Synthesis and Structure–Activity Relationship Study of Chemical Probes as Hypoxia Induced Factor-1α/Malate Dehydrogenase 2 Inhibitors

Abstract

A structure-activity relationship study of hypoxia inducible factor-1α inhibitor 3-aminobenzoic acid-based chemical probes, which were previously identified to bind to mitochondrial malate dehydrogenase 2, was performed to provide a better understanding of the pharmacological effects of LW6 and its relation to hypoxia inducible factor-1α (HIF-1α) and malate dehydrogenase 2 (MDH2). A variety of multifunctional probes including the benzophenone or the trifluoromethyl diazirine for photoaffinity labeling and click reaction were prepared and evaluated for their biological activity using a cell-based HRE-luciferase assay as well as a MDH2 assay in human colorectal cancer HCT116 cells. Among them, the diazirine probe 4a showed strong inhibitory activity against both HIF-1α and MDH2. Significantly, the inhibitory effect of the probes on HIF-1α activity was consistent with that of the MDH2 enzyme assay, which was further confirmed by the effect on in vitro binding activity to recombinant human MDH2, oxygen consumption, ATP production, and AMP activated protein kinase (AMPK) activation. Competitive binding modes of LW6 and probe 4a to MDH2 were also demonstrated.

A Novel Malate Dehydrogenase 2 Inhibitor Suppresses Hypoxia-Inducible Factor-1 by Regulating Mitochondrial Respiration

“…MDH2 enzyme activity was determined by oxaloacetate-dependent NADH oxidation assays as previously described [ 15 ]. The reaction was performed in 100 mM potassium phosphate buffer (pH 7.4) with 0.25 nM rhMDH2, 200 μM oxaloacetic acid, and 200 μM NADH.…”

“…Cells were incubated for 12 h in the presence or absence of drugs under normoxic or hypoxic condition. After removing the supernatant, luciferase activity was measured using a Dual-Luciferase Assay System (Promega, Madison, WI, USA), according to the manufacturer’s instructions, with a Victor™ X Light luminescence reader [ 15 ].…”

“…Previously, we developed HIF-1α inhibitor LW6 from an aryloxyacetylamino benzoic acid scaffold [ 11 – 13 ] and showed that LW6 inhibits accumulation of HIF-1α and expression of its target genes in colorectal cancer cells. Using multifunctional chemical probes of LW6, we determined that it directly targets malate dehydrogenase 2 (MDH2) [ 14 , 15 ], which is involved in the regulation of HIF-1α accumulation under hypoxia.…”

Hypoxia‐inducible factor‐1 (HIF‐1) inhibitors from the last decade (2007 to 2016): A “structure–activity relationship” perspective

“…Calcineurin B homologous protein 1 (CHP1) was another molecular target protein identified as a target of LW6 in other studies. [23][24][25][26] In another study, we found that heat shock protein (HSP) 70 was a target protein of the phase I clinical candidate adamantyl-based derivative IDF-11774. 27 LW6-based chemical probes were selected from a SAR study of various chemical probes synthesized in our lab.…”

The novel hypoxia-inducible factor-1α inhibitor IDF-11774 regulates cancer metabolism, thereby suppressing tumor growth

“…36 For bioluminescence imaging assays, IDF-11774 (50 mg/kg) was administered orally to Balb/c nude mice bearing tumors (100 mm 3 ) formed from HCT116 cells (1 × 10 7 ) expressing HRE-luciferase by subcutaneous injection. After intraperitoneal injection of d -luciferin, bioluminescence images of each tumor were analyzed using the IVIS Lumina II Luminescence Imaging System (Caliper Life Science, Alameda, CA, USA) and Living Image Software (Caliper Life Science).…”