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NPI-0052 is a potent 20S proteasome inhibitor in Phase I clinical trials for the treatment of cancer. Two classes of analogs with unique in vitro profiles have emerged: NPI-0052 and analogs with halogen leaving groups, and less potent, non-leaving group (NLG) analogs. The X-ray crystal structure of the NPI-0052-proteasome complex indicates that the chlorine atom is eliminated upon binding. To further elucidate the role of chlorine, biochemical, in vitro and in vivo studies were conducted on NPI-0052 and NLG analogs. We first evaluated the reversibility of proteasome inhibition by measuring proteolytic activity before and after attempted removal of the ligand. NPI-0052 completely inhibited all three proteolytic sites with no recovery of enzyme activity after 12h dialysis, indicating a very slow enzyme off-rate. We then measured dissociation constants (Ki) and rates of inactivation (kinact) for the three proteolytic subunits. Product formation in the presence of varying concentrations of NPI-0052 exhibited non-linearity for all three sites, indicative of slow, tight inhibition kinetics and consistent with a two-step binding mechanism. In the first, reversible step, the inhibitor is recognized by the active site with Ki values of 23 nM, 97 nM and >1 µM for the CT-L, trypsin-like (T-L) and caspase-like (C-L) sites, respectively. Once bound, the inhibitor reacts at the active site to completely inactivate the enzyme, as reflected by kinact=0.01s-1, 0.003s-1 and >0.02s-1 for the CT-L, T-L and C-L sites. Next, inhibition of CT-L activity was measured in human multiple myeloma (MM) RPMI 8226 cells after 1h treatment with NPI-0052 or NLG analogs NPI-0047 and NPI-2063. NPI-0052 inhibited CT-L activity by 94% at 10 nM, while 1000-fold higher concentrations of NPI-0047 and NPI-2063 resulted in only 65% and 87% inhibition, respectively. To compare the effect of time of drug exposure, MM.1S cells were treated with NPI-0047 (IC50 at 24h: 1 µM) for up to 6h or NPI-0052 (IC50 at 24h: 7 nM) for 1h, followed by drug washout. Treatment with NPI-0052 resulted in ~35% cell death and ~75% inhibition of CT-L activity versus ~7% and ~1%, respectively, for NPI-0047. Longer NPI-0047 treatment times (6h) were more effective (~30% cell death and 15% inhibition of CT-L activity). Finally, in vivo studies in mice indicated that NPI-0052 is a more potent inhibitor of whole blood CT-L activity than NPI-0047. The collective biochemical, in vitro and in vivo biological data indicate that the chlorine atom of NPI-0052 imparts a distinct biological profile compared to NLG analogs.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA