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

Lead Research Organisation: University of Leeds
Department Name: Ctr for Plant Sciences


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.

Technical Summary

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.

Planned Impact

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.


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Description We have used nuclear magnetic resonance (NMR) spectroscopy to characterise the structure of the amino-terminal (N-terminal) half (DI/DII) of the auxin co-receptor protein AXR3 both in isolation and in complex with the receptor TIR1 and the plant hormone auxin. Our results show that auxin triggers a global binding event between the intrinsically disordered AXR3 DI/DII protein and the TIR1 receptor. The early events of the complex formation were distinguished with the synthetic auxin cvxIAA, which has restricted access to the auxin binding pocket. Crucially, we show that cvxIAA triggers the auxin-dependent binding event between AXR3 with TIR1, before the degron core is fully docked. This indicates auxin is likely to have a role in complex formation which precedes the final binding state and represents an encounter complex. This provides an important insight into the formation of this crucial molecular interaction in plants, which regulates almost every aspect of plant development. Our work highlights the possibility of additional target sites at the interface of these proteins for the development of more selective auxinic herbicides and growth regulators to help protect food security and the environment. Manuscripts are currently being prepared for the publication of these exciting findings.
Exploitation Route We anticipate that our finding will eventually to used in the rational design of new agrochemicals.
Sectors Agriculture, Food and Drink,Chemicals,Environment

Description Member of the Advisory Committee of the UK Plant Science Federation
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
Description Beeckman lab collaboration 
Organisation Flanders Institute for Biotechnology
Country Belgium 
Sector Charity/Non Profit 
PI Contribution We have provided research materials and shared data ahead of publication. We performed biochemical experiments for the papers listed below.
Collaborator Contribution They have provided research materials and shared data ahead of publication.
Impact De Rybel, B., Audenaert, D., Xuan, W., Overvoorde, P., Strader, L.C., Kepinski, S., Hoye, R., Brisbois, R., Parizot, B., Vanneste, S., Liu, X., Gilday, A., Graham, I.A., Nguyen, L., Jansen, L., Njo, M.F., Inzé, D., Bartel, B., Beeckman, T. (2012) A role for the root cap in root branching revealed by the non-auxin probe naxillin. Nature Chemical Biology 8: 798-805 De Rybel, B., Audenaert, D., Beeckmann, T., Kepinski, S. (2009) The past, present and future of chemical biology in auxin research. ACS Chemical Biology 4(12), 987-998
Start Year 2009
Description Estelle lab collaboration 
Organisation University of California, San Diego (UCSD)
Country United States 
Sector Academic/University 
PI Contribution We have provided research materials and shared data ahead of publication. We performed biochemical/biophysical experiments for the papers listed below.
Collaborator Contribution They have provided research materials and shared data ahead of publication.
Impact alderon Villalobos, L-I., Lee, S., Armitage, L., Parry, G., Mao, H., De Oliveira, C., Ivetac, A., Brandt, W., McCammonn, A., Zheng, N., Napier, R., Kepinski, S., Estelle, M. (2012) TIR1/AFBs and Aux/IAAs constitute a combinatorial co-receptor system to perceive auxin with differential sensitivities. Nature Chemical Biology 8: 477-485
Start Year 2007
Description Ferro lab collaboration 
Organisation University of Bonn
Country Germany 
Sector Academic/University 
PI Contribution We have shared data in advance of publication and discussed the significance of the calculation and analysis performed by the Ferro lab.
Collaborator Contribution Quantum chemical analysis of our data.
Impact Lee, S., Sundaram, S., Armitage, L., Evans, J.P., Hawkes, T., Kepinski, S., Ferro, N., Napier, R.M. (2014) Defining binding efficiency and specificity of auxins for SCF(TIR1/AFB)-Aux/IAA co- receptor complex formation. ACS Chem Biol. 2014 Mar 21;9(3):673-82
Start Year 2012
Description Friml Collaboration 
Organisation Institute of Science and Technology Austria
Country Austria 
Sector Academic/University 
PI Contribution We share an interest in gravitropism and PIN biology with this group. We have supplied the Friml group we molecular genetic tools.
Collaborator Contribution The Friml lab has hosted PDRAs from my group to allow them to use the vertical-stage confocal microscope they have at IST, Vienna
Impact Papers are being prepared
Start Year 2013
Description Hayashi lab collaboration 
Organisation Okayama University of Science
Country Japan 
Sector Academic/University 
PI Contribution We have provided research materials and shared data ahead of publication. We performed biochemical experiments for the papers listed below.
Collaborator Contribution They have provided research materials and shared data ahead of publication.
Impact Hayashi, K., Neve, J., M., Hirose, M., Kuboki, A., Shimada, Y., Kepinski, S., Nozaki, H. (2012) Rational design of an auxin antagonist of the SCFTIR1 auxin receptor complex. ACS Chemical Biology 7: 590-98 Hayashi, K., Tan, X., Zheng, N,. Hatate, T., Kimura,Y., Kepinski, S., Nozaki. H. (2008) Small- molecule agonists and antagonists of F-box protein-substrate interactions in auxin perception and signaling. Proc. Natl. Acad. Sci. U S A 105, 5632-5637
Start Year 2007
Description Discovery Zone 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact The feedback from schools is always positive.

Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012,2013,2014,2015
Description UK Plant Science Federation Funding Working Group 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact The activity of this working group is ongoing.
Year(s) Of Engagement Activity 2014