Mutational profile in previously treated patients with chronic lymphocytic leukemia progression on acalabrutinib or ibrutinib.

ABSTRACT: 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 patients with relapsed/refractory CLL 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 acalabrutinib-treated (66%) and 11 ibrutinib-treated patients (37%; median variant allele fraction [VAF], 16.1% vs 15.6%, respectively). 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, whereas neither mutation occurred with ibrutinib. L528W and A428D comutations presented in 1 ibrutinib-treated patient. Preexisting TP53 mutations were present in 25 acalabrutinib-treated (53.2%) and 16 ibrutinib-treated patients (53.3%) 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 1 ibrutinib-treated patient had emergent TP53/BTK comutations. Emergent PLCG2 mutations occurred in 3 acalabrutinib-treated (6%) and 6 ibrutinib-treated patients (20%). One acalabrutinib-treated patient and 4 ibrutinib-treated patients had emergent BTK/PLCG2 comutations. Although common BTK C481 mutations were observed with both treatments, patterns of mutation and comutation frequency, mutation VAF, and uncommon BTK variants varied with acalabrutinib (T474I and E41V) and ibrutinib (L528W and A428D) in this patient population. The trial was registered at www.clinicaltrials.gov as #NCT02477696.

Immunoproteasomal Inhibition With ONX-0914 Attenuates Atherosclerosis and Reduces White Adipose Tissue Mass and Metabolic Syndrome in Mice.

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 ß5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit ß1i/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.

Identification and Characterization of ACP-5862, the Major Circulating Active Metabolite of Acalabrutinib: Both Are Potent and Selective Covalent Bruton Tyrosine Kinase Inhibitors .

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.

A humanized yeast proteasome identifies unique binding modes of inhibitors for the immunosubunit ß5i.

Inhibition of the immunoproteasome subunit ß5i alleviates autoimmune diseases in preclinical studies and represents a promising new anti-inflammatory therapy. However, the lack of structural data on the human immunoproteasome still hampers drug design. Here, we systematically determined the potency of seven α' ß' epoxyketone inhibitors with varying N-caps and P3-stereochemistry for mouse/human ß5c/ß5i and found pronounced differences in their subunit and species selectivity. Using X-ray crystallography, the compounds were analyzed for their modes of binding to chimeric yeast proteasomes that incorporate key parts of human ß5c, human ß5i or mouse ß5i and the neighboring ß6 subunit. The structural data reveal exceptional conformations for the most selective human ß5i inhibitors and highlight subtle structural differences as the major reason for the observed species selectivity. Altogether, the presented results validate the humanized yeast proteasome as a powerful tool for structure-based development of ß5i inhibitors with potential clinical applications.

Two-Step Bioorthogonal Activity-Based Protein Profiling of Individual Human Proteasome Catalytic Sites.

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 ß1i, ß2i, ß5c, and ß5i 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 ß1c proved incapable of penetrating the cell membrane, but effectively labels ß1c in vitro. The compound developed for ß2c proved not selective, but its azide-containing analogue LU-002c proved effective in labeling of ß2c 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.

Discovery of quinoline-based irreversible BTK inhibitors.

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.

Proteasome Subunit Selective Activity-Based Probes Report on Proteasome Core Particle Composition in a Native Polyacrylamide Gel Electrophoresis Fluorescence-Resonance Energy Transfer Assay.

Most mammalian tissues contain a single proteasome species: constitutive proteasomes. Tissues able to express, next to the constitutive proteasome catalytic activities (ß1c, ß2c, ß5c), the three homologous activities, ß1i, ß2i and ß5i, may contain numerous distinct proteasome particles: immunoproteasomes (composed of ß1i, ß2i and ß5i) and mixed proteasomes containing a mix of these activities. This work describes the development of new subunit-selective activity-based probes and their use in an activity-based protein profiling assay that allows the detection of various proteasome particles. Tissue extracts are treated with subunit-specific probes bearing distinct fluorophores and subunit-specific inhibitors. The samples are resolved by native polyacrylamide gel electrophoresis, after which fluorescence-resonance energy transfer (FRET) reports on the nature of proteasomes present.

The immunoproteasomes are key to regulate myokines and MHC class I expression in idiopathic inflammatory myopathies.

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, ß1i or ß5i 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 ß1i or ß5i. 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 ß1i or ß5i. Selective inhibition or depletion of ß5i amplified the TNF-α or IFN-γ mediated expression of cytokines/chemokines (myokines) in myoblasts. Furthermore, we demonstrated that specific inhibitors of ß1i or ß5i 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.

A Set of Activity-Based Probes to Visualize Human (Immuno)proteasome Activities.

Proteasomes are therapeutic targets for various cancers and autoimmune diseases. Constitutively expressed proteasomes have three active sites, ß1c, ß2c, and ß5c. Lymphoid tissues also express the immunoproteasome subunits ß1i, ß2i, and ß5i. Rapid and simultaneous measurement of the activity of these catalytic subunits would assist in the discovery of new inhibitors, improve analysis of proteasome inhibitors in clinical trials, and simplify analysis of subunit expression. In this work, we present a cocktail of activity-based probes that enables simultaneous gel-based detection of all six catalytic human proteasome subunits. We used this cocktail to develop specific inhibitors for ß1c, ß2c, ß5c, and ß2i, to compare the active-site specificity of clinical proteasome inhibitors, and to demonstrate that many hematologic malignancies predominantly express immunoproteasomes. Furthermore, we show that selective and complete inhibition of ß5i and ß1i is cytotoxic to primary cells from acute lymphocytic leukemia (ALL) patients.

Systematic Analyses of Substrate Preferences of 20S Proteasomes Using Peptidic Epoxyketone Inhibitors.

Cleavage analyses of 20S proteasomes with natural or synthetic substrates allowed to infer the substrate specificities of the active sites and paved the way for the rational design of high-affinity proteasome inhibitors. However, details of cleavage preferences remained enigmatic due to the lack of appropriate structural data. In a unique approach, we here systematically examined substrate specificities of yeast and human proteasomes using irreversibly acting α',ß'epoxyketone (ep) inhibitors. Biochemical and structural analyses provide unique insights into the substrate preferences of the distinct active sites and highlight differences between proteasome types that may be considered in future inhibitor design efforts. (1) For steric reasons, epoxyketones with Val or Ile at the P1 position are weak inhibitors of all active sites. (2) Identification of the ß2c selective compound Ac-LAE-ep represents a promising starting point for the development of compounds that discriminate between ß2c and ß2i. (3) The compound Ac-LAA-ep was found to favor subunit ß5c over ß5i by three orders of magnitude. (4) Yeast ß1 and human ß1c subunits preferentially bind Asp and Leu in their S1 pockets, while Glu and large hydrophobic residues are not accepted. (5) Exceptional structural features in the ß1/2 substrate binding channel give rise to the ß1 selectivity of compounds featuring Pro at the P3 site. Altogether, 23 different epoxyketone inhibitors, five proteasome mutants, and 43 crystal structures served to delineate a detailed picture of the substrate and ligand specificities of proteasomes and will further guide drug development efforts toward subunit-specific proteasome inhibitors for applications as diverse as cancer and autoimmune disorders.

The novel ß2-selective proteasome inhibitor LU-102 synergizes with bortezomib and carfilzomib to overcome proteasome inhibitor resistance of myeloma cells.

Proteasome inhibitor resistance is a challenge for myeloma therapy. Bortezomib targets the ß5 and ß1 activity, but not the ß2 activity of the proteasome. Bortezomib-resistant myeloma cells down-regulate the activation status of the unfolded protein response, and up-regulate ß2 proteasome activity. To improve proteasome inhibition in bortezomib-resistant myeloma and to achieve more efficient UPR activation, we have developed LU-102, a selective inhibitor of the ß2 proteasome activity. LU-102 inhibited the ß2 activity in intact myeloma cells at low micromolar concentrations without relevant co-inhibition of ß1 and ß5 proteasome subunits. In proteasome inhibitor-resistant myeloma cells, significantly more potent proteasome inhibition was achieved by bortezomib or carfilzomib in combination with LU-102, compared to bortezomib/carfilzomib alone, resulting in highly synergistic cytotoxic activity of the drug combination via endoplasmatic reticulum stress-induced apoptosis. Combining bortezomib/carfilzomib with LU-102 significantly prolonged proteasome inhibition and increased activation of the unfolded protein response and IRE1-a activity. IRE1-α has recently been shown to control myeloma cell differentiation and bortezomib sensitivity (Leung-Hagesteijn, Cancer Cell 24:3, 289-304). Thus, ß2-selective proteasome inhibition by LU-102 in combination with bortezomib or carfilzomib results in synergistic proteasome inhibition, activation of the unfolded protein response, and cytotoxicity, and overcomes bortezomib/carfilzomib resistance in myeloma cells in vitro.

Proteasome inhibitors with photocontrolled activity.

Proteasome inhibitors are widely used in cancer treatment as chemotherapeutic agents. However, their employment often results in severe side effects, due to their non-specific cytotoxicity towards healthy tissue. This problem might be overcome by using a photopharmacological approach, that is, by attaining external, dynamic, spatiotemporal photocontrol over the activity of a cytotoxic agent, achieved by the introduction of a photoswitchable moiety into its molecular structure. Here we describe the design, synthesis, and activity of photoswitchable proteasome inhibitors. Substantial differences in proteasome inhibitory activity in cell extracts were observed before and after irradiation with light. The presented results show potential for the development of chemotherapeutic agents that can be switched on and off with light, constituting a new strategy for spatiotemporally modulating proteasomal activity.

A combined solid- and solution-phase approach provides convenient access to analogues of the calcium-dependent lipopeptide antibiotics.

The calcium-dependent lipopeptide antibiotics represent a promising new class of antimicrobials for use in combating drug-resistant bacteria. At present, daptomycin is the only such lipopeptide used clinically and displays potent antimicrobial activity against a number of pathogenic Gram-positive bacteria. Given the increasing need for new antibiotics, practical synthetic access to unnatural analogues of daptomycin and related antimicrobial lipopeptides is of value. We here report an efficient synthetic route combining solid- and solution-phase techniques that allows for the rapid preparation of daptomycin analogues. Using this approach, four such analogues, including two enantiomeric variants, were synthesized and their antimicrobial activities and hydrolytic stabilities evaluated.

Exploring dual electrophiles in peptide-based proteasome inhibitors: carbonyls and epoxides.

Peptide epoxyketones are potent and selective proteasome inhibitors. Selectivity is governed by the epoxyketone dual electrophilic warhead, which reacts with the N-terminal threonine 1,2-amino alcohol uniquely present in proteasome active sites. We studied a series of C-terminally modified oligopeptides featuring adjacent electrophiles based on the epoxyketone warhead. We found that the carbonyl moiety in the natural warhead is essential, but that the adjacent epoxide can be replaced by a carbonyl, though with considerable loss of activity.

Proteasome Inhibition in Multiple Myeloma: Head-to-Head Comparison of Currently Available Proteasome Inhibitors.

Proteasome inhibitors (PIs) are a backbone of multiple myeloma (MM) therapy. The proteasome harbors six proteolytically active subunits (ß1, ß2, ß5), while ß5 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 ß1 or ß2 with ß5 activity achieves meaningful functional proteasome inhibition and cytotoxicity, while the selective ß2/ß5 inhibition of both constitutive and immunoproteasome is the most cytotoxic. In the long-term setting, selective inhibition of ß5 subunit is sufficient to induce cytotoxicity in PI-sensitive, but not in PI-resistant MM, and the ß5/ß2 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.

Structure-Based Design of Inhibitors Selective for Human Proteasome ß2c or ß2i Subunits.

Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal relevance of individual proteasome active sites. Whereas the inhibitors for the ß1c, ß1i, ß5c, and ß5i subunits exploit the differences in the substrate-binding channels identified by X-ray crystallography, compounds selectively targeting ß2c or ß2i 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 ß2c- and ß2i-selective compounds LU-002c (4; IC50 ß2c: 8 nM, IC50 ß2i/ß2c: 40-fold) and LU-002i (5; IC50 ß2i: 220 nM, IC50 ß2c/ß2i: 45-fold), respectively. Co-crystal structures with ß2 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 ß2 substrate-binding channels and to complete the set of subunit-selective proteasome inhibitors.

Amelioration of autoimmunity with an inhibitor selectively targeting all active centres of the immunoproteasome.

BACKGROUND AND PURPOSE: Multicatalytic endopeptidase complex-like-1 (ß2i), low molecular mass polypeptide (LMP) 2 (ß1i) and LMP7 (ß5i) 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 APPROACH: The 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 RESULTS: LU-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 IMPLICATIONS: This 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.

Inhibition of the Proteasome ß2 Site Sensitizes Triple-Negative Breast Cancer Cells to ß5 Inhibitors and Suppresses Nrf1 Activation.

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 ß5 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 ß1 and ß2 to demonstrate that inhibiting a second site of the proteasome, particularly the ß2 site, sensitizes cell lines to Btz and Cfz in vitro and in vivo. Inhibiting both ß5 and ß2 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 ß5 and ß2 inhibitors for the treatment of solid tumors.

Proteasome activity regulates CD8+ T lymphocyte metabolism and fate specification.

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.

Structure-Based Design of ß5c Selective Inhibitors of Human Constitutive Proteasomes.

This work reports the development of highly potent and selective inhibitors of the ß5c 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 ß1c/i, ß2c/i, and ß5i 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 ß5c in cells expressing both constitutive proteasomes and immunoproteasomes.