Aldehyde dehydrogenase 3A1 (ALDH3A1) plays an important role in many cellular oxidative processes, including
cancer chemoresistance, by metabolizing activated forms of oxazaphosphorine drugs such as
cyclophosphamide (CP) and its analogues, such as
mafosfamide (MF),
ifosfamide (IFM), and
4-hydroperoxycyclophosphamide (4-HPCP). Compounds that can selectively target ALDH3A1 could permit delineation of its roles in these processes and could restore chemosensitivity in
cancer cells that express this
isoenzyme. Here we report the detailed kinetic and structural characterization of an ALDH3A1-selective inhibitor, CB29, previously identified in a high-throughput screen. Kinetic and crystallographic studies demonstrate that CB29 binds within the
aldehyde substrate-binding site of ALDH3A1. Cellular proliferation of ALDH3A1-expressing
lung adenocarcinoma (A549) and
glioblastoma (SF767) cell lines, as well as ALDH3A1 non-expressing lung fibroblast (CCD-13Lu) cells, is unaffected by treatment with CB29 and its analogues alone. However, sensitivity toward the anti-proliferative effects of
mafosfamide is enhanced by treatment with CB29 and its analogue in the
tumor cells. In contrast, the sensitivity of CCD-13Lu cells toward
mafosfamide was unaffected by the addition of these same compounds. CB29 is chemically distinct from the previously reported small-molecule inhibitors of ALDH
isoenzymes and does not inhibit ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, or ALDH2
isoenzymes at concentrations up to 250 μM. Thus, CB29 is a novel small molecule inhibitor of ALDH3A1, which might be useful as a chemical tool to delineate the role of ALDH3A1 in numerous metabolic pathways, including sensitizing ALDH3A1-positive
cancer cells to oxazaphosphorines.