cells into a pluripotent state and aids the growth and maintenance of human pluripotent stem cells (hPSCs).

HIF-1α inhibitors: Synthesis and biological evaluation of novel moracin O and P analogues

The natural products moracins O and P exhibited potent in vitro inhibitory activity against hypoxia-inducible factor (HIF-1), which is a key mediator during adaptation of cancer cells to tumour hypoxia. Systematic variations of the structures of benzofuran type moracins were made and structure-activity relationship analysis showed the importance of the 2-arylbenzofuran ring and the (R)-configuration of the core scaffold. Further evaluation of the representative compound 5 showed its inhibitory effect on HIF-1α protein accumulation and target gene expression under hypoxia.

The first total synthesis of moracin O and moracin P, and establishment of the absolute configuration of moracin O

The first total synthesis of the naturally occurring benzofurans, moracins O and P was achieved using a Sonogashira cross coupling reaction followed by in situ cyclization, and the absolute configuration of natural moracin O was established.

Synthesis and structure-activity relationship of (E)-phenoxyacrylic amide derivatives as hypoxia-inducible factor (HIF) 1α inhibitors.

A series of (E)-phenoxyacrylic amide derivatives were synthesized and evaluated as hypoxia inducible factor (HIF) 1α inhibitors. The present structure-activity relationship study on this series identified the morpholinoethyl containing ester 4p as a potent inhibitor of HIF-1α under hypoxic conditions (IC50=0.12 μM in a cell-based HRE reporter assay) in HCT116 cells. The representative compound 4p suppressed hypoxia-induced HIF-1α accumulation and targeted gene expression in a dose-dependent manner. The effect of HIF-1α inhibition by 4p was further demonstrated by its inhibitory activity on in vitro tube formation and migration of cells, which may be valuable for development of novel therapeutics for cancer and tumor angiogenesis.

A novel class of highly potent multidrug resistance reversal agents: disubstituted adamantyl derivatives.

Novel disubstituted adamantyl derivatives were synthesized and evaluated in a P-glycoprotein dependent multidrug resistance cancer cell line. The hit to lead optimization provided potent MDR reversal agents. Some potent adamantyl derivatives were more than 10-fold more potent than verapamil without considerable intrinsic cytotoxicity. The 3-trifluorophenyl derivative 14f did not affect the metabolism of CYP450 3A4, whereas most of MDR revertants had a weak inhibitory effect.

Effect of coumarin derivative-mediated inhibition of P-glycoprotein on oral bioavailability and therapeutic efficacy of paclitaxel.

Since P-glycoprotein (P-gp) acts as a barrier to intestinal absorption of various drugs, P-gp inhibitors have been studied as oral absorption enhancers of P-gp substrate drugs. Here, we investigated the in vitro and in vivo effects of a novel coumarin derivative (LL-348) for its P-gp inhibitory activity. With LL-348, accumulation of daunomycin (DNM) increased 270% and efflux of DNM decreased 63% compared to that of DNM alone. Paclitaxel (PTX, 25mg/kg) after oral administration with LL-348 (5mg/kg), the optimal dose of LL-348 as an oral absorption enhancer of PTX, improved the relative bioavailability (RB) of PTX to 961%. In a xenograft animal model, PTX (40mg/kg) treated with LL-348 (10mg/kg) significantly increased the efficacy of PTX. The results collectively demonstrate that LL-348 can provide a therapeutic benefit in the oral absorption of P-gp substrate anticancer drugs.

Recent Advances in Protein Tyrosine Phosphatase Detection Using Chemical Probes

Protein tyrosine phosphatases (PTPs) are key regulatory enzymes in signal transduction pathways and their aberrancy has been implicated in various diseases such as cancers, metabolic syndrome, and autoimmune disorders. In spite of its great importance, determination of the functional significance of PTPs remains a major challenge, and efficient methodologies are needed to specifically delineate PTP functions. Besides the strategy to use potent and selective PTP inhibitors to study the physiological roles of the enzymes, measurement of PTP activities using specific PTP substrates or activity-based probes (ABPs) has been reported. This review focused on the recent development of small molecular probes to detect PTP activities, consisting of five sub-categories: 1. Conventional fluorescent substrates; 2. Ratiometric fluorescent PTP substrates; 3. Fluorescence substrates with selectivity to a single PTP or a class of PTPs; 4. ABPs specific for PTPs; and 5. A real-time imaging of PTP-substrate complex using a small molecule PTP probe which, offers a measurement of a real-time activity of a certain PTP in cells.