Didemnin B (DB) is a cyclic depsipeptide with a variety of biologic effects, including potent antiviral, antitumor, and immunosuppresive activities. Although its mechanism of action has been attributed to inhibition of DNA and protein synthesis, the exact cellular site of interaction has not been previously defined. Since DB is strongly antiproliferative in Nb2 node lymphoma cells, we investigated potential DB binding sites in these cells, using [3H]-DB (2.7 mCi/mg) as the radiolabeled ligand. Time course studies with Nb2 cells showed that steady state [3H]-DB binding was attained after 4 h. Scatchard analysis with resting cells yielded a Kd of 180 nM (200 ng/ml), and 7 × 106 binding sites/cell. The IC50 of DB inhibition of ongoing protein and DNA synthesis in Nb2 cells, measured 24 h after prolactin (PRL) stimulation, was also in the range of 100 ng/ml. Didemnin analogs, with alterations at critical amino acid residues, inhibited the synthesis of DNA and protein and competed with [3H]-DB binding with the same rank order of potency. This implies that this binding site may mediate the inhibition of macromolecule synthesis. Subcellular fractionation of [3H]-DB labeled Nb2 cells revealed that specific binding occured predominantly in the 100,000 g cytosolic fraction. Comparison with cyclophilin and the FK506 binding protein, both cytosolic receptors, suggests that the DB binding site may also belong to the family of immunophilins.
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