Biochemical and Cellular Characterization of the Novel Proteasome Inhibitor PR-171.

Recent clinical studies have identified the proteasome as an important therapeutic target for hematologic malignances. The proteasome inhibitor, bortezomib, has been approved for the treatment of relapsed or refractory multiple myeloma and ongoing clinical trials suggests a potential benefit for the treatment of Non-Hodgkin’s lymphoma. PR-171 is a novel epoxomicin derivative that is a potent and irreversible inhibitor of the human proteasome. It inhibits the chymotrypsin-like activity of purified human 20S proteasome with a k_inact/K_i of 34,000 M⁻¹s⁻¹ and is >300-fold selective over the other proteasome catalytic activities. In addition, PR-171 has been found to exhibit minimal activity in a broad diversity panel of biochemical assays that includes 67 receptor/ligand and 37 enzyme assays. PR-171 retains its potency for inhibition of the proteasome chymotrypsin-like activity in mammalian cells displaying IC₅₀ values <10 nM in multiple tumor cell lines. Although PR-171 is a covalent irreversible inhibitor, proteasome activity in cells recovers with a t_1/2 of approximately 24 hr after removal of the compound. This recovery is likely due to induction of de novo synthesis since other proteasome inhibitors have been shown to promote transcription of multiple proteasome subunit genes. The cellular consequences of proteasome inhibition by PR-171 include accumulation of polyubiquitinated proteins, cell cycle arrest, and induction of apoptosis. Potent cytotoxic activity of PR-171 is observed across a broad panel of human tumor cell lines (IC₅₀ range: 2–40 nM). Pulsatile exposure studies designed to mimic the anticipated drug exposure in vivo have demonstrated that cytotoxicity is dependent upon the magnitude and duration of proteasome inhibition. These studies have also shown that hematological tumor lines are most sensitive to brief PR-171 exposure, with solid tumor lines exhibiting intermediate sensitivity and non-transformed cells being the least sensitive to such treatment. Finally, PR-171 has been found to retain its cytotoxic potential on cells made resistant to bortezomib in vitro. These studies motivate the clinical investigation of PR-171 in hematological malignances.