Biochemical and computational studies towards selective inhibition of the immunoproteasome

The proteasome pathway degrades >90% of cytosolic proteins deemed redundant, misfolded or toxic, thereby influencing key regulatory pathways including: cell cycle control, DNA repair and apoptosis. As such, proteasome inhibitors (PI) have exhibited broad therapeutic applications, particularly for multiple myeloma and mantle cell lymphoma with 3 inhibitors gaining FDA approval. However, covalent binding and lack of targeted action cause severe toxicity. Upon stimulation by inflammatory cytokines, constitutive proteasome (CP) active sites β1c, β2c and β5c are replaced with corresponding β1i, β2i and β5i subunits; forming the immunoproteasome (IP). The abundant CP is required for regular cell function, however due to upregulation in diseased states selective IP inhibition is associated with an increased therapeutic index. Recent identification of structural differences between CP and IP specificity pockets (S1-4) allows structure-based drug design. The cyclic peptides argyrin A and F exhibit potent, reversible CP inhibition with mechanisms distinct to existing therapeutics. In this project, argyrin B inhibition and binding interactions between the CP and IP are investigated, using purified enzyme assays alongside computational molecular modelling. Kinetic assays revealed argyrin B IC_50 values of 146.5 μM and 8.76 μM at β1c and β1i, respectively; a 16-fold difference with statistical significance. Whilst argyrin B also showed slight preference towards β5i over β5c, with low micromolar IC_50 values. The same trends were supported by Ki values and molecular docking estimated binding energies. AutoDock and FRED simulations suggest increased β1i S1 pocket hydrophobicity, T21S and G97H substitutions from β1c to β1i as key towards favourable β1i binding. At β5c, small, hydrophobic characteristics of S2 become polar in β5i that enhances argyrin B interactions. These findings facilitate design of further IP selective inhibitors, whilst the identification of the first known β1i selective and non-covalent PI shows great therapeutic potential with reduced toxicity proposed in comparison to existing therapeutics

Argyrin B a non-competitive inhibitor of the human immunoproteasome exhibiting preference for β1i

Inhibitors of the proteasome have found broad therapeutic applications however, they show severe toxicity due to the abundance of proteasomes in healthy cells. In contrast, inhibitors of the immunoproteasome, which is upregulated during disease states, are less toxic and have increased therapeutic potential including against autoimmune disorders. In this project, we report argyrin B, a natural product cyclic peptide to be a reversible, non-competitive inhibitor of the immunoproteasome. Argyrin B showed selective inhibition of the β5i and β1i sites of the immunoproteasome over the β5c and β1c sites of the constitutive proteasome with nearly 20-fold selective inhibition of β1i over the homologous β1c. Molecular modelling attributes the β1i over β1c selectivity to the small hydrophobic S1 pocket of β1i and β5i over β5c to site-specific amino acid variations that enable additional bonding interactions and stabilization of the binding conformation. These findings facilitate the design of immunoproteasome selective and reversible inhibitors that may have a greater therapeutic potential and lower toxicity

Association Between Schizophrenia-Related Polygenic Liability and the Occurrence and Level of Mood-Incongruent Psychotic Symptoms in Bipolar Disorder

Importance Bipolar disorder (BD) overlaps schizophrenia in its clinical presentation and genetic liability. Alternative approaches to patient stratification beyond current diagnostic categories are needed to understand the underlying disease processes/mechanisms. Objectives To investigate the relationship between common-variant liability for schizophrenia, indexed by polygenic risk scores (PRS) and psychotic presentations of BD, using clinical descriptions which consider both occurrence and level of mood-incongruent psychotic features. Design Case-control design: using multinomial logistic regression, to estimate differential associations of PRS across categories of cases and controls. Settings & Participants 4399 BD cases, 2966 (67%) female, mean age-at-interview 46 [sd 12] years, from the BD Research Network (BDRN) were included in the final analyses. For comparison genotypic data for 4976 schizophrenia cases and 9012 controls from the Type-1 diabetes genetics consortium and Generation Scotland were included. Exposure Standardised PRS, calculated using alleles with an association p-value threshold < 0.05 in the second Psychiatric Genomics Consortium genome-wide association study of schizophrenia, adjusted for the first 10 population principal components and genotyping-platform. Main outcome measure Multinomial logit models estimated PRS associations with BD stratified by (1) Research Diagnostic Criteria (RDC) BD subtypes (2) Lifetime occurrence of psychosis.(3) Lifetime mood-incongruent psychotic features and (4) ordinal logistic regression examined PRS associations across levels of mood-incongruence. Ratings were derived from the Schedule for Clinical Assessment in Neuropsychiatry interview (SCAN) and the Bipolar Affective Disorder Dimension Scale (BADDS). Results Across clinical phenotypes, there was an exposure-response gradient with the strongest PRS association for schizophrenia (RR=1.94, (95% C.I. 1.86, 2.01)), then schizoaffective BD (RR=1.37, (95% C.I. 1.22, 1.54)), BD I (RR= 1.30, (95% C.I. 1.24, 1.36)) and BD II (RR=1.04, (95% C.I. 0.97, 1.11)). Within BD cases, there was an effect gradient, indexed by the nature of psychosis, with prominent mood-incongruent psychotic features having the strongest association (RR=1.46, (95% C.I. 1.36, 1.57)), followed by mood-congruent psychosis (RR= 1.24, (95% C.I. 1.17, 1.33)) and lastly, BD cases with no history of psychosis (RR=1.09, (95% C.I. 1.04, 1.15)). Conclusion We show for the first time a polygenic-risk gradient, across schizophrenia and bipolar disorder, indexed by the occurrence and level of mood-incongruent psychotic symptoms

Childhood bullying, paranoid thinking, and the misappraisal of social threat: trouble at school

Background:Experiences of bullying predict the development of paranoia in school-age adolescents. While many instances of psychotic phenomena are transitory, maintained victimization can lead to increasingly distressing paranoid thinking. Furthermore, paranoid thinkers perceive threat in neutral social stimuli and are vigilant for environmental risk. Aims:The present paper investigated the association between different forms of bullying and paranoid thinking, and the extent to which school-age paranoid thinkers overestimate threat in interpersonal situations. Methods: Two hundred and thirty participants, aged between eleven and fourteen, were recruited from one secondary school in the UK. Participants completed a series of questionnaires hosted on the Bristol Online Survey tool. All data were collected in a classroom setting in quiet and standardized conditions. Results: A significant and positive relationship was found between experiences of bullying and paranoid thinking: greater severity of bullying predicted more distressing paranoid thinking. Further, paranoid thinking mediated the relationship between bullying and overestimation of threat in neutral social stimuli. Conclusion: Exposure to bullying is associated with distressing paranoid thinking and subsequent misappraisal of threat. As paranoid thinkers experience real and overestimated threat, the phenomena may persist

Hypoxia Sensitive Metal β-Ketoiminate Complexes Showing Induced Single Strand DNA Breaks and Cancer Cell Death by Apoptosis

A series of ruthenium and iridium complexes have been synthesised and characterised with 20 novel crystal structures discussed. The library of β-ketoiminate complexes has been shown to be active against MCF-7 (human breast carcino-ma), HT-29 (human colon carcinoma), A2780 (human ovarian carcinoma) and A2780cis (cisplatin resistant human ovarian carcinoma) cell lines, with selected complexes being more than three times as active as cisplatin against the A2780cis cell line. Complexes have also been shown to be highly active under hypoxic conditions, with the activities of some complexes increasing with a decrease in O2 concentration. The enzyme thioredoxin reductase is over-expressed in cancer cells and complexes reported herein have the advantage of inhibiting this enzyme, with IC50 values measured in the nanomolar range. The anti-cancer activity of these complexes was further investigated to determine whether activity is due to effects on cellular growth or cell survival. The complexes were found to induce significant cancer cell death by apoptosis with levels induced correlating closely with activity in chemosensitivity studies. As a possible cause of cell death, the ability of the complexes to induce damage to cellular DNA was also assessed. The complexes failed to induce double strand DNA break or DNA crosslinking but induced significant levels of single DNA strand breaks indi-cating a different mechanism of action to cisplatin

Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η6-p-cym)RuCl(PPh3)2]+, allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N′-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin-ruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 μM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 μM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 μM in DU-145 and IC50 = 11.33 μM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 μM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 μM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides

An impinging remnant meniscus causing early polyethylene failure in total knee arthroplasty: a case report

The management of patients with an apparently normal functional total knee arthroplasty (TKA) suffering from unexplained persistent pain and swelling is a challenging issue. The usual causes of pain after total knee replacement are well known, but there are a small number of patients in whom its aetiology is obscure. Malfunction due to soft tissue impingement has rarely been reported. A patient with an unusual case of posterior soft tissue impingement secondary to a trapped posterior horn of a remnant medial meniscus after TKA and responsible for severe early polyethylene wear, is reported. The diagnosis was confirmed by arthroscopy. Treatment was performed by arthrotomy. The meniscus remnant was removed followed by total synovectomy and isolated exchange of the polyethylene insert. To our knowledge, this is the first well-documented case reporting this association

Working with Young People Who Offend : An Examination of the Literature Regarding Violence, Substance Misuse and Harmful Sexual Behaviour

This paper presents a review of the recent literature relating to effective practice with young people displaying harmful sexual behaviour (HSB), violence or risky substance misuse. The intention is to build upon and update the 2007 literature review Research and practice in risk assessment and risk management of children and young people engaging in offending behaviour, funded by the Risk Management Authority (RMA) and carried out by the Scottish Centre for Crime and Justice Research (SCCJR)

Effects of plasma magnesium and prolactin on quantitative ultrasound measurements of heel bone among schizophrenic patients

<p>Abstract</p> <p>Background</p> <p>Osteoporosis is a bone disease that can reduce both bone mass and bone strength. It can cause serious fractures of bones, along with causing significant and even devastating physical, psychological and financial consequences for patients and their family members. Many reports have revealed that the prevalence of decreased bone density is higher in schizophrenic patients than in the non-psychological diseased population. The previous report of our group revealed that chronic schizophrenia patients have poorer BUA levels since they were young as compared to the general community population. Hyperprolactinemia and antipsychotics are reported to be among the risk factors for osteoporosis in chronic schizophrenic patients.</p> <p>Methods</p> <p>93 schizophrenic patients with severely poor adjusted BUA values and 93 age and gender matched patients with normal adjusted BUA values from a previous survey study were selected. Data were collected via questionnaires and via reviews of antipsychotic medications. Blood samples were drawn, and serum levels of prolactin, estradiol, testosterone, magnesium, calcium, phosphate, osteocalcin, Cross-linked N-teleopeptide of type I collagen (NTX), thyroid hormone and parathyroid hormone were checked. The association between BUA levels and serum levels of the above items, along with the type of received antipsychotic medication, was evaluated.</p> <p>Results</p> <p>There was no significant association found between reduced BUA levels and serum prolactin, calcium, phosphate, osteocalcin, NTX, thyroid stimulating hormone and parathyroid hormone levels. There was also no association between BUA levels and types of currently received antipsychotics. There was no association between BUA levels and menstruation condition in female patients. Hypermagnesemia had a borderline association with classical and combined (classical and atypical) antipsychotic medications in male patients. Nevertheless, hypermagnesemia is a significant protective factor of reduced BUA levels in female patients. Hyperprolactinemia had a significant association with classical and combined antipsychotic medications in female patients. Hyperprolactinemia, however, provides a protective effect on reduced BUA levels in male patients. There was no significant association found between serum prolactin level and the type of antipsychotic medication received.</p> <p>Conclusions</p> <p>The results of this study are in contrast with literature that has reported an association between bone mass and serum prolactin levels, serum magnesium levels and type of received antipsychotics. Further study to investigate the pathophysiological process and the association between bone mass and serum prolactin level, serum magnesium level and specific antipsychotics is necessary.</p