Halogen and non-halogen leaving groups enhance the potency of β-lactone proteasome inhibitors: further investigation into the role of the halogen of NPI-0052 (salinosporamide A)

3256 NPI-0052 (Salinosporamide A) is a potent 20S proteasome inhibitor in Phase I clinical trials for the treatment of cancer. Two classes of analogs with unique profiles have emerged: NPI-0052 and analogs bearing a halogen leaving group (LG), and less potent, non-leaving group (NLG) analogs. We previously demonstrated that halogen LG analogs are ~1 log unit more potent inhibitors of isolated 20S proteasomes than their NLG congeners, and ~3 log units more potent in whole cell assays. In this study, we investigated mechanisms whereby the LG may provide enhanced potency to proteasome inhibitors of this class. First, we synthesized non-halogen LG analogs to supplement halogen LG and NLG analogs, as well as 3 H-NPI-0052 and deschloro NLG analog 3 H-NPI-0047. Analogs were evaluated for inhibition of chymotrypsin-like (CT-L), trypsin-like (T-L) and caspase-like (C-L) activities of isolated rabbit 20S proteasomes. All LG analogs (halogen and non-halogen) showed a similar range of potencies, being up to 10-fold more potent as inhibitors of CT-L activity than NLG analogs. Analogs were then evaluated for cytotoxicity and inhibition of CT-L activity in human multiple myeloma cell line RPMI-8226. LG analogs were ~2-3 log units more potent (cytotoxic) than NLG analogs. Moreover, treatment with LG analogs (10-50 nM) resulted in > 90% inhibition of CT-L activity, whereas concentrations of ≥ 10µM were required for the NLG analogs to achieve a similar degree of inhibition. The results suggest that it is the LG property , as opposed to identity , that enhances potency. Next, we evaluated reversibility of proteasome inhibition by measuring CT-L activity before and after attempted removal of the ligand by dialysis for up to 24 h. LG analogs inhibited CT-L activity with no recovery after 24 h. In the case of NLG analogs, CT-L activity partially recovered after 5 h and was completely restored after 24 h. Uptake studies in PC-3 cells with 3 H-NPI-0052 and 3 H-NPI-0047 revealed that both compounds rapidly enter cells by a combination of diffusion and carrier-mediated transport. The carrier-mediated transport appears to be a saturable process that is linear over ~ 30 sec. The collective contributions of proteasome inhibition, reversibility of proteasome inhibition, and cell uptake to the potency of this unique class of proteasome inhibitor will be presented.