Adenosine A2a receptor screening with polymer-encapsulated nanodiscs

This article is based on a poster originally authored by Philip Rawlins, Jim Reid, William Lee, Holly Madden, Soumya Dhanavade, James Craswell, Satya Bhamidimarri, Anna Hopkins, Joe Mason, Marieke Lamers, Andrew Ratcliffe, Stefanie Reich and Trevor Askwith, which was presented at ELRIG Drug Discovery 2024 in affiliation with Domainex Ltd.

This poster is being hosted on this website in its raw form, without modifications. It has not undergone peer review but has been reviewed to meet AZoNetwork's editorial quality standards. The information contained is for informational purposes only and should not be considered validated by independent peer assessment.

Introduction

• As an alternative to the traditional detergent-based extraction methods, the recent development of amphipathic co-polymers offers the possibility for detergent-free purification of integral membrane proteins without stripping away the native lipid bilayer.
• Amphipathic polymers, such as SMA, DIBMA, AASTY, and related polymers, can insert directly into cell membranes and spontaneously form nanodisc structures of native membrane lipids encapsulated by polymer (polymer-lipid-particles, PoLiPa). This allows membrane proteins to be extracted without removing them from the native lipid environment.
• We showcase the extraction and purification of the Adenosine A2a G-protein coupled receptor (GPCR) and the subsequent use of a Spectral Shift binding assay for fragment screening.

Polymer lipid particles

Solubilization of lipid membranes

• Amphipathic polymers insert directly into cell membranes
• The polymer self-assembles into discs of the lipid bilayer that allow the solubilization of membrane proteins, maintaining the native lipid environment
• Detergent-free

Purification of polymer solubilized membrane proteins

• Compatible with conventional chromatography methods (e.g., affinity, size exclusion)
• Polymer is not required in subsequent purification steps
• Generic solubilization conditions mean similar purification conditions can be used for any membrane protein

Applications

• Antibody generation, ELISAs, structural studies (CryoEM), and biophysical studies (GCI [Creoptix/Malvern], Spectral Shift, nanoDSF [NanoTemper])

Fig 1. Description of amphipathic polymer extraction. Image Credit: Image courtesy of Philip Rawlins et al., in partnership with ELRIG (UK) Ltd.

Adenosine A2a receptor purification

• The A2a receptor was expressed in insect cells using the baculoviral expression system.
• Extracted membrane fractions were solubilized using either detergent or selected polymer from the solubilization screen, and the proteins were purified using Ni-NTA affinity and size exclusion chromatography
• Purification yields (purity > 90 %) are comparable for detergent and Polymer formulations. DLS confirms protein to be monodisperse.

Protein was suitable for downstream applications

Fig 2. PoLiPa workflow (left) and Western blots of solubilized membrane fractions. Image Credit: Image courtesy of Philip Rawlins et al., in partnership with ELRIG (UK) Ltd.

Fig 3. Size exclusion chromatography (left), DLS (left- inset) and SDS-PAGE (right) of A2a in either detergent or polymer formulation. Image Credit: Image courtesy of Philip Rawlins et al., in partnership with ELRIG (UK) Ltd.

Adenosine A2a receptor spectral shift assay

Protein validation via nanoDSF

• The receptor had a single inflection point, consistent with a single folded species and known antagonists increased the melting temperature by +3 to +13 °C, indicating the receptor is physiologically folded

Spectral shift assay (NanoTemper)

• Spectral Shift (NanoTemper) is a biophysical assay that directly measures molecular interactions in solution.
• Binding is detected when ligands cause very subtle changes in the emission spectra of a fluorescent dye attached to a molecular target.
• A Spectral Shift assay was developed using an NTA-labelling kit on the Dianthus instrument (Nanotemper)
• Reproducible concentration-response curves for four known antagonists were obtained.
• The K_D value for ZM241283 was weaker than expected due to it reaching the tight-binding limit of the assay.

Fig 4. NanoDSF data for A2aR protein samples in the presence and absence of tool compounds. Image Credit: Image courtesy of Philip Rawlins et al., in partnership with ELRIG (UK) Ltd.

Fig 5. Schematic of Spectral Shift emission. Image Credit: Image courtesy of Philip Rawlins et al., in partnership with ELRIG (UK) Ltd.

Fig 6. Affinity binding curves for four known A2aR small molecules (left) and summary data table (right). Image Credit: Image courtesy of Philip Rawlins et al., in partnership with ELRIG (UK) Ltd.

Adenosine A2a receptor fragment screen

Fragment screening

• The Domainex fragment library (960 fragments) was screened at 250 μM against adenosine A2A receptor using spectral shift.
• A 9 % hit rate was obtained using a user-specified threshold

Fig 7. Data for 160 fragments in duplicate. Image Credit: Image courtesy of Philip Rawlins et al., in partnership with ELRIG (UK) Ltd.

Fig 8. Analysis of the fragment screening data. Image Credit: Image courtesy of Philip Rawlins et al., in partnership with ELRIG (UK) Ltd.

Fragment affinities

• 32 fragment hits were selected from the fragment screen based on their binding signal and heavy atom count (HAC)
• One fragment hit gave a binding curve that increased spectral shift rather than decreased, suggesting the binding mode or site of this hit may be different
• More than 30 fragment hits were identified with ligand efficiencies (LE) greater than 0.3 (LE = 1.37*(pKd/HAC)

Fig 9. Example binding curves (left) and plot of Fragment Hit Ligand Efficiencies versus pKd (right). Image Credit: Image courtesy of Philip Rawlins et al., in partnership with ELRIG (UK) Ltd.

Conclusions

• We have prepared a polymer-encapsulated nanodisc (Polymer-lipid-particles - PoLiPa) for the adenosine A2a GPCR with high purity and yield.
• This is the first fragment screen with a membrane protein purified in a Polymer-Encapsulated Nanodisc.
• Ligand-efficient fragment hits have been identified, and work is ongoing to develop compound SAR via plate-based chemistry (D2B) and to determine the crystal structures of the fragment hits

About Domainex

Domainex is a fully integrated drug discovery CRO based near Cambridge, UK. It serves pharmaceutical, biotechnology, academic and patient foundations globally. Domainex’s drug discovery service business was established in 2001 and since that time has continued to expand to serve a wider range of international clients including UCB, FORMA Therapeutics, St George’s University, and The Institute of Cancer Research.

About ELRIG (UK) Ltd.

The European Laboratory Research & Innovation Group (ELRIG) is a leading European not-for-profit organization that exists to provide outstanding scientific content to the life science community. The foundation of the organization is based on the use and application of automation, robotics and instrumentation in life science laboratories, but over time, we have evolved to respond to the needs of biopharma by developing scientific programmes that focus on cutting-edge research areas that have the potential to revolutionize drug discovery.

Comprised of a global community of over 12,000 life science professionals, participating in our events, whether it be at one of our scientific conferences or one of our networking meetings, will enable any of our community to exchange information, within disciplines and across academic and biopharmaceutical organizations, on an open access basis, as all our events are free-of-charge to attend!

Our values

Our values are to always ensure the highest quality of content and that content will be made readily accessible to all, and that we will always be an inclusive organization, serving a diverse scientific network. In addition, ELRIG will always be a volunteer led organization, run by and for the life sciences community, on a not-for-profit basis.

Our purpose

ELRIG is a company whose purpose is to bring the life science and drug discovery communities together to learn, share, connect, innovate and collaborate, on an open access basis. We achieve this through the provision of world class conferences, networking events, webinars and digital content.

Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.