Applications of Mass Spectrometry to Membrane Protein Drug Development

Lead Research Organisation: University of Oxford
Department Name: Oxford Chemistry

Abstract

Membrane proteins constitute the largest class of current drug targets. With many atomic structures now coming to the fore, including human protein targets, we have an unprecedented opportunity to study and rationalise the action of drugs on these important targets. One of the reasons why structures of these important proteins are only just becoming available is the inherent properties of these complexes that have hindered their study over the years; specifically their low expression levels and poor solubility contribute to the practical challenges that had to be overcome.
Now that atomic structures and mechanistic insights are emerging,new methods are therefore required to assess their drug-binding properties and to contribute to data gleaned from other biophysical methods. Mass spectrometry is one such method that is providing fascinating insight into the properties of membrane proteins. Without the complication of protective coatings, required for solubility, the gas phase can be an ideal environment for the study membrane proteins, liberated from the bilayer, but often with critical lipid binding maintained.
We are keen to develop further to our initial findings that lipids can modulate the properties of membrane proteins by extending these observations to the role of lipids and drugs simultaneously. We have selected three different themes, all of high strategic importance for drug development, described briefly here.
The ability of bacteria to resist the challenge of antibiotics is a global threat to human health. Bacteria use a variety of mechanisms to do this including through pumps embedded in cell membranes to actively expel drugs or by controlling import of drugs through pores, formed by proteins in their outer cell-envelopes. One objective of our research programme is to apply mass spectrometry to gain new insight into these two mechanisms. Specifically, we will study the roles of lipids that play in holding open pores for drug import and in speeding up the transport of drugs out through pumps.
Since many drugs now target membrane proteins a plethora of unwanted side effects are occurring through indiscriminate binding to other membrane proteins. These include binding of antipsychotic drugs to the human glucose transporter, anti HIV drugs to proteins involved in ageing and anticancer drugs to multidrug resistance pumps and to a human transporter of unknown function. A second objective is therefore to develop and apply mass spectrometry methods to study the unwanted side effects of drug binding to these targets.
A third objective of our research programme involves the study of complexes involved in the assembly and synthesis of the armoury that protects the bacterial cell envelope. Among these complexes, one folds and assembles the pore proteins and the other orchestrates the formation of peptidoglycan layer. Both are therefore attractive targets for antibiotic intervention. For example, if we could uncover new targets that prevent folding and assembly of the pore proteins we could increase their synthesis to improve drug import. Similarly if we could control synthesis of the protective peptidoglycan layer we could affect the survival of bacteria, their division and the import of drugs.
Overall therefore this research programme will have a number of beneficiaries. Firstly, it will benefit those using mass spectrometry by uncovering new methods to gain insight into the structural biology of membrane proteins. Secondly, the research outcomes will be of interest to pharmaceutical companies and biotechnology industries, particularly those with a focus on membrane protein as targets in human health or nanotechnology devices. In the longer-term interpreting mechanisms of drug resistance, unravelling the unwanted side-effects of drugs and understanding of the cell-wall biosynthesis of pathogenic bacteria will impact global human health.

Technical Summary

Our research objective is to develop and apply mass spectrometry (MS) to drug development for membrane proteins. Specifically we will deliver new insight into drug resistance phenomena, off-pathway side-effects of drugs that bind to membrane proteins and new assemblies for antibiotic targeting. We will first look at the mechanisms operating in multi-drug resistance (MDR) efflux pumps and outer membrane porins. Together these complexes control the influx and efflux of antibiotics into, and out of, cells. Using MS, coupled with ion mobility, the simultaneous binding of drugs and lipids can be probed and their effects on the population of different conformers determined. We will use the solute carrier family of membrane proteins, also involved in MDR, to develop our drug binding strategies. Our second theme is to study the mechanism of off-pathway drug binding to membrane protein targets. To do so we will have to overcome one of the major challenges in the field: that of maintaining the stability of membrane protein drug complexes in the absence of the lipid bilayer. A third major area is characterising membrane protein assemblies that could serve as new targets for antibiotics. Here we will study complexes involved in outer membrane protein biogenesis in E. coli, specifically the beta-barrel assembly machine complex, and the lipid II flippase and elongation machinery. Both complexes play critical roles in the synthesis and assembly of bacterial cell wall components; their subunit composition, overall architecture and functional mechanisms, however, remain unclear. We will monitor oligomeric state, interaction partners and lipid binding properties of these dynamic membrane embedded machineries. Through well-established links with pharmaceutical and biotechnology companies, as well as a close working relationship with the MS industry we will be in an excellent position to exploit our research findings.

Planned Impact

Academic Community
The research described in my proposal, to develop and apply mass spectrometry (MS) to membrane proteins for drug development, will uncover many facets of the biophysics and mechanics of membrane proteins. Specifically it will inform the academic community about the synergistic role of lipids and membrane proteins, their modulation by small molecule inhibitors and their assembly into larger complexes. It will also uncover new information about the composition of key complexes involved in the synthesis of petidoglycan and in the assembly and folding of outer membrane proteins.
Our research programme will also contribute to further our understanding of the application of MS to membrane proteins and their gas phase properties. Studying drug binding in this phase, and using unfolding trajectories to inform on the stability of membrane proteins and their complexes, has not been achieved previously. Success in this area will further our understanding of this important property and will likely prove highly translational. The outcomes of this research will be published in high profile journals and will therefore highlight to the academic community the myriad of opportunities that are available for the study of membrane protein targets using MS.
In summary these academic advances will cross the traditional boundaries of chemistry, membrane protein biophysics, biochemistry and structural biology and contribute to an increased understanding of membrane proteins in the gas phase of the MS.
Economic and societal impacts The outputs of this research programme will contribute to the global competitiveness of the United Kingdom, specifically by the creation of a new spin-out company (OMASS) based on the MS of membrane proteins. This company will undertake collaborative projects based on the investigation of membrane protein targets. A number of contracts have been discussed with Amgen, Genentech, Oxford Nanopore and Roche. Two employees have been recruited to market the company and to carry out this commercial project based research. This has therefore created new employment opportunities for the UK. The PI will remain 100% research active acting as a consultant to the company but will not be involved in the day-to-day running.
Impact in membrane protein research using MS for drug development will undoubtedly have a positive effect on the competitiveness of the spin-out company and will benefit to the UK economy. Ultimately our international standing in the science and technology industries could well be affected by these developments.
For the MS industry, progress in the design and implementation of modifications for specialised research has been a hallmark of our success.
Impact on global health.
The research outlined in this proposal also has the possibility of enhance the quality of health, particularly with respect to the direct benefits gleaned from increased understanding of multidrug resistance and the unwanted side effects of drugs that bind to membrane proteins. To the best of our knowledge this is the first time this has been attempted using MS and the results will have wide ranging impact. It is often difficult to assess small molecule binding to membrane protein targets using traditional structural biology approaches, such as crystallography, primarily due to the heterogeneity of lipid, detergent and drug binding as well as the instability of many membrane protein drug complexes when isolated from the lipid bilayer.
Impact on Structural biologists
One of the other impacts for the structural biology community is the ability to monitor reconstitution of heterogeneous complexes. This has been a long-term difficulty in structural biology and is becoming increasingly apparent with the transformative developments in Cryo-EM technology. The research in this proposal in which reconstitution is monitored by MS could have far-research consequences for structural biology.

Publications

10 25 50
 
Title Back cover journal illustration 
Description Back cover journal illustration Angewandte Chemie 
Type Of Art Artwork 
Year Produced 2017 
Impact Increased interest in research 
 
Description Address Scottish Parliament
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Canadian Institute for Advanced Research (CIFAR)
Geographic Reach North America 
Policy Influence Type Participation in a advisory committee
 
Description President, Royal Society of Chemistry
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Covid 19
Amount £20,000 (GBP)
Organisation University of Oxford 
Sector Academic/University
Country United Kingdom
Start 02/2020 
End 12/2020
 
Description MRC Confidence in Concept Round 4
Amount £33,835 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 08/2018
 
Title 'Nativeomics' 
Description A top-down method, 'nativeomics', unifying 'omics' (lipidomics, proteomics, metabolomics) analysis with native mass spectrometry. 
Type Of Material Improvements to research infrastructure 
Year Produced 2020 
Provided To Others? Yes  
Impact Facilitates identification of ligands bound to membrane protein assemblies. 
 
Title HDX Mass Spectrometry 
Description Development of Hydrogen-Deuterium Exchange Mass Spectrometry to probe the conformational dynamics of a lipid sorting protein, LptDE, induced by lipids and antibiotics. 
Type Of Material Improvements to research infrastructure 
Year Produced 2021 
Provided To Others? Yes  
Impact As above 
 
Title MS approach - annular and lipid binding to membrane proteins. 
Description A mass spectrometry based approach to distinguish annular and specific lipid binding to membrane proteins 
Type Of Material Improvements to research infrastructure 
Year Produced 2019 
Provided To Others? Yes  
Impact Facilitates study of membrane proteins interactions with surrounding lipids and co-factors. 
 
Title Sonicated Lipid Vesicle MS 'SOLVE-MS' 
Description A protocol that enables direct ejection of protein complexes from membranes for analysis by native MS 
Type Of Material Improvements to research infrastructure 
Year Produced 2020 
Provided To Others? Yes  
Impact Permits the study of membrane proteins directly from the membrane without the use of chemicals and allows identification of many of the loosely associated cofactors, lipids and subunits often lost with traditional methods. 
 
Title Assembly and regulation of the chlorhexidine specific efflux pump AceI 
Description Raw Mass Spectrometry data for main figures and supplementary figures for the journal article "Assembly and regulation of the chlorhexidine-specific efflux pump AceI". 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
Impact Information available to other users. 
URL https://figshare.com/articles/Assembly_and_regulation_of_the_chlorhexidine_specific_efflux_pump_AceI...
 
Title Combining Native and 'omics' mass spectrometry to identify endogenous ligands bound to membrane proteins - RAW DATA 
Description Raw mass spectrometry data. Figures and Extended Data forCombining Native and 'omics' mass spectrometry to identify endogenous ligands bound to membrane proteinsMain Figures 1,2,3Extended Data 3,4,5Data for supplementary figures available on request from corresponding authors 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
Impact Data available to other users 
URL https://figshare.com/articles/Combining_Native_and_omics_mass_spectrometry_to_identify_endogenous_li...
 
Description Andrew Kruse Laboratory 
Organisation Harvard University
Department Harvard Medical School
Country United States 
Sector Academic/University 
PI Contribution Used native mass spectrometry to pin-point the effects of agonists, antagonists and membrane lipids on the oligomeric states of human sigma-1 receptor.
Collaborator Contribution Provided sigma-1 receptor and its ligands
Impact Manuscript under preparation for joint publication.
Start Year 2018
 
Description Bharat Laboratory 
Organisation University of Oxford
Department Sir William Dunn School of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution Generated mass spectrometry data
Collaborator Contribution Provided proteins
Impact Generated mass spectra of CdrA protein alone and with a nanobody. Nanobodies targeting the tip of cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt pre-existing biofilms in conjunction with bactericidal antibiotics to prevent or treat problematic, chronic bacterial infections".
Start Year 2020
 
Description David Julius 
Organisation University of California, San Francisco
Department Department of Physiology
Country United States 
Sector Academic/University 
PI Contribution Mass spectrometry data
Collaborator Contribution TRP Channels
Impact Informative mass spectra
Start Year 2016
 
Description Hongjin Zheng Laboratory 
Organisation University of Colorado
Country United States 
Sector Academic/University 
PI Contribution Used native mass spectrometry to investigate the role of lipids on the structure and function of YbtPQ membrane transporter from uropathogenic E. coli.
Collaborator Contribution Provided proteins
Impact No outputs yet
Start Year 2020
 
Description Kelly Nguyen Laboratory 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Used native mass spectrometry to investigate subunit interactions and oligomeric state of human shelterin complex
Collaborator Contribution Provided proteins
Impact No outputs yet
Start Year 2020
 
Description Lewis Laboratory 
Organisation Northeastern University - Boston
Country United States 
Sector Academic/University 
PI Contribution Generated mass spectrometry data
Collaborator Contribution Provided antibiotics
Impact Mass spectra of antibiotics binding to Bam complex; ajoint publication under review at Nature
Start Year 2019
 
Description Li Laboratory 
Organisation University of Hong Kong
Country Hong Kong 
Sector Academic/University 
PI Contribution Generated mass spectrometry data
Collaborator Contribution Provided antibiotics
Impact Mass spectra of antibiotics binding to MurJ and MraY proteins
Start Year 2019
 
Description Liz Carpenter 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Generated mass spectra
Collaborator Contribution Donation of proteins
Impact Publication in Cell (2018)
Start Year 2016
 
Description Matthew Higgins 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Not applicable
Collaborator Contribution HDX/MS
Impact Paper in Nature Microbiology (2018)
Start Year 2018
 
Description Michel Laboratory 
Organisation Max Planck Society
Department Max Planck Institute of Biophysics
Country Germany 
Sector Charity/Non Profit 
PI Contribution Generated mass spectrometry data
Collaborator Contribution Provided proteins and reagents
Impact Mass spectra of lipid binding to PfMATE protein and AI-2 binding to TqsA
Start Year 2019
 
Description Naismith Laboratory 
Organisation Rosalind Franklin Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Generated mass spectrometry data
Collaborator Contribution Provided proteins
Impact Generated mass spectra of wild type tyrosine kinase, Wzc protein and several tyrosine mutants. Enabled phosphoproteomics analysis of wild type to identify phosphorylation sites.
Start Year 2021
 
Description Pepys Laboratory, Centre for Amyloidosis and Acute Phase Proteins 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Generated mass spectrometry data
Collaborator Contribution Provided proteins and reagents
Impact Mass spectra of drug binding to human CRP protein
Start Year 2020
 
Description Simon Newstead 
Organisation University of Oxford
Department Medical Sciences Division
Country United Kingdom 
Sector Academic/University 
PI Contribution Generated mass spectra
Collaborator Contribution Donation of proteins
Impact Informative mass spectra
Start Year 2016
 
Description Van den Berg Laboratory 
Organisation Newcastle University
Country United Kingdom 
Sector Academic/University 
PI Contribution Used native mass spectrometry to characterize enzyme-generated fructo-oligosaccharides and their binding to the SusCD transporter.
Collaborator Contribution Provided ligands and proteins
Impact Mass spectra of lipid binding to membrane proteins; Publication in Nature Communications (2021).
Start Year 2020
 
Description Vollmer Laboratory 
Organisation Newcastle University
Country United Kingdom 
Sector Academic/University 
PI Contribution Generated mass spectrometry data
Collaborator Contribution Provided plasmids and reagents
Impact Mass spectra of lipid binding to membrane proteins; joint publication under review at journal Chem.
Start Year 2018
 
Description Yu 
Organisation Case Western Reserve University
Country United States 
Sector Academic/University 
PI Contribution Generated mass spectrometry data
Collaborator Contribution Provided plasmids and reagents
Impact Joint publication in PNAS journal.
Start Year 2018
 
Title Detection of membrane protein-therapeutic agent complexes by mass spectrometry 
Description Mass spectrometry as a tool for drug discovery 
IP Reference WO2012172378 
Protection Patent granted
Year Protection Granted
Licensed No
Impact Interest from industry
 
Company Name OMass Technologies 
Description Mass spectrometry techniques for biotherapeutics/membrane proteins 
Year Established 2016 
Impact Interest and contracts from industry / interest from investors
Website https://www.omasstech.com/
 
Description 6th Molecular Microbiology Conference, Newcastle 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Engaging with scientific community
Year(s) Of Engagement Activity 2019
 
Description American Society for Mass Spectrometry Online Reboot 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Fostered interest in research
Year(s) Of Engagement Activity 2020
 
Description Annual Biophysical Society Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Annual lecture to Biophysical Society Members
Year(s) Of Engagement Activity 2019
 
Description Astra-Zeneca Medal Award Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Astra-Zeneca Medal Award Lecture
Year(s) Of Engagement Activity 2016
 
Description Baddiley Lecture, University of Newcastle 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Dissemination of research results
Year(s) Of Engagement Activity 2017
 
Description Bashour Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Reporting research and engaging with scientific community
Year(s) Of Engagement Activity 2020
 
Description Dame Anne McLaren Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Bring research to different audiences
Year(s) Of Engagement Activity 2018
 
Description Drug Target Review 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Article on using native mass spectrometry to inform drug discovery.
Year(s) Of Engagement Activity 2017
URL https://www.drugtargetreview.com/article/23594/drug-target-review-issue-2-2017/
 
Description Empowering Women in Chemistry Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Encourage and engage with women in Chemistry.
Year(s) Of Engagement Activity 2019
 
Description Female leaders in Science Workshop 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Workshop to discuss role of women in science leadership and provide information and advice.
Year(s) Of Engagement Activity 2019
 
Description Hans Krebs Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Increased interest in MS research
Year(s) Of Engagement Activity 2017
 
Description Harry G Day Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Named lecture at University of Indiana
Year(s) Of Engagement Activity 2016
 
Description Host: Kuggie Vallee Distinguished Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Support of prominent women in biomedical science and discussion of some of the contemporary challenges for women building scientific careers in different parts of the world.
Year(s) Of Engagement Activity 2019
 
Description Jani Reddy Bolla - Case Western 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact To share information.
Year(s) Of Engagement Activity 2018
 
Description Jani Reddy Bolla Secunderabad 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact To share information.
Year(s) Of Engagement Activity 2018
 
Description Joe L Franklin Memorial Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Memorial lecture in honour of Joe L Franklin, RICE, Houston Texas.
Year(s) Of Engagement Activity 2018
 
Description Mabel Fitzgerald Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Bring research to different audiences
Year(s) Of Engagement Activity 2018
 
Description Oxford Chemistry Alumni Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact To discuss Chemical Sciences and vision for future.
Year(s) Of Engagement Activity 2019,2020
 
Description Peter Garland Lecture, Dundee 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Dissemination of research results
Year(s) Of Engagement Activity 2017
 
Description Rayson Huang Visiting Lecture, Hong Kong 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Lecture to disseminate current research.
Year(s) Of Engagement Activity 2019
 
Description Structural mass spectrometry of membrane proteins 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Engaging with mass spectrometry community.
Year(s) Of Engagement Activity 2019
 
Description The Making of a Scientist 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact A film to illustrate different role models and foster greater awareness to encourage more women into science.
Year(s) Of Engagement Activity 2018
 
Description Torbern Bergmann Award Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Award lecture
Year(s) Of Engagement Activity 2016
 
Description Webster Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Bring research to audiences
Year(s) Of Engagement Activity 2018