110 111 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 Soft CondensedMatter Group Beamline B23 Asthmamedication offers a potential new treatment for dementia Related publication: Townsend D. J., Mala B., Hughes E., Hussain R., Siligardi G., Fullwood N. J. &Middleton D. A. Circular Dichroism Spectroscopy Identifies the β-Adrenoceptor Agonist Salbutamol As a Direct Inhibitor of Tau Filament Formation in vitro . ACS Chem. Neurosci. 11 , 2104–2116 (2020). DOI: 10.1021/acschemneuro.0c00154 Publication keywords: Alzheimer’s disease; Tau; Amyloid; β-Adrenoceptor; Salbutamol; Dobutamine; Circular Dichroism Spectroscopy A lzheimer’s disease is aneurological condition that affects the largeageingpopulation.There is currentlyno cure for thedisease,which is associatedwith the formation of large protein plaques in the brain that kill neurons. A group of scientists led by Professor Middleton of Lancaster University, with the help of Diamond Light Source's Circular Dichroism beamline (B23) team, observed that the asthma drug salbutamol disrupted the formation of tau protein deposits that would otherwise accumulate in the brain. Salbutamol could, therefore, be a potential treatment for Alzheimer’s disease. The team conducted the laboratory study using various techniques. These revealed that salbutamol, by interacting with the tau protein, substantially prevented the formation of abnormal tangled clumps that at the early stages are thought to damage brain neurons and progressively induce dementia. In this work, high-throughput screening of libraries of generic chemicals enabled the identification of adrenalin as a successful candidate that precluded the formation of tau deposits. Subsequent screening of several drugs with similar chemical features identified salbutamol. Developing new drugs is slow and very expensive. It is far quicker and cheaper to repurpose clinically approved drugs for other diseases. This more economical approach can be applied to other neurological disorders (such as Parkinson’s and Huntington’s diseases) and any other disorder associatedwith protein deposits, such as type II diabetes, TTR amyloidosis and cancer. Alzheimer’s disease (AD) is classed by the World Health Organisation as a globalhealthpriorityaffecting47millionpeopleworldwide.Withan increasingly aging population, this figure is expected to triple to over 130 million cases by the year 2050, with an economic burden of $0.8 trillion 1 .The increase in the incidence of AD is compounded by the lack of a significant breakthrough in drug therapies in the past 40 years and no successful disease modifying treatment since its discovery in 1907 2 . Currently, only four drugs have been approved for use though none provide disease modifying therapeutic treatments. AD is characterised pathologically by the deposition of amyloid-β (Aβ) and tau proteins in the brain as insoluble amyloid fibrils and neurofibrillar tangles (NFT), respectively. The formation of NFT, which leads to neuron destabilisation and ultimately cell death, is thought to occur after the aggregation of Aβ and the related inflammatory response in AD. It has been known for a long time that the distribution of tau pathology in AD correlates much better with the clinical severity of AD than the distribution of Aβ plaques 3 . Finally, the central role of tau in neurodegeneration is evident in diseases such as primary age-related tauopathy where Aβ plaques are absent. One therapeutic approach for AD is to block or impair Aβ amyloid formation or tau NFT deposition. A common first step toward identifying potential inhibitors is in vitro screening of compounds that reduce tau or Aβ self-assembly kinetics and yield. The amyloid-sensitive fluorescent dye thioflavin T (ThT) is a convenient tool that is amenable to high-throughput screening but can report false positives when compounds compete for the same binding sites as the dye. Circular dichroism (CD) spectroscopy is an alternative approach, which reports directly on the structural changes of the protein as it undergoes aggregation and is therefore not prone to the ThT-type errors, but a high-throughput analysis is more challenging. High-throughput synchrotron radiation circular dichroism (HT-SRCD) has beenusedasanovelprimaryscreeningplatformtocomparethe inhibitoryeffects of a small library of drug-like compounds against tau filamentous assembly. B23 HT-SRCD has been developed as a powerful new method for the rapid analysis of protein structures under a range of different conditions 4 . A library of 88 drug and drug-like compounds, covering a broad range of chemical structures and indications, was selected. With large numbers of spectra to process and analyse, a multivariate approach of a principal component analysis was used to ascertain which compounds were most effective at stabilising tau in its native unaggregated structure. HT-SRCD indicates epinephrine to inhibit tau aggregation invitro and is a good starting point fromwhich to identify chemically similar compounds that may have more favourable properties in vivo . Figure 1a shows the principal component (PC) scores plot for the HT-SRCD spectra obtained 3 h after initiating tau aggregation. A PC trajectory plot, representing the change in the spectra between 4 and 6 h (Fig. 1b) reveals two samples that overlap or lie close to the control data for tau under nonaggregating conditions. These correspond to (±)-epinephrine hydrochloride (Fig. 1c) and (−)-epinephrine bitartrate. All other spectra (represented by arrows outside of thecircledregion in(Fig.1b) indicatedapartialorfullaggregationoftaubetween 1 and 6 h, were not representative of tau in its unaggregated and aggregated structures. Follow-up benchtop CD spectra of tau in the presence of epinephrine confirmed that the compound inhibited tau aggregation (Fig. 1d). B23HT-SRCDindicatesthatepinephrineissuperiortoallotherdrugsscreened, which cover a wide chemical space, in its ability to inhibit tau aggregation in vitro and is therefore a good starting point from which to identify chemically similar compounds that may have more favourable properties in vivo . Of the four investigated drugs chemically similar of epinephrine (Fig. 2), the CD data suggest that salbutamol impairs tau aggregation and stabilises tau in a structure close to its initial native state. By contrast, dobutamine appears to not have a significant effect on the rate of tau structural modification. Inthepresenceofheparin,tauaggregates into insolubleaggregatesafter24h andthevisualisationbynegativestainingtransmissionelectronmicroscopy(TEM) reveals a loose mesh of interwoven filaments with a typical amyloidmorphology, consistingofnetworksof longunbranchedfibresfrom500nmto1μm(Fig.3).The treatment of tau with salbutamol results in the deposition of fibrillar structures with different morphologies to the untreated tau. The TEM images concur with ThTandCDand indicatethatsalbutamol interfereswiththefibrilformationoftau. In summary, the in vitro ability of salbutamol to inhibit tau amyloidosis has been demonstrated providing the basis for further in vitro and eventually a full in vivo evaluation as a potential therapeutic for AD. References: 1. Improving Healthcare for People Living with Dementia − Coverage, Quality and Costs Now and in the Future - https://www.alz.co.uk/research/World Alzheimer Report 2016.pdf 2. Lane C. A. et al. Alzheimer’s disease. Eur. J. Neurol. 25 , 59–70 (2018). DOI: 10.1111/ene.13439 3. Arriagada P.V. et al. Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer’s disease. Neurology 42 , 631–639 (1992). DOI: 10.1212/wnl.42.3.631 4. Hussain R. et al. High-throughput SRCD usingmulti-well plates and its applications. Sci. Rep. 6 , 38028 (2016). DOI: 10.1038/srep38028 Corresponding author: Prof. Giuliano Siligardi, Diamond Light Source,
[email protected] Figure 1: HT-SRCD of drug-like compounds for inhibition of tau aggregation. (a) PC score plot of variance after 6 h incubation. Red circled for the compounds having greatest inhibitory effect, and the cyan circled for partially or full aggregated tau; (b) PC trajectory plot of spectral change, 1-6 h. Red arrows represent tau spectra non-aggregated, cyan arrows largest inhibitory effect of epinephrine (EPN(1)) and (EPN(2)). (C) HT-SRCD spectra of tau with EPN(1) (blue/green), tau under nonaggregating conditions t = 0 (black; n=10) and with heparin after 6 h (red; n=6). (D) CD spectra of heparin-induced tau aggregation (black) and with EPN(1) (red). Figure 2: (a) Far-UV CD spectra of tau (20 μM) after incubation for 0 (black) and 5 h (red) with equimolar concentrations of four catecholamine-derived β2-adrenergic receptor agonist drugs. The double arrows represent the increase signal at 218 nm consistent with β-sheet formation from the spectra of tau alone at 0 and 5 h. For salbutamol, an additional spectrum is shown after 24 h of incubation (blue); (b) Kinetics of tau aggregation with the four drugs, monitored at 218 nm; (c) Percent change in the secondary structure of tau with dobutamine and salbutamol incubated for 5 h. Figure 3: Negatively stained TEM images of tau (20 μM) aggregates formed in the presence of heparin (5 μM) after 24 h of incubation at 37 ° C. (a) and with the addition of 20 μM salbutamol; (b) Three different regions of the TEM grids are shown for each sample group.