Design,synthesis, and biological evaluation of some novel naphthoquinone-glycine/β-alanine anilide derivatives as noncovalent proteasome inhibitors

A series of novel noncovalent glycine/β-alanine anilide derivatives possessing 2-chloronaphthoquinone structure as a pharmacophoric unit were designed, synthesized, and evaluated for their antiproliferative and antiproteasomal activities against MCF-7 cell line, in vitro. According to biological activity results, all the target compounds showed antiproliferative activity in the range of IC₅₀ = 7.10 ± 0.10–41.08 ± 0.14 μM and most of them exhibited inhibitory efficacy with varying ratios against the three catalytic subunits (β1, β2, and β5) presenting caspase-like (C-L), trypsin-like (T-L) and chymotrypsin-like (ChT-L) activities of proteasome. The antiproteasomal activity evaluations revealed that compounds preferentially inhibited the β5 subunit compared with β1 and β2 subunits of the proteasome. Among the compounds, compounds 7 and 9 showed the highest antiproliferative activity with an IC₅₀ value of 7.10 ± 0.10 and 7.43 ± 0.25 μM, respectively. Additionally, compound 7 displayed comparable potency to PI-083 lead compound in terms of β5 antiproteasomal activity with an inhibition percentage of 34.67 at 10 μM. This compound showed an IC₅₀ value of 32.30 ± 0.45 μM against β5 subunit. Furthermore, molecular modeling studies of the most active compound 7 revealed key interactions with β5 subunit. The results suggest that this class of compounds may be beneficial for the development of new potent proteasome inhibitors.     

The polyglutamine neurodegenerative protein ataxin 3 regulates aggresome formation

The polyglutamine-containing neurodegenerative protein ataxin 3 (AT3) has deubiquitylating activity and binds ubiquitin chains with a preference for chains of four or more ubiquitins. Here we characterize the deubiquitylating activity of AT3 in vitro and show it trims/edits K48-linked ubiquitin chains. AT3 also edits polyubiquitylated (125)I-lysozyme and decreases its degradation by proteasomes. Cellular studies show that endogenous AT3 colocalizes with aggresomes and preaggresome particles of the misfolded cystic fibrosis transmembrane regulator (CFTR) mutant CFTRDeltaF508 and associates with histone deacetylase 6 and dynein, proteins required for aggresome formation and transport of misfolded protein. Small interfering RNA knockdown of AT3 greatly reduces aggresomes formed by CFTRDeltaF508, demonstrating a critical role of AT3 in this process. Wild-type AT3 restores aggresome formation; however, AT3 with mutations in the active site or ubiquitin interacting motifs cannot restore aggresome formation in AT3 knockdown cells. These same mutations decrease the association of AT3 and dynein. These data indicate that the deubiquitylating activity of AT3 and its ubiquitin interacting motifs as well play essential roles in CFTRDeltaF508 aggresome formation.     

Molecular basis of Japanese variants of pyrimidine 5'-nucleotidase deficiency

The type-I isoform of pyrimidine 5'-nucleotidase (P5N-I) has an important role in the catabolism of pyrimidine mononucleotides during erythroid maturation. Two alternatively spliced forms of P5N-I mRNA have been identified, and we found another alternatively spliced form in reticulocytes, which included an additional 87-bp sequence. The sequence is located 6.2-kb downstream of the exon 2 and 2.7-kb upstream of the exon 3 sequence; consequently, the P5N-I gene encodes 11 exons, which span approximately 48 kb. We identified five novel mutations in nine families with P5N-I deficiency: two missense mutations (425C, 721C), one splice mutation (339C), one 1-bp insertion (251-insA-252) and one 9-bp deletion (del 192-200). All patients were homozygous for each mutation. The mutant P5N-I with 721C (G241R) had lower affinity for cytidine monophosphate, suggesting that Gly241 is important for substrate binding. Haplotype analysis showed that 721C, which had been identified in five unrelated families, was a founder mutation. The mutant P5N was then expressed in Cos-7. The degradation of P5N with 425C (L142P) was significantly faster than a wild-type control, and proteasome inhibitors restored the stability of L142P. These data suggest that L142P increases susceptibility to the degradation by the ubiquitin-proteasome pathway.     

Hepatitis C core protein impairs insulin downstream signalling and regulatory role of IGFBP-1 expression

Chronic infection with hepatitis C virus (HCV), mainly genotype 1, has been shown to be associated with insulin resistance and type 2 diabetes. The mechanisms underlying this association are partly understood. Increased levels of tumor necrosis factor (TNF)-α occurring in HCV infection have an important role in HCV-mediated insulin resistance; however, other direct effects of HCV core protein on disrupting insulin signalling have been suggested. The insulin receptor substrate (IRS) proteins are key players in insulin signal transduction and are the major substrates of the insulin receptor. To further elucidate the direct effect of HCV core protein on insulin signalling. We studied the direct effects of HCV core protein in two cell lines transfected with HCV core protein. We found several impairments in the insulin signalling cascade which could be attributed to a significant proteasomal degradation of IRS-1 protein, in a dose-dependent way. In addition, our data show that liver cells transfected by HCV core protein show a marked attenuation of the regulatory inhibitory role of insulin on insulin growth factor binding protein-1 (IGFBP-1) expression. Since IGFBP-1 may have a role in glucose regulation and hepatic insulin sensitivity, this effect of HCV core protein can contribute to insulin resistance in chronic HCV infection. Our data suggest that the degradation of IRS-1 by HCV core protein translates to impaired ability of insulin to inhibit the expression of the target gene IGFBP-1 in the liver and may serve as a novel mechanism for insulin resistance and hyperglycaemia.     

Novel biological therapies for the treatment of multiple myeloma

The therapeutic management of multiple myeloma (MM) for the last several decades has mainly involved regimens based on use of glucocorticoids and cytotoxic chemotherapeutics. Despite progress in delineating the activity of such regimens, at either conventional or high doses, MM has remained an incurable disease, without substantial improvement in the median overall survival. This has sparked major interest in the development of novel therapies that in part capitalize on recent advances in our understanding of the biology of MM, including the molecular mechanisms by which MM cell-host bone marrow (BM) interactions regulate tumor-cell growth, survival, and drug resistance in the BM milieu. The development of in vitro and in vivo models of MM-stromal interactions has allowed not only for better characterization of these molecular phenomena but also for identification of specific therapeutic strategies to overcome these interactions and achieve an enhanced anti-MM effect, even against MM resistant to conventional therapies. Herein, we review the latest progress in the development of these novel anti-MM therapies, with major focus on therapies which have translated from preclinical evaluation to clinical application, including thalidomide and its more potent immunomodulatory (IMiD) derivatives, the first-in-class proteasome inhibitor bortezomib (formerly known as PS-341), and arsenic trioxide (As2O3).     

Proteasome inhibitor therapy for antibody-mediated rejection

AMR is being recognized with increasing efficiency, but continues to present a significant threat to renal allograft survival. Traditional therapies for AMR (IVIG, plasmapheresis, rituximab, and antilymphocyte preparations) in general have provided inconsistent results and do not deplete the source of antibody production, viz., the mature plasma cell. Recently, the first plasma cell-targeted therapy in humans has been developed using bortezomib (a first in class PI) for AMR treatment in kidney transplant recipients. Initial experience with bortezomib involved treatment of refractory AMR. Subsequently, the efficacy of bortezomib in primary therapy for AMR was demonstrated. In a multicenter collaborative effort, the initial results with bortezomib in AMR have been confirmed and expanded to pediatric and adult heart transplant recipients. More recently, results from a prospective, staged desensitization trial has shown that bortezomib alone can substantially reduce anti-HLA antibody levels. These results demonstrate the significant potential of proteasome inhibition in addressing humoral barriers. However, the major advantage of proteasome inhibition lies in the numerous potential strategies for achieving synergy.     

蛋白酶体亚基LMP 2在稽留流产的蜕膜和绒毛组织中的表达

目的探讨蛋白酶体亚基LMP-2在稽留流产中的作用。方法 用免疫组化方法研究LMP-2在201例稽留流产中的48例绒毛和45例脱膜与114例正常早孕人工流产中的56例绒毛和58例蜕膜中的表达。结果LMP-2在稽留流产的绒毛和蜕膜中的表达均低于正常早孕人工流产的绒毛和蜕膜,二者间的表达强度差异有统计学意义（P〈0.05）。结论 LMP-2在稽留流产中绒毛与蜕膜的表达减弱提示泛素-蛋白酶体通路介导的细胞蛋白降解参与调控滋养层细胞的生长过程。     

Unique anti‐myeloma activity by thiazolidine‐2,4‐dione compounds with Pim inhibiting activity

Proviral Integrations of Moloney virus 2 (PIM2) is overexpressed in multiple myeloma (MM) cells, and regarded as an important therapeutic target. Here, we aimed to validate the therapeutic efficacy of different types of PIM inhibitors against MM cells for their possible clinical application. Intriguingly, the thiazolidine‐2,4‐dione‐family compounds SMI‐16a and SMI‐4a reduced PIM2 protein levels and impaired MM cell survival preferentially in acidic conditions, in contrast to other types of PIM inhibitors, including AZD1208, CX‐6258 and PIM447. SMI‐16a also suppressed the drug efflux function of breast cancer resistance protein, minimized the sizes of side populations and reduced in vitro colony‐forming capacity and in vivo tumourigenic activity in MM cells, suggesting impairment of their clonogenic capacity. PIM2 is known to be subject to ubiquitination‐independent proteasomal degradation. Consistent with this, the proteasome inhibitors bortezomib and carfilzomib increased PIM2 protein levels in MM cells without affecting its mRNA levels. However, SMI‐16a mitigated the PIM2 protein increase and cooperatively enhanced anti‐MM effects in combination with carfilzomib. Collectively, the thiazolidine‐2,4‐dione‐family compounds SMI‐16a and SMI‐4a uniquely reduce PIM2 protein in MM cells, which may contribute to their profound efficacy in addition to their immediate kinase inhibition. Their combination with proteasome inhibitors is envisioned.