Antitumor Activity of PR-171, a Novel Irreversible Inhibitor of the Proteasome Susan D. Demo, Christopher J. Kirk, Monette A. Aujay, Tonia J. Buchholz, Maya Dajee, Mark N. Ho, Jing Jiang, Guy J. Laidig, Evan R. Lewis, Francesco Parlati, Kevin D. Shenk, Mark S. Smyth, Congcong M. Sun, Marcy K. Vallone, Tina M. Woo, Christopher J. Molineaux, and Mark K. Bennett Proteolix, Inc., South San Francisco, California Abstract Clinical studies with bortezomib have validated the protea- some as a therapeutic target for the treatment of multiple myeloma and non-Hodgkin’s lymphoma. However, significant toxicities have restricted the intensity of bortezomib dosing. Here we describe the antitumor activity of PR-171, a novel epoxyketone-based irreversible proteasome inhibitor that is currently in clinical development. In comparison to bortezo- mib, PR-171 exhibits equal potency but greater selectivity for the chymotrypsin-like activity of the proteasome. In cell culture, PR-171 is more cytotoxic than bortezomib following brief treatments that mimic the in vivo pharmacokinetics of both molecules. Hematologic tumor cells exhibit the greatest sensitivity to brief exposure, whereas solid tumor cells and nontransformed cell types are less sensitive to such treat- ments. Cellular consequences of PR-171 treatment include the accumulation of proteasome substrates and induction of cell cycle arrest and/or apoptosis. Administration of PR-171 to animals results in the dose-dependent inhibition of the chymotrypsin-like proteasome activity in all tissues examined with the exception of the brain. PR-171 is well tolerated when administered for either 2 or 5 consecutive days at doses resulting in >80% proteasome inhibition in blood and most tissues. In human tumor xenograft models, PR-171 mediates an antitumor response that is both dose and schedule dependent. The antitumor efficacy of PR-171 delivered on 2 consecutive days is stronger than that of bortezomib administered on its clinical dosing schedule. These studies show the tolerability, efficacy, and dosing flexibility of PR-171 and provide validation for the clinical testing of PR-171 in the treatment of hematologic malignancies using dose-intensive schedules. [Cancer Res 2007;67(13):6383–91] Introduction The proteasome is a multicatalytic protease complex that is responsible for the ubiquitin-dependent turnover of cellular proteins (1–3). Proteasome substrates include misfolded or misassembled proteins as well as short-lived components of signaling cascades that regulate cell proliferation and survival pathways. Inhibition of the proteasome results in the accumulation of these substrate proteins and leads to cell death (4). The catalytic core of the proteasome includes three proteolytic activities that are commonly described by their substrate selectivities (5): chymo- trypsin-like, trypsin-like, and caspase-like. Each proteasome active site uses the side chain hydroxyl group of an NH 2 -terminal threonine as the catalytic nucleophile, a mechanism that distinguishes the proteasome from other cellular proteases (3). Clinical validation of the proteasome as a therapeutic target in oncology has been provided by the dipeptide boronic acid bortezomib (also known as PS-341 or Velcade; refs. 4, 6). Bortezomib is a covalent, slowly reversible inhibitor that primarily targets the chymotrypsin-like activity of the proteasome (7). Bortezomib has proven efficacious as a single agent in multiple myeloma (8) and some forms of non-Hodgkin’s lymphoma (NHL; refs. 9, 10). The cellular mechanism(s) responsible for the clinical efficacy of bortezomib remain unclear, but may include disruption of cell adhesion– and cytokine-dependent survival pathways, in part through suppression of NF-nB activity (11, 12), inhibition of angiogenesis (13), and/or activation of a misfolded protein stress response (14, 15). Although the clinical success of bortezomib is encouraging, a significant fraction of patients remain refractory to treatment (8–10). Furthermore, a number of toxicities including painful peripheral neuropathy (16) and thrombocytopenia (17) have restricted bortezomib to a biweekly day 1/day 4 dosing schedule that allows full recovery of proteasome activity between doses (18, 19). Therefore, clinical evaluation of additional proteasome inhibitor classes is warranted. Two irreversible proteasome inhibitors are currently under development: (a ) salinosporamide A (NPI-0052), a natural product related to lactacytsin (20–22) and (b) PR-171, a modified peptide related to the natural product epoxomicin. Epoxomicin was identified based on its in vivo antitumor activity (23) and subsequently shown to be a potent and selective inhibitor of the proteasome (24). Epoxomicin and its analogues are comprised of two key elements: a peptide portion that selectively binds in the substrate binding pocket(s) of the proteasome with high affinity and an epoxyketone pharmacophore that stereospe- cifically interacts with the catalytic threonine residue to irreversibly inhibit enzyme activity. X-ray crystallography has shown that epoxomicin forms a dual covalent morpholino adduct with the proteasome that requires the close juxtaposition of both the side chain hydroxyl and a-amino groups of the active site threonine residue (25). This unique mechanism imparts a high degree of specificity to the proteasome relative to the active sites of other protease classes. Medicinal chemistry efforts focused on increasing the potency and chymotrypsin-like selectivity of epoxomicin resulted in the identification of YU-101 (26), a synthetic tetrapeptide epoxyketone analogue. PR-171, a derivative of YU-101 with improved pharma- ceutical properties, is currently under evaluation in phase I clinical Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). S.D. Demo and C.J. Kirk contributed equally to this work. Requests for reprints: Mark K. Bennett, Proteolix, Inc., South San Francisco, CA 94080. E-mail: mkbennett@proteolix.com. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-4086 www.aacrjournals.org 6383 Cancer Res 2007; 67: (13). 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