The identification and characterisation of novel inhibitors of the 17β-HSD10 enzyme for the treatment of Alzheimer's disease

In 2015, an estimated 46.8 million people were living with dementia, a number predicted to increase to 74.7 million by 2030 and 131.5 million by 2050. Whilst there are numerous causes for the development of dementia, Alzheimer’s disease is by far the most common, accounting for approximately 50-70% of all cases. Current therapeutic agents against Alzheimer’s disease are palliative in nature, managing symptoms without addressing the underlying cause and thus disease progression and patient death remain a certainty. Whilst the main underlying cause for the development of Alzheimer’s disease was originally thought to be an abnormal deposition of insoluble amyloid-β peptide derived plaques within the brain, the failure of several high-profile therapeutic agents, which were shown to reduce the plaque burden without improving cognition, has recently prompted a shift in focus to soluble oligomeric forms of amyloid-β peptide. Such soluble oligomers have been shown to be toxic in their own right and to precede plaque deposition. Soluble amyloid-β oligomers have been identified in various subcellular compartments, including the mitochondria, where they form a complex with the 17β-HSD10 enzyme resulting in cytotoxicity. Interestingly, hallmarks of this toxicity have been shown to be dependent on the catalytic activity of the 17β-HSD10 enzyme, suggesting two therapeutic approaches may hold merit in treating Alzheimer’s disease: disrupting the interaction between the 17β-HSD10 enzyme and amyloid-β peptide, or directly inhibiting the catalytic activity of the 17β-HSD10 enzyme. In 2006, Frentizole was identified as a small molecule capable of disrupting the 17β-HSD10/amyloid interaction. The work described herein details the generation of a robust screening assay allowing the catalytic activity of the 17β-HSD10 enzyme to be measured in vitro. This assay was subsequently employed for small molecule screening using two methodologies; first in a targeted approach using compounds derived from the Frentizole core scaffold, and second in an explorative manner using a diverse library of compounds supplied by the National Cancer Institute. As a result, a range of novel small molecule inhibitors of the 17β-HSD10 enzyme have been identified and the most promising characterised in terms of potency and mechanism of action. De-selection assays were developed to allow the efficient triage of hit compounds and work was begun on a cellular based assay which would allow the ability of compounds of interest to reverse a disease relevant phenotype to be assessed in a cellular environment. As such, we now have a number of hit compounds which will form the basis for the generation of subsequent series of derivatives with improved potency and specificity, as well as the robust assays required to measure such criteria, potentially leading to the generation of novel therapeutic agents against Alzheimer’s disease

A scan for genes associated with cancer mortality and longevity in pedigree dog breeds.

Selective breeding of the domestic dog (Canis lupus familiaris) rigidly retains desirable features, and could inadvertently fix disease-causing variants within a breed. We combine phenotypic data from > 72,000 dogs with a large genotypic dataset to search for genes associated with cancer mortality and longevity in pedigree dog breeds. We validated previous findings that breeds with higher average body weight have higher cancer mortality rates and lower life expectancy. We identified a significant positive correlation between life span and cancer mortality residuals corrected for body weight, implying that long-lived breeds die more frequently from cancer compared to short-lived breeds. We replicated a number of known genetic associations with body weight (IGF1, GHR, CD36, SMAD2 and IGF2BP2). Subsequently, we identified five genetic variants in known cancer-related genes (located within SIPA1, ADCY7 and ARNT2) that could be associated with cancer mortality residuals corrected for confounding factors. One putative genetic variant was marginally significantly associated with longevity residuals that had been corrected for the effects of body weight; this genetic variant is located within PRDX1, a peroxiredoxin that belongs to an emerging class of pro-longevity associated genes. This research should be considered as an exploratory analysis to uncover associations between genes and longevity/cancer mortality

Condensation properties of stress granules and processing bodies are compromised in Myotonic Dystrophy Type 1

RNA regulation in mammalian cells requires complex physical compartmentalisation, using structures thought to be formed by liquid-liquid phase separation. Disruption of these structures is implicated in numerous degenerative diseases. Myotonic dystrophy type 1 (DM1) is a multi-systemic trinucleotide repeat disorder resulting from an expansion of nucleotides CTG (CTGexp) in the DNA encoding DM1 protein kinase (DMPK). The cellular hallmark of DM1 is the formation of nuclear foci that contain expanded DMPK RNA (CUGexp) (with thymine instead of uracil). We report here the deregulation of stress granules (SGs) and processing bodies (P-bodies), two cytoplasmic structures key for mRNA regulation, in cell culture models of DM1. Alterations to the rates of formation and dispersal of SGs suggest an altered ability of cells to respond to stress associated with DM1, while changes to the structure and dynamics of SGs and P-bodies suggest that a widespread alteration to the biophysical properties of cellular structures is a consequence of the presence of CUGexp RNA.</p

6-benzothiazolyl ureas, thioureas and guanidines are potent inhibitors of ABAD/17β-HSD10 and potential drugs for Alzheimer's disease treatment : design, synthesis and in vitro evaluation

Background : The mitochondrial enzyme amyloid beta-binding alcohol dehydrogenase (ABAD) also known as 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) has been connected with the pathogenesis of Alzheimer’s disease (AD). ABAD/ 17β-HSD10 is a binding site for the amyloid-beta peptide (Aβ) inside the mitochondrial matrix where it exacerbates Aβ toxicity. Interaction between these two proteins triggers a series of events leading to mitochondrial dysfunction as seen in AD. Methods : As ABAD’s enzymatic activity is required for mediating Aβ toxicity, its inhibition presents a promising strategy for AD treatment. In this study, a series of new benzothiazolylurea analogues have been prepared and evaluated in vitro for their potency to inhibit ABAD/ 17β-HSD10 enzymatic activity. The most potent compounds have also been tested for their cytotoxic properties and their ability to permeate through blood-brain barrier has been predicted. To explain the structure-activity relationship QSAR and pharmacophore studies have been performed. Results and Conclusions : Compound 12 was identified being the most promising hit compound with good inhibitory activity (IC50 = 3.06 ± 0.40µM) and acceptable cytotoxicity profile comparable to the parent compound of frentizole. The satisfactory physical-chemical properties suggesting its capability to permeate through BBB make compound 12 a novel lead structure for further development and biological assessment.PostprintPeer reviewe

Stiffness is more than just duration and severity: A qualitative exploration in people with rheumatoid arthritis

Objective. Stiffness is internationally recognized as an important indicator of inflammatory activity in RA but is poorly understood and difficult to measure. The aim of this study was to explore the experience of stiffness from the patient perspective. Methods. Semi-structured interviews conducted with 16 RA patients were analysed independently by researchers and pat.ient partners using inductive thematic analysis. Results. Six themes were identified. Part of having RA identified stiffness as a normal consequence of RA, perceived as associated with disease-related aspects such as fluctuating disease activity, other RA symptoms and disease duration. Local and widespread highlighted stiffness occurring not only in joints, but also over the whole body, being more widespread during the morning or flare. Linked to behaviour and environment illustrated factors that influence stiffness, including movement, medications and weather. Highly variable captured the fluctuating nature of stiffness within and between patients and in relation to temporality, duration and intensity. Impacts on daily life emphasized the effect of stiffness on a range of domains, including physical function, quality of life, psychological well-being, activities of daily living and participation in work and leisure activities. Requires self-management detailed self-management strategies targeting both the symptom and its consequences. Conclusion. Patients’ experiences of stiffness were varied, complex and not exclusive to the morning period. Importantly, stiffness was reported in terms of impact rather than the traditional measurement concepts of severity or duration. Based on these findings, further research is needed to develop a patient-centred measure that adequately reflects inflammatory activity

Design, synthesis and in vitro evaluation of benzothiazole-based ureas as potential ABAD/17β-HSD10 modulators for Alzheimer’s disease treatment

This work was supported by the Ministry of Health of the Czech Republic (no. NV15-28967A), Charles University in Prague (no. GAUK B-CH/992214, SVV 260 291) and the Alzheimer’s Society (specifically The Barcopel Foundation). This research is part-funded by the MSD Scottish Life Sciences fund.Amyloid-beta peptide (Aβ) has been recognized to interact with numerous proteins, which may lead to pathological changes in cell metabolism of Alzheimer’s disease (AD) patients. One such known metabolic enzyme is mitochondrial amyloid-binding alcohol dehydrogenase (ABAD), also known as 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10). Altered enzyme function caused by the Aβ-ABAD interaction, was previously shown to cause mitochondrial distress and a consequent cytotoxic effect, therefore providing a feasible target in AD drug development. Based on previous frentizole derivatives studies, we report two novel series of benzothiazolyl ureas along with novel insights into the structure and activity relationships for inhibition of ABAD. Two compounds ( 37 , 39 ) were identified as potent ABAD inhibitors, where compound 39 exhibited comparable cytotoxicity with the frentizole standard; however, one-fold higher cytotoxicity than the parent riluzole standard. The calculated and experimental physical chemical properties of the most potent compounds showed promising features for blood-brain barrier penetration.PostprintPeer reviewe

General practitioners’ perspectives on campaigns to promote rapid help-seeking behaviour at the onset of rheumatoid arthritis

Objective. To explore general practitioners’ (GPs’ ) perspectives on public health campaigns to encourage people with the early symptoms of rheumatoid arthritis (RA) to seek medical help rapidly. Design. Nineteen GPs participated in four semistructured focus groups. Focus groups were audio-recorded, transcribed verbatim, and analysed using thematic analysis. Results. GPs recognised the need for the early treatment of RA and identified that facilitating appropriate access to care was important. However, not all held the view that a delay in help seeking was a clinically significant issue. Furthermore, many were concerned that the early symptoms of RA were often non-specific, and that current knowledge about the nature of symptoms at disease onset was inadequate to inform the content of a help-seeking campaign. They argued that a campaign might not be able to specifically target those who need to present urgently. Poorly designed campaigns were suggested to have a negative impact on GPs’ workloads, and would “clog up” the referral pathway for genuine cases of RA. Conclusions. GPs were supportive of strategies to improve access to Rheumatological care and increase public awareness of RA symptoms. However, they have identified important issues that need to be considered in developing a public health campaign that forms part of an overall strategy to reduce time to treatment for patients with new onset RA. This study highlights the value of gaining GPs’ perspectives before launching health promotion campaigns

The summer undergraduate research experience as a work-integrated learning opportunity and potential pathway to publication in psychology

© 2019 Golding, Breen, Krause and Allen. Unlike disciplines which focus on skill development from year one of a bachelor's degree, training in psychology in Australia follows the scientist-practitioner model. According to this model, an undergraduate psychology degree should focus on the scientific principles underpinning the discipline and provide a foundation for the development of professional skills in graduate school. However, most Australian psychology undergraduates do not continue into graduate school, and concerns have been raised about their lack of applied skills and work-readiness. Work-integrated learning (WIL) refers to strategies aimed at providing students with practical experiences (e.g., fieldwork, placements, and internships) directly related to their course of study. The objective of WIL is to increase work-readiness. Accreditation standards coupled with the norms of the discipline have historically prevented the inclusion of typical WIL experiences in Australian undergraduate psychology degrees. However, one particular type of WIL activity-the undergraduate research experience (URE)-is particularly suited to psychology. In a typical URE, students collaborate with faculty to conduct research designed to make an original contribution to their field. The current study is a qualitative investigation of stakeholder perceptions of a competitive summer URE program ran from 2012 to 2016. Six faculty members and seven undergraduate students were engaged in semi-structured interviews about their URE experiences. Constructed themes broadly reflected the benefits and challenges of the program and included work-readiness and additional research experience, networking and teamwork, publication, quality of experience and equity of opportunities. Faculty members and students spoke favorably of their UREs in most cases, although issues of administration and financial concerns were mentioned consistently, as were concerns about the length, timing, and nature of projects. Students reported skill development and networking as two of the key benefits of their participation in the program, and article publication was seen as particularly beneficial to career prospects. Our findings suggest that student co-authored publications resulting from UREs are possible, but careful thought is required to optimize their likelihood. Overall, this research adds to a growing literature suggesting that UREs can confer a range of benefits to Australian psychology schools related to increased research capacity and student satisfaction

Broadly neutralizing antibodies from an individual that naturally cleared multiple hepatitis c virus infections uncover molecular determinants for E2 targeting and vaccine design

Cumulative evidence supports a role for neutralizing antibodies contributing to spontaneous viral clearance during acute hepatitis C virus (HCV) infection. Information on the timing and specificity of the B cell response associated with clearance is crucial to inform vaccine design. From an individual who cleared three sequential HCV infections with genotypes 1b, 1a and 3a strains, respectively, we employed peripheral B cells to isolate and characterize neutralizing human monoclonal antibodies (HMAbs) to HCV after the genotype 1 infections. The majority of isolated antibodies, designated as HMAbs 212, target conformational epitopes on the envelope glycoprotein E2 and bound broadly to genotype 1–6 E1E2 proteins. Further, some of these antibodies showed neutralization potential against cultured genotype 1–6 viruses. Competition studies with defined broadly neutralizing HCV HMAbs to epitopes in distinct clusters, designated antigenic domains B, C, D and E, revealed that the selected HMAbs compete with B, C and D HMAbs, previously isolated from subjects with chronic HCV infections. Epitope mapping studies revealed domain B and C specificity of these HMAbs 212. Sequential serum samples from the studied subject inhibited the binding of HMAbs 212 to autologous E2 and blocked a representative domain D HMAb. The specificity of this antibody response appears similar to that observed during chronic infection, suggesting that the timing and affinity maturation of the antibody response are the critical determinants in successful and repeated viral clearance. While additional studies should be performed for individuals with clearance or persistence of HCV, our results define epitope determinants for antibody E2 targeting with important implications for the development of a B cell vaccine.</div

Small-molecule inhibition of METTL3 as a strategy against myeloid leukaemia.

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5-7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy