Applications of Mass Spectrometry to Membrane Protein Drug Development

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.

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.

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.