Reference CTP-SAI-074
Functional and Pharmacological Characterization of Insect Inositol-1,4,5-triphosphate Receptors
Reference CTP-SAI-074
Functional and Pharmacological Characterization of Insect Inositol-1,4,5-triphosphate Receptors
Prof. Graham Ladds (Dept. Pharmacology, Cambridge University),
Dr Taufiq Rahman (Dept. Pharmacology, Cambridge University),
James Goodchild (Syngenta, Jealott’s Hill Int. Res. Stn, Bracknell, UK),
Dr Federico Dapiaggi (Syngenta, Stein, Switzerland).
BACKGROUND
The delivery of novel means of protecting crops against damage caused by insect pests is essential for sustainable agriculture. There is both a need to identify new protein targets and to better resolve the molecular interactions between target proteins and their effector ligands to tackle the spread of target-based resistance. The recent development of crop-protection products which deliver their insecticidal effects by modulating ryanodine receptor (RyR) function has highlighted the potential of intracellular Ca2+signaling as a target for insect control. The success of these products, which are based on a class of insecticides collectively known as diamides, has been limited by the surprisingly rapid emergence of target-based resistance in the field. The RyR is a huge protein, so it is conceivable that structural variants with high resistance potential were already occurring naturally in insect pest populations before these populations were ever exposed to diamides. The IP3-receptor is significantly smaller in size yet shares similarities with RyR in terms of structure, function, and regulatory mechanisms. In this project multidisciplinary project we will combine molecular biology, cell biology, bioinformatics and electrophysiology with an aim to generate tools and methods that will enable some key questions relating to the potential of IP3Rs as novel insecticide targets to be addressed.
OBJECTIVES AND APPROACHES
1. Develop a cell-based assay to study the IP3R from a pest insect such as Plutella xylostella. (0-15 months) We have access, within the Cambridge Cell Signalling group, to a HEK293 cell line which is null for the three human IP3R (HEK-IP3R null). We will clone the insect IP3R into pcDNA3.1 and express this in the HEK-IP3R null cell line and assay for histamine/carbachol induced Ca2+ release. Both histamine and carbachol activate Gq-coupled receptors to stimulate IP3 production. Using fluorescent dyes (furo-8 etc) we will assay insect IP3R activity. Once the system has been successfully established, a HEK cell line will be developed that expresses the insect IP3R stably within the cell. Compounds known to target the RyR will then be assayed for selectivity against the IP3R.
2. SDM pharmacology studies to identify residue positions in druggable sites representing key differences between species. (12-30 months). We will use bioinformatics to compare sequences from target and non-target organisms to identify residues with potential for achieving target selectivity. Key sites will be mutated within the receptors which will be expressed in the HEK-IP3R null cell line and assayed as described in 1. In silico modelling, docking and atomistic molecular dynamic (MD) simulations will be performed to probe the IP3R binding sites and compared to human IP3Rs.
3. Assess the potential for targeting the insect IP3R. (18-30 months). Using the cell lines developed in 1, we will screen a series of compounds (supplied by Syngenta) against the insect IP3Rs. This will enable the development of new chemical scaffolds to target the IP3R.
4. Electrophysiological investigations of insect and vertebrate IP3Rs. (24-48 months) We will express tagged versions of insect and vertebrate IP3Rs to enable electrophysiological (at single channel level) investigations using nuclear (on-nucleus or excised-mode) patch-clamp recording, or even pull-down purification and reconstitution in planar lipid bilayers. Such methods could be used to characterise basic biophysical properties of insect IP3R relative to a mammalian reference (e.g., rat IP3R1) and may allow further profiling of active compounds (tested in 2 and 3) in terms of their species selectivity or involvement in mechanisms of Ca2+ regulation including IP3-dependent and IP3-independent Ca2+inhibition, regulation by ATP, channel inactivation etc. We will also consider co-expression of an insect IP3R in HEK-IP3R null cells with a Ca2+-activated Cl-- channel such as anoctamin-1 to report IP3R-mediated calcium release. Such cells would be compatible with the whole-cell patch-clamp method which could provide a relatively straightforward way to investigate the effects of compounds. It should also be possible to explore the effects of endogenous modulators to some degree by altering the concentrations of Ca2+, Mg2+and ATP in the pipette solution.
The Dept. of Pharmacology at Univ. Cambridge is a world-leading authority on cellular calcium signalling and has broad expertise in techniques for visualising the contributions of proteins involved in Ca-signalling pathways at the sub-cellular and molecular level. Extending this experience to the study of insect IP3Rs would provide key insight into the role of these ion-channels in the response to insecticides and evaluate their potential as insecticide targets.
PRIMARY LOCATION OF THIS PHD
The student will be registered with the University of Cambridge and based in the Department of Pharmacology, Tennis Court Road, Cambridge.
TRAINING
Students will have access to training in key bioscience areas (bioinformatics, statistics and mathematics) to enhance employability and research capability.
The CTP – SAI (https://www.ctp-sai.org) is a groundbreaking partnership between leading businesses, charities and research providers offering outstanding training for the agri-food sector
All CTP-SAI students will receive the Leadership and Management training provided by MDS (www.mds-ltd.co.uk) and will create their Personal Development Plan (PDP) to identify their development needs and areas of strength. Each student will receive individual coaching and mentoring with regards to their career plans and skills development (in addition to the scientific project supervision).
INDUSTRIAL PLACEMENT
Placements are a key feature of CTP and UKRI-BBSRC expects all doctoral candidates on a CTP programme to undertake a placement. Placements can be in the form of research placements (3-18 months duration) or used more flexibly for experiential learning of professional skills for business and/or entrepreneurship. All placements are developed in collaboration between the partners with input from the doctoral candidate.
APPLICATION AND ELIGIBILITY
Contact Prof. Graham Ladds (Email: grl30@cam.ac.uk) for an informal discussion on the research content of this PhD.
This studentship will begin in October 2024. The successful candidate should have (or expect to have) an Honours Degree (or equivalent) with a minimum of 2.1 in Plant Science, Applied Statistics, or other related science subjects. Students with an appropriate Masters degree are particularly encouraged to apply.
We welcome UK, EU, and international applicants. Candidates whose first language is not English must provide evidence that their English language is sufficient to meet the specific demands of their study. Candidates should check the requirements for each host organization they are applying to, but IELTS 6.5 (with no component below 6.0) or equivalent is usually the minimum standard.
This studentship is for four years and is fully funded in line with UKRI-BBSRC standard rates. These were for 2023/24, an annual maintenance stipend of £18,622, fee support of £4,596, a research training support grant of £5,000 and conference and UK fieldwork expenses of £300.
To be classed as a home student, candidates must meet the following criteria:
Be a UK National (meeting residency requirements), or
Have settled status, or
Have pre-settled status (meeting residency requirements), or
Have indefinite leave to remain or enter
If a candidate does not meet the criteria above, they would be classed as an international student and must demonstrate the ability to meet the supplement in fees required for an international student.
Anyone interested should complete the online application form before the deadline of 7th January 2024. Interviews will be held during January 2024.
Please contact recruitment-ctp-sai@niab.com for further application details.
