Brostallicin (PNU-166196) is a synthetic alpha-bromoacrylic, second-generation
DNA minor groove binder structurally related to
distamycin A, presently in Phase II trials in Europe and the United States. The compound shows broad antitumor activity in preclinical models and dramatically reduced in vitro myelotoxicity in human hematopoietic progenitor cells compared with that of other minor groove binders.
Brostallicin showed a 3-fold higher activity in
melphalan-resistant L1210 murine
leukemia cells than in the parental line (IC(50) = 0.46 and 1.45 ng/ml, respectively) under conditions in which the cytotoxicity of conventional
antitumor agents was either unaffected or reduced. This
melphalan-resistant cell line has increased levels of
glutathione (GSH) in comparison with the parental cells. Conversely, GSH depletion by
buthionine sulfoximine in a human ovarian
carcinoma cell line (A2780) significantly decreased both the cytotoxic and the proapoptotic effects of
brostallicin. In one experiment, human
glutathione S-transferase pi (GST-pi)
cDNA was transfected into A2780 cells, and four clones of A2780 with different expression levels of GST-pi were generated (i.e., two clones with high and two clones with low GST-pi expression). A 2-3-fold increase in GST-pi levels resulted in a 2-3-fold increase in cytotoxic activity of
brostallicin. Similar results were obtained for GST-pi-transfected human
breast carcinoma cells (MCF-7).
Brostallicin showed 5.8-fold increased cytotoxicity in GST-pi-transfected versus empty vector-transfected cells with low GST-pi expression. In an in vivo experiment, A2780 clones were implanted into nude mice. The antitumor activity of
brostallicin was higher in the GST-pi-overexpressing
tumors without increased toxicity. Regarding the mechanism of action,
brostallicin interacts reversibly with the
DNA minor groove TA-rich sequences but appears unreactive in classical in vitro
DNA alkylation assays. We speculated that an intracellular reactive nucleophilic species, e.g., GSH, could react with the alpha-bromoacrylamide moiety functions. Experiments on the interaction with plasmid
DNA showed a change of the
DNA topology from supercoiled to circular form (nicking) in the presence of GSH, whereas no change was found in its absence. In vitro incubations of
brostallicin were performed with the human recombinant GST
isoenzymes A1-1, M1-1, and P1-1 (alpha, mu and pi
isoenzymes, respectively) in the presence of GSH. The decrease in
brostallicin levels was monitored in these incubations; the rate of loss (and therefore
brostallicin metabolism) was significantly higher for the M1-1 and P1-1
isoenzymes than for the A1-1
isoenzyme.