Award details

Next generation auxins and anti-auxins : principles for binding and design

Reference	BB/L010623/1
Principal Investigator / Supervisor	Professor Stefan Kepinski
Co-Investigators / Co-Supervisors
Institution	University of Leeds
Department	Ctr for Plant Sciences
Funding type	Research
Value (£)	358,503
Status	Completed
Type	Research Grant
Start date	31/03/2014
End date	30/03/2017
Duration	36 months

Abstract

We will quantify the initial molecular events in auxin binding and, with the aid of chemometrics, develop principles for the development of next-generation selective auxins, both agonists and antagonists. Surface plasmon resonance will be used to measure on- and off-rates for TIR1 and AFB5 in the absence of co-receptor to define binding to this pocket alone. Isothermal titration calorimetry will be used to determine thermodynamic parameter differences in deltaH, deltaG and deltaS between TIR1 and AFB5. Values measured will be combined with compound chemometrics. We believe that the TIR1/AFBs may have auxin-dependent prolyl cis-trans isomerase activity. NMR will be used to clarify degron transitions in solution and bound. Importantly, the structure of full-length Aux/IAA proteins will be determined to report on the endogenous disposition of the degron. We will add co-receptor degron peptides to measure thermodynamics associated with cis-trans isomerisation. Stapled peptides will be used to measure TIR1/AFB5 isomer preferences by SPR. Having determined detailed information on the two initial steps of co-receptor assembly we will refine our chemometric ligand classifications and combine the information to create a platform for discovery of TIR1/AFB selective compounds. We will screen a knowledge-led selection of the Syngenta compound library to add robustness to existing datasets and test for early compound predictions. Adding fragment bin screening with SPR will add naive information about each distinct binding pocket to create distinct pharmacophoric models based on receptor binding alone. With these anchor-sites defined, selective anti-auxins will be designed elaborated in chemical space outside the TIR1/AFB binding pocket using what is learnt from degron presentation. Anti-auxin synthesis (Prof Hayashi) and evaluation will follow, using both biophysical and in vivo plant assays and novel reporter lines generated to provide target-specific readouts.

Summary

Context: Plant growth and development are dynamically controlled by hormones. Hormones are mobile signalling molecules which coordinate growth in response to environmental cues. Auxin is a hormone and is involved in almost every part of a plant's life, from embryo to wood. In order for auxin to trigger responses it needs a receptor, a protein to which it binds in a very specific and defined way. Auxin binding acts as a molecular switch, initiating a chain of events that leads to changes in which whole groupings of the plant's genes are switched on or off to change developmental decisions. We have been studying a protein called TIR1 as an auxin receptor, along with members of its family called AFB proteins. We have shown that some auxins (there are many) are selective for one or other receptor family member. Aims and objectives: This proposal describes a set of experiments that allows us to specify and quantify the changes between family members. In turn, this allows us to describe the special features on each type of auxin which determine specificity and allows us to start to understand the molecular rules defining this specificity. Auxins are also valuable agrochemicals. In their main application as herbicides they already present a certain element of selectivity, killing broad-leaved weeds in preference to cereals. However, we now know that there are more layers of selectivity to be exploited. This makes it imperative that we learn much more detail about the rules of specificity if we are to design a new generation of selective plant growth regulators. Our project sets out a number of complimentary lines of experimentation to investigate in great detail the features which differentiate AFB5, for example, from TIR1. We will use the latest biophysical techniques to measure the speeds of binding and the energy changes on binding. By comparing these values and comparing them with computer-driven calculations of the auxin molecules themselves, we will be able to derive design features specific for each template. Further, we know that when auxin binds to its receptor, this interaction creates a binding site for a second protein, a co-receptor. We believe that the TIR1 receptor acts as an enzyme to modify the shape of the co-receptor during binding. We will investigate this hypothesis and add kinetic details of this second part of the co-receptor assembly into our molecular models. With the two primary stages completed, we will have a matrix of detailed information about what makes a molecule an auxin and how they are selective and we will use this as a platform for designing new auxins and anti-auxins. These will be made by colleagues and tested for efficacy and selectivity. Potential applications and benefits: Examples of agricultural use of auxins include treatments to flowers, fruits and nuts and as selective weedkillers to kill broadleaved plants, not cereals and grasses and are of great agricultural value. Up to now, millions of compounds have been made and screened to find the chemicals we use. This project will measure in fine detail the very special interactions made by auxins at their several, but specific target sites. From this information we will start to define rules for new and more selective auxinic agrochemicals because, so far, agriculture has exploited only auxin analogues. Our technologies will enable us to add selective auxin antagonists (anti-auxins) into the toolkit. The aim is to create a new generation of safe, selective and low dosage agricultural compounds.

Impact Summary

Our primary route to exploitation will be in collaboration and under the guidance of Syngenta working with our University technology transfer offices . The PI and Co-I have a long-established relationship with Syngenta Jealott's Hill. Some of the key resources provided in the previous IPA project are still in use under agreement (e.g. recombinant baculovirus lines) and we value this relationship highly. A new Agreement for access and confidentiality, covering IP generated in this project by individual parties and joint discovery, and covering rights for exploitation by all partners will be drawn up if, and as soon as, the project is supported (as noted in the letter of support from Syngenta). We expect the project to create a novel platform from which new auxinics may be predicted or designed. This platform, based on the many quantitative datasets generated by the PI and Co-I and the chemometric analyses contributed by Ferro (Bonn), will be tested through the synthetic chemistry of Hayashi. These third parties bring much foreground IP to the partnership and this will be recognised. The platform itself may have high exploitation value and we would work closely with our University technology transfer offices to ensure that the partners recognise all subsequent income, but our primary drive will be to make the research work available. It is expected that most parts of the raw research findings, synthetic procedures, understanding of plant processes will be published in refereed journals without undue delay. Syngenta have always supported open access publication of jointly supported research results. The algorithms generated by Ferro may need special consideration, but this will be primarily an agreement between IPTC Bonn and Syngenta, although the PI and Co-I will be involved because the algorithms will be fed by the data from this project. Beneficiaries include not only Syngenta, but other agrochemical companies as more is learnt and published about the principal target sites for auxinic herbicides. Managing resistance to herbicides is becoming an increasing problem worldwide. Auxinics are amongst the oldest lineages of herbicides and resistance is widespread, but local in nature. Indeed, the next generation of GM herbicide tolerant crops will target auxin (oxyacetic acid) metabolism (Dow AgroSciences) on top of glyphosate resistance (Monsanto), showing that there is confidence that there remain considerable commercial potential in auxinic products. Nevertheless, all stakeholders need to be wise to, and manage these products for the future and greater knowledge of the protein target sites will be invaluable. Further, from our work, novel routes to selective auxinic resistance can be envisaged such that new generations of herbicide tolerant crops may be realised through (natural variation using markers, TILLED breeding selections etc., as well as GM). We will put forward additional project proposals to follow such opportunities. Given the utility of auxins as agrochemicals (in e.g. fruit farming as well as herbicides), beneficiaries further downline include farmers and growers, and ultimately all those with responsibility towards Food Security. Herbicides are essential for sustaining and growing the productivity of all managed crops (outside the organics sector), The PI and co-I will ensure we take the research and its more general importance out to public and non-specialist audiences in order to contribute to awareness on the issues of herbicides in agriculture and of the importance of blue-skies research for agriculture and food security. This project will help establish the leadership for this research in the UK, and with the primary route to exploitation benefitting the UK economy through Syngenta and lead partners. Part of our role as leaders will be to ensure that the PDRAs and students who become associated with the work are trained for drug discovery in the agricultural sector.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Plant Science, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/L009366/1 Next generation auxins and anti-auxins : principles for binding and design

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