Unconventional chemistry to generate enabling reporting tools to be used in mono- and poly-ADP-ribosylation biology

Lead Research Organisation: Queen's University of Belfast
Department Name: Sch of Pharmacy

Abstract

Biologists working in the field of protein modifications are in need of new tools to interrogate the cellular events and mechanisms which are linked to the transfer of adenosine diphosphate (ADP) ribose units from nicotinamide adenine dinucleotide (NAD) on proteins. Through this transfer, NAD plays a pivotal role in the regulation of DNA repair, stress resistance and cell death, linking metabolism to cell survival. In mammalian cells, mono-ADP ribosylation reactions are catalysed by mono-ADP-ribosyltransferases (MART) which transfer one ADP-ribosyl unit on proteins' side chains as means of regulating the activity of the latter. Whilst having been under intense scrutiny for over a decade because of their importance in cellular homeostasis, cell division, cellular "ageing" and cancer, both the MART enzymes and their biological targets have largely remained elusive. This is mainly due to the lack of effective and specific techniques to study that biology, more particularly the limited access to specific antibodies. A key factor to identify ADP-ribosylated proteins in blot experiments and mass spectroscopy analyses is to have access to specific antibodies which can detect and report the presence of such modified proteins in functional assays. The current challenges in preparing such antibodies come from the fact that mono-ADP ribosylated peptides must remain stable to degrading enzymes for as long as the immune response is actively fighting the foreign entity and creating antibodies. To access modified proteins which look like mono-ADP ribosylated proteins but which are more stable, NAD analogues which are good substrates for MART must be synthesised. Additionally, the modifications thus introduced must be sufficiently minimal to induce the generation of antibodies which will also recognise the "true" ADP-ribosylated protein targets. This latter aspect is critical if these antibodies are to become widely used in MAR biology.
The biology of polymeric ADP-ribosylation of proteins, catalysed by ADP-ribosyl polymerases (PARP) has been more readily studied due to the development of a broad range of finely-tuned biological tools, such as highly efficient antibodies and in situ detection assays. In human, five members of the PARP family are DNA binding enzymes activated by breaks to the DNA and are critical to the base excision repair process, with the sixth member (PARP13) being a newly identified mRNA binding protein. Despite extensive research leading to major drug discovery programs, poly(ADP-ribose) (PAR), as a chemical entity, has retained numerous levels of unexplored complexity which are critical to understanding its cellular properties. More specifically, the recent identification of four different poly(ADP-ribose) binding motifs have revolutionised how the decoding of this "appendage" by its partner-proteins can be explained. The limitations now reside in accessing functional and structurally defined short strands of poly-ADP-ribose that would help probe and rationalise the recognition and recruitment events elicited by the binding partners for these structurally different epitopes.
Therefore, substantial progress could be achieved in the field of ADP-ribosylation if specific antibodies to MAR-ylation could be generated and stable structure-specific PAR fragments became more widely available. To access this type of tools, novel chemistry must be devised to access the required functionalities in a modular manner.
We are the only synthetic laboratory that has specialised in phosphorus and nucleoside chemistry where the use of unconventional solvents and mechanochemistry allows for high synthetic efficacy in the preparation of NAD and ADP-ribose type molecules. Here, we will synthesise NAD derivatives which will be used to raise antibodies specific to protein MAR-ylation and PAR fragments with reporting properties to enable structural and functional analyses of the poly-ADP-ribolome.

Technical Summary

In the field of ADP-ribosylation, current limiting factors include a lack of specific antibodies that detect proteins which have been mono-ADP-ribosylated and a lack of chemically defined short polymeric ADP-ribosyl fragments to enable functional analyses of the poly-ADP-ribolome.
The biology associated with polymeric ADP-ribosylation catalysed by polymerases (PARP) has been extensively studied following the identification in the 1960s of PARP1, one of the six polymerases belonging to the ADP-ribosyltransferase family. In contrast, the nine mono-ADP-ribosyltransferases (MART) and their modified protein targets have largely remained elusive. Key to this delay is the lack of antibodies that are specific for mono-ADP-ribosylated proteins and that report the presence of such functionalisation in blot assays. Similarly, poly(ADP-ribose) (PAR), as a structurally complex appendage, remains challenging in terms of studying its functional diversity. The present limitation is due to the fact that short strands of poly-ADP-ribose that can probe the poly-ADPR's specific protein binding mechanisms are not available.
Substantial progress could be achieved, if specific antibodies to mono-ADP-ribosylated peptides could be generated and if stable poly-ADP-ribosyl fragments became available. To achieve this, novel chemical tools need to be devised.
We have developed effective phosphorus and nucleoside chemistry which depends on the use of ionic liquids and mechanochemistry to synthesise NAD and ADP-ribose derivatives, which we will apply to address these needs. Biologists, experts in the field, will use the novel NAD derivatives to raise antibodies specific to protein MAR-ylation and will gain access to ADP-ribosyl fragments to investigate how these units control the macromolecular recruitment and recognition processes by protein partners. As a result of this work, new therapeutic opportunities are likely to emerge as new functional biology will be revealed.

Planned Impact

We have developed an expertise equal to none in handling phosphorus reagents in ionic liquids and this has placed us in a position whereby we can address, otherwise challenging, chemistry in high yields with excellent overall atom efficiency under sustainable recycling conditions. Combining this expertise to mechanochemistry has been our most recent success and its implementation to address the present biological challenges will further exemplify the leading aspect of our approach to advanced phosphorus and nucleoside chemistry.
Through this synthetic effort, we will greatly enhance the UK knowledge economy by generating new basic knowledge (e.g. functionalisable phosphorus and new chemical conversions and processes; new structural and functional ADP ribose related biology) and novel technology (e.g. novel chemical reporting entities; scalable chemical processes; novel antibodies, new biological assays) and thus provide a means to further advance the field of nucleotide chemistry and (Mono-Poly)ADP-ribosyl biology, with wide ranging impacts on health and ageing-related diseases.
Due to the breadth of the PAR and MAR research fields, this work will contribute to the international academic advancement by addressing some of the current limitations encountered worldwide in terms of functional tools. It is also anticipated that this work will provide strong evidence to support future grant applications to international funding organisations such as the EC-Horizon 2020 or the World Cancer Research Fund International and the Human Frontier Science Program.
Critically, this work will have an impact in both academia and industry as the tools which will be developed will become main-stream entities for research in this field, and could potentially be commercialised. It is anticipated that national companies like Bioserv UK and Cambridge Bioscience (antibody manufacturers) and international companies like Jena Biosciences (manufacturer of nucleotide-based research tools), Sigma-Aldrich and Invitrogen will welcome the opportunity to generate yet untapped market products of this type.
Finally, we also aim to demonstrate that one can embed scalability and atom/energy efficiency at the onset of the syntheses' design in projects where biological research is the main driving force. Here, this work will demonstrate how one can implement green chemistry and sustainable approaches to enable biology-driven projects. This will further strengthen the leading role of the UK in achieving sustainability through research.

Publications

10 25 50
 
Description We have developed new means to prepare small molecules that are to be used by biologists who wish to better understand the chemistry of ADP-ribosylation and the effect of post-translational protein modification by NAD.
Exploitation Route publication
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology

 
Description Following on from this work on ADPR-derivatives, new opportunities have arisen to work with a Nucleic Acid manufacturing company, in the US. This is interest arose from our demonstrated know-how in the handling of hydrolytically unstable nucleoside phosphoramidites.
First Year Of Impact 2015
Sector Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description BBSRC- research grant
Amount £439,435 (GBP)
Funding ID BB/N001842/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2016 
End 12/2018
 
Description Industrial grant
Amount £125,000 (GBP)
Organisation ChromaDex 
Sector Private
Country United States
Start 06/2015 
End 05/2017
 
Title new NAD chemical tools 
Description NAD precursors which increase NAD bioavailability and manipulate enzymatic activity through PARylation or deaceylation, along with changing in the cellular redox capacity. New NAD derivatives for antibodies development (later part not yet completed) 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2015 
Provided To Others? Yes  
Impact cellular processes have been uncovered using these novel metabolites 
 
Description ChromaDex 
Organisation ChromaDex
Country United States 
Sector Private 
PI Contribution Synthetic know how on nucleosides and nucleotides
Collaborator Contribution Starting materials and expensive reagents
Impact New untraced tical and new potential biologically active nucleosides
Start Year 2011
 
Description Professor C. Brenner- University of Iowa 
Organisation University of Iowa
Department Department of Biochemistry
Country United States 
Sector Academic/University 
PI Contribution Synthesis of chemical entities to probe the NAD metaoblome; Hosting of QUB team member at the university of Iowa; 2 x1 visiting fellows (QUB PhD students) hosted in the Brenner lab.
Collaborator Contribution Cell work; animal work; access to facilties and know-how.
Impact publication; fellowship; mobility ofresearchers
Start Year 2014
 
Description Professor M. Ziegler Collaboration 
Organisation University of Bergen
Country Norway 
Sector Academic/University 
PI Contribution Provide chemical entities to probe the NAD metaoblome
Collaborator Contribution evaluation in cell lines
Impact publication Sept 2015
Start Year 2015
 
Description collaboration with Innovative DNA Technology 
Organisation Integrated DNA Technologies
Country United States 
Sector Private 
PI Contribution Work on ionic liquids stabilised phosphoramidite
Collaborator Contribution New reagents on which to apply ionic liquids technology
Impact NDA in place. Only chemistry
Start Year 2014