Development of the Proteasome Inhibitor PS-341JULIAN ADAMSMillennium Pharmaceuticals, Inc., Cambridge, Massachusetts, USAKey Words. Antineoplastic agents · Boronic acids · Enzyme inhibitors (administration and dose) · NF-κB (antagonists and inhibitors) · Hematologic neoplasms (drug therapy) · Multienzyme complexes (antagonists and inhibitors) · 26S Proteasome · Multiple myelomaABSTRACTOver the last decade, the critical role of the protea-some in cell-cycle regulation has become increasinglyapparent. The proteasome, a multicatalytic protease pre-sent in all eukaryotic cells, is the primary component ofthe protein degradation pathway of the cell. By degrad-ing regulatory proteins (or their inhibitors), the protea-some serves as a central conduit for many cellularregulatory signals and, thus, is a novel target for thera-peutic drugs. PS-341 is a small molecule that is a potentand selective inhibitor of the proteasome. In vitro andmouse xenograft studies of PS-341 have shown anti-tumor activity in a variety of tumor types, includingmyeloma, chronic lymphocytic leukemia, prostate cancer,pancreatic cancer, and colon cancer, among others.Although PS-341 rapidly leaves the vascular compart-ment, a novel pharmacodynamic assay has shown thatinhibition of proteasome—the biologic target—is dosedependent and reversible. These studies provided therationale for a twice-weekly dosing schedule employed inongoing clinical studies. Phase I trials in a variety oftumor types have shown PS-341 to be well tolerated, andphase II trials in several hematologic malignancies andsolid tumor types are now in progress. Efficacy andsafety data from the most advanced of these, a phase IImulticenter trial in myeloma, will be available in early2002. The Oncologist 2002;7:9-16The Oncologist 2002;7:9-16www.TheOncologist.comCorrespondence: Julian Adams, Ph.D., Millennium Pharmaceuticals, Inc., 75 Sidney Street, Cambridge, Massachusetts02139, USA. Telephone: 617-551-3674; Fax: 617-679-7370; e-mail: [email protected] Received October 5, 2001; acceptedfor publication November 21, 2001. ©AlphaMed Press 1083-7159/2002/$5.00/0INTRODUCTIONIn addition to the recycling of damaged or obsolete pro-teins, protein degradation is a mechanism for controlling theavailability of regulatory proteins in the cell. The 26S pro-teasome is a primary component of the protein degradationpathway of the cell (over 80% of all cellular proteins areprocessed by this enzyme), and the proteasome’s rapid andirreversible elimination of targeted proteins is key to theactivation or repression of many cellular processes, includ-ing cell-cycle progression and apoptosis. This fundamentalrole for the proteasome singles it out as a unique target foranticancer therapy, and PS-341 is the first proteasomeinhibitor to enter human trials. In preclinical studies, thispotent and specific inhibitor has shown significant antitumoractivity as a single agent and in combination with othercytotoxic drugs [1-6]. PS-341 treatment has also proven tobe well tolerated in phase I trials at doses that result in sig-nificant proteasome inhibition and show preliminary evi-dence of biologic activity, justifying a large-scale phase IIprogram in a variety of tumor types, both solid and hemato-logic [7, 8]. This review provides a summary of the clinicalexperience with PS-341, as well as the preclinical rationalethat supports the potential of PS-341 as a novel treatment forhematologic and solid tumor malignancies.THE PROTEASOME AND PROTEASOME INHIBITIONThe proteasome is a large, multiprotein particle—pre-sent in both the cytoplasm and the nucleus of all eukaryoticcells—composed of two functional components: a 20Score catalytic complex and a 19S regulatory subunit.Proteins that are to be degraded are marked with ubiquitinchains, which bind to a receptor on the 19S complex (Fig.1). Once recognized by the regulatory complex, the ubiq-uitin chain is removed and the protein denatured in prepa-ration for degradation. The protease activity resides in achannel at the center of the 20S complex, which is formedfrom four stacked, multiprotein rings. The outer α subunitrings form a narrow channel that allows only denaturedproteins to enter the catalytic chamber formed by the cen-tral β subunit rings [9-11]. Inside the catalytic chamber,proteins are surrounded by six protease-active sites (threeon each β subunit ring). The proteasome protease functionssimilarly to serine proteases but is unique since it relies ona threonine residue in the active site. Proteins processed bythe proteasome are reduced to small polypeptides 3 to 22residues in length [12].Proteolysis by the 26S proteasome is a fundamentalmetabolic process, and complete blockade of the protea-some activity with an inhibitor results in death for cells andorganisms. Many laboratories have demonstrated theeffects of proteasome inhibition on the stability of variouscell-cycle regulatory proteins, especially those that areshort lived [1, 5, 13-15]. Cyclins, cyclin-dependent kinaseinhibitors, and tumor suppressors (e.g., cyclin B1,p21Waf1/Cip1, p27, p53) are all substrates for the ubiquitin-proteasome pathway (Table 1), and inhibiting their requisitedegradation has been clearly implicated in sensitizing cellsto apoptosis [16-18]. Camptothecin-bound topoisomerase Icleavable complexes are also substrates for the proteasome[19]; inhibition of the proteasome thus stabilizes these 10Development of the Proteasome Inhibitor PS-341LysLysProtein substrateNH2HNUbOnIsopeptidasesATPDegradedproteins26SProteasomecomplexUbOUb OHATPAMPPPiE1–SHE1–S UbOOE2–SHE2–SUbE3Figure 1. Ubiquitin-proteasome protein degradation. Prior to degradation, doomed proteins are first marked on specific lysine residueswith a polyubiquitin chain. Marking the protein is a three-step process in which ubiquitin is activated by a ubiquitin-activating enzyme (E1),then transferred to a ubiquitin-conjugating enzyme (E2). E3s (the ubiquitin-protein ligases) recognize degradation motifs on specific sub-strates, and catalyze the transfer of ubiquitin from the E2to the target. These polyubiquitinated substrates are then recognized and degradedby the 26S proteasome.Table 1. Cell cycle–related proteins degraded by the