Development and anticancer properties of Up284, a spirocyclic candidate ADRM1/RPN13 inhibitor

Abstract

Bortezomib has been successful for treatment of multiple myeloma, but not against solid tumors, and toxicities of neuropathy, thrombocytopenia and the emergence of resistance have triggered efforts to find alternative proteasome inhibitors. Bis-benzylidine piperidones such as RA190 covalently bind ADRM1/RPN13, a ubiquitin receptor that supports recognition of polyubiquitinated substrates of the proteasome and their subsequent deububiqutination and degradation. While these candidate RPN13 inhibitors (iRPN13) show promising anticancer activity in mouse models of cancer, they have suboptimal drug-like properties. Here we describe Up284, a novel candidate iRPN13 possessing a central spiro-carbon ring in place of RA190’s problematic piperidone core. Cell lines derived from diverse cancer types (ovarian, triple negative breast, colon, cervical and prostate cancers, multiple myeloma and glioblastoma) were sensitive to Up284, including several lines resistant to bortezomib or cisplatin. Up284 and cisplatin showed synergistic cytotoxicity in vitro. Up284-induced cytotoxicity was associated with mitochondrial dysfunction, elevated levels of reactive oxygen species, accumulation of very high molecular weight polyubiquitinated protein aggregates, an unfolded protein response and the early onset of apoptosis. Up284 and RA190, but not bortezomib, enhanced antigen presentation in vitro. Up284 cleared from plasma in a few hours and accumulated in major organs by 24 h. A single dose of Up284, when administered to mice intra peritoneally or orally, inhibited proteasome function in both muscle and tumor for >48 h. Up284 was well tolerated by mice in repeat dose studies. Up284 demonstrated therapeutic activity in xenograft, syngeneic and genetically-engineered murine models of ovarian cancer.

Preclinical studies of RA475, a guanidine-substituted spirocyclic candidate RPN13/ADRM1 inhibitor for treatment of ovarian cancer

“…Likewise, RA475 induced apoptosis in the triple negative breast cancer line MDA-MB-468 ( S6 Fig ). This is consistent with Up284 and other prior iRPN13 [ 37 ].…”

“…Biotinylated Up284 (Up284B) covalently binds to RPN13 in direct labeling assays in vitro, and Up284 also competes with biotinylated prototype iRPN13, RA190B, for binding to RPN13 [ 37 ]. Since RA475 cannot be biotinylated on the central ring nitrogen like RA190 or Up284, we resorted to a competition assay.…”

“…No obvious adverse effects were observed during this PK study. The average RA475 plasma concentrations data for IV, IP, and PO dosed groups ( S10 – S12 Tables) is graphically presented in Fig 4 , and may be compared to published data for Up284 [ 37 ]. Pharmacokinetic parameters were calculated from mean concentration-time data using non-compartmental methods in Phoenix WinNonlin version 8.3 (Certara, Princeton, NJ) ( S13 Table ).…”

“…The dose normalized AUC 8h for RA475 was significantly lower than the AUC 8h for Up284 via the IP route of administration. In summary, the calculated oral and IP bioavailability for RA475 was 1.1% and 103% respectively ( S13 Table ), which contrasts values of 23% and 87% respectively for Up284 [ 37 ], suggesting the guanine moiety produces a profound reduction in the oral bioavailability of RA475. This is consistent with the minimal stabilization of UbFL after oral delivery of 40 mg/Kg RA475 ( Fig 3F ), and compared to the robust (>20-fold) stabilization observed upon 40 mg/Kg delivered IP ( Fig 3E ), or in vitro with 1.25 μM of either RA475 or Up284 ( Fig 3D ).…”

“…One recent approach was to replace the central piperidone ring with a more constrained 3,5 spirocyclic core [ 36 ]. Addition of nitrile electron withdrawing groups on both side rings produced the most effective 3,5 spirocyclic candidate, Up284 [ 37 ]. Here we sought to improve key drug-like properties of this molecule by adding a guanidine moiety to its central ring nitrogen, and examine its impact on the pharmaco-kinetic/dynamic profile of this new compound, RA475, and its therapeutic activity in murine models of ovarian cancer ( Fig 1 ).…”