Identification of a nanomolar affinity α-synuclein fibril imaging probe by ultra-high throughput in silico screening

Ultra-high throughput in silico screening identified molecules that bind to α-synuclein fibrils, which were analyzed by photo-crosslinking, structure-activity studies, and radioligand binding to validate this approach for finding imaging probes.

Introduction a-Synuclein (aS) is a 140 amino acid, intrinsically disordered protein which is abundantly expressed at the presynaptic termini of central nervous system neurons. 1,2 When bound to membranes, aS takes on a partially helical structure. 3,4 Although the function of aS is not fully understood, its localization, along with knockout and overexpression studies, suggest that alongside synapsin, VAMP2 and others, aS plays a role in maintaining synaptic vesicle reserve pools, neurotransmitter release and synapse function and plasticity. 1, 5,6 Conversely, the pathological role of aS is well documented, where neuronal inclusions, comprised principally of b-sheet rich brillar aS termed Lewy bodies (LBs) and Lewy neurites (LNs), have long served as a post-mortem hallmark of Parkinson's disease (PD). 7,8 Furthermore, similar aggregates can be observed in dementia with Lewy bodies (DLB), while a second form of aggregate has been identied in multiple system atrophy (MSA). 9 Compared to LBs and LNs which occur in neurons of the substantia nigra, aS aggregates associated with MSA are found in oligodendrocytes of white matter tracts and are referred to as glia cell inclusions (GCIs). 9 However, the inability to track bril formation and localization in living patients has hindered the development of robust correlations between bril distribution and PD, DLB, or MSA progression and prevents aS brils from serving as useful clinical markers.
To date, diagnosis of PD has relied heavily on the presentation of clinical symptoms, primarily motor decits. Although these symptoms are effective in tracking progression at later stages of these diseases, they are not observed until a substantial degree of neuronal loss has occurred. 10 Moreover, these symptoms are not exclusive to PD, but are observed for other Parkinsonian syndromes. 11 Therefore, since differences in the presence and localization of brillar aS have already been established, methods for tracking deposits in patients could clarify diagnosis. Over the past decade, a breakthrough in the clinical evaluation of Alzheimer's disease (AD) was enabled by in vivo imaging of protein deposits with positron emission tomography (PET). [12][13][14][15][16][17][18] The development of specic amyloid b (Ab) and tau PET probes has allowed researchers to determine that the formation of Ab aggregates precedes disease onset while tau-based neurobrillary tangles (NFTs) occur later in disease progression. 19 These promising results from imaging studies in AD patients have generated interest in the development of PET tracers to image aS brils and improve the diagnosis of PD.
Despite these advancements, the development of imaging probes with the requisite specicity for use in PD research and diagnosis has been challenging. In addition to LBs and LNs, PD patients also frequently present neuronal aggregates comprised of Ab and tau. 20 Although our laboratories and others have successfully identied compounds with moderate binding affinities for employment as PET imaging agents, no compound to date has displayed sufficiently high affinity and specicity for use as a radioligand. 21,22 Following publication of the rst solidstate NMR (ssNMR) structure of aS brils by Rienstra and colleagues, we demonstrated that through the combined use of computational docking, competition radioligand binding assays, and photo-crosslinking mass spectrometry we were able to describe distinct binding sites for several previously developed compounds. 23,27 Here, we explore the utility of another computational approach, exemplar-based in silico screening, in an effort to develop a molecule that potently and specically binds to aS brils. 28 Through the application of this method we have identied a molecular scaffold, conrmed its binding site through photo-crosslinking, and used structure-activity relationship (SAR) studies to identify members of this compound series that have nanomolar affinity for aS brils with moderate specicity for aS over Ab brils and other neuronal proteins. We go on to demonstrate the potential for development of this molecule as a PET probe through binding aS deposits in mouse brain tissue using a radiolabeled analog of the identied molecule. Finally, we provide SAR analysis based on the ssNMR structure used in our original in silico screening as well as several cryo-electron microscopy (cryo-EM) structures that have become available since we initiated these studies. [29][30][31][32][33] Results

Exemplar-based in silico screen
Through methods developed by Karanicolas and colleagues, using the Rosetta Modeling Suite, exemplars can be facilely generated for any protein of interest given an input structure. 34 An exemplar is a pseudoligand designed to be an ideal molecular complement to a surface exposed pocket on a protein of interest (Fig. 1). 28,34 Following selection of an anchor residue, the protein is cast onto a three-dimensional grid, and grid points that correspond to the protein pocket are "chemotyped" by the adjacent functional features on the protein. That is, exemplar grid points are assigned characteristics that are complementary to the protein surface based on whether the protein surface presents hydrogen bond donating or accepting moieties or a hydrophobic patch. The exemplar therefore represents perfect compatibility with the protein surface, but contains no chemical bonds. Subsequently, scored molecular alignment of compound structures from a database allows for ultra-high throughput in silico screening to identify molecular architectures that satisfy the chemical features (i.e. hydrogen bonding/accepting positions) captured by the exemplar. 28 In order to assess the potential of using an exemplar-based approach for aS PET probe development, we targeted sites that had been identied through our prior efforts as the binding sites for existing aS bril radioligands (Fig. 1). 23 In particular, two compounds, [ 3 H]Tg-190b and [ 3 H]BF2846 (ESI, Fig. S8 †), selectively bind to Site 2 (residues Y39-S42-T44, conrmed through photo-crosslinking in Hsieh et al. 23 ) and Site 9 (residues G86-F94-K96, conrmed through ClX1 photocrosslinking in ESI, Fig. S6 †) respectively, and can be utilized for in vitro competition binding assays to screen compounds identied in silico. Therefore, we chose these two sites as our initial targets. Using the ssNMR structure (PDB ID 2N0A) deposited by Rienstra and colleagues, pocket templates were generated using residues 44 and 86 of the central strand of the bril as anchor points for Sites 2 and 9 respectively. The exemplar for each site was screened using Align-It against $10 million commercially available, lead-like molecules from the ZINC15 database. [24][25][26] For each site, the 50 molecules scored as having the best alignment to each exemplar pseudoligand were retained (ESI, Fig. S2 and S3 †). From this set of in silico hits, 17 compounds were purchased based on considerations of scaffold diversity.
These compounds were employed in a screening assay to determine their relative affinities for specic sites on aS brils. Of the set of molecules selected, two molecules were able to displace Site 2 radioligand [ 3 H]Tg-190b ( Fig. 2A).
Competitive binding experiments revealed that compounds 2 and 6 displayed inhibitory concentrations (IC 50 s) of 490 nM and 9.49 nM, respectively, against the Site 2 radioligand (Fig. 2b). 35 (Bottom) Workflow for identifying small molecule binders. 23 A zoom in on the Site 2 exemplar is shown on the left with compound 6 docked in the conformation identified from the ZINC 15 database using Align-It. [24][25][26] Lead compound binds to site 2 and does not affect aggregation Following the identication of two potential lead compounds, we decided to further investigate the more potent binder to conrm that the compound (1) binds to the target site on aS brils and that (2) the compound is non-perturbing and does not affect the aggregation of aS. We believe that a suitable imaging compound should not perturb the aggregation process, either by increasing or decreasing the amount of brils or altering their conformations. Therefore, a derivative of compound 6 was synthesized with a photo-crosslinking group (ClX2) to conrm the binding location (ESI, Scheme S2 †). Following incubation of compound ClX2 with aS brils and irradiation with 365 nm light, the sample was analyzed by whole protein and trypsin digest mass spectrometry (ESI, Fig. S7 †). Analysis of the resulting data revealed a mass shi corresponding to crosslinking of one molecule of ClX2, suggesting that most of the compound binds at a single site. Furthermore, analysis of the digested sample conrmed that crosslinking could be observed on peptides corresponding to the target site, Site 2, initially used for the in silico screen (ESI, Fig. S7 †).
To conrm that the probe does not affect the aggregation state or propensity of aS, we generated uorescently labeled aS for use in uorescence polarization (FP) assays. By attaching uorescein-maleimide to a Y136C mutant aS construct, FP can be used to monitor bril aggregation and gross conformational changes, as well as the compound's effect on these processes, as previously described. 36,37 Compound 6 did not show any signicant effect on the rate of aggregation or on the stability of aS brils (ESI, Fig. S4 and S5 †).

SAR screen to improve affinity/specicity
In order to improve the affinity and selectivity of compound 6, we conducted a SAR screen by culling structurally similar compounds from the Mcule library via a similarity search. 38 In total, 39 molecules representing derivatives of the core scaffold 26 (Fig. 3, top) were used to determine the impact of various substituents on the A and C rings as well as replacement of the B ring with other 5-membered heterocycles. A subset of these compounds representing discreet changes to the core scaffold are shown in Fig. 3, along with their % bound of the Site 2 ( Fig. 3, bottom) radioligand. The structures and competition . ANOVA was performed with Dunnett's multiple comparisons test, where *P < 0.0332, **P < 0.0021, ***P < 0.0002 and ****P < 0.0001. . ANOVA was performed with Dunnett's multiple comparisons test, where *P < 0.0332, **P < 0.0021, ***P < 0.0002 and ****P < 0.0001. The full set of structures and binding data for all compounds tested in the SAR screen are given in ESI Fig. S9 and S10. † binding data for the full set of compounds are shown in ESI Fig. S9 and S10. † Several aspects of the SAR are clear. Changes to the A ring, particularly para substitution, are well-tolerated (19,28,31,34,39,40), and in many cases improve binding relative to molecules that are otherwise identical (19 or 31 vs. 24). Replacement of the A ring with a pyridine heterocycle (21) did not improve binding; nor did replacement with a furan or thiophene group (20 and 22, ESI, Fig. S9 Fig. S9 and S10, † 32, 35, 37, 44, 50), but moderate to strong binding is seen for compounds with electron donating methyl (28, 40, and 52) and methoxy (19, 24, 31, 34 and 39) groups. Lastly, we nd that compounds with substituents at the a-carbon of the central glycolic amide bind poorly (Fig. 3 23 and ESI, Fig. S9 † 35), possibly due to a break in the planarity of the molecule.
Screening of the SAR library (Fig. S10 †) was analyzed with one-way ANOVA and compounds showing the highest affinity for aS brils (p < 0.0001) were selected for in-depth characterization. For these compounds, we measured their affinity for both Site 2 and Site 9 in aS through multi-point competitive binding assays with [ 3 H]Tg-190b and [ 3 H]BF2846 (Table 1). Compound 28 was found to have a higher affinity than compound 6, with some apparent site-specicity.

Synthesis and characterization of radioligand
The SAR studies and IC 50 determinations identied compound 28, a close analog of original hit 6, as the most potent binder. Thus, we set out to synthesize and further characterize an iodinated derivative, compound 61, as well as its radiolabeled isotopolog [ 125 I]61. Chloroacetamide 60 was synthesized by reaction of iodo-isoxazolamine 58 and 2-chloroacetyl chloride, and subsequently coupled with 3,4-dimethylphenol to obtain iodo-derivative 61 with an overall yield of 27% (Scheme 1). In order to obtain the precursor for radioiodination, compound 28 was synthesized in a similar manner starting from bromoisoxazolamine 57, and subsequent stannylation of 28 yielded precursor 62. Radioiodination was then successfully achieved at room temperature to obtain [ 125 I]61 for in vitro binding experiments.
First, we conrmed that replacement of the uorine or bromine in compounds 6 and 28 with an iodine did not perturb the relative affinity for Site 2 (Fig. 4a) (Fig. 4b). We also demonstrated that 61 has a $5-fold selectivity (Fig. 4c) for aS brils over amyloid b (using brils of the 42 amino acid Ab42 peptide). While these results are promising, it should be noted that the levels of non-specic binding were relatively high. Crosslinking with CLX2 followed by labeling with a uorescent azide demonstrated binding to aS brils, but also to many other proteins in mouse brain lysate (ESI, Fig. S13 †). The potential efficacy of this radioligand as an imaging probe was then tested by in vitro autoradiography studies. Images were obtained by incubating 10 mm thick sections of sarkosyl-insoluble fraction from 15 month old PD mouse model (A53T) with [ 125 I]61 and subsequent exposure to storage phosphor screens. The autoradiograms and quantication thereof are shown in Fig. 5. The images clearly demonstrate binding of [ 125 I]61, and this binding can be blocked by co-incubation with unlabeled 61. Autoradiograms of the entire brain section from A53T and C6C3F1/J control mouse were also obtained (ESI Fig. S14 †). While there is increased signal in A53T mouse brain in aS-rich regions compared with the control, the non-ideal physicochemical properties of this radioligand make it unsuitable to accurately assess binding on whole brain sections.

Discussion
The development of a PET tracer to image PD is a high priority in the eld of radiopharmaceutical research, since a radioligand that binds to aS could greatly improve the clinical diagnosis of PD. Despite signicant academic and industrial effort in recent years, a PET tracer for aS with high affinity and selectivity has yet to be developed. In this work, we utilized a combination of exemplar-based in silico screening and radioligand binding studies to identify several compounds that bind to aS brils with nanomolar affinity and with moderate selectivity over Ab brils. We are currently working to optimize 61 for in vivo PET studies. With this in mind, several aspects of our results are worth considering in more depth, including the implications of the SAR study for compound optimization, the importance of the ssNMR structure among various currently available aS bril structures, and the relevance of models based on these structures to brils in tissue.
The SAR study reveals that features of all three rings and the unsubstituted nature of the central amide unit are important for high affinity binding. An examination of docked compound 6 in the aS 2N0A structure (Fig. 6) reveals how several of these trends can be rationalized. The narrowness of the cavity is consistent with the fact that methyl substituents in compounds such as 23 generate non-planar structures which cause steric binding, ortho substituents weaken binding (as in 30). These clash with the sidechains of Y39 and T44 (Fig. 6b). While meta, and especially para, substituents on the C ring improve binding, ortho substituents would clash with the protein backbone at K43/S42. On the A ring, para halogenation improves Site 2 affinity, but this seems to be a steric effect, rather than an electronic effect, since electron-donating methyl substituents (19) at this position are also tolerated and diuorination in 39 and 40 does not dramatically improve binding. However, it is possible that there are counterbalancing electronic effects on interactions of the A ring p system with the Y39 and T44 sidechains. Finally, the fact that the compounds with a 1,2,4-oxadiazole B ring do not bind well indicates that the SAR around this ring system is subtle and may depend on effects on the acidity of the heteroatoms, altering their hydrogen bonding ability with the T44 sidechain or even the backbone. Certainly, additional experiments with a tailored compound set will be necessary to fully understand the binding mode; we will also employ modeling that accounts for multiple rotamers and the various structures of aS brils that are now available.
The exemplar in silico screen was performed with the 2N0A ssNMR structure, which was the only aS bril structure available at the time that this study was initiated. Since then, an additional ssNMR structure and several cryo-electron microscopy (cryo-EM) structures have become available, including structures of brils isolated from MSA patients. [29][30][31][32][33] Furthermore, biochemical and biophysical techniques have also characterized molecular differences between in vitro brillar preparations and between patient samples from the major synucleinopathies. 39,40 It is notable that the Site 2 fold is similar (1 nM) in sarkosyl-insoluble fraction from A53T mouse brain. Nonspecific binding was defined using unlabeled 61 (100 nM). (b) Quantification of autoradiograms (n ¼ 4); t-test was performed, where *P < 0.0332, **P < 0.0021, ***P < 0.0002 and ****P < 0.0001. Fig. 6 Model of compound 6 bound to Site 2. Two views of an equilibrated structure of compound 6 in the 2N0A Site 2 binding pocket, highlighting steric fit. Compound 6 was docked into the structure by alignment to the Site 2 exemplar and the structure minimized as described in ESI. † to 2N0A Site 2 in some of these structures, but different in others ( Fig. 7 and ESI, Fig. S15/S16 †). We modeled the binding of each Table 1 compound in 2N0A Site 2 using a standard Autodock 4.2 procedure or by alignment to the original exemplar using Shape-It followed by minimization in PyRosetta where the total ligand score (TLS) of each molecule was computed. We determined correlations between the simulated DDG values, computed from PyRosetta TLS values or Autodock binding energies (BEs), and DDG values derived from the experimental IC 50 values ( Fig. 7 and ESI Fig. S14 †). We found that PyRosetta TLS correlated signicantly better than Autodock BE (R 2 (TLS) ¼ 0.90, R 2 (BE) ¼ 0.55). In addition to superior correlation, PyRosetta provided an accurate ranking of relative affinities of the compound set, whereas Autodock only correctly predicted the highest and lowest affinity compounds (ESI, Table S1 †). While the small size of the data set does not allow us to conclude that the TLS correlations can reliably predict the affinities of new compounds, we believe that they can provide evidence that a given binding site structure is a good model for interpreting SAR data.
We performed PyRosetta-based docking and TLS calculations for the compounds in Table 1 with the Site 2 pocket from six of the available aS bril structures. For these alternative structures, one can see that some, but not all, provide a good correlation between DDG values derived from the IC 50 values and their calculated Site 2 TLS (ESI, Fig. S16 †). The variability of these correlations indicates that some of the structures are better models for interpreting SAR data than others. In particular, one of the MSA patient-derived structures (6XYQ) shows a correlation comparable to 2N0A with a similar rank ordering of ligands. In spite of a somewhat different overall fold, the alignment of the Site 2 region for the 6XYQ (MSA) and 2N0A (ssNMR) structures is quite good as one can see from an alignment of the two sets of PDB coordinates and an overlay of the coordinates with the cryo-EM density map (Fig. 7). This implies that the 2N0A structure in the Site 2 region may be representative of aS brils in MSA patients and raises the exciting possibility that our compounds will have similar affinities in MSA patient tissue.
We expect that multiple patient-derived cryo-EM aS bril structures will be reported, as they have been for tau and Ab, using material derived from human patients with Alzheimer's disease or related neuropathies. [41][42][43] With such structures available, an essential question for drug design will be whether they accurately represent the conformation (or conformations assuming that varying polymorphs underlie differences in synucleinopathies, as seen for tau) adopted by brils in vivo. SAR studies performed with patient tissue using the compound classes identied by our approach can be used in conjunction with correlations like those reported here to determine which of the structures best represents the conformations of the brils in vivo. Subsequent optimization for in vivo PET imaging will certainly focus on improvement of solubility, removal of probable metabolic liabilities, blood-brain barrier penetration, and selectivity against Ab and tau brils. While some of this optimization will rely on empirical knowledge of the pharmacokinetic properties of functional groups, it will also be guided by computational modeling of candidate compounds bound to the most relevant structures of aS brils as well as counterselection using structures of Ab and tau brils.
In summary, we have used an ultra-high throughput in silico screen to identify lead compounds for PET imaging of aS brils in PD and related synucleinopathies. Lead compound 61 has a 1 nM affinity for aS brils with demonstrated animal tissue binding, and the correlations shown in Fig. 7 imply that our compound class may be particularly suited for imaging in MSA patients. Moreover, our approach is unique and represents a new paradigm for radioligand development. The exemplar strategy is particularly well-suited to the problem of identifying ligands for shallow surfaces where no existing binding partner or enzyme substrate can provide a starting point for molecular design. Future implementations of these methods will also include pre-screening the compound library to remove compounds with unfavorable physico-chemical properties, which could help to avoid non-specic binding of the sort observed for compound 61. This computational approach, coupled with screening of commercially available compounds in competition assays with radioligands, allows one to rapidly develop SAR for a given binding site. For a case such as the aS bril, with multiple shallow binding sites, conrmation of site-specicity using photo-crosslinking also plays an important role. While we had previously established a set of site-specic ligands for competition assays, a de novo approach would also be possible in which in silico hits are converted to either radiolabeled or photo-crosslinkable compounds to establish binding sites and affinities. Indeed, the azidation reaction used to generate compound ClX1 for conrming the Site 9 selectivity of BF2846 (ESI, Fig. S6 †) is nicely suited to this purpose. These initial lead compounds could then be used in high throughput assays with other unlabeled compounds as demonstrated in our work. Thus, the paradigm for imaging probe development established here should be broadly applicable to other bril forming proteins, which are increasingly recognized as targets not just in PD, but also in other therapeutic areas with unmet molecular imaging needs such as amyotrophic lateral sclerosis (ALS) or neuroinammation.

Exemplar generation and in silico compound screen
The ZINC15, lead-like, commercially available compound database, consisting of $10 million molecules was used for the initial screen against the three sites. 24 Exemplars were extracted from all residues in the PDB 2N0A ssNMR structure using default parameters in Rosetta and residues 156, 163 and 198 provided the best representations of the previously describes Sites 2, 3/13 and 9, respectively. 23 Molecular alignments of target molecules to each selected exemplar were performed using Align-It which reduces each molecule/exemplar to a set of pharmacophores and reports a Tanimoto coefficient for each alignment, capturing both agreements in molecular features and their alignment in three-dimensional space. 25 The top 30 compounds as quantied by the Tanimoto coefficient from each search were retained and a subset of compounds from each search was selected by hand for experimentation. Since the compounds identied for Site 3/13 were either too small or too similar to compounds previously explored, 23 select compounds from the Site 2 and Site 9 screens were used in subsequent experimental screens and the full set of compounds can be found in ESI Fig. S2 and S3. †

Screening compound library
Compounds 1-17 were purchased from vendors that were listed on the ZINC15 compound library. In order to screen the purchased compounds for aS binding, 100 nM of each compound was incubated for 1 h at 37 C with 100 nM aS brils and [ 3 H]Tg-190b (6 nM) or [ 3 H]BF2846 (3 nM) in 50 mM Tris-HCl, pH 7.4. Total binding was measured in the absence of competitor and nonspecic binding was determined in reactions containing unlabeled Tg-190b (1 mM) or BF2846 (0.5 mM). Aer incubation, bound and free radioligand were separated by vacuum ltration, followed by washing with buffer containing 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl. Filters containing the bound ligand were mixed with 3 mL of scintillation cocktail (MicroScint-20, PerkinElmer, Inc.) and counted aer 12 h of incubation on a MicroBeta System (PerkinElmer, Inc.). All data points were measured in triplicate. Screening of compounds 18-56 for the SAR study was performed in a similar fashion.

Saturation binding assay
aS (50 nM) or Ab42 (100 nM) brils were incubated for 1 h at 37 C with increasing concentrations of [ 125 I]61 in 50 mM Tris-HCl, pH 7.4, in a total volume of 150 mL. Nonspecic binding was determined in a duplicate set of binding reactions containing 2 mM non-radioactive 61. Aer incubation, bound and free radioligand were separated by vacuum ltration through Whatman GF/C lters in a 24-sample harvester system, followed by washing with buffer containing 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl. Filters containing the bound ligand were counted immediately on 2470 WIZARD Automatic Gamma Counter. All data points were collected in triplicate. The equilibrium dissociation constant (K d ) and the maximal number of binding sites (B max ) were determined by tting the data to the equation Y ¼ B max Â X/(K d + X), using GraphPad Prism soware.

In vitro autoradiography on sarkosyl-insoluble fraction
The sarkosyl-insoluble fraction was obtained from brain homogenates of A53T (B6C3-Tg(Prnp-SNCA*A53T)83Vle/J) mice from The Jackson Laboratory. Frozen sections were thawed and incubated (1 h at room temperature) with 40% ethanol in PBS containing either [ 125 I]61 (1 nM) alone or [ 125 I]61 (1 nM) with 100 nM unlabeled 61. Aer incubation, sections were washed in ice-cold 40% ethanol in PBS (3 Â 1 min), followed by a wash in ice-cold DI water (1 min). Sections were dried in a stream of air, exposed to a storage phosphor screen (GE Healthcare) and the screen was imaged on a Typhoon FLA 7000 phosphor imager (GE Healthcare). Autoradiography images were quantied using Fujilm Multi Gauge soware.

SAR correlation calculations
Two dimensional compounds were converted to three dimensional structures using openbabel. 44 Compound 6 was aligned to each of the bril architectures via their respective exemplars using Shape-It from Silicos-It. 26 Derivatives were then aligned to the parent compound using Align-It, also from Silicos-It. Complexes were minimized using MinMover in PyRosetta where sidechain optimization was performed on the residues comprising Site 2 (residues 38-46), whereas backbone optimization was globally disallowed. Minimized complexes were scored using the beta_nov16 score function and the total ligand scores (TLSs) for the ligands in their minimized orientations were computed. 45 Rosetta TLS values in REU were converted to DDG values in kcal mol À1 using the previously reported scaling factor of 2.94. 46 Corresponding Autodock binding energy (BE) calculations were performed using a previously published protocol. 23 Experimental DDG values were determined by using compound 52 as a reference. Computed DDG values from Autodock for the 2N0A structure and from PyRosetta for 2N0A, 6XYQ, and several other aS bril structures were linearly regressed with the experimental DDG values to determine correlations. Details of all of these calculations are found in ESI † with comparisons of the other brils shown in Fig. S15/ S16. †

Conflicts of interest
There are no conicts to declare.