Chronic lymphocytic leukemia (CLL) progression during Bruton tyrosine kinase (BTK) inhibitor treatment is typically characterized by emergent B-cell receptor pathway mutations. Using peripheral blood samples from relapsed/refractory CLL patients in ELEVATE-RR (NCT02477696) (median 2 prior therapies), we report clonal evolution data for patients progressing on acalabrutinib or ibrutinib (median follow-up 41 months). Paired (baseline and progression) samples were available for 47 (excluding 1 Richter) acalabrutinib-treated and 30 (excluding 6 Richter) ibrutinib-treated patients. At progression, emergent BTK mutations were observed in 31 (66%) acalabrutinib-treated and 11 (37%) ibrutinib-treated patients (median variant allele fraction [VAF]: 16.1% vs 15.6%). BTK C481S mutations were most common in both groups; T474I (n = 9; 8 co-occurring with C481) and the novel E41V mutation within the pleckstrin homology domain of BTK (n = 1) occurred with acalabrutinib, while neither mutation occurred with ibrutinib. L528W and A428D co-mutations presented in one ibrutinib-treated patient. Pre-existing TP53 mutations were present in 25 (53.2%) acalabrutinib-treated and 16 (53.3%) ibrutinib-treated patients at screening. Emergent TP53 mutations occurred with acalabrutinib and ibrutinib (13% vs 7%; median VAF: 6.0% vs 37.3%, respectively). Six acalabrutinib-treated patients and one ibrutinib-treated patient had emergent TP53/BTK co-mutations. Emergent PLCG2 mutations occurred in 3 (6%) acalabrutinib-treated and 6 (20%) ibrutinib-treated patients. One acalabrutinib-treated patient and 4 ibrutinib-treated patients had emergent BTK/PLCG2 co-mutations. While common BTK C481 mutations were observed with both treatments, patterns of mutation and co-mutation frequency, mutation VAF, and uncommon BTK variants varied with acalabrutinib (T474I and E41V) and ibrutinib (L528W, A428D) in this patient population.

BACKGROUND: Atherosclerosis is the major underlying pathology of cardiovascular disease and is driven by dyslipidemia and inflammation. Inhibition of the immunoproteasome, a proteasome variant that is predominantly expressed by immune cells and plays an important role in antigen presentation, has been shown to have immunosuppressive effects. METHODS: We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit β5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit β1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr –/– and APOE*3-Leiden.CETP mice. RESULTS: ONX-0914 treatment significantly reduced atherosclerosis, reduced dendritic cell and macrophage levels and their activation, as well as the levels of antigen-experienced T cells during early plaque formation, and Th1 cells in advanced atherosclerosis in young and aged mice in various immune compartments. Additionally, ONX-0914 treatment led to a strong reduction in white adipose tissue mass and adipocyte progenitors, which coincided with neutrophil and macrophage accumulation in white adipose tissue. ONX-0914 reduced intestinal triglyceride uptake and gastric emptying, likely contributing to the reduction in white adipose tissue mass, as ONX-0914 did not increase energy expenditure or reduce total food intake. Concomitant with the reduction in white adipose tissue mass upon ONX-0914 treatment, we observed improvements in markers of metabolic syndrome, including lowered plasma triglyceride levels, insulin levels, and fasting blood glucose. CONCLUSIONS: We propose that immunoproteasomal inhibition reduces 3 major causes underlying cardiovascular disease, dyslipidemia, metabolic syndrome, and inflammation and is a new target in drug development for atherosclerosis treatment.

Introduction: Acalabrutinib (Acala) is a highly selective, next-generation covalent Bruton tyrosine kinase inhibitor (BTKi) approved for CLL. In ELEVATE-RR (NCT02477696) at a median follow-up of 41 mo, Acala demonstrated noninferior progression-free survival with fewer cardiovascular adverse events versus ibrutinib (Ibr) in patients (pts) with relapsed/refractory (R/R) CLL. Disease progression on covalent BTKis is often characterized by acquisition of B-cell receptor pathway mutations, but no data have compared mutational profiles of Acala versus Ibr. We report clonal evolution data in pts with CLL progressing on Acala versus Ibr in ELEVATE-RR. Methods: Peripheral blood samples at baseline and relapse from pts in ELEVATE-RR were used. DNA was extracted from enriched CD19+ cells (RoboSep) and subjected to a 50-gene sequencing assay panel with a sensitivity cutoff for BTK and PLCG2 resistance–associated mutations at 0.5% variant allele fraction (VAF). Forty-eight other CLL-associated genes were assessed at 1%–2% VAF. Results: Paired (baseline and progression) samples were available for 47 (excluding 1 Richter) and 30 (excluding 6 Richter) pts in the Acala and Ibr groups, respectively. At progression, emergent BTK mutations were seen in 31 (66%) Acala versus 11 (37%) Ibr pts (P = 0.02) (Figure 1; median VAF: 5.7 vs. 5.8). Emergent PLCG2 mutations occurred in 3 (6%) Acala vs. 6 (20%) Ibr pts (P = 0.14). Only 1 Acala pt had co-occurrence of BTK and PLCG2 mutations versus 4 Ibr pts. BTK C481S, C481Y, and C481R mutations occurred at similar frequency in both groups; a novel E41V mutation within the pleckstrin homology domain of BTK (median VAF: 16%) was seen in 1 Acala pt. L528W and A428D co-mutations were observed in 1 Ibr pt. Six Acala pts had TP53 and BTK co-mutations. Emergent TP53 mutations were seen in both groups (13% [Acala] vs. 7% [Ibr], P = 0.47; median VAF: 5% [Acala] versus 37% [Ibr]). Only 2 Ibr pts had TP53 mutations (1 had TP53/BTK co-mutation). No statistical difference was seen in the proportions of Acala versus Ibr pts who acquired BTK mutations among pts with del(17p) (39% vs. 64%; P = 0.18), del(11q) (77% vs. 46%; P = 0.07), complex karyotype (58% vs. 73%; P = 0.48), unmutated IGHV (90% vs. 100%; P = 0.55), or trisomy 12 positivity (3% vs. 18%; P = 0.16). Additional mutations (Acala vs. Ibr) included DNMT3A (5 vs. 1 pts), TET2 (1 pt for each), and NRAS (1 pt; Acala only). The research was funded by: AstraZeneca Keywords: Chronic Lymphocytic Leukemia (CLL), Genomics, Epigenomics, and Other -Omics, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. J. A. Woyach Consultant or advisory role: Abbvie, AstraZeneca, BeiGene, Genentech, Janssen, Merck, Loxo/Lilly, Newave, Pharmacyclics Research funding: Abbvie, Janssen, Karyopharm Therapeutics, Loxo/Lilly, Pharmacyclics, Schrodinger D. Jones Research funding: Abbvie, Acerta/AstraZeneca, Pharmacyclics, Novartis, MingSight, Other remuneration: The Ohio State University: High sensitivity BTK mutation profiling W. Jurczak Consultant or advisory role: Abbvie, AstraZeneca, BeiGene, Lilly, Roche, Takeda Research funding: Abbvie, AstraZeneca, BeiGene, Janssen, Lilly, Roche, Takeda T. Robak Consultant or advisory role: AstraZeneca, BeiGene, Janssen Oncology Honoraria: AstraZeneca, BeiGene, Janssen Research funding: AstraZeneca, BeiGene, Janssen A. Illés Employment or leadership position: University of Debrecen Honoraria: Janssen, Celgene, Novartis, Pfizer, Takeda, Roche Research funding: Takeda, Seattle Genetics A. P. Kater Consultant or advisory role: AstraZeneca, BMS, Roche/Genentech, Janssen, AbbVie, LAVA Research funding: AstraZeneca, BMS, Roche/Genentech, Janssen, AbbVie Other remuneration: Janssen, LAVA, AbbVie, AstraZeneca P. Ghia Honoraria: AbbVie, AstraZeneca, Janssen, BMS, MSD, Loxo Oncology/Lilly, and Roche Research funding: AbbVie, AstraZeneca, Janssen, and BMS J. C. Byrd Consultant or advisory role: Janssen, Novartis, Syndax, Newave, AstraZeneca, Kura, Vincerx, Trilrom, Abbvie Stock ownership: Vincerx Research funding: Zencor, Pharmacyclics Educational grants: Janssen, Novartis Other remuneration: Ohio State University J. F. Seymour Consultant or advisory role: AbbVie, AstraZeneca, Celgene, Genentech, Genor Bio, Gilead, Janssen, Morphosys, Roche, Sunesis, TG Therapeutics Research funding: Abbvie, Celgene, Janssen, Roche Other remuneration: Abbvie, Celgene, Roche, TG Therapetics G. De Jesus Employment or leadership position: AstraZeneca Stock ownership: AstraZeneca R. Lai Employment or leadership position: AstraZeneca G. de Bruin Employment or leadership position: Acerta Pharma B.V. A. Butturini Employment or leadership position: AstraZeneca Stock ownership: AstraZeneca, Amgen, Roche S. Rule Employment or leadership position: AstraZeneca V. Munugalavadla Employment or leadership position: AstraZeneca Stock ownership: AstraZeneca; Gilead Sciences

Acalabrutinib is a covalent Bruton tyrosine kinase (BTK) inhibitor approved for relapsed/refractory mantle cell lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma. A major metabolite of acalabrutinib (M27, ACP-5862) was observed in human plasma circulation. Subsequently, the metabolite was purified from an in vitro biosynthetic reaction and shown by nuclear magnetic resonance spectroscopy to be a pyrrolidine ring-opened ketone/amide. Synthesis confirmed its structure, and covalent inhibition of wild-type BTK was observed in a biochemical kinase assay. A twofold lower potency than acalabrutinib was observed but with similar high kinase selectivity. Like acalabrutinib, ACP-5862 was the most selective toward BTK relative to ibrutinib and zanubrutinib. Because of the potency, ACP-5862 covalent binding properties, and potential contribution to clinical efficacy of acalabrutinib, factors influencing acalabrutinib clearance and ACP-5862 formation and clearance were assessed. rCYP (recombinant cytochrome P450) reaction phenotyping indicated that CYP3A4 was responsible for ACP-5862 formation and metabolism. ACP-5862 formation Km (Michaelis constant) and Vmax were 2.78 μM and 4.13 pmol/pmol CYP3A/min, respectively. ACP-5862 intrinsic clearance was 23.6 μL/min per mg. Acalabrutinib weakly inhibited CYP2C8, CYP2C9, and CYP3A4, and ACP-5862 weakly inhibited CYP2C9 and CYP2C19; other cytochrome P450s, UGTs (uridine 5'-diphospho-glucuronosyltransferases), and aldehyde oxidase were not inhibited. Neither parent nor ACP-5862 strongly induced CYP1A2, CYP2B6, or CYP3A4 mRNA. Acalabrutinib and ACP-5862 were substrates of multidrug resistance protein 1 and breast cancer resistance protein but not OATP1B1 or OATP1B3. Our work indicates that ACP-5862 may contribute to clinical efficacy in acalabrutinib-treated patients and illustrates how proactive metabolite characterization allows timely assessment of drug-drug interactions and potential contributions of metabolites to pharmacological activity. SIGNIFICANCE STATEMENT: This work characterized the major metabolite of acalabrutinib, ACP-5862. Its contribution to the pharmacological activity of acalabrutinib was assessed based on covalent Bruton tyrosine kinase binding kinetics, kinase selectivity, and potency in cellular assays. The metabolic clearance and in vitro drug-drug interaction potential were also evaluated for both acalabrutinib and ACP-5862. The current data suggest that ACP-5862 may contribute to the clinical efficacy observed in acalabrutinib-treated patients and demonstrates the value of proactive metabolite identification and pharmacological characterization.

Bruton tyrosine kinase (BTK) is an important target in oncology and (auto)immunity. Various BTK inhibitors have been approved or are currently in clinical development. A novel BTK inhibitor series was developed starting with a quinazoline core. Moving from a quinazoline to a quinoline core provided a handle for selectivity for BTK over EGFR and resulted in the identification of potent and selective BTK inhibitors with good potency in human whole blood assay. Furthermore, proof of concept of this series for BTK inhibition was shown in an in vivo mouse model using one of the compounds identified.

Bioorthogonal chemistry allows the selective modification of biomolecules in complex biological samples. One application of this methodology is in two-step activity-based protein profiling (ABPP), a methodology that is particularly attractive where direct ABPP using fluorescent or biotinylated probes is ineffective. Herein we describe a set of norbornene-modified, mechanism-based proteasome inhibitors aimed to be selective for each of the six catalytic sites of human constitutive proteasomes and immunoproteasomes. The probes developed for β1i, β2i, β5c, and β5i proved to be useful two-step ABPs that effectively label their developed proteasome subunits in both Raji cell extracts and living Raji cells through inverse-electron-demand Diels-Alder (iEDDA) ligation. The compound developed for β1c proved incapable of penetrating the cell membrane, but effectively labels β1c in vitro. The compound developed for β2c proved not selective, but its azide-containing analogue LU-002c proved effective in labeling of β2c via azide-alkyne click ligation chemistry both in vitro and in situ. In total, our results contribute to the growing list of proteasome activity tools to include five subunit-selective activity-based proteasome probes, four of which report on proteasome activities in living cells.

Background: Bruton tyrosine kinase (BTK) is a validated target for B-cell malignancies. The BTK inhibitor ibrutinib was approved in chronic lymphocytic leukemia, mantle cell lymphoma (MCL), and Waldenstrom macroglobulinemia. Acalabrutinib is a potent, highly selective, covalent BTK inhibitor with minimal off-target activity; it received accelerated FDA approval in October 2017 for the treatment of patients with MCL having ≥1 prior therapy. In addition to the approved covalent BTK inhibitors ibrutinib and acalabrutinib, clinical data in B-cell malignancies are available for spebrutinib (CC-292), tirabrutinib (ONO/GS-4059) and zanubrutinib (BGB-3111). We performed biochemical and cellular profiling of these 5 BTK inhibitors, investigating potency and selectivity.

Acalabrutinib is a potent and highly selective, oral covalent inhibitor of Bruton tyrosine kinase (BTK) that received accelerated approval for relapsed/refractory mantle cell lymphoma by the United States Food and Drug Administration in October 2017.ADME studies in humans, rat and dog, revealed extensive metabolism of acalabrutinib. The major circulating metabolite (M27, ACP-5862) was produced by CYP3A oxidation, resulting in a pyrrolidine ring-opened metabolite, with the butynamide electrophile still present. We investigated the on-target BTK inhibition and kinase selectivity profile of ACP-5862 using the same biochemical and cellular assays previously employed to profile acalabrutinib.1The apparent BTK IC50 was determined over time using the LanthaScreen assay. Results indicate that ACP-5862, like acalabrutinib, is a covalent inhibitor of BTK. Binding kinetics derived from IC50 over time data, indicated that acalabrutinib and ACP-5862 have similar affinity (KI), however the BTK inactivation rate (kinact) for ACP-5862 was half that relative to acalabrutinib. This indicated a covalent BTK inhibition potency for ACP-5862 that was 2-fold lower than acalabrutinib.The overall kinome inhibition profile at 1 µM using KINOMEscan (Eurofins DiscoverX), as well as IC50 determinations on closely related kinases with a Cys in the same position as Cys481 in BTK (ITK, TXK, TEC, BMX, EGFR, ERBB2, ERBB4, BLK, JAK3), both revealed that the kinase selectivity profiles of acalabrutinib and ACP-5862 were similar.On-target inhibition in B cells was investigated measuring inhibition of B-cell antigen receptor-mediated activation of CD69 cell surface expression on human peripheral B cells using human peripheral blood mononuclear cells and human whole blood (hWB). The EC50 for anti-IgD-induced CD69 expression in hWB was 64 ± 6 nM for ACP-5862, compared to 9.2 ± 4.4 nM for acalabrutinib. The hWB EC90, representing near complete inhibition of BTK by acalabrutinib and ACP-5862 was observed at 72 ± 20 nM and 544 ± 376 nM, respectively.In conclusion, the results indicate that ACP-5862, the major metabolite of acalabrutinib, has intrinsic BTK inhibitory activity and a similar kinase selectivity profile as acalabrutinib. The contribution of ACP-5862 to on-target covalent inhibition of BTK in humans is unclear at present, but is most likely limited, since the EC90 in hWB for ACP-5862 approximated observed plasma Cmax in humans dosed with 100 mg acalabrutinib. The relative contribution of acalabrutinib and ACP-5862 to BTK pharmacodynamics is under further investigation.Terry Podoll and J. Greg Slatter are former Acerta Pharma employees.Reference1. Barf T, Covey T, Izumi R, et al. Acalabrutinib (ACP-196): A covalent Bruton tyrosine kinase inhibitor with a differentiated selectivity and in vivo potency profile. J Pharmacol Exp Ther. 2017;363(2):240-252.Citation Format: Allard Kaptein, Terry Podoll, Gerjan de Bruin, Maaike Emmelot-van Hoek, Anouk de Jong, Bart van Lith, Niels Hoogenboom, Tjeerd Barf, Joseph Ware, J. Greg Slatter. Preclinical pharmacological profiling of ACP-5862, the major metabolite of the covalent BTK inhibitor acalabrutinib, displays intrinsic BTK inhibitory activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2194.

Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal relevance of individual proteasome active sites. Whereas the inhibitors for the β1c, β1i, β5c, and β5i subunits exploit the differences in the substrate-binding channels identified by X-ray crystallography, compounds selectively targeting β2c or β2i could not yet be rationally designed because of the high structural similarity of these two subunits. Here, we report the development, chemical synthesis, and biological screening of a compound library that led to the identification of the β2c- and β2i-selective compounds LU-002c (4; IC50 β2c: 8 nM, IC50 β2i/β2c: 40-fold) and LU-002i (5; IC50 β2i: 220 nM, IC50 β2c/β2i: 45-fold), respectively. Co-crystal structures with β2 humanized yeast proteasomes visualize protein–ligand interactions crucial for subunit specificity. Altogether, organic syntheses, activity-based protein profiling, yeast mutagenesis, and structural biology allowed us to decipher significant differences of β2 substrate-binding channels and to complete the set of subunit-selective proteasome inhibitors.

During an immune response, CD8(+) T lymphocytes can undergo asymmetric division, giving rise to daughter cells that exhibit distinct tendencies to adopt terminal effector and memory cell fates. Here we show that "pre-effector" and "pre-memory" cells resulting from the first CD8(+) T cell division in vivo exhibited low and high rates of endogenous proteasome activity, respectively. Pharmacologic reduction of proteasome activity in CD8(+) T cells early during differentiation resulted in acquisition of terminal effector cell characteristics, whereas enhancement of proteasome activity conferred attributes of memory lymphocytes. Transcriptomic and proteomic analyses revealed that modulating proteasome activity in CD8(+) T cells affected cellular metabolism. These metabolic changes were mediated, in part, through differential expression of Myc, a transcription factor that controls glycolysis and metabolic reprogramming. Taken together, these results demonstrate that proteasome activity is an important regulator of CD8(+) T cell fate and raise the possibility that increasing proteasome activity may be a useful therapeutic strategy to enhance the generation of memory lymphocytes.

Most known β2-selective proteasome inhibitors suffer from relatively poor cell permeability as the result of a net positive charge caused by the basic moiety at P1. In this paper, we describe the synthesis of oligopeptide vinyl sulfones that contain different amino acids bearing amino groups with reduced basicity at P1 and/or P3. For this, we developed the first enantioselective synthesis of lysine(4-ene) and lysine(4-yne). These amino acids, as well as histidine and diaminopropionic-acid-glycine, were incorporated at the P1 and/or P3 positions of oligopeptide vinyl sulfones. All inhibitors were found to inhibit β2, but with a loss of potency compared to our most potent and selective β2 inhibitor, LU-102. These results notwithstanding, our results provide important insights for the future design of β2-selective proteasome inhibitors.

Background and PurposeMulticatalytic endopeptidase complex‐like‐1 (β2i), low molecular mass polypeptide (LMP) 2 (β1i) and LMP7 (β5i) are the proteolytically active subunits of the immunoproteasome, a special type of proteasome mainly expressed in haematopoietic cells. Targeting LMP7 has been shown to be therapeutically effective in preclinical models of autoimmune diseases. In this study, we investigated the selectivity and biological activity of LU‐005i, a recently described inhibitor of the immunoproteasome.Experimental ApproachThe specificity of LU‐005i and other immunoproteasome‐selective inhibitors was characterized using fluorogenic peptide substrates. The effect of proteasome inhibition on cytokine release was investigated in endotoxin‐stimulated mouse splenocytes or human peripheral blood mononuclear cells (PBMCs). The effect of proteasome inhibition on inflammatory bowel disease in the dextran sulfate sodium (DSS)‐induced colitis model was assessed by measuring weight loss and colon length.Key ResultsLU‐005i is the first human and mouse immunoproteasome‐selective inhibitor that targets all three proteolytically active immunoproteasome subunits. LU‐005i inhibited cytokine secretion from endotoxin‐stimulated mouse splenocytes or human PBMCs. Furthermore, differentiation of naïve T helper cells to T helper 17 cells was impaired in the presence of LU‐005i. Additionally, LU‐005i ameliorated DSS‐induced colitis.Conclusion and ImplicationsThis study with a novel pan‐immunoproteasome inhibitor substantiates that the immunoproteasome is a promising drug target for the treatment of inflammatory diseases and that exclusive inhibition of LMP7 is not necessary for therapeutic effectiveness. Our results will promote the design of new generations of immunoproteasome inhibitors with optimal therapeutic efficacy for clinical use in the treatment of autoimmunity and cancer.

The proteasome inhibitors carfilzomib (Cfz) and bortezomib (Btz) are used successfully to treat multiple myeloma, but have not shown clinical efficacy in solid tumors. Here we show that clinically achievable inhibition of the β5 site of the proteasome by Cfz and Btz does not result in loss of viability of triple-negative breast cancer cell lines. We use site-specific inhibitors and CRISPR-mediated genetic inactivation of β1 and β2 to demonstrate that inhibiting a second site of the proteasome, particularly the β2 site, sensitizes cell lines to Btz and Cfz in vitro and in vivo. Inhibiting both β5 and β2 suppresses production of the soluble, active form of the transcription factor Nrf1 and prevents the recovery of proteasome activity through induction of new proteasomes. These findings provide a strong rationale for the development of dual β5 and β2 inhibitors for the treatment of solid tumors.

Proteasome inhibitors (PIs) are a backbone of multiple myeloma (MM) therapy. The proteasome harbors six proteolytically active subunits (β1, β2, β5), while β5 was identified as rate-limiting and is a primary target of clinically available PIs. The most effective pattern of subunit inhibition provided by these PIs for cytotoxic activity in MM is unknown. A head-to-head comparison of clinically available PIs shows that in the clinically relevant setting only the co-inhibition of β1 or β2 with β5 activity achieves meaningful functional proteasome inhibition and cytotoxicity, while the selective β2/β5 inhibition of both constitutive and immunoproteasome is the most cytotoxic. In the long-term setting, selective inhibition of β5 subunit is sufficient to induce cytotoxicity in PI-sensitive, but not in PI-resistant MM, and the β5/β2 co-inhibition is the most cytotoxic in PI-resistant MM. These results give a rational basis for selecting individual PIs for the treatment of MM.

Idiopathic inflammatory myopathies (IIMs) are diseases with muscle weakness, morphologically characterized by inflammatory infiltration and increased expression of MHC class I molecule on myofibers. Immunoproteasome, as a proteolytic complex that shapes the repertoire of antigenic peptides, has been previously demonstrated to be over-expressed in IIMs at mRNA level. In this study, we investigated the expression and the function of the immunoproteasome in IIMs in more detail. As shown by immunofluorescence staining, expression of relevant players of the immunoproteasome was detectable in the inflamed skeletal muscle tissue from IIM patients. In fact, two subunits of the immunoproteasome, β1i or β5i were upregulated in sporadic inclusion body myositis, immune-mediated necrotizing myopathies and dermatomyositis muscle biopsies and co-localized with the MHC class I expressing myofibers. Double immunofluorescence revealed that both myofibers and muscle infiltrating cells, including CD8+ T-cells and CD68 + macrophages in IIMs expressed β1i or β5i. In addition, we have also investigated the role of the immunoproteasome in myoblasts during in vitro inflammatory conditions. Using human primary myoblasts cultures we found that pro-inflammatory cytokines, TNF-α or IFN-γ upregulate β1i or β5i. Selective inhibition or depletion of β5i amplified the TNF-α or IFN-γ mediated expression of cytokines/chemokines (myokines) in myoblasts. Furthermore, we demonstrated that specific inhibitors of β1i or β5i reduced the cell surface expression of MHC class I in myoblasts induced by IFN-γ. Taken together, our data suggest that the immunoproteasome is involved in pathologic MHC class I expression and maintenance of myokine production in IIMs. Thus, induction of the immunoproteasome was identified as a pathomechanism underlying inflammation in IIMs.

This work reports the development of highly potent and selective inhibitors of the β5c catalytic activity of human constitutive proteasomes. The work describes the design principles, large hydrophobic P3 residue and small hydrophobic P1 residue, that led to the synthesis of a panel of peptide epoxyketones; their evaluation and the selection of the most promising compounds for further analyses. Structure-activity relationships detail how in a logical order the β1c/i, β2c/i, and β5i activities became resistant to inhibition as compounds were diversified stepwise. The most effective compounds were obtained as a mixture of cis- and trans-biscyclohexyl isomers, and enantioselective synthesis resolved this issue. Studies on yeast proteasome structures complexed with some of the compounds provide a rationale for the potency and specificity. Substitution of the N-terminus in the most potent compound for a more soluble equivalent led to a cell-permeable molecule that selectively and efficiently blocks β5c in cells expressing both constitutive proteasomes and immunoproteasomes.

The incorporation of adamantylalanine and carboranylalanine at the P2 site of bortezomib is well tolerated and provided potent cell permeable proteasome inhibitors with increased off-rates compared to bortezomib. Adamantylalanine and carboranylalanine were synthesized enantioselectively by an asymmetric Strecker reaction on Ellmans tert-butyl sulfinimines.