Construction of a High-Throughput Hydrogen Deuterium Exchange Mass Spectrometry Platform

Lead Research Organisation: University of Dundee
Department Name: Cellular Medicine

Abstract

Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) is a structural biology technique which excels in the investigation of protein interactions. All proteins have amide groups along their polypeptide backbone which routinely undergo a phenomenon of solvent exchange - where the hydrogen of the amide swaps with the hydrogen of the surrounding solvent, typically water. The rate of this exchange is inextricably linked to the structural environment of that amino acid on the polypeptide backbone. Areas of protein which are rigid, densely packed and highly structured have a much slower solvent exchange rate than disordered, flexible and loosely packed parts of the protein. Additionally, the creating of new bonds through the creation of new interaction interfaces, whether that be protein: protein interactions, or protein: drug, protein: membrane, or protein: nucleic acid, will all alter the solvent exchange rate.

The measurement solvent exchange is achieved through the exposure of a protein to a deuterated solvent. When a solvent exchange event occurs, the protein's hydrogen atom is swapped for deuterium, and thus the mass of the amino acid is altered. Using mass spectrometry equipment, we can detect this increase in mass, and by incubating the protein with deuterium for multiple time points, we can measure the solvent exchange rate. The solvent exchange rate can then be manipulated by e.g. adding a drug which interacts with the protein, and determine where on the protein the solvent exchange rate is altered, thus locate where on a protein a drug, binding partner, or lipid membrane is interacting.

Determining how proteins interact with other entities can provide insights into their mechanisms, and can rationalise how diseases may alter their structural properties and impact their function. Likewise, determining where drugs interact with proteins can guide their development and inform how they may be altered to be more potent, or have fewer side-effects.

HDX-MS has numerous advantages over other technologies which may also provide structural insights into how proteins interact with other agents. For example, the technique of x-ray crystallography is highly dependent on having a rigid protein structure, so proteins which are intrinsically disordered cannot be investigated. Another technology, cryo-electron microscopy, can overcome some of these limitations, but it is a very slow technique for screening molecules.

This proposal is for a LEAP HDX Extended System automated sample handling robot, attached to a Waters Synapt XS HDMS Mass Spectrometer. Together these two pieces of equipment work seamlessly to produce the highest quality of HDX-MS data, allowing for the most complex and cutting-edge applications of the technique, while also ensuring the greatest efficiency of mass spectrometry capacity.

The LEAP HDX Extended System allows for highly reproducible data collection and sample preparation. HDX-MS experiments are extremely sensitive to temperature fluctuations and incubation timing, making sample preparation ideally conducted by robotics. Additionally, the work-flow of chromatography involves multiple steps, again ideally conducted through automation to ensure reproducibility. Furthermore, the nature of HDX-MS experiments requires numerous samples being analysed individually on a single instrument, and a robotically handled sample injection system allows for a maximisation of useful data collection, especially through the night and over weekends. The Waters Synapt XS HDMS Mass Spectrometer is well suited for HDMS. Due to the temperature constraints on HDX samples, the ability to resolve peptides by chromatography is limited. The ion-mobility ability of the Synapt XS compensates for this when analysing complex mixtures of proteins. Additionally, the ability to use alternative fragmentation methods allows for more cutting edge HDX-MS experiments to be conducted which increase the resolution of the technique.

Technical Summary

Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) is a technique which now firmly embedded within the biosciences. Its broad applicability to diverse investigations into protein structure and protein interactions provide utility in a wide array of investigations. One underutilised aspect of HDX-MS however is the screening of small molecule interactions. Although this instrument will be available to all users for any HDX-MS application, the core aim is deploying HDX-MS to structurally screen compound series.

Currently, most small molecule drug interactions are investigated structurally using x-ray crystallography. This process has numerous limitations - primarily that there are proteins that are incapable of crystallising. Furthermore, the addition of a small molecule drug may alter the protein structure sufficiently to cause protein crystals, once eventually obtained, to collapse and dissolve. There is also the potential for crystallisation constructs, crystal contacts, and non-biological solvent conditions to create binding artefacts, with drugs binding in pockets or with cofactors that would not be found in a biologically relevant condition. While cryo-electron microscopy holds promise, it is currently more costly than crystallography, and there are intractable limitations in both protein targets and automation.

HDX-MS offers an attractive alternative for small molecule drug discovery. The use of automated sample handling systems such as the LEAP system, routinely used in the biopharmaceutical industry, provides the combination of increased sensitivity, throughput, reproducibility and precision - all of which facilitate the medium-throughput screening of small molecule therapeutics.
Key aspects of this system are:

- Both sample, quench and UPLC temperature control.
- Automated sample injections allowing more efficient mass spec. instrument use.
- Compatible with all electro-spray source mass spec.

Publications

10 25 50
 
Description We have been able to successfully use this instrument for a number of different and diverse projects. A very important project was determining how a new class of drugs, capable of stimulating nerve growth and wound healing, bind to their target.
Exploitation Route This work highlights the ability of this technique for routine drug screening, especially for complex mechanisms of action, which might not be suitable for other techniques.
Sectors Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

 
Title HDX-MS of Activators and Inhibitors of PI3Kalpha 
Description Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development. This also applies to the PI 3-kinase (PI3K) signalling pathway which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation1-3. Here we report on the discovery of UCL-TRO-1938 (further referred to as 1938), a small molecule activator of the PI3Ka isoform, a critical effector of growth factor signalling. 1938 allosterically activates PI3Ka through a unique mechanism, by enhancing multiple steps of the PI3Ka catalytic cycle, and causes both local and global conformational changes in the PI3Ka structure. This compound is selective for PI3Ka over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia reperfusion injury and, upon local administration, enhances nerve regeneration following nerve crush. This study identifies a unique chemical tool to directly probe PI3Ka signalling and a novel approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes, through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development. 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
Impact Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development. This also applies to the PI 3-kinase (PI3K) signalling pathway which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation1-3. Here we report on the discovery of UCL-TRO-1938 (further referred to as 1938), a small molecule activator of the PI3Ka isoform, a critical effector of growth factor signalling. 1938 allosterically activates PI3Ka through a unique mechanism, by enhancing multiple steps of the PI3Ka catalytic cycle, and causes both local and global conformational changes in the PI3Ka structure. This compound is selective for PI3Ka over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia reperfusion injury and, upon local administration, enhances nerve regeneration following nerve crush. This study identifies a unique chemical tool to directly probe PI3Ka signalling and a novel approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes, through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development. 
URL https://www.ebi.ac.uk/pride/archive/projects/PXD037721
 
Description A small molecule PI3Ka activator in cardioprotection and neuroregeneration 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution HDX-MS determination of a novel small molecule binding to a oncogenic target
Collaborator Contribution Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development. This also applies to the PI 3-kinase (PI3K) signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report on the discovery of UCL-TRO-1938 (further referred to as 1938), a small molecule activator of the PI3Ka isoform, a critical effector of growth factor signalling. 1938 allosterically activates PI3Ka through a unique mechanism, by enhancing multiple steps of the PI3Ka catalytic cycle, and causes both local and global conformational changes in the PI3Ka structure. This compound is selective for PI3Ka over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia reperfusion injury and, upon local administration, enhances nerve regeneration following nerve crush. This study identifies a unique chemical tool to directly probe PI3Ka signalling and a novel approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes, through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.
Impact A small molecule PI3Ka activator in cardioprotection and neuroregeneration Paper in Press
Start Year 2022
 
Description A small molecule PI3Ka activator in cardioprotection and neuroregeneration 
Organisation University College London
Department UCL Cancer Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution HDX-MS determination of a novel small molecule binding to a oncogenic target
Collaborator Contribution Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development. This also applies to the PI 3-kinase (PI3K) signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report on the discovery of UCL-TRO-1938 (further referred to as 1938), a small molecule activator of the PI3Ka isoform, a critical effector of growth factor signalling. 1938 allosterically activates PI3Ka through a unique mechanism, by enhancing multiple steps of the PI3Ka catalytic cycle, and causes both local and global conformational changes in the PI3Ka structure. This compound is selective for PI3Ka over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia reperfusion injury and, upon local administration, enhances nerve regeneration following nerve crush. This study identifies a unique chemical tool to directly probe PI3Ka signalling and a novel approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes, through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.
Impact A small molecule PI3Ka activator in cardioprotection and neuroregeneration Paper in Press
Start Year 2022