Lipid Nanoparticles for Membrane Protein Target Drug Discovery

Membrane proteins are essential in a plethora of physiological processes and perturbation of their function often leads to disease. As such, membrane proteins are the target for over 50% of pharmaceuticals. Their location within the lipid bilayer has meant that the study of their structure and function still lags behind that of easier to isolate, soluble proteins; this has hindered progress in drug design and development. The application of styrene maleic acid (SMA) polymers to isolate proteins directly from cell membranes, forming SMA lipid particles (SMALPs) where the proteins retain their lipid bilayer environment makes them potentially more suitable for structural and functional studies and could be used for drug development purposes. The aim of this project was to develop polymer-lipid particle approaches for biophysical assays such as surface plasmon resonance (SPR) or microscale thermophoresis (MST) suitable for medium to high throughput screening. To achieve this aim, two model proteins were used, Atm1 and LeuT. Atm1 is an ATP Binding Cassette (ABC) transporter and LeuT is a prokaryotic homologue of neurotransmitter transporters of the neurotransmitter: sodium symporter (NSS) family. Recombinant Atm1 and LeuT were expressed in E. coli cells, extracted, and purified using several different polymers and stability and function investigated. Immobilisation studies for SPR were undertaken, which found that amine-coupling an anti-his antibody to the sensor chip and then binding the his-tagged SMALP-LeuT to that antibody was the most effective method for immobilisation. However, the level of immobilisation was too low to detect small molecule binding. Initial studies with MST showed SMALP-LeuT could be successfully labelled with a fluorophore at the his tag. Detection of leucine binding was highly variable and poorly reproducible. However, binding of the drug desipramine to SMALP-LeuT could be successfully measured and a dose-response curve obtained. Chemical modification of the SMA polymer was tested, to potentially enable immobilisation or labelling of the polymer rather than the protein. Sulfhydryl groups were added to SMA forming SMA-SH, and the modified polymer was able to solubilise and purify proteins. Future work would look at labelling with biotin for immobilisation. Molecular biology approaches to add a SNAP-tag to the Atm1 protein was also started. The SNAP-tag enables site specific labelling with a range of different probes. Finally, two series of novel SMA polymer variants were tested to see if they offered any improvement over SMA2000. The first series were partially esterified SMA polymers, and the second series were benzylamine modified SMA polymers. Whilst all of the novel polymers were able to solubilise and purify membrane proteins, they did not offer an improvement over SMA2000, and were even more sensitive to divalent cations

Examining the stability of membrane proteins within SMALPs

Amphipathic co-polymers such as styrene-maleic acid (SMA) have gained popularity over the last few years due to their ability and ease of use in solubilising and purifying membrane proteins in comparison to conventional methods of extraction such as detergents. SMA2000 is widely used for membrane protein studies and is considered as the optimal polymer for this technique. In this study a side-by-side comparison of SMA2000 with the polymer SZ30010 was carried out as both these polymers have similar styrene:maleic acid ratios and average molecular weights. Ability to solubilise, purify and stabilise membrane proteins was tested using three structurally different membrane proteins. Our results show that both polymers can be used to extract membrane proteins at a comparable efficiency to conventional detergent dodecylmaltoside (DDM). SZ30010 was found to give a similar protein yield and, SMALP disc size as SMA2000, and both polymers offered an increased purity and increased thermostability compared to DDM. Further investigation was conducted to investigate SMALP sensitivity to divalent cations. It was found that the sensitivity is polymer specific and not dependent on the protein encapsulated. Neither is it affected by the concentration of SMALPs. Larger divalent cations such as Co2+ and Zn2+ resulted in an increased sensitivity

Membrane protein extraction and purification using styrene-maleic acid (SMA) co-polymer:effect of variations in polymer structure

The use of styrene maleic acid (SMA) co-polymers to extract and purify transmembrane proteins, whilst retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene to maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA) which vary in size and shape were used. Our results show that several polymers can be used to extract membrane proteins comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular weight (7.5-10 kDa) is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification SMA 2000 was found to be the best choice for yield, purity and function. However the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications

Membrane protein extraction and purification using partially-esterified SMA polymers

Styrene maleic acid (SMA) polymers have proven to be very successful for the extraction of membrane proteins, forming SMA lipid particles (SMALPs), which maintain a lipid bilayer around the membrane protein. SMALP-encapsulated membrane proteins can be used for functional and structural studies. The SMALP approach allows retention of important protein-annular lipid interactions, exerts lateral pressure, and offers greater stability than traditional detergent solubilisation. However, SMA polymer does have some limitations, including a sensitivity to divalent cations and low pH, an absorbance spectrum that overlaps with many proteins, and possible restrictions on protein conformational change. Various modified polymers have been developed to try to overcome these challenges, but no clear solution has been found. A series of partially-esterified variants of SMA (SMA 2625, SMA 1440 and SMA 17352) has previously been shown to be highly effective for solubilisation of plant and cyanobacterial thylakoid membranes. It was hypothesised that the partial esterification of maleic acid groups would increase tolerance to divalent cations. Therefore, these partially-esterified polymers were tested for the solubilisation of lipids and membrane proteins, and their tolerance to magnesium ions. It was found that all partially esterified polymers were capable of solubilising and purifying a range of membrane proteins, but the yield of protein was lower with SMA 1440, and the degree of purity was lower for both SMA1440 and SMA17352. SMA2625 performed comparably to SMA 2000. SMA 1440 also showed an increased sensitivity to divalent cations. Thus, it appears the interactions between SMA and divalent cations are more complex than proposed and require further investigation

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Effects of pre-operative isolation on postoperative pulmonary complications after elective surgery: an international prospective cohort study an international prospective cohort study

We aimed to determine the impact of pre-operative isolation on postoperative pulmonary complications after elective surgery during the global SARS-CoV-2 pandemic. We performed an international prospective cohort study including patients undergoing elective surgery in October 2020. Isolation was defined as the period before surgery during which patients did not leave their house or receive visitors from outside their household. The primary outcome was postoperative pulmonary complications, adjusted in multivariable models for measured confounders. Pre-defined sub-group analyses were performed for the primary outcome. A total of 96,454 patients from 114 countries were included and overall, 26,948 (27.9%) patients isolated before surgery. Postoperative pulmonary complications were recorded in 1947 (2.0%) patients of which 227 (11.7%) were associated with SARS-CoV-2 infection. Patients who isolated pre-operatively were older, had more respiratory comorbidities and were more commonly from areas of high SARS-CoV-2 incidence and high-income countries. Although the overall rates of postoperative pulmonary complications were similar in those that isolated and those that did not (2.1% vs 2.0%, respectively), isolation was associated with higher rates of postoperative pulmonary complications after adjustment (adjusted OR 1.20, 95%CI 1.05–1.36, p = 0.005). Sensitivity analyses revealed no further differences when patients were categorised by: pre-operative testing; use of COVID-19-free pathways; or community SARS-CoV-2 prevalence. The rate of postoperative pulmonary complications increased with periods of isolation longer than 3 days, with an OR (95%CI) at 4–7 days or ≥ 8 days of 1.25 (1.04–1.48), p = 0.015 and 1.31 (1.11–1.55), p = 0.001, respectively. Isolation before elective surgery might be associated with a small but clinically important increased risk of postoperative pulmonary complications. Longer periods of isolation showed no reduction in the risk of postoperative pulmonary complications. These findings have significant implications for global provision of elective surgical care. We aimed to determine the impact of pre-operative isolation on postoperative pulmonary complications after elective surgery during the global SARS-CoV-2 pandemic. We performed an international prospective cohort study including patients undergoing elective surgery in October 2020. Isolation was defined as the period before surgery during which patients did not leave their house or receive visitors from outside their household. The primary outcome was postoperative pulmonary complications, adjusted in multivariable models for measured confounders. Pre-defined sub-group analyses were performed for the primary outcome. A total of 96,454 patients from 114 countries were included and overall, 26,948 (27.9%) patients isolated before surgery. Postoperative pulmonary complications were recorded in 1947 (2.0%) patients of which 227 (11.7%) were associated with SARS-CoV-2 infection. Patients who isolated pre-operatively were older, had more respiratory comorbidities and were more commonly from areas of high SARS-CoV-2 incidence and high-income countries. Although the overall rates of postoperative pulmonary complications were similar in those that isolated and those that did not (2.1% vs 2.0%, respectively), isolation was associated with higher rates of postoperative pulmonary complications after adjustment (adjusted OR 1.20, 95%CI 1.05–1.36, p = 0.005). Sensitivity analyses revealed no further differences when patients were categorised by: pre-operative testing; use of COVID-19-free pathways; or community SARS-CoV-2 prevalence. The rate of postoperative pulmonary complications increased with periods of isolation longer than 3 days, with an OR (95%CI) at 4–7 days or ≥ 8 days of 1.25 (1.04–1.48), p = 0.015 and 1.31 (1.11–1.55), p = 0.001, respectively. Isolation before elective surgery might be associated with a small but clinically important increased risk of postoperative pulmonary complications. Longer periods of isolation showed no reduction in the risk of postoperative pulmonary complications. These findings have significant implications for global provision of elective surgical care