Monitoring Tubulin PolymerizationUsing Nano-Differential ScanningFluorimetry

To monitor tubulin polymerization, we used ananoDSF instrument from NanoTemper Technologies, which enables trackingof the protein’s intrinsic fluorescence at 330 and 350 nm acrossa temperature range of 15–95 °C. The ratio of these twosignals (F₃₅₀/F₃₃₀) reflects the exposure of the protein’s tryptophan side-chainaromatic groups to the solvent, allowing for the observation of proteindenaturation upon heating the sample. We have noticed that the dimerizationinterface of tubulin contains several tryptophan residues, potentiallyenabling the use of nanoDSF not only to monitor tubulin denaturationbut also to observe the formation of MTs. To test this hypothesis,we subjected tubulin samples at varying concentrations to heatingusing nanoDSF in a polymerization buffer, wherein tubulin has thecapability to polymerize upon reaching physiological temperatures(FigureB,C). At alow subcritical concentration of tubulin, we detected only a singletransition around 62 °C, indicative of tubulin denaturation denotedfurther asT_m. Consistently, at higherconcentrations of tubulin, two additional transitions emerged on thethermogram. The first transition, occurring between 26 and 39 °C(depending on tubulin concentration), displayed a signal oppositethat of tubulin denaturation, suggesting that tryptophan side-chainaromatic groups were concealed from the solvent rather than exposed.This event most probably corresponds to tubulin polymerization. Thesubsequent transition, occurring between 50 and 55 °C, matchedthe magnitude of signal change of the first transition but was oppositein direction, strongly suggesting it correlates with the depolymerizationof MTs. These transitions are also accompanied by changes in sampleturbidity at 350 nm, with an increase followed by a decrease, returningto baseline around 55 °C (FigureE). The polymer state of tubulin under nanoDSF experimentalconditions was confirmed using TEM at different temperatures (FigureD), thus validatingnanoDSF as a tool to study the impact of compounds on tubulin polymerization.Henceforth, we denote the temperatures corresponding to the minimaand maxima of the first derivative of theF₃₅₀/F₃₃₀ ratio as the apparent temperaturesof tubulin polymerization (T_poly) anddepolymerization (T_depoly), respectively.

Given that tubulin has multiple binding sites influencing its polymerization,we evaluated the interaction of tubulin with three MTAsVinblastine(VBL), Taxol (TXL), and Mebendazole (MBZ)each binding to distinctsites (FigureF).This was done to assess the capability of the nanoDSF assay to detectinteractions between tubulin and MTAs. Therefore, tubulin samplesin the presence of increasing concentrations of MBZ, VBL, and TXLwere subjected to heating from 15 to 80 °C using the nanoDSFinstrument. This induced markedly distinct alterations in tubulinpolymerization and denaturation profiles (FigureG–I). A gradual increase in MBZ concentrationresulted in a notable shift ofT_poly tohigher temperatures without affectingT_depoly andT_m (FigureG,J), until MBZ reached a concentration of100 μM. At this concentration, MBZ entirely inhibited MT formation,resulting in the disappearance of the first two transitions (FigureG, yellow curve).

Contrary to MBZ, VBL is able not only to inhibit tubulin polymerizationin substoichiometric amounts but also to decrease the temperatureof depolymerization (FigureH,K). VBL is known to sequester tubulin into spiral structures.Therefore, the observed changes inT_poly andT_depoly values and the shallowerpeak slopes may indicate structural transitions between MTs and spiralsrather than dimers. This also explains the increase in the heightof the denaturation peak since it would induce the exposure to thesolvent of tryptophans not only from the tubulin core but also fromthe dimerization interface. Moreover, the marked increase in the denaturationtemperature may not result directly from ligand binding but ratherfrom mutual stabilization of tubulin dimers when arranged within spiralstructures.

Unlike MBZ and VBL, TXLknown to promoteMT formationshiftsT_poly andT_depoly in opposite directions (refer toFigureI,L). At a certainTXL concentration, thecharacteristic transitions become indistinguishable:T_poly drops below 15 °C, while the depolymerizationtransition merges with the denaturation peak. Thus, similarly to VBL,high concentrations of TXL lead to an increase in the height of thedenaturation peak, likely reflecting the unfolding of tubulin thatremains assembled in MTs at the onset of denaturation. Despite thissimilarity, the denaturation profiles of tubulin differ markedly betweenTXL and VBL. Specifically, a detailed examination of tubulin denaturationpeaks with increasing concentrations of VBL reveals a sequence whereinitially, the peak’s magnitude decreases, followed by thepeak becoming asymmetric, and ultimately, it increases in amplitudeand shifts to higher temperatures. In contrast, TXL leads to a gradualincrease in both the amplitude andT_m oftubulin’s symmetric denaturation peak. This may reflect differencesin site accessibility, which in turn couldinfluence the dynamics of compound exchange between free and boundstates, thereby differentially affectingtubulin denaturation.

Ultimately, the apparent affinity constantsof compounds couldbe independently estimated by analyzing both theT_m shift and the alterationsin fluorescence signal at 15 °C,which is particularly advantageous under experimental conditions whereconventional reference methods, like ITC, are ineffective (FigureM–O). Moreover,the nanoDSF assay is easier to set up and requires significantly lessmaterial than ITC.

Application of Nano-Differential ScanningFluorimetry for MicrotubuleTargeting Agent Screening

Thus, tracking temperature-inducedtubulin polymerization with nanoDSF enables the detection of shiftsin the polymerization temperature (ΔT_poly) across a broad concentration range with high sensitivity. Thisapproach facilitates the qualitative assessment of MTAs’ effectson tubulin polymerization, allowing for their comparative evaluationbased on this criterion. Additionally, further heating reveals theimpact of MTAs on tubulin’s thermostability (ΔT_m). By measuring these two distinct parameters,the first directly related to the tubulin function targeted by MTAsand the second reflecting the structural influence of MTAs on tubulin,this approach emerges as highly promising for MTA screening. AdvancednanoDSF instruments, such as the automated Prometheus NT.Plex, areequipped to conduct high-throughput screening of chemical librariesfor compounds targeting tubulin. To evaluate the efficacy of our novelMTA screening methodology, we applied it, as a proof of concept, tothe PCL, which comprises 1520 approved drugs (FigureA–C). SinceT_poly is highly sensitive to tubulin concentration (FigureB,C), each run includeda control sample, and the polymerization temperature shifts (ΔT_poly) were calculated relative to this control.This approach helped to minimize the variability of ΔT_poly values across experiments. Notably,T_poly among 67 control samples followed a normaldistribution, with a standard deviation of 0.5 °C, while thestandard deviation ofT_poly in runs withouthits containing one control and 23 molecules was twice as low, atjust 0.25 °C.

We observed that in the presence of 20 compounds (1.3% of PCL),tubulin exhibited no polymerization, suggesting either a completeinhibition of MT formation or the initiation of MT formation at temperaturesbelow 15 °C. These compounds are henceforth categorized as stronghits. Among these, nine are already known as MTAs (Table), three are suspected of havingMTA activity (Auranofin (AUF), Ebselen (EBS), and Riboflavin (RBF)),and eight (Aprepitant (APT), Benzarone (BZ), Benzbromarone (BZB),Benziodarone (BZI), Bithionol (BTN), Hexachlorophene (HCP), Nifedipine(NFD), and Nisoldipine (NSD)) are newly identified as exhibiting MTAactivity, previously unreported. For the remaining 1500 drugs, tubulinpolymerization occurred, enabling their classification based on twometrics: ΔT_poly and ΔT_m. The initial findings, along with 1D and 2Ddistributions of these metrics, are depicted inFigureB–D with standard deviation equalto 0.7 and 0.2 °C for ΔT_poly and ΔT_m, respectively. Both valuesdemonstrate Laplace distribution; compounds causing a ΔT_poly shift greater than 2 °C were designatedas hits withp < 0.0004. Those with a 1 °C< ΔT_poly < 2 °C are alsoconsidered potential MTAs, termed weak hits (p <0.02), meriting further investigation.

Among both strong and weak hits, we discovered that 30 compoundsare classified within the therapeutic category targeting metabolism;26 are utilized in infectious diseases, 17 in endocrinology, and 17in the treatment of CNS disorders (seeFigureE,F). When examining the therapeutic effectsof the drugs that influence tubulin polymerization, we identified27 compounds with antifungal properties, 19 with antineoplastic effects,16 with antibacterial activity, and 9 each with anti-inflammatory,antipsychotic, and anthelmintic effects. As expected, the main knowntarget of those hits was tubulin; still, more than 75% of hits hasanother protein listed as the “main” target (FigureG). Therefore, tubulinshould also be considered a significant target for these compounds,which could explain the molecular mechanism of action of some compounds,or the side effects associated with the clinical use of these molecules.

To assess whether compounds with MTA activity also demonstratecytotoxic effects, cell survival assays were conducted on the humanU87MG glioblastoma cell lines at varying concentrations of some identifiedcompounds. Our analysis revealed that for approximately 50% of thesecompounds (54 molecules), the IC₅₀ value was less than40 μM, while for about 20% (19 compounds), it was under 10 μM(FigureH–J,Tables S1–S8). The IC₅₀ valuesshowed no significant correlation with the change in the tubulin polymerizationtemperature (ΔT_poly) (FigureJ).

Structure–ActivityRelationship of Some Newly IdentifiedMicrotubule Targeting Agents

Furthermore, we analyzed thestructural similarity among all of the hit compounds. To achieve this,we initially computed a structure similarity matrix detailing thepairwise distances between compounds, utilizing “Morgan Connectivity”fingerprints. Subsequently, we derived a cluster hierarchy based onthis matrix (FigureA) and reorganized the structure similarity matrix in accordancewith the identified clustering, excluding compounds with minimal structuralresemblance (FigureB, hits with small structural similarity are listed inTable S8). This process enabled us to identifyand better visualize several clusters of molecules with analogousstructures within the hits (FigureA,B). Thus, various small clusters that include bothpreviously identified and novel MTAs are identified (FigureA,B,F–L,Tables S3–S7), two prominent clustersare highlighted, composed of established MT inhibitors: carbendazim(FigureD,Table S2) and phenothiazine (PTZ) derivatives(FigureE,Table S1). In the last cluster, we identifiedtwo pairs of molecules, perphenazine (PPZ) and fluphenazine (FPh),as well as chlorpromazine (CPZ) and triflupromazine (TFZ), whereinthe substitution of a chlorine atom (–Cl) at the second positionof the PTZ scaffold with a trifluoromethyl group (−CF₃) (seeFigureD)leads to a significant increase in ΔT_poly. To gain deeper understanding of this phenomenon, we employed funnelmetadynamics to simulate the docking of these four molecules, alongwith colchicine as a control, into the colchicine binding site ofβ-tubulinalso recognized as the binding site for PTZderivatives. The docking of colchicineto β-tubulin resulted in a center-of-mass position that wasconsistent with the X-ray crystallographic data (data not shown).Next, a comparative analysis of the two pairs of compounds showedthat the introduction of trifluoromethyl groups generally alteredboth the position and affinity of the molecules (FigureA,B). In the CPZ–TFZpair, the difference was most pronounced, with the trifluoromethylgroup penetrating deep into the protein cavity and “dragging”the entire molecule with it. In the PPZ–FPh pair, the trifluoromethylgroup also played a key role in forming effective contacts withinthe hydrophobic region of the β-sheet near the colchicine bindingsite. According to our calculations, the affinity of FPh was significantlyhigher than that of PPZ. Comparison of TFZ and FPh suggests that theirinhibition efficiencies arise from different mechanisms: while FPhexhibits high affinity, TFZ binding leads to substantial rearrangementsin the interfacial interactions between α- and β-tubulinsubunits.

New Approach for MicrotubuleTargeting Agent Screening

MTAs are an important class ofcompounds widely used in the treatmentof various diseases, including antifungal, antibacterial, antihelminthic,and antineoplastic therapies. Moreover, there is a growing body ofevidence that MT stabilizing MTAs could be used for treatment of braindisorders., Despite MTAs being considered a relativelyold class of anticancer drugs, some of which are perceived as no longer“trendy”, new therapeutic approaches based on MTAs continueto be proposed for cancer treatment. However,due to the development of drug resistance and significant side effectsassociated with these drugs, there is a constant need for more efficientand specific compounds that target tubulin polymerization. Numerousefforts have been made to develop MTA screening methods. In 2016,the team of Klassen et al. developed an assay of antitubulin drugsscreening based on catch-and-release electrospray ionization massspectrometry. They concluded that thedeveloped assay could be applied for anticancer drug screening andfor ranking the affinities of compounds to tubulin. Still, they didnot apply this new assay to “real” approved chemicallibraries. Moreover, the affinities of compounds are not always directlycorrelated with the anticancer activity of the molecules. While atleast six distinct binding sites for tubulin inhibitors are currentlyknown, each influencing tubulin polymerization in different ways,recent virtual screening studies have expanded the number of potentialbinding sites to 27. To validate thevirtual screening findings, the development of the functional MTAtest is still needed. This encouraged the team of Stefano Di Fioreto develop a SNAP-tag-based screening assay for the analysis of MTdynamics and cell cycle progression. Unfortunately,like the previous assay, it was tested only on a small number of molecules;however, it follows MT functions, making it more appropriate for newMTA screening. The main disadvantage of the proposed assay is thatit follows the impact of tested compounds in the cells wherein itis very difficult to separate the direct impact of the molecules ontubulin polymerization from indirect perturbation of the cellularcytoskeleton through molecules binding to some other targets thatperturb cell cycle and thus impact the cytoskeleton. Finally, to thebest of our knowledge, until now, there has been no functional high-throughputassay for MTA screening applied to diverse chemical libraries exceptin silico virtual screenings− sometimes followed by further in vitro validation.

The new nanoDSF screening assay introducedin this study not only overcomes the limitations of previous methodsbut also introduces additional advantages that are crucial for accuratelydetermining the mode of action of compounds. First, nanoDSF servesas an in vitro functional assay within a simplified environment, enablingthe ranking of compounds by their effect on tubulin polymerization,as indicated by shifts inT_poly. Second,nanoDSF incorporates several internal controls that enhance the assay’sreliability. The initial fluorescence measurement confirms the correcttubulin concentration, essential sinceT_poly is concentration dependent. Additionally, this assay enables thedetermination of tubulin’sT_m,thereby not only calculating ΔT_m for each compound but also ensuring the proper folding state oftubulin at each run. This feature is particularly vital for automatedscreenings, where maintaining the stability of such “fragile”proteins as tubulin over extended periods in plates is a key concern.Furthermore, by employing varying concentrations of compounds, itis feasible to ascertain the apparent association constants of hitswith tubulin based on both the fluorescence signal at a fixed temperatureand the denaturation temperature shift. Unlike previous MTA screeningassays, we validated our method on a library of 1520 approved compoundsand successfully identified every known MTA in that set Colchicine,Docetaxel, Fenbendazole, Mebendazole, Paclitaxel, Podophyllotoxin,Albendazole, Griseofulvin, Nocodazole, Oxibendazole, Oxfendazole,Parbendazole, and Triclabendazole.

Compounds with HighestMicrotubule Targeting Agent Activity

Through a novel nanoDSFscreening assay, we identified approximately95 compounds with MTA activity within the PCL. Among these, 20 compoundsfully inhibited temperature-induced tubulin polymerization under theexperimental conditions (Table). Some achieved this by directly inhibiting polymerization,while others promoted polymerization at lower temperatures. Notably,recognized MTAs such as Paclitaxel, Docetaxel, Podophyllotoxin, Colchicine,Fenbendazole, and Mebendazole were among those that prevented temperature-inducedpolymerization entirely. Additionally, our study confirmed that artificialestrogens like Hexestrol and Dienestrol, as well as the antisepticThiomersal, also impacted tubulin polymerization, consistent withprevious reports of their direct effects on tubulin.,

Over half of the strong hits were identified as MTAs for thefirst time through this screening. Among these, Auranofin (AUF), Ebselen(EBS), and Riboflavin (RBF) had previously been shown to affect thecytoskeleton despite the absence of direct evidence for tubulin binding.Thus, AUF, an antirheumatic gold complex, inhibits neutrophil activationby markedly reducing the number of centriole-associated MTs and obstructingphagocytosis in human polymorphonuclear leukocytes, likely througha mechanism that involves MT dysregulation., AUF is increasingly recognized as a potential anticancer agent;by serving as an inhibitor of both thioredoxin reductase and proteasome,it induces oxidative stress and triggers apoptosis in models of NSCLC. EBS, an organoselenium compound that mimicsglutathione peroxidase, has been explored as a neuroprotectant inischemia and conditions linked to oxidative stress. Research demonstrates its ability to destabilize MTs inskin melanocytes and inhibit tumor growth through the suppressionof 6-phosphogluconate dehydrogenase activity.− Deficienciesin RBF (vitamin B2) transport are linked to disturbances in MT dynamics;however, these disruptions can be alleviated through the administrationof RBF in cellular models. Furthermore,combining RBF with chemotherapy has been suggested as a strategy toreduce side effects and enhance therapeutic outcomes.

Eight of the identified strong hits were previouslyunrecognizedin their association with tubulin, marking them as novel discoveries.Notably, a family of benzofurans (Benzbromarone (BZB), Benzarone (BZ),and Benziodarone (BZI)FigureI) has been demonstrated to directly and effectively modulatetubulin polymerization, aligning with previous findings that BZ inhibitstumor growth in vitro. Intriguingly,BZ and BZB both were used in the treatment of gout akin to CLHawell-known MTA. While BZB is believed to act through uric acid reuptake,CLH disrupts inflammasome assembly at the cytoskeletal level., The revelation that benzofurans may also interact with MTs introducesan additional dimension to our understanding of their anticancer andanti-inflammatory properties. Among the novel MTAs identified is APT,a GPCR inhibitor is extensively used in chemotherapy to prevent commonside effects like nausea. Remarkably, APT is also attributed withthe antitumor properties of its own., BTN and HCP(FigureL) are fungicidesfrom a class of bridged diphenyl compounds with cytotoxic and antiproliferativeaction in cancer cell lines., Our findings revealthat several dihydropyridines, approved for managing angina, alsointerfere with MT assembly. Notably, Nifedipine (NFD) and Nisoldipine(NSD) (FigureG) demonstratedthe most significant impact on tubulin. While calcium channel blockerslike NFD have been previously noted to enhance the sensitivity ofdrug-resistant cancer cell lines to PTX, our research marks the first instance of identifying these medicationsas MTAs.

Collectively, the strong hits identified in this studypresentcompelling cases for drug repurposing, echoing the findings of priorresearch. Specifically, compounds such as AUF, EBS, APT, BZ, BZB,and HCP exhibit antitumor activity in vitro.,,,,, NFD shows potentialin reversing drug resistance in cancer cells, while RBF and BTN enhance the effectiveness of existing anticancerdrugs., Additionally, BZI and NFD represent modificationsof molecules with established antineoplastic properties, further underscoringtheir potential for repurposing in cancer therapy.

Structure–ActivityRelationship

Materials

Human glioblastoma cells were obtained fromATCC (Gaithersburg, MD, USA). Compounds used in the cytotoxicity assaywere from PCL, MedChemTronica (Bergkällavägen, Sweden),or Sigma (St Louis, MO, USA).

Tubulin Purification

Tubulin was purified from lambbrains by ammonium sulfate fractionation and ion-exchange chromatographyand stored in liquid nitrogen as described. Tubulin concentration was determined at 275 nm with an extinctioncoefficient of 109,000 M^–1 cm^–1 in 6 M guanidine hydrochloride.

Turbidimetry and DifferentialScanning Fluorimetry Assays

Aliquots of tubulin were passedthrough a larger (1 × 10 cm)gravity column of Sephadex G25 equilibrated with 20 mM sodium phosphatebuffer, 1 mM EGTA, 10 mM MgCl₂, 3.4 M glycerol, and 0.1mM GTP, pH 6.5 (PEMGT buffer) or 20 mM Tris, 1 mM MgCl₂, 0.1 mM GTP, pH 6.5 for nonpolymerizing conditions. For MTA bindingassays, VBL, MBZ, and TXL were used at concentrations up to 100 μM,with tubulin at 15 or 10 μM, respectively. Each capillary wasloaded with 10 μL of the sample. Fluorescence and turbidimetrymeasurements were performed on a nanoDSF Prometheus NT.Plex systemequipped with backscattering optics from 15 to 80 °C, using 10%excitation power and a temperature ramp of 1 K/min.

Nano-DifferentialScanning Fluorimetry Screening

FornanoDSF screening of 1520 FDA-approved compounds, 50 nL aliquots in100% DMSO were dispensed into 384-well microplates and stored at −80°C. Prior to nanoDSF measurements, 10 μL of 10 μMtubulin in PEMGT buffer was added to each well in a 24-well lane andmixed thoroughly by pipetting. The final compound concentration wasapproximately 50 μM in 0.5% DMSO. Plates were briefly centrifugedto eliminate air bubbles, and the samples were transferred to standardDSF-grade capillaries mounted on a 24-capillary rack. All measurementswere performed on a nanoDSF Prometheus NT.Plex instrument from 20to 75 °C, with 10% excitation power and a heating rate of 1 K/min.

Isothermal Titration Calorimetry

Binding of MTA totubulin was probed using a MicroCal iTC200 instrument (MicroCal, Northampton,MA, USA, now part of Malvern Instruments Ltd., Malvern, UK) in PEMGTbuffer. To prevent tubulin polymerization, the cell temperature wasmaintained at 10 °C. Tubulin was loaded into the calorimetriccell at a concentration of 20 μM, and MTA was titrated witha syringe at 250 μM.

Transmission Electron Microscopy

Samples were adsorbedonto 200 mesh Formvar carbon-coated copper grids, stained with 2%(w/v) uranyl acetate, and blotted to dryness. Grids were observedusing a JEOL JEM-1220 transmission electron microscope operated at80 kV. Magnifications used range from 60,000× to 120,000×.To ensure that MTs do not disassemble during adsorption, this stepwas performed in a thermostated room at 37 °C. The same stepwas performed for grids at 4 and 80 °C.

Cell Culture, Cytotoxicity,and Proliferation Assays

Glioblastoma cell culture routines,viability, and proliferationassays were performed as previously described. U87MG cells were maintained in complete MEM media supplementedwith 10% FBS and 2 mMl-glutamine (Invitrogen, Paris, France).For cytotoxicity assays, cells were counted and plated in 96-wellflat-bottom plates (50,000 cells/mL, 5000 cells per well). After 24h, the cells were treated with increasing concentrations of the MTAs(0, 1, 5, 10, and 20–40 μM) in a vehicle solution, containing0.05% DMSO. All concentrations were done in triplicates. The survivingcells were quantified after 72 h by the tetrazolium bromide MTT-assay,according to the manufacturer’s instructions. After cell lysis,the optical density was measured at 600 nm using a Multiskan MS Thermoplate reader (LabSystems, Waltham, MA, USA). Cell viability was expressedas a percentage of survival, using cells treated with the vehiclesolution as 100%, and the IC₅₀ values were calculated byusing the Chou and Talalay linearization method.

Funnel Metadynamics

We employedthree freely availablemodern force fields, including Amber19sb. All ligands were parametrizedusing acpype, with atom point charges derived from ab initio 6-31Gcalculations utilizing psiresp. Additionalparameters were assigned according to the GAFF2 force field. GDP wasparametrized in the same manner. The structure of tubulin alpha wasmodeled based on the coordinates from PDB ID4o2b. Protonation states of residues were predicted using PROPKAand manually verified. The system wasthen solvated in a triclinic box with periodic boundary conditionsby using TIP3P water molecules. To neutralize the system and achievean ionic strength of 0.15 M, Na⁺ and Cl^– ions were added. Energy minimization was performed using 5000 stepsof the steepest descent method. The equilibration phase comprisedseven steps. Initially, a 100 ps NVT simulation was conducted, applyingpositional restraints of 1000 kJ/(mol nm²) to the heavyatoms. Temperature coupling was maintained with a velocity rescalethermostat. This was followed by fiverounds of NPT equilibration, each lasting 100 ps, during which restraintstrength was gradually reduced: 1000, 500, 200, 100, and 10 kJ/(molnm²). Pressure coupling was achieved using a stochasticbarostat.

The funnel metadynamicssetup was modeled after the approach described by Raniolo and Limongelli. A metadynamic potential of 0.5 kJ/mol was appliedevery 500 steps. Two collective variables were employed: first, thedistance between the COM of CLH in its binding site and a referencepoint is 20 Å away from its position. This variable projectedthe ligand along the funnel line. Second, the perpendicular distanceof the ligand from this line. A correction to the binding free energywas applied to account for the entropic contribution due to the funnel-shapedrestraint, following the equation provided in Raniolo and Limongelli. The correction factor for the cylinder was calculatedas 1.59 kcal/mol. Final binding free energies were reported with errorestimates, following the method of Bhakat and Söderhjelm, using a statistical analysis window of 1000ns.

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Supplementary Materials

Data Availability Statement

All data availableupon request.