Deadenylase enzymes as potential novel drug targets in osteoporosis, bone disease, and repair

Lead Research Organisation: University of Nottingham
Department Name: Sch of Pharmacy

Abstract

In the UK, women over 45 years old spend more time in hospital due to osteoporosis than heart disease, diabetes or breast cancer. Common hip, wrist, and spinal bone fractures due to decreased bone density are a huge financial cost to the NHS and impact significantly on morbidity and the quality of life, in particular of women and the elderly. Although several options are available for the prevention and treatment of osteoporosis, currently available drugs are not always effective. Moreover, not all patients tolerate the available medications due to side-effects.
If successful, our project is the first step towards novel drugs of the future that may be useful for the treatment of osteoporosis, bone disease and repair. These future drugs will have more desirable characteristics compared to current medications. They will be available as tablets, have fewer side-effects and enhance bone formation directly by stimulating bone-forming cells. The idea behind the selection of the enzyme target in bone-forming cells comes from genetically engineered mice with heavy bones and studying the relevant enzymes in great detail. We will search for drug-like molecules by screening a large collection of thousands of compounds using robots. In parallel, we will use high-performance computer clusters to screen a database with information of up to 1 million compounds using ?cheminformatics?. We will then optimise our hits using synthetic chemistry and test the activity of our compounds by looking at bone formation in cell culture dishes. If successful, our research will identify molecules that may represent candidates for future drug development.

Technical Summary

In this proof of concept project, we shall explore the development of small-molecule inhibitors of deadenylase enzymes as a novel therapeutic approach for bone-related diseases, such as osteoporosis. We hypothesise that small-molecule inhibitors of deadenylase enzymes involved in mRNA degradation lead to increased osteoblast activity, will enhance bone density in osteoporotic bone, and promote the healing of bone fractures. The basis for this hypothesis is the observation that the CNOT7 deadenylase is a repressor of osteoblast cell activity as exemplified by the observation that CNOT7-null mice have increased bone density. Similar effects on bone are observed in mice lacking the Tob protein, which interacts with the CNOT7 gene product as a positive regulator. The CNOT7 protein is a poly(A) ribonuclease involved in cytoplasmic mRNA degradation that acts as a repressor of bone morphogenic protein-mediated signalling in osteoblasts.
We aim to identify small-molecule deadenylase inhibitors using an interdisciplinary approach that brings together expertise in the biochemistry and cell biology of gene regulation, the biology of osteoblast cells and bone tissue, and screening capabilities and medicinal chemistry at the University of Nottingham. We shall use complementary approaches to maximise the potential of identifying small-molecule inhibitors. In addition to virtual ligand screening based on high-resolution crystal structures that are available in the public domain, we will develop and deploy a fluorescent deadenylase assay screen of an extensive, chemically diverse library of small drug-like molecules using robotics. Hit compounds will be verified and their selectivity determined using secondary biochemical and cell-based screens with a range of deadenylase enzymes. Hit-to-lead conversion medicinal chemistry will be carried out and lead compounds will be profiled extensively in terms of target and cell potency, selectivity, specificity, and pharmacological action, including the use of human and rodent osteoblast cells. If successful, this strategy will identify lead compounds that can modulate bone formation in cell-based models in a manner that suggests potential therapeutic use, and that may represent candidates for future drug development. Furthermore, such compounds will have much wider use as chemical probes in molecular and cellular medicine.
 
Description Studentship MRC DTP
Amount £85,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2015 
End 04/2019
 
Title New reagents 
Description A series of compounds were generated and characterised in terms of their inhibitory activity versus the human Caf1/CNOT7 and PARN enzymes. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact These compounds received interest from a number of academic groups in Europe and the US. It resulted in the formal exchange of reagents with one group in Germany and possible future collaborations were discussed with the National Cancer Institute, USA. 
URL https://www.ncbi.nlm.nih.gov/pubmed/26299350
 
Title Novel activities for known chemical entities 
Description We disclosed novel activities for five known chemical entities. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Proof-of-principle that deadenylase enzymes, which are potential targets in osteoporosis and bone repair, can be targeted by small-molecule inhibitors. 
URL https://www.ncbi.nlm.nih.gov/pubmed/24170810
 
Title Screening Assay 
Description Screening assay for the discovery and characterisation of small-molecule inhibitors of deadenyase enzymes 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Publication Discovery of novel activities of known chemical entities Characterisation of new activities of novel and known chemical entities 
URL http://nar.oxfordjournals.org/content/early/2013/10/28/nar.gkt972.full
 
Title pQE80L-CNOT6L 
Description DNA product (plasmid): Prokaryotic expression vector PQE80L containing a CNOT6L cDNA that is codon optimised for expression in Escherichia coli. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This plasmid made it possible to express and purify the Ccr4/CNOT6L protein, which is used to assess the selectivity of drug-like inhibitors of the Caf1/CNOT7 enzyme. 
 
Title pQE80L-CNOT7 
Description DNA product (plasmid): Prokaryotic expression vector PQE80L containing a CNOT7 cDNA that is codon optimised for expression in Escherichia coli. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This plasmid made it possible to express and purify the Caf1/CNOT7 protein used in a biochemical screening assay to identify drug-like inhibitors of this enzyme. 
 
Description Collaboration China 
Organisation Nankai University
Country China 
Sector Academic/University 
PI Contribution My lab provided materials - compounds produced as part of the MRC award.
Collaborator Contribution The partner produced large amounts of the protein target, carried out crystallisation, and further structural analysis is in progress.
Impact The collaboration is ongoing; there are currently no outputs.
Start Year 2016
 
Description Collaboration Heidelberg 
Organisation Heidelberg University
Department Center for Molecular Biology (ZMBH)
Country Germany 
Sector Academic/University 
PI Contribution Provided unique compounds synthesised in the course of the grant for testing on Trypanosomes.
Collaborator Contribution Partners will carry out the bulk of the work. The effect of deadenylase inhibitors on trypanosome biology will be studied.
Impact N/A
Start Year 2015
 
Description Collaboration NCI 
Organisation National Cancer Institute (NCI)
Country United States 
Sector Public 
PI Contribution Antibodies and DNA expression plasmids that were generated in the course of the grant were shared to investigate the involvement of the CNOT7 ribonuclease in breast cancer metastasis. Contributions to experimental design were made. Following this collaboration, which validated the CNOT7 nuclease as a potential drug target for the prevention of breast cancer metastasis, follow-up funding was sought from two cancer charities (Breast Cancer Now, Worldwide Cancer Research; unsuccessful).
Collaborator Contribution The bulk of the work was carried out by the partners. A mouse model was used to study the involvement of the CNOT7 ribonuclease in breast cancer metastasis. CNOT7 was genetically validated as a potential target to reduce the metastatic potential of breast cancer cells. Following this collaboration, which validated the CNOT7 nuclease as a potential drug target for the prevention of breast cancer metastasis, follow-up funding was sought from two cancer charities. The applications were supported by a letter-of-support from the collaborator, who has access to NCI core facilities for PK/PD development in model organisms.
Impact Publication Faraji F, Hu Y, Yang HH, Lee MP, Winkler GS, Hafner M, et al. (2016) Post-transcriptional Control of Tumor Cell Autonomous Metastatic Potential by CCR4-NOT Deadenylase CNOT7. PLoS Genet 12(1): e1005820. doi:10.1371/journal.pgen.1005820
Start Year 2011
 
Description University Online Press Release 2011 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? Yes
Type Of Presentation Paper Presentation
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact A press release was written with the University's Media Relations Manager and approved by the funder. The piece was published on the home page of the University of Nottingham under the heading 'Latest news'.
http://www.nottingham.ac.uk/news/pressreleases/2011/november/drug-hunters-rebuild-our-bones.aspx

The news item was also published on the home page of the School of Pharmacy, University of Nottingham.
http://www.nottingham.ac.uk/pharmacy/news/news2011/new-treatment-for-fragile-bones.aspx


While the press release was not picked up by (local) press, many members of the pubic have viewed the news items. No evaluation of page views was completed.
Year(s) Of Engagement Activity 2011