Fig 1.
Cytotoxicity of Up284 against cell lines of diverse cancer origin.
A. Chemical structures of key compounds. B. The cytotoxicity of Up284 for a broad panel of cancer cell lines. Briefly each cell line (~2500–10,000 cells/well depending on the cell type) was treated with titrations of Up284 in triplicate and incubated for 72 h, and the cell viability was then measured using MTT reagent. The data were plotted and IC50 analyzed by curve fitting in Graphpad Prism.
Table 1.
Structural features of substituted cyclic piperidones and their cytotoxicity.
Structures and synthesis of the compounds is described in S1 File. Toxicity to HeLa or SKOV3 cells by IC50 by MTT assay is shown in μM. * as amide.
Fig 2.
Biotin labeling of Up284 and its binding to RPN13 in cell lysates.
A) E. coli Rosetta 2(DE3) bacterial cell lysate expressing RPN13 (tagged with 6His at both termini, expected MW 46.8KDa) was treated with biotinylated RA190 (RA190B), Up284 (Up284B) or inactive compound Up108 (Up108B) at 50 μM for the period of 45 min at 4°C and subjected to SDS-PAGE and transfer to PVDF membrane. The membrane was probed with HRP-Streptavidin (upper panel) and anti-RPN13 (lower panel). B) Same as in A, but examining dose dependency of labeling of RPN13 in 2DE3 bacterial cell lysate expressing 6His-RPN13. C) A2780 cell lysate was pretreated with streptavidin beads for 45 min at 0°C to remove non-specific biotinylated proteins. Next, the lysates were treated with Up284B or RA190B at 0.5 μM to 10 μM for 45 min at 0°C and subjected to SDS-PAGE, PVDF membrane transfer analysis and probed with HRP-streptavidin. D) As in C, but lysate of human ovarian cancer cell line ES2 was treated with Up284B (1–25 μM) for 45 min at 0°C. E) As in C, but lysate of HeLa cells was pre-cleared with streptavidin beads. Aliquots of pre-cleared lysate were pre-treated with the indicated compounds (20 μM) for 45 min at 0°C and then each incubated with RA190B (40 μM) for 45 min at 0°C. To detect labeling, the lysates (40 μg) were subject to SDS-PAGE, PVDF membrane transfer, and then probed with HRP-streptavidin. The membrane was stained with Ponceau solution to confirm equal loading. F) SKOV3 cells were treated with the indicated compounds (5 μM) for 1 h, and then total cellular DUB activity determined by measuring Ub-AMC cleavage over time. NEM (1 mM) used as positive control.
Fig 3.
Up284 thermostabilizes RPN13 and promotes antigen presentation.
A and B) SKOV3 cells were treated with DMSO or 10 μM Up284 for 30 min then washed with PBS, trypsinized, pelleted, and resuspended in PBS supplemented with Halt protease inhibitor cocktail (Thermo Scientific). Resuspended cells were aliquoted into PCR strips. Cells were incubated in a thermal cycler (Bio-Rad) over the indicated gradients of temperature for 4 min, followed by incubation at 25°C for 3 min. Cells were snap-frozen in liquid nitrogen and subjected to three freeze–thaw cycles. Samples were briefly vortexed and centrifuged at 16,000 g for 20 min at 4°C. Cleared cell lysates were mixed with a 1/3 volume of 6×Laemmli sample buffer. After boiling, cell lysates were resolved in SDS–PAGE followed by immunoblotting for RPN13, GAPDH, RPN2 and USP14. C) TC1-OVA cells were plated (125,000 cells/well) in a 6 well plate in 2 mL/well in complete medium. After incubation for 24 h at 37°C, cells were treated with Up284, RA190 or bortezomib (0.25 μM) or vehicle for 12 h. The cells were then washed with PBS, fixed and stained for 1 h at RT with APC-labeled monoclonal anti-mouse H-2Kb bound to SIINFEKL antibody (25-D1.16, Biolegend), and binding was analyzed by flow cytometry.
Fig 4.
Downstream effects of Up284 treatment.
A) ES2 cells were treated with each compound for 12 h at indicated doses and then lysates subjected to Western blot analysis and using anti-ubiquitin antibody, or anti-actin as the loading control. B) As in A, but ES2 cells were treated for 4 h with 1 μM of the indicated compounds. Lysates were analyzed by Western blot using anti-ubiquitin antibody, or anti-tubulin as the loading control. C) As in B, but HeLa cells were treated for 1 h with 1 μM or 5 μM Up284. D) ES2 cells were treated with DMSO, or 1 μM Up284 or Bortezomib for 4 h. Total RNA was isolated and analyzed for CHOP10 expression by qRT-PCR. E) ES2 cells were treated with Up284 or Up285 at the indicated doses for 12 h and cellular levels of ROS were measured using Amplex Red/HRP fluorescence (571/585 nm) measurement. F) Ovarian cancer cell lines TOV21G or ES2 treated with compounds (Up284, Bortezomib, cisplatin or combination) at indicated doses for 12 h. The cells were stained with PE-Annexin and 7-AAD for 20 min in the dark and analyzed by flow cytometry. G-H) Impact on mitochondrial was assessed using the Seahorse XF instrument in ES2 cells upon treatment with Up284 (0.5 μM) or DMSO for 12 h. G) Oxygen consumption rate (OCR) was measured following sequential additions of Oligomycin, FCCP, Rotenone and Antimycin A in to the culture media. H) OCR and extracellular acidification rate (ECAR) were measured by Seahorse XF analyzer after treatment with Up284 (0.5 μM) or DMSO. Data are representative of 3 independent experiments, and 9 biologic replicates per conditions used.
Fig 5.
Pharmacodynamics and Pharmacokinetics of Up284 in mice.
A) ES2 cells stably transduced with the 4UbFL reporter plasmid were treated with compounds at indicated doses for 4 h. The cells were then washed, lysed and their luminescence measured by a luminometer after addition of luciferin. B) Female CD1 mice electroporated with 4UbFL plasmid in their leg muscle. After 48 h, the mice were injected with luciferin, imaged by IVIS for their basal bioluminescence and randomized into two groups of five. The groups of mice were treated IP with one dose of either vehicle or Up284 (40mg/kg) and imaged for bioluminescence at 4 h, 24 h and 48 h by IVIS. C) As in B, CD1 mice (n = 5) were treated PO with one dose of vehicle, Up284 (40mg/kg), or Ixazomib (10mg/kg). D) Up284 tissue distribution. Female CD1 mice (n = 3) were treated with one IV dose of Up284 (40mg/kg) and euthanized after 24 h with collection of plasma and tissues including brain, colon, heart, kidney, liver, lung and spleen. Up284 levels in tissue samples were analyzed by HPLC. E) Up284 pharmacokinetics. Male CD1 mice (n = 4) were treated with Up284 IV (5mg/kg), IP (20mg/kg) or PO (50mg/kg). Blood was collected at indicated time points thereafter and analyzed for Up284 levels by HPLC.
Fig 6.
Impact of Up284 treatment upon tumor growth in xenograft, spontaneous and syngeneic mouse models of ovarian cancer.
A-C) Female nude mice were inoculated IP with ES2-luc cells, a human ovarian cancer line expressing luciferase. Tumor growth was followed upon IP treatment with Up284 (n = 7, 10 mg/kg every 3 days for 2 weeks) or vehicle (25% β-hydroxypropyl cyclodextrin in water, n = 6) by IVIS imaging of bioluminescence (A-B), and survival was monitored (C); D-F) Female C57BL6 mice bearing spontaneous genetically engineered mouse model show tumor control with Up284 (n = 7, 20 mg/kg every 3 days delivered IP) as compared to vehicle treatment (n = 7) (D-E), and extended survival (F); G-H) Female C57BL6 mice bearing intraperitoneal syngeneic ID8-luc ovarian cancer model show tumor control with Up284 (n = 7, 20 mg/kg every 3 days, IP) as compared to vehicle treatment (n = 7). I) In vivo stabilization of 4UbFL reporter by Up284 (20 mg/kg, 1 dose delivered IP) or Bortezomib (0.75 mg/kg, 1 dose delivered IP) was followed by IVIS imaging in nude mice with established IP tumor derived from ES2 cells stably expressing 4UbFL (ES2-4UbFL) and concurrently in the leg muscle of the same animal electroporated with the 4UbFL reporter plasmid (n = 5). The IP tumor tissue and electroporated normal muscle tissue, being spatially distinct, were identified with separate regions of interest (ROI) after simultaneous imaging with IVIS.