Development of Novel Epoxyketone-Based Proteasome Inhibitors as a Strategy To Overcome Cancer Resistance to Carfilzomib and Bortezomib

Abstract

Over the past 15 years, proteasome inhibitors (PIs), namely bortezomib, carfilzomib (Cfz) and ixazomib, have significantly improved the overall survival and quality-of-life for multiple myeloma (MM) patients. However, a significant portion of MM patients do not respond to PI therapies. Drug resistance is present either de novo or acquired after prolonged therapy through mechanisms that remain poorly defined. The lack of a clear understanding of clinical PI resistance has hampered the development of next-generation PI drugs to treat MM patients who no longer respond to currently available therapies. Here, we designed and synthesized novel epoxyketone-based PIs by structural modifications at the P1' site. We show that a Cfz analog, 9, harboring a hydroxyl substituent at its P1' position was highly cytotoxic against cancer cell lines displaying de novo or acquired resistance to Cfz. These results suggest that peptide epoxyketones incorporating P1'-targeting moieties may have the potential to bypass resistance mechanisms associated with Cfz and to provide additional clinical options for patients resistant to Cfz.

Figure 1.

(A) Structures of proteasome inhibitors in clinical use. The gray-colored markings denote the functional groups that are proposed to form favorable interactions with the specificity pockets (S1–4) of a proteasome catalytic subunit. (B) Schematic representation of a prototypical proteasome substrate or substrate-like inhibitor bound to a proteasome catalytic subunit showing the unprimed residues (P1, P2, P3, and P4) located N-terminal to the cleavage site of the proteasome catalytic subunit (shown as an arrow) and the primed P1′ residue located C-terminal to the cleavage site.

Figure 2.

Ubiquitin-proteasome system (UPS) and its association with various hallmarks of cancer.

Figure 3.

(A) Structures of UK101 and UK102. (B) Comparison of the cytotoxic potency (72-h IC₅₀ values) of H727 and H23 cells to carfilzomib, bortezomib, MG-132, UK101, UK102, and lactacystin. Data are reported as the mean ± SD.

Figure 4.

(A) Effects of carfilzomib (Cfz) on the viability of an RPMI8266 cell line with acquired Cfz resistance (RPMI8266/CfzR) in comparison to the parental cell line. Cell viability was measured by MTS assay after 72 h drug treatment (left). Immunoblotting analysis showing a marked increase of P-gp expression in RPMI8226 Cfz-resistant cells in comparison to parental cells (right). (B) Comparison of the sensitivity (72-h IC₅₀ values) of RPMI8226 parental and Cfz-resistant cells to Cfz, epoxomicin, UK101, and UK102. Data are reported as the mean ± SD. For epoxomicin and carfilzomib, the SD values were obtained from three independent experiments. For UK101 and UK102, the SD values were from nonlinear regression analysis using three replicates.

Figure 5.

Effects of carfilzomib (Cfz), UK101, and UK102 on cell viability of primary MM samples from 14 different donors, six from Btz/Cfz-naïve patients and eight from patients relapsed on Btz therapy. Primary MM cells were treated with Cfz (A, 50 nM), UK101 (B, 10 μM), or UK102 (C, 10 μM) for 48 h. Cell viability was measured using an ATP-based luminescent assay.

Figure 6.

(A) Predicted docking models of UK101 and carfilzomib (Cfz) bound to β5 or β1i. The location of UK101’s TBDMS group positioned within putative P1′ pockets is highlighted using a purple-colored circle. β5 (PDB ID: 3UNB) and β1i (PDB ID: 3UNF) from mammalian 20S proteasomes were used as templates. In cartoon presentation, β1i (gray, PDB ID: 3UNF) was superposed to β5 (yellow, PDB ID: 3UNB) and only different amino acid residues are shown in stick model. (B) Comparison of UK101 and 5(UK101-OH) in terms of their potency (IC₅₀ values) against proteasome chymotrypsin-like activity (in RPMI8226 cell lysate), β1i/LMP2 catalytic activity (in 20S purified human immunoproteasome), and against H23, H727, and Cfz-resistant RPMI8226 cells as measured by MTS cell viability assay. Data are reported as the mean ± SD. Docking model of UK101-OH bound to β1i (PDB ID: 3UNF). The P1′-OH of UK101-OH (5) is perfectly positioned to form hydrogen bonds with Ser168 and Ser21.

Figure 7.

Potency (IC₅₀ values) for compounds with various substitutions at the P1′site against proteasome chymotrypsin-like activity (RPMI8226 cell lysate), β1i/LMP2 activity (purified human 20S immunoproteasome), and cell viability of H23, H727, and Cfz-resistant RPMI8226 cells. Data reported as the mean ± SD (carfilzomib, n = 3 independent experiments) or from a single experiment (3 replicates, 7, 9, and 12).

Figure 8.

(A) Schematic depicting the rapid metabolism of Cfz by microsomal epoxide hydrolase (mEH) to the inactive diol. (B) Quantification of the remaining levels of Cfz or 9 following the incubation with rat liver homogenate containing active mEH and peptidase activities for 5, 10, and 20 min, respectively. Data presented as mean ± SD.

Scheme 1.. Synthesis Scheme of 5 (UK101-OH) and 9 (Cfz-OH)^a

^aReagents and conditions. (A) (a) (i) CH₃PO(OCH₃)₂, n-BuLi, THF, −78 °C, 2 h, Boc-serine methylester, THF, −78 °C, 3 h, 55%; (ii) Aqueous solution of formaldehyde, K₂CO₃, H₂O, rt, 4 h, 59%; (iii) TBDMS-Cl, imidazole, DCM, rt, 30 min, 71% or iodomethane, Ag₂O, MeCN, rt, 24 h, 51%; (b) benzonitrile, H₂O₂, DIEA, MeOH, 0 °C, 3 h, 35–41%; (c) heptanoic acid, HBTU, HOBt, DIEA, DCM, rt, 18 h, 82%; (d) (i) H₂, Pd/C, methanol, 1 h, (ii) Boc-deprotected 2a, HBTU, HOBt, DIEA, DCM, rt, 18 h, 91%; (e) TBAF, DCM, 2 h, 70%. (B) (a) (i) Phenylalanine benzyl ester hydrochloride, HBTU, HOBt, DIEA, DCM, rt, 18 h, 81%; (ii) TFA, DCM, rt, 1 h then evaporation and drying, Boc-homoPhe-OH, HBTU, HOBt, DIEA, DCM, rt, 18 h, 79%; (iii) TFA, DCM, rt, 1 h then evaporate and dried, morpholin-4-yl-acetic acid hydrochloride, HBTU, HOBt, DIEA, DCM, rt, 18 h, 65%; (b) (i) H₂/Pd, C, methanol, 1 h; (ii) 2a or 2b, HBTU, HOBt, DIEA, DCM, rt, 18 h, 45% and 48%, respectively; (c) TBAF, DCM, 2 h, 64%; (d) MOM-Cl or MEM-Cl or methanesulfonyl chloride, DIEA, DCM, overnight, 60–64%.