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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., Competing Interests: Disclosures None.

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 K m (Michaelis constant) and V max 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., (Copyright © 2022 by The Author(s).)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background and Purpose: Multicatalytic 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 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., (© 2017 The British Pharmacological Society.)

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.

The Institute of Medicine report Improving Diagnosis in Health Care called for tools to monitor physicians' diagnostic process. We addressed this need by developing a tool for clinicians to record and analyze their diagnostic process. The tool was a secure web application in which clinicians used a structured grading system to assess the relative impact of clinical, laboratory, and neuroimaging data for every new diagnosis. Four neurohospitalists used the tool for 6.5 months on a general neurology ward service at a single tertiary-level teaching hospital. Process measures of tool use included number of diagnoses entered, time spent on each data entry, and concordance of diagnoses compared to the medical record. We also aggregated the data across clinicians to examine the average process scores across common inpatient disorders. The 4 clinicians entered 254 new diagnoses that took approximately 3 minutes per patient. In 50 randomly chosen cases, the neurohospitalists' diagnoses entered into the tool agreed with 92% of diagnoses in the medical record, which was better than the agreement between billing code and medical record diagnoses (74%). The diagnostic process varied across disease categories, showing a spectrum of clinical-dominant (eg, headache), laboratory-dominant (eg, encephalitis), and neuroimaging-dominant (eg, stroke) disorders. This study demonstrated the feasibility of a clinician-driven diagnostic process monitoring system, along with preliminary characterization of the process for common disorders. The tracking of diagnostic process has the potential to promote reflection on clinical practice, deconstruct neurologists' clinical decision-making, and improve health-care safety., Competing Interests: Declaration of Conflicting Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.