Isothermal titration calorimetry instrumentation for structural biology, biological mechanisms and drug discovery

Lead Research Organisation: King's College London
Department Name: Randall Div of Cell and Molecular Biophy

Abstract

Biomolecular interactions and characterisations form the basis of a plethora of research projects at King's, from fundamental investigations of molecular mechanisms to more applied research with medical and social impact. There is an increasing need to improve the precision and accuracy of these characterisations to achieve a detailed understanding of health and disease pathways and mechanisms, to facilitate the translation of this science towards outcomes that will benefit society. One of the key techniques used for such investigations is isothermal titration calorimetry (ITC), which provides a universal approach to study in detail how molecules are able to interact with one another, for example to understand which potential drug is able to block its target more efficiently, how bacteria develop resistance to antibiotics, or to explain the fundamental bases of life. For the many groups at King's, spread across four Faculties, there is a clear and urgent demand to improve upon the results that we can currently produce, and enable the characterisation and/or identification of new biochemical reactions or molecular assemblies that play a key role in a number of fields, including host-pathogen interactions, natural product biosynthesis, ageing, cardiovascular research, cancer, chemical biology and bioengineering, neurological disorders, signalling pathways, asthma and allergy. The instrumentation requested here holds promise of transforming the science of a wide user-base community at King's.
King's is a leading international research institution across a range of biological disciplines, thus the potential for this investment of local provision of ITC instrumentation to add value is enormous. The ITC instrumentations will be part of the Wellcome Trust-supported Centre for Biomolecular Spectroscopy and will operate in synergy with other methodologies, integrating with other biophysical and structural biology methods and promoting exchanges with other in-house infrastructure and platforms (e.g. mass spectrometry, 'omics' platforms, imaging). The setting within the Centre will also facilitate usage by researchers from other academic institutions and/or industries, widening the breadth of the science that will be performed.

Equipped with the latest ITC instrumentation, the Centre will contribute to the elucidation of molecular mechanisms of fundamental bioscience and permit state-of-the-art characterisation of biological therapeutics, to tackle strategic challenges in line with the BBSRC remit and priorities. The new instrument will strengthen the strategic mission of the Centre for Biomolecular Spectroscopy to deliver the highest quality data for internationally competitive research, train new generations of scientists in cutting edge methodologies, promote exchange of ideas and foster new interdisciplinary approaches (with academia and industries) for solving fundamental biological questions, addressing new therapeutic and societal challenges and providing benefits for human health.

Technical Summary

Isothermal Titration Calorimetry (ITC) is a powerful, invaluable and unique tool to determining the molecular nature of non-covalent interactions involved in biological processes. A single ITC experiment enables the characterisation of a complete set of thermodynamic parameters - enthalpy, entropy and free energy - which are intimately related to the mechanism of molecular association, in addition to an accurate stoichiometry of the molecular association. As ITC measures the intrinsic heat effect for a given molecular event, it requires no labelling or immobilisation, thereby mimicking a more 'natural environment' and circumventing the potential artefacts, time, cost, or experimental difficulties associated with these modifications. Many groups at King's require a state-of-the-art ITC instrumentation with automated capabilities to tackle key mechanistic biological problems and facilitate translational approaches. Furthermore groups at King's have been developing innovative methodologies for the biosciences involving ITC, namely enzymatic characterisations and fragment-based screening pipelines, which require the latest instrumentation.

The existing instrument at King's no longer has the technical capability to respond to current and anticipated demands. The latest ITC technology requested here will fill this deficit, as it enjoys significantly improved power-response times, is designed to execute applications unattainable on old calorimeters, and will feature automation accessories. It will therefore provide a genuine step change in the activities at King's by enabling: (i) the measurement of a much broader range of interactions; (ii) the simultaneous characterisation of kinetics and thermodynamics parameters; (iii) the development and use of new methodologies, potentially leading to new industrial partnerships; (iv) the realisation of projects limited by amount of material or lower enthalpies; (v) medium throughput applications (e.g. in drug discovery).

Planned Impact

The latest state-of-the-art ITC instrumentation will have the major impact of conferring an essential technology to the existing range of internationally excellent structural biology and biophysics infrastructure at King's. We expect that the new instrument will play a key role in facilitating new synergies and collaborations between scientists from different disciplines and institutions.

The impact will be in the first place specific for the research conducted by each PI. With research addressing a large breadth of discovery science to more applied projects, the new equipment has the potential to benefit a large number of academic researchers, clinicians, pharmaceutical and biotechnology companies, and influence public policies (e.g. on microbe resistance to antibiotics, antifungal vaccines, life-style interventions etc.).

Furthermore there will be considerable industrial interest as ITC is a very well established technique to investigate affinities and thermodynamics of small molecules binding to potential therapeutic targets, and automation has made these investigations possible at larger scales. Our research will have practical applications that will inform drug development and our work will be applied directly in our effort to develop new therapeutics, e.g. novel anti-IgE therapeutics in asthma (Sutton/McDonnell, patent WO2017/211928A1 with UCB), inhibitors of p38-TAB interaction in heart ischemia (De Nicola/Marber with GSK), use of C. albicans secreted peptide for vaccination (Naglik, US Patent No.: 9,969,796) and natural products with antibiotic properties (Barry).

Furthermore, the new ITC-based technologies led by groups at King's will have the potential to facilitate collaborations and partnerships with industries. For instance new ITC methodologies developed by Conte in collaboration with Malvern Panalytical (manufacturer of the Microcal PEAQ ITC requested here) are likely to generate further exchanges, benefitting both parties (e.g. development of software packages for complex mathematical analysis of enzymatic measurements etc.).

As part of the Centre for Biomolecular Spectroscopy, the new ITC instrument will play an important part in the training of undergraduates and postgraduates, including those funded by the BBSRC London Interdisciplinary Bioscience Consortium Doctoral programme (LIDo). King's is a very much research-led teaching university and this area will capture the interest and educate a new generation of scientists to cutting edge biophysical quantitative methodologies. Postdoctoral scientists will also receive training on the latest state-of-the-art instrument, leading to better job prospects.
The Centre for Biomolecular spectroscopy every year hosts students from schools that have a poor record of progression to Universities (e.g. through the King's STARS - Science Training for Aspiring Research Scientists scheme), to raise the ambitions of these students, inspire and encourage them to pursue biomedical research. Additional activities are listed in the pathway to impact. King's has been ranked 5th in the world in the 2019 THE University Impact Rankings, an award created to recognise and celebrate the social and economic contribution of universities, and applicant's labs will take full advantage of King's network and support in place for such activities (e.g. K+, a widening participation scheme to support University applications for sixth-formers from local non-selective state schools).

Each applicant is involved in outreach activities and communication with the public, and few examples of successful initiatives are described in the Pathway to impact. We envisage that the Centre will use some of its own budget (and sponsorships) to develop public engagement events, not only a scientific open day symposium but also a programme of educational and/or art events including panel discussions and hand-on workshops, and take it on the road to reach communities in London and the UK.

Publications

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Description We are using this isothermal titration calorimetry methodology to understand exactly how macromolecules interact and how they behave, for a wide range of biological and biotechnology applications, and to facilitate drug discovery projects. The instrument's puchase, delivery and installation were significantly delayed because of COVID: the instrument was purchased in June 2022 and delivered in July 2022. We are therefore at an early stage yet in terms of discoveries and outcomes.
Exploitation Route The methodologies used and developed here can be then applied to other systems. Outcome of the work can also be used by other scientisist in the field to advance knowledge and enable new applications.
Sectors Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description A dual DLS and SEC-MALS instrumentation to characterize protein oligomerization for structural and mechanistic biology
Amount £205,223 (GBP)
Funding ID BB/V01966X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2021 
End 05/2022
 
Description iSENSE DNA HORIZON-EIC-2021-PATHFINDEROPEN-01
Amount € 3,000,419 (EUR)
Funding ID HORIZON-EIC-2021-PATHFINDEROPEN-01 
Organisation Uppsala University 
Sector Academic/University
Country Sweden
Start 03/2022 
End 03/2025
 
Title ITC to measure enzyme kinetics and inhibition 
Description With this award we will develop fast, convenient and reliable methodologies to investigate how enzymes work and how they can be regulated by drugs or other molecules in general. This will employ a technique called isothermal titration calorimetry and uses the heat released or absorbed in every reaction as an internal probe: monitoring the amount of heat exchanged over time is correlated to the number of molecules that take part of the reaction . Because of the intrinsic properties of molecular reactions, calorimetry methods are not limited by specific molecular characteristics, and are potentially applicable to a wide range of systems. We started working on this methodology in 2018, and have a forst publication in 2019 (indicated belwo) - however the methodology needs to be extended to other systems, and this is the current on-going work that is yet to be published. 
Type Of Material Improvements to research infrastructure 
Year Produced 2019 
Provided To Others? Yes  
Impact Enzymes are essential components for life. They catalyse vital biological processes, constitute the majority of current pharmacological targets and are heavily utilised in other industries, including food, biotechnologies, detergents. Understanding how enzymes work and how they can be modulated by drugs or inhibitors is therefore paramount both for the fundamental understanding of processes governing life and underlying industrial activities. Many enzymes however are difficult to study with traditional methods that often introduce variables that impact on the accuracy of the kinetic determination and often require high amounts of protein and/or substrates. With these new research tools we will able to address key questions on how enzymes work and how they can be regulated. The impact of enzyme research to biology, chemistry, biotechnology, biomedicine and pharmacology give to the development of these new protocols a potential impact for scientific research and for the wide society.