Calmidazolium Chloride and Its Complex with Serum Albumin Prevent

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Calmidazolium chloride and its complex with serum albumin prevent Huntingtin exon1 aggregation Virender Singh, Rama Nagesh Venkata Krishna Deepak, Bhaswati Sengupta, Abhayraj S. Joshi, Hao Fan, Pratik Sen, and Ashwani Kumar Thakur Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00380 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 9, 2018

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Molecular Pharmaceutics

Calmidazolium chloride and its complex with serum albumin prevent Huntingtin exon1 aggregation Virender Singh†, R. N.V. Krishna Deepak‡, Bhaswati Sengupta€, Abhayraj S. Joshi†, Hao Fan‡§, Pratik Sen€ and Ashwani Kumar Thakur†* †

Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur – 208016, India

€

Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur – 208016, India ‡

§

Bioinformatics Institute, 30 Biopolis Street, Matrix #07-01, Singapore 138671

Department of Biological Sciences, National University of Singapore, 117545 Singapore

ACS Paragon Plus Environment

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

Huntington’s disease (HD) is a genetic disorder caused by a CAG expansion mutation in Huntingtin gene leading to polyglutamine (polyQ) expansion in the N-terminus side of Huntingtin (Httex1) protein. Neurodegeneration in HD is linked to aggregates formed by Httex1 bearing expanded polyQ. Initiation and elongation steps of Httex1 aggregation are potential target steps for the discovery of therapeutic molecules, for yet untreatable and cruel, HD. Here we report Httex1 aggregation inhibition by calmidazolium chloride (CLC) by acting on the initial aggregation event. Being hydrophobic, CLC was adsorbed to the vial surface and could not sustain inhibition effect for longer duration. Usage of bovine serum albumin (BSA) prevented its adsorption by forming BSA-CLC complex. This complex showed improved Httex1 aggregation inhibition by interacting with the aggregation initiator, NT17 part of Httex1. Further, a biocompatible CLC loaded BSA nanoparticles were made which reduced the polyQ aggregates in HD-150Q cells.

KEYWORDS Huntington’s disease, polyglutamine aggregation, calmidazolium chloride, adsorption, serum albumin 1

Introduction With an increase in life expectancy over the years, there is an unexpected surge in the

incidence of age-related ailments particularly, neurodegenerative diseases.1,

2

Huntington’s

disease (HD) is one of the late age onset disease caused by the CAG triplet expansion mutation in the exon 1 of huntingtin gene.3,

4

This results in expanded polyglutamine (polyQ) in

Huntingtin (Httex1) protein, which forms aggregates in neurons and causes cellular toxicity.3 Also, recent studies have elucidated the role of the flanking N-terminus-17 amino acid (NT17) of


2

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Httex1 to accelerate the polyQ aggregation kinetics. NT17 tends to form an amphipathic helix which self-associates to alter polyQ aggregation pathway towards non-nucleated pathway via oligomer formation. Thus polyQ-rich Httex1 peptide aggregation and its aggregation steps are promising therapeutic targets in HD.5-8 Reducing its aggregation with peptide inhibitors like NT17, QBP1 and P42 is known to mitigate the cellular toxicity.9-11 These inhibitors are yet to see clinical developments due to difficulty in delivery across blood brain barrier (BBB).12 Structure based design research on small molecule inhibitors is limited in HD due to the intrinsic disorder of Httex1.13, 14 Based on the research on other intrinsically disordered peptides like amyloid-β15 and synuclein16, aromatic interactions (π•••π stacking) are proved to play an important role in the aggregation inhibition.16-19 Considering the similarities in some of the steps of aggregation among aggregating proteins and peptides yielding oligomer, protofibril and fibril formation,20 we selected a molecule with known anti-aggregation effect on other amyloid-forming proteins. Taking aromatic interaction-mediated inhibition as a reference, we chose to test calmidazolium chloride (CLC) which is known to modulate amyloid-β (1-40) aggregation to prevent mature fiber formation.21 CLC is a hydrophobic molecule and carries a positive charge with imidazolium ring at the centre and a large extended hydrophobic region at the periphery. It is a known calmodulin (CaM) antagonist22, 23 and also blocks transient receptor potential cation channel subfamily V member 1 (TRPV1) channel to alter calcium transport to relieve pain.24 CaM is one of the known targets in HD which binds to mutant huntingtin (mHtt) and regulates transglutaminase 2 dependent crosslinking of Htt.25 Testing a molecule which could show bispecific property i.e. target Httex1 aggregation as well as block CaM association with mHtt and transglutaminase might have high therapeutic value.25, 26


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In this article, we show the ability of a hydrophobic molecule calmidazolium chloride (CLC) to prevent Httex1 aggregation. To overcome the problem of loss of CLC to vial surface adsorption, we developed serum albumin-based novel assay where serum albumin (bovine or human origin) forms a complex with CLC to prevent its adsorption to the vial surface. With the help of biophysical techniques and molecular docking, we characterized the serum albumin-CLC complex formation. This complex shows better inhibition of Httex1 aggregation by forming a ternary complex with NT17 of Httex1 peptide. For cell culture studies, we have synthesized and characterized the biocompatible CLC loaded serum albumin nanoparticles. We have demonstrated that these nanoparticles are able to reduce the polyQ aggregate burden in HD150Q cells, suggesting their potential for HD therapeutic and could be evaluated in in preclinical studies. 2

Materials and methods

2.1

Materials

Calmidazolium

chloride

(CLC,

1-

[Bis(4-chlorophenyl)methyl]-3-[2,4-dichloro-β-(2,4-

dichlorobenzyloxy)phenethyl]imidazolium chloride) was purchased from Sigma. Httex1 (MATLEKLMKAFESLKSFQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQPPP PPPPPPPKK) and Q35 (KKQQQQQQQQQQQQQQQQQQQQQQQQQQ QQQQQQQQQKK) peptides were purchased from Keck biotechnology resource laboratory, Yale University, New Haven, CT, USA. NT17 (MATLEKLMKAFESLKSF) peptide, bovine serum albumin (BSA), Human serum albumin (HSA), coumarin-343, 4-dimethylamino pyridine (DMAP), N,Ndicyclohexylcarbodiimide,

trifluoroacetic

acid

(TFA),

1,1,1,3,3,3-Hexafluoro-2-Propanol

(HFIP), sodium azide, formic acid, Dimethyl sulfoxide (DMSO)-d6, Ponasterone A (PNST), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

(MTT),

4',6-diamidino-2-


4

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phenylindole (DAPI), p-nitrophenyl coumarin ester (NPCE) and dialysis membrane tubing (12 kDa cutoff), were purchased from Sigma-Aldrich (Bangalore, India). Acetonitrile (HPLC grade), centrifugal filter units (Amicon Ultra, 10 kDa cutoff), ethanol (99.9% purity), triton-X 100, glutaraldehyde (25% aqueous solution), DMSO, di-sodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from Merck, India. N6,2′-O-Dibutyryladenosine 3′,5′cyclic monophosphate sodium salt (dbcAMP) was purchased from Sisco Research Laboratories (SRL). Trypsin was purchased from New England Biolabs. All chemicals were used as received. In all of the experiments, 0.22 µm filtered MilliQ water (18 MΩ cm) was used unless stated otherwise. 2.2

Peptide disaggregation

All three peptides i.e. Httex1, Q35 and NT17 peptides were purified, lyophilized and disaggregated as per published protocols.7 Briefly, 0.1 mg of peptide was dissolved in 1 mL of TFA: HFIP (1:1) and kept at room temperature for 12 h in dark. The solvent was evaporated under the purge of nitrogen and vacuum dried in the desiccator for 2 h for vacuum drying. 2.3

Reverse phase-High performance liquid chromatography (RP-HPLC) analysis

A disaggregated peptide was dissolved in acidified water (pH 3 adjusted with TFA) followed by centrifugation at 303,824 × g (Thermo S80-AT2 fixed angle rotor) for 4 h at 4 °C. Peptide aggregation reaction was carried out in phosphate buffer saline (PBS, pH 7.4) with 0.05% (v/v) sodium azide as an anti-microbial agent. Sample was incubated at 37 °C in a microcentrifuge tube (polypropylene, Tarsons). The peptide aggregation reaction was set up in the absence and presence of 100 µM CLC. The CLC stock (1 M) was prepared in DMSO and final concentration of DMSO was kept at 10% (v/v) in aggregation reaction. For BSA-CLC complex formation, constituents were added in the following order: first acidified water (pH 3 adjusted with TFA)


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was added followed by PBS, 0.05% (v/v) sodium azide, 5 µM BSA and lastly 100 µM CLC. The mixture was mixed gently and then peptide was added. Httex1 peptide aggregation reaction was monitored by taking small aliquots of sample intermittently at different time points. Samples were centrifuged at 119,500 × g for 30 min at 4 °C. Top 70% of supernatant was taken and formic acid (final concentration 20% v/v) was added to stop the ongoing aggregation reaction. Samples were analyzed using RP-HPLC at 215 nm wavelength on Agilent HPLC 1260 infinity system. ZORBAX eclipse plus C18 rapid resolution column (4.6 × 100 mm, 3.5 µm) was used with column thermostat set at 25 oC. Water and acetonitrile with 0.05% (v/v) TFA were used as mobile phase at 1 mL min-1 flow rate. To determine the effect on the initial phase of aggregation, Httex1 aggregation was set at three different concentrations (15 µM, 20 µM and 25 µM) in the presence of CLC (100 µM). For comparison of the delay in aggregation rate, reaction was monitored for initial 30% loss of monomer and concentration was plotted against square of time (s2). The slopes obtained from the plot define the aggregation rate for initial phase of Httex1 aggregation. 2.4

CLC recovery from Httex1 aggregates

For determining the presence of CLC in the Httex1 aggregates, Httex1 was allowed to aggregate in the presence of CLC (100 µM). Aggregates were washed eight times with water (resuspending aggregates and centrifuging intermittently) till all loosely bound CLC was removed. After 8th wash, pellet was dissolved in formic acid (20% v/v) to dissolve all aggregates. CLC was characterized by using RP-HPLC. 2.5

Vial surface adsorption assay

To monitor CLC loss to the vial surface, 100 µM CLC was added to PBS (pH 7.4) and incubated at 37 °C. Samples were analyzed using RP-HPLC. The two group comparison was


6

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performed using unpaired student’s t-test and the difference was considered statistically significant when p < 0.05. 2.6

Dynamic light scattering (DLS)

Particle size and diffusion coefficient analysis was performed on Malvern Zetasizer Nano ZS90 equipped with He–Ne laser operating at a wavelength of 633 nm and the scattering angle of 90°. Both buffer and peptide solution were filtered through 0.22 µm syringe filter before mixing. All the DLS measurements were performed at 25 ºC. The size of a particle is calculated from the translational diffusion coefficient by using the Stokes Einstein equation:

= η

(1)

where d(H)- hydrodynamic diameter; D- translational diffusion coefficient; k- Boltzmann’s constant; T- absolute temperature; η- viscosity. 2.7

Tryptophan fluorescence quenching measurements

The fluorescence intensities were recorded with a Perkin-Elmer LS 55 fluorescence spectrometer using low volume cuvette (120 µL) with 1 cm path length. The tryptophan was excited at λex 295 nm with slit width of 10-nm for excitation and 5-nm for emission slit and scan speed was kept 500 nm min-1. BSA concentration was kept 1 µM for tryptophan quenching. CLC dissolved in DMSO was titrated to 130 µL protein solution (PBS pH 7.4); to give a final CLC concentration in the range 0-20 µM. Fluorescence measurements were recorded at 25 oC. The dilution at the end of titration was 2.3% (v/v) and accounted for the calculation of ligand concentration. To confirm no interference from CLC in tryptophan fluorescence, UV absorbance spectra was recorded for 1 M CLC and found to be negligible at 295 nm. The change in fluorescence emission intensity was measured immediately after mixing CLC or DMSO to the protein. A normalized fluorescence spectrum for BSA was plotted for CLC titrations. Data was


7


Page 8 of 38

analyzed by plotting the relative fluorescence intensities (F0 − F)/F0 at 354 nm versus increasing concentrations of CLC (µM). Here, F0 and F are the fluorescence intensities of BSA in the absence and presence of CLC, respectively. Three independent titration experiments were performed and data was plotted as mean ± SD. Equilibrium Dissociation constant (Kd) was determined using one site saturation module of simple ligand binding tool of Sigma plot 12.0. For BSA and BSA-CLC complex interaction with NT17 peptide, slit width was kept at 10 nm for excitation, 7 nm for emission and scan speed was 600 nm min-1. BSA-CLC complex was prepared by adding 5 µM CLC to 1 µM BSA (PBS pH 7.4, 5% DMSO). BSA-CLC complex was titrated with NT17 peptide and change in tryptophan fluorescence was monitored. For determining NT17 interaction with only BSA, 1 µM BSA was titrated with NT17 solution and change in tryptophan fluorescence was monitored. 2.8

Fluorescence correlation spectroscopy (FCS)

An in-house assembled FCS instrument was used for all measurements. It consists of an inverted confocal microscope (Olympus IX-71, Japan) equipped with a 60X water immersion objective with numerical aperture (NA) 1.2 (UplanSApo, Olympus, Japan) to focus the 405 nm excitation light, from a 5 mW CW laser source (PSU-III-FDA, Optoelectronics Tech. Co. Ltd., China). The protein sample was put on the top of a coverslip (Blue Star, Polar Industrial Corporation) placed on the sample platform. The laser light was focused into the sample at a distance of 40 µm from the upper surface of the coverslip. The emitted photons were collected by the same objective and focused on a multimode fiber patch cord of 25 µm diameter (M67L01 25 µm 0.10 NA, ThorLabs, USA) after passing through a dichroic mirror (ZT405rdc, Chroma Tech. Corp., USA) and an emission filter (FSQ-GG455, USA). A fluorescence signal was then directed towards a photon-counting module (SPCM-AQRH-13-FC, Excelitas Tech. Inc., Canada) through


8

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the fiber patch chord and then to a correlator card (Flex99OEM-12/E, Correlator.com, USA) to generate the autocorrelation function, G(τ). Finally, autocorrelation curves were displayed using LabView® (National Instruments, USA) software. Details of autocorrelation function and equations are provided in the supporting information. The samples were prepared in 50 mM PBS (pH 7.4) and the concentration of NPCE tagged HSA was kept at 50 nM. All the measurements were done at 298 ± 1 K. Data analysis was performed as described earlier.27-30 2.9

Computational docking of CLC to BSA

The structure of calmidazolium (CLC; ZINC 4262457) was obtained from the ZINC12 database.31, 32 To identify the possible binding sites in BSA, CLC was computationally docked to BSA (PDB: 4F5S)33 using SwissDock.34-36 Using the web server of SwissDock (http://www.swissdock.ch/docking), the docking was done for BSA without specifying particular binding site to avoid any sampling bias. SwissDock performs calculations in the CHARMM force field. The most favorable BSA-CLC binding mode was interpreted on the basis of the cluster with the best FullFitness score. A greater negative score represents a better fit for a favorable binding mode. The binding conformation of CLC within the binding pocket in BSA identified using SwissDock was refined by further docking, using GOLD.37 GOLD uses a genetic algorithm to explore the full range of ligand conformational flexibility.37 2.10 Synthesis and purification of BSA nanoparticles BSA nanoparticles were synthesized as per published protocol with minor modifications.38 BSA was dissolved in water (pH 8.5) at a concentration of 50 mg mL-1. To 500 µL of BSA, 2 mL of ethanol was added at the rate of 1 mL min-1 under constant stirring at 800 rpm at 25 °C. The suspension was allowed to stir for 20 min and 8% glutaraldehyde (0.6 µL per mg of BSA) was added. The suspension was stirred continuously for another 18-20 h. The resulting albumin


9


Page 10 of 38

nanoparticles were purified by three cycles of ultracentrifugation (16,000 × g, 20 min, 25 °C) followed by redispersion of the pellet in 2.5 mL water. Each redispersion step was performed in an ultrasonication bath (MSE 150 ultrasonicator) for 5 min. Finally, empty BSA nanoparticles (ENPs) were resuspended in 2.5 mL water (0.02% sodium azide) and stored at 4 °C. For CLC loaded BSA nanoparticles (CNPs), CLC (100 µM) was dissolved in ethanol and added to BSA solution as described for empty nanoparticles. To optimize maximum encapsulation and loading, different concentrations of CLC were used i.e. 25 µM, 200 µM and 200 µM. Similarly, two different concentrations of BSA were tested i.e. 50 mg ml-1 and 75 mg mL-1. 2.11 Scanning electron microscopy BSA nanoparticles (8 mg mL-1) were diluted 500 times with 0.2 µm filtered water. An aliquot was placed on a glass coverslip pasted on carbon tape attached to the copper stub and air-dried at room temperature overnight. The samples were sputtered with gold for 60 s under argon gas atmosphere (automatic sputter coater) and analyzed with a field emission electron microscope with an upper detector (FE-SEM, JSM-7100F; JEOL) at 10 kV. The size of the BSA-NPs was examined using the ImageJ software. 2.12 In vitro CLC release An aliquot of 5 mg of CLC nanoparticles (CNP) were suspended in 300 µl of PBS pH 7.4 (0.05% v/v sodium azide) in 1.5 mL microcentrifuge tube. The tube was incubated at 37 °C under constant stirring at 175 rpm. The sample was withdrawn at 24 h and centrifuged at 16,000 × g for 20 min. Supernatant was analyzed using RP-HPLC. For trypsin digestion of CNPs, trypsin was used at concentration of 1.25 µg mg-1 of CNP. Released CLC was analyzed after 24 h using RP-HPLC.


10

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2.13 Cell viability assay Cell viability in the presence of various concentrations of CLC and BSA nanoparticles was analyzed using MTT assay. The HD-150Q (kindly gifted by Dr. Nihar Ranjan Jana, NBRC, India) cells were seeded (2,500 cells/well) in a 96-well plate. After 24 h, cells were treated with 1 mM dbcAMP for differentiation. To induce expression of mHtt150Q-EGFP, cells were incubated with 10 µM PNST for 24 h. Neuro-2a cells were incubated with ENPs and CNPs (800 µg mL-1 concentration) after differentiation and tNmhHtt-150Q-EGFP expression. After 24 h treatment, 20 µL MTT solution was added in each well and the incubation was continued for another 4 h at 37 oC. After 4 h, the medium was removed from wells carefully and 200 µL DMSO was added to dissolve formazan crystals. The absorbance was measured at 570 nm using Perkin-Elmer plate reader and cell viability was represented as a percentage relative to the untreated control cells. 2.14 Effect of CLC on Htt aggregation in HD-150Q cells In vitro effect of CLC against aggregation was analyzed in Neuro-2a cells expressing tNhttEGFP-150Q (HD-150Q).39 2,500 HD-150Q cells were seeded in each well of 24 well plate containing a glass coverslip. After 24 h, cells were treated with 1 mM dbcAMP for differentiation. To induce expression of tNhtt-EGFP-150Q, cells were incubated with 10 µM PNST for 24 h. After PNST treatment, cells were incubated with only medium (control), ENPs (control) and CNPs (250 µg mL-1 CLC concentration) for 24 h. On the following day, cells were fixed using 4% paraformaldehyde, stained with DAPI and put on a glass slide for microscopic analysis. Images were captured using 10× and 40× objectives using ultra violet (UV) and green filters, merged using adobe photoshop CS6. For quantification of tNhtt-EGFP-150Q aggregates, images were analyzed using ImageJ software (V1.50i). For each image, total number of green


11


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fluorescence protein (GFP) positive puncta was counted representing total number of aggregates. Similarly, total number of DAPI stained nucleus were counted representing total number of cells. Results were represented as percentage of cells containing aggregates obtained by dividing GFP puncta count by DAPI count. 2.15 Hemolytic assay Blood was collected from three healthy mice in anticoagulant coated AcCuvet-Plus K3-EDTA tube as per approved protocol (IITK/IAEC/2017/1071). Blood was immediately centrifuged at 1,000 × g for 10 min at 4 oC and plasma along with the buffy coat was aspirated carefully. Red blood cells (RBC) were washed thrice with 0.9% saline. RBCs were then diluted to 1/10th of their original volume (10%v/v) in PBS pH 7.4. 60 µL of RBCs suspension (10% v/v) was then added to 140 µL PBS (pH 7.4) followed by 100 µL nanoparticles to obtain 2% (v/v) RBCs suspension. PBS pH 7.4 was used as a negative control and 1% Triton-X 100 was used as a positive control (100% hemolysis). RBCs suspension containing ENPs and CNPs (800 µg mL-1) was incubated at 37 oC for 1 h and centrifuged at 1,000 × g for 10 min to separate RBCs. 200 µL of supernatant from each tube was transferred to separate wells in a 96-well plate and absorbance was measured at 540 nm. Hemolysis was characterized by disappearance of the RBCs pellet and appearance of free hemoglobin in the supernatant. % =

!"#$ %& "'&

&#"#$ %& "'& !"#$ %& "'&

( × 100

(2)

2.16 Statistics Statistics were performed in Sigma plot 12.0. For two-sample comparison, unpaired student ttest was performed. For all pairwise multiple comparisons and comparisons against control, the differences between the groups were evaluated with one-way analysis of variance (ANOVA) followed by Holm-Sidak post-hoc test. p < 0.05 was considered to be statistically significant.


12

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3

Results and discussion

3.1

CLC modulates Httex1 aggregation

Calmidazolium chloride (CLC), a hydrophobic molecule was used here to check its aggregation inhibitory potential against Httex1 (Figure 1a). Literature suggests that targeting amphipathic face of NT17 domain of Httex1 may prevents its aggregation.6, 40 Thus the use of hydrophobic CLC might help it to bind to the hydrophobic face, probably via aromatic interactions and might abrogate Httex1 aggregation. The Httex1 peptide follows non-nucleated spontaneous aggregation kinetics via oligomer formation,7 as a first step (Figure 1b) towards complete aggregation. CLC significantly delayed this aggregation of the Httex1 peptide for initial 15 h but inhibition effect receded thereafter.

Figure 1. Calmidazolium chloride (CLC) modulates Httex1 aggregation.


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(a) Chemical structure of CLC (ZINC 4262457). (b) Httex1 peptide aggregation inhibition by CLC. Httex1 peptide (20 µM) aggregation kinetics in the absence (black) and presence (red) of 100 µM CLC (n = 4). Aliquots of sample were centrifuged at respective time point and monomer content of Httex1 peptide in the absence and presence of CLC was determined using RP-HPLC. (c) Aggregation rate of Httex1 at different concentrations in the presence of 100 µM CLC. Plot represents the first 30% of Httex1 aggregation reaction and slope represents the corresponding aggregation rate. (d) Detection of the presence of CLC in Httex1 aggregates using RP-HPLC. Aggregates of Httex1 incubated with CLC were washed 8 times with water and final aggregates were solubilized in formic acid (20% v/v). CLC released from aggregates was analyzed by RPHPLC and representative chromatogram is shown with CLC peak at 12.6 minutes. Httex1 alone follows downhill aggregation kinetics7 but the presence of CLC probably added a kinetic barrier to this spontaneous aggregation. The effect induced by CLC was further confirmed by determining the aggregation rate for 30% of initial aggregation at different concentration of Httex1. At 15 µM concentration of Httex1, the presence of CLC resulted in slower aggregation rate compared to that of 20 µM and 25 µM concentrations (Figure 1c). These results hint towards the possible interaction of CLC with Httex1 during an early stage of aggregation reaction. Interestingly, CLC failed to inhibit aggregation of Q35 peptide which was without NT17 domain (Figure S1). This corroborates well with the published literature where no aggregation inhibition was observed on polyQ in the presence of CLC.21 This suggests towards NT17 interaction mediated anti-aggregation activity of CLC. Similar action was earlier reported for peptide inhibitors.7 Further to confirm possible interaction of CLC with Httex1 before its spontaneous aggregation, we checked CLC co-localization with aggregates. The presence of CLC in formic acid solubilized aggregates confirmed CLC Httex1 interaction (Figure 1d).


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Similar inhibition trends and inhibitor co-localization with Httex1 aggregates were reported for peptide aggregation inhibitors.6 Thus, CLC interacts with the Httex1 peptide to target the initial event (NT17 peptide self-association) during its spontaneous aggregation. But, aggregation inhibition observed was short lived and the effect was lost after the initial delay. 3.2

CLC adsorption to vial surface diminish its aggregation inhibitory activity

We observed only 7-10 µM of CLC in the supernatant of Httex1 reaction after 3 h (Figure 2a). In addition, CLC recovered from the aggregates accounted for partial CLC amount, which prompted us to look for the cause responsible for its loss. Literature suggests that hydrophobic molecules tend to adsorb nonspecifically to the vial (plastic/glass) surface.41, 42

Figure 2. CLC adsorption to the vial surface.


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(a) CLC loss to vial surface during Httex1 peptide aggregation reaction. Soluble CLC content was determined by RP-HPLC at each time point (n = 2). (b) CLC loss to the vial surface adsorption in PBS (pH 7.4). CLC solubilized in 100% DMSO served as a positive control with no loss to the vial surface. Adsorbed CLC was recovered with DMSO wash from the vial, previously incubated with CLC in PBS (n = 4, * p < 0.05, ns- not significant, pairwise comparison was done using one-way ANOVA with Holm-Sidak post-hoc test). (c) Schematic representation of CLC adsorption to the vial surface. Red circles represent CLC. (d) The particle size of CLC in the aqueous solution shown as number percent was determined by DLS. CLC was used at three different concentrations. Being highly hydrophobic, we suspected that CLC might not be stable in solution for long and might adsorb to the hydrophobic surface of reaction vial. To investigate this, CLC was separately incubated in the vial containing 100% DMSO, an ideal solvent for solubilizing hydrophobic molecules. No loss of CLC was observed from the solution after 24 h of incubation in DMSO while 80% of CLC was lost when incubated in PBS (Figure 2b). CLC adsorption to the vial surface was further confirmed when vial surface was rinsed with DMSO and the lost CLC was recovered. Due to hydrophobic nature of CLC, it preferred the vial surface over solution (Figure 2c). Adsorption kinetics showed a rapid adsorption till 5 h followed by a decline after 10 h (Figure S2). This is in congruence with Httex1 aggregation inhibition data wherein CLC lost its activity at the later stages of aggregation reaction (Figure 1b). CLC formed sub-micron sized particles in PBS as observed by DLS (Figure 1d), similar to other hydrophobic molecules under similar conditions.43 To our surprise, adsorption of CLC did not take place in water pH 5.5 and phosphate buffer pH 7 (without salt) (Figure S3a). However, adsorption took place in both PBS (pH 7) with 150 mM NaCl and water (pH 5.5) containing 150


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mM NaCl. This was also confirmed by the increase in particle size and scattered light intensity (kcps) (Figure S3b). Thus ionic strength contributes majorly to CLC surface adsorption. The presence of NaCl resulted in the formation of large particle size in aqueous solution indicating self-assembly of CLC. This was further confirmed by 1H NMR, where aryl protons of CLC showed upfield shift, indicating π-π stacking induced self-association of CLC when present in aqueous solution (Figure S4). Although the self-association phenomenon of the small molecule is not completely understood, it was reported for few dyes and many lipophilic molecules.44-46,43 Thus data suggests that the observed loss in inhibition potency of CLC could be attributed to the non-availability of CLC in solution. Since it was unavoidable to omit salt from the buffer for the present study to mimic in vivo conditions, we tested other ways to avoid vial surface adsorption of CLC. First, we tested the vial with hydrophilic surface modification or coating with protein to avoid adsorption. A similar loss of CLC was observed in case of eppendorf LobindTM vial and BSA coated vial in comparison to tarson microcentrifuge vial used in this study (Figure 3a). Coating vial surface with BSA is a well-known technique to prevent protein surface adsorption but it failed in the case of CLC. This could be due to possible loss of CLC to the adsorbed BSA on the vial surface. To check this hypothesis, we used BSA in solution to have free BSA molecules to bind to the adsorption prone CLC. A significant decrease was observed in CLC adsorption to the vial at 5 µM and 100 µM BSA concentrations (Figure 3a). Even, 5 µM BSA was able to diminish CLC vial surface adsorption. The observed phenomenon was specific to BSA, as lysozyme could not prevent adsorption. Serum albumin was effective irrespective of the origin of protein i.e. bovine (BSA) or human (HSA) (Figure S5). The presence or absence of fatty acid did not affect the antiadsorption property of BSA (Figure S5). Also, For 100 µM CLC, 1 µM BSA prevented 60%


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adsorption after 96 h at 37 °C and 5 µM prevented 80% surface adsorption and preferred for further experiments (Figure S6). BSA:CLC complex in the ratio of 1:10 is capable of preventing adsorption completely while BSA:CLC ratio of 1:50 prevented adsorption partially.

Figure 3. Serum albumin prevents CLC adsorption to the vial surface. (a) Effect of vial surface modification (chemical and physical) and the addition of protein in solution on CLC adsorption after 24 h of incubation. CLC content in solution was determined using RP-HPLC (n = 3). *p < 0.05, ns- not significant, all pairwise comparisons were done using one-way ANOVA with Holm-Sidak post-hoc test. (b) Particle size (diameter in nanometer) of CLC, BSA and BSA-CLC in PBS was determined by DLS. Change in hydrodynamic diameter of


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BSA-CLC complex formation shown as number percent. (c) Intrinsic tryptophan fluorescence quenching by CLC binding to BSA. Decrease in fluorescence emission of BSA (1 µM) on titration with CLC. (d) Effect of increasing concentration of CLC on BSA tryptophan fluorescence. Relative fluorescence intensity represented as (,- − , ⁄ ,- . Dissociation constant (Kd) was determined using Sigma plot 12.0 (n = 3, mean ± SD). 3.3

CLC complex with BSA rescues its vial surface adsorption

DLS data revealed the emergence of a new peak with an intermediate size when compared to BSA and CLC alone (Figure 3b). This could be due to the complex formation which was further characterized by combination of biophysical and computational methods. 1H NMR spectroscopy showed the changes in the chemical shifts of amide hydrogen and aryl protons of BSA with increasing CLC concentration (Figure S7). It could be possible that aromatic ring of CLC interacts with either aromatic amino acids through π-π interaction or with positively charged residues of BSA through cation•••π interactions. Extent of this interaction was further revealed by the fluorescence quenching of tryptophan of serum albumin (Figure S8). Titration of BSA with CLC resulted in a gradual decrease in emission intensity with increasing CLC concentration, indicating intrinsic fluorescence quenching due to binding of CLC (Figure 3c). Also, a slight blue shift in the emission maximum of both BSA and HSA indicates binding of CLC that resulted in a more hydrophobic environment around tryptophan.47, 48 It yielded binding constant (Kd) of 0.78 ± 0.06 µM for BSA-CLC complex (Figure 3d). Similar tryptophan fluorescence quenching was observed for HSA (Figure S9a) with Kd of 2.48 ± 0.42 µM for HSA-CLC complex (Figure S9b) representing weak CLC binding. The small difference in Kd for HSA and BSA could be due to the availability of different binding sites for CLC.49


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Computational docking analysis of CLC binding to serum albumin

Putative binding pockets for CLC on BSA were identified by docking using the SwissDock web server.36 Docking predictions primarily place CLC at the inter-domain interfaces of BSA with a majority of docked poses clustered within the cleft formed by domain I and III (22/32 clusters). A small number of clusters (5/32) also occupy a pocket formed by the regions of domains I, II and III (Figure 4a). We refer to the former as the S1 pocket and the latter S2. It is interesting to note that both the S1 and S2 pockets are highly-polar with side chains of many polar and charged residues projecting into them. As ~70% of CLC docked clusters are predicted within the S1 pocket, we identified S1 to be the primary binding site for CLC and refined its binding conformation using GOLD.37 The resultant best-scoring conformation of CLC in the S1 pocket of BSA is shown (Figure 4b) with some of the key interactions highlighted in Figure 4c.

Figure 4. Serum albumin-CLC molecular docking. (a) Clusters of docked conformations of CLC (shown in sphere representation) on BSA (shown in ribbon representation) obtained following docking using SwissDock. The regions of the BSA colored red, green, and blue correspond to the domain I, II, and III, respectively. The clusters shown in yellow and magenta colors correspond to CLC binding modes within the S1 and S2


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pockets, respectively. (b) The refined binding conformation of CLC (shown in sphere representation and yellow color) in the S1 pocket of BSA with a GoldScore of 77.33 (Top 10 average GoldScore = 70.55). (c) Close-up of CLC (ball-and-stick) in the S1 pocket of BSA showing residues forming non-covalent interactions with CLC. Cation•••π and π•••π interactions between CLC and residues of S1 pocket of BSA are shown using dotted lines. From the docking results, it could be suggested that the interaction of CLC with the S1 pocket of BSA can be stabilized by non-covalent interactions such as cation•••π, between the basic residues projecting into the S1 pocket (R458, R435, R185, R144 and K114) and the aromatic moieties of CLC. These basic residues also form multiple polar contacts with the chlorine atoms of CLC. The cationic imidazolium moiety of CLC could also favorably interact with Glu424, potentially forming a salt-bridge under physiological conditions. Also, His145 of BSA interacts with CLC through π•••π interaction. Taken together, experimental and docking results clearly indicate that serum albumin forms a complex with CLC in aqueous solution. Complex is stabilized by multiple non-covalent interactions with binding affinity in the range of micromolar concentration. This complex abrogates the adsorption of CLC to the vial surface, thus making it available in solution and may act on Httex1 to suppress aggregation. 3.5

Inhibition of Httex1 peptide aggregation by BSA-CLC complex

To ascertain that CLC in complex with BSA still retains the aggregation inhibitory property, Httex1 aggregation was set in the absence or presence of CLC, BSA and BSA-CLC complex. A delay in aggregation kinetics of Httex1 was observed in the presence of CLC, BSA and BSACLC complex (Figure 5a). Interestingly, a twenty-five fold decrease in Httex1 aggregation was found in the presence of BSA-CLC complex as compared to only tenfold decrease for BSA alone


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and a four-fold decrease in case of CLC alone. Aggregation inhibition by serum albumin is not surprising as it is earlier shown to directly interact with the amyloid-β and inhibit its aggregation.50-52

Figure 5. Serum albumin-CLC complex delays Httex1 peptide aggregation. (a) Aggregation kinetics of Httex1 peptide (20 µM) in the absence (black) and presence of 100 µM CLC (green), 5 µM BSA (red) and 100 µM CLC + 5 µM BSA (complex- blue). At each time point, an aliquot was centrifuged and Httex1 monomer content was determined in supernatant using RP-HPLC (n = 4). (b) Comparison of diffusion coefficient of Httex1 in the absence and presence of BSA-CLC complex. Diffusion coefficient of Httex1 alone and in the presence of BSA-CLC complex at 0 h and 48 h were determined by DLS (n = 4). *p < 0.05,


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statistical analysis was done using T-test. (c) Fluorescence correlation spectroscopy (FCS) of fluorophore (NPCE) tagged serum albumin (HSA) in absence and presence of CLC. Normalized autocorrelation functions of NPCE tagged HSA at increasing concentrations of CLC. (d) FCS of NPCE-tagged HSA-CLC complex in the absence and presence of NT17 peptide. Normalized autocorrelation functions of NPCE tagged HSA in complex with CLC at increasing concentrations of NT17. The presence of BSA-CLC complex reduces the aggregate formation ability of Httex1 and results in the formation of smaller assemblies as shown by large diffusion coefficient (Figure 5b). Both CLC alone and BSA-CLC complex showed an impact on the initial aggregation phase (Figure 5a). It is known that NT17 domain flanking polyQ forms an amphipathic helix which triggers oligomerization event to accelerate the aggregation kinetics of Httex1.7 Inhibiting this event at the time of NT17 self-association diminishes the ability of Httex1 to form aggregates.6, 53 So, in order to get more details on the behavior of serum albumin-CLC complex in presence of NT17 peptide in solution, fluorescence correlation spectroscopy (FCS) was used. For FCS study, we used HSA instead of BSA as the latter cannot be tagged with a single fluorophore. It is also to note here that the three-dimensional structure of HSA and BSA is very similar. The autocorrelation curves of HSA tagged with NPCE were recorded at different CLC concentrations (Figure 5c). The single diffusion equation fails to fit the data. Instead, we observed two diffusion components, one for unbound HSA and the other for HSA-CLC complex. There was a regular increment of the diffusion time for HSA-CLC complex with increasing concentration of CLC (Figure 5c). Also, percent fraction of HSA-CLC complex was found to increase with increasing CLC concentration (Figure S10a). Further, we checked the direct interaction of NT17 with serum albumin-CLC via formation of a ternary complex using FCS. We used NT17 peptide for FCS


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experiment as it does not aggregate and serves a good model peptide for mechanistic studies. At constant HSA (50 nM) and CLC (5 µM) concentration, an increase in diffusion time of HSA with increasing concentration of NT17 was observed (Figure 5d), which indicate the formation of the ternary HSA-CLC-NT17 complex. Here to note that for 20 µM of NT17 the diffusion time of the ternary complex (HSA:50 nM-CLC:5 µM-NT17:20 µM) is 7835 ± 635 µs, whereas for the HSA-CLC complex of the same composition, the value of the diffusion time is found to be 4335 ± 360 µs. Percent fraction of such HSA-CLC-NT17 complex was also found to increase with increasing NT17 concentration (Figure S10b). Formation of this ternary HSA-CLC-NT17 complex might be responsible for the inhibitory effect seen in the case of Httex1. Since NT17 participate in the oligomer formation to accelerate the aggregation, non-availability of NT17 for oligomerization due to ternary complex formation possibly delayed the aggregation kinetics. In order to capture one of the binding modes of NT17 peptide to BSA and BSA-CLC complex, we used α-helical NT17 peptide for docking.54-56 Four out of the top five resultant transformations occupied the S1 pocket, similar to CLC, while the other was found docked in the S2 pocket. The best-scoring transformation (NT17 in S1) was further refined using FiberDock57 and is illustrated in Figure S11a. Interestingly, in the presence of CLC, NT17 peptide tends to occupy S2 pocket formed by domain I, II and III together (Figure S11b). NT17 peptide binding to BSA involves different interactions i.e. salt-bridge: K15NT17•••D450BSA, cation•••π: F11NT17•••R217BSA, F11NT17•••K294BSA and multiple inter-molecule hydrophobic contacts. This binding mode could be one of the ways by which BSA-CLC complex interacts with the NT17 peptide of Httex1 to make ternary complex. Based on our experimental data and docking observations, it could be safely concluded that BSA-CLC complex is capable of forming a ternary complex to prevent Httex1 aggregation. CLC


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in free and serum albumin bound state (BSA-CLC complex) prevents NT17 self-association probably by targeting the amphipathic helix of NT17.40, 58 Interaction of CLC and BSA-CLC with NT17 renders it to form oligomers which in turn inhibit Httex1 aggregation. However, further experimental validation is required to prove this interaction and complete inhibition mechanism. 3.6

BSA nanoparticles as formulation of CLC

Due to hydrophobic nature of CLC, use in cell culture assays and in vivo studies is limited due to non-specific adsorption and possible membrane disruption. Further, we used 10% DMSO (v/v) for in vitro studies and at this concentration DMSO is known to be toxic to mammalian cells.59 To avoid these problems, we encapsulated CLC in BSA nanoparticles. Since CLC showed the binding affinity for BSA, we opted for the albumin nanoparticle-based approach for the preparation of CLC loaded BSA nanoparticle formulation. Further, the formulation in the form of nanoparticles might show better solution stability as compared to BSA-CLC complex alone.60 CLC loaded BSA nanoparticles were prepared by an established desolvation technique followed by glutaraldehyde cross-linking (Figure S12a,b).38 We obtained nanoparticles with a Z-average size of 124 ± 2 nm for empty nanoparticles (ENP) and 134 ± 4 nm for CLC loaded nanoparticles (CNP) (Table S1). Both ENPs and CNPs showed PDI < 0.1 and zeta potential of 22 and -15, respectively. BSA nanoparticles showed stability with no comparable change in size and PDI over 15 days when stored at 4 oC (Table S1). Zeta potential showed slight change for both ENP and CNP after 15 days of storage. Both empty (Figure 6a) and loaded nanoparticles (Figure 6b) obtained were spherical in shape as shown by SEM. An average size for ENPs and CNPs was 99 ± 17 and 120 ± 14 respectively, which matches with the hydrodynamic size determined by DLS (Figure 6c). CLC loaded nanoparticles showed significantly large size in both DLS and SEM as compared to


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ENPs. Also, the resultant BSA nanoparticles were auto-fluorescent with both green and red fluorescence (Figure 6) due to electronic transitions (π•••π* of C=C bond and n•••π* of C=N bond.61

Figure 6. Characterization of BSA nanoparticles. Scanning electron microscopy image of (a) empty BSA nanoparticles (ENP) and (b) CLC loaded BSA nanoparticles (CNP). Inset shows single near-spherical BSA nanoparticle. Scale bar is 1 µm (10,000× magnification) and for the inset scale is 100 nm (50,000× magnification). (c) Average particle size (d.nm) of nanoparticles (ENPs and CNPs) based on SEM image analysis using ImageJ. n = 60, *p < 0.05, comparison was done using T-test. (d) Auto-fluorescent CLC loaded BSA nanoparticles showing both green and red fluorescence. (e) CLC loading on BSA nanoparticles confirmed using RP-HPLC analysis. Representative chromatograms of CLC, ENPs, and CNPs are shown. The peak at ~15 min represents CLC which is present in CNPs and


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absent in ENPs. (f) Effect of trypsin on CLC release from BSA nanoparticles. CLC released from CNPs after 24 h of incubation in the absence and presence of trypsin was determined using RP-HPLC (n = 2). *p < 0.05, comparison was done using T-test. Different concentrations of CLC and BSA were optimized in order to improve encapsulation efficiency. We could achieve 23% loading efficiency at 100 µM CLC concentration and 50 mg mL-1 BSA concentration, with 2 µg CLC per mg of BSA in nanoparticles (Table S2). The BSA nanoparticles showed a change in secondary structure when compared to free BSA as observed by CD and FTIR spectroscopy (Figure S13a). On the contrary, ENP and CNP showed little change in secondary structure, which indicates CLC loading did not alter BSA structure to a large extent (Figure S13a). CLC loading into CNPs was confirmed by dissolving CNPs in DMSO and analyzing the supernatant by RP-HPLC (Figure 6e). To simulate in vivo release of CLC, CNPs were incubated at 37 °C with shaking (175 rpm) in the absence and presence of trypsin. We observed 4% CLC release from CNPs in 24 h while CLC release increased by twofold when CNPs were digested with trypsin (Figure 6f). 3.7

Effect of CNPs on Httex1 aggregation in HD-150Q cells

To check the applicability of CNPs to the in vivo studies, we tested their biocompatibility and efficacy in Huntington’s disease mammalian cell culture model. After successful synthesis and characterization of CNPs, the toxicity of CNPs to HD-150Q cells was determined by MTT assay. CNPs showed no significant cellular toxicity on HD-150Q (Figure 7a). Also, both ENPs and CNPs showed good hemocompatibility (Figure 7b). In case of positive control Triton-X 100, complete hemolysis was observed as seen by red colored supernatant due to complete hemoglobin release due to membrane disruption by the surfactant (Figure 7b inset). No hemolysis was observed for isotonic PBS (negative control) and RBCs formed pellet after


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centrifugation (Figure 7b inset). Similar to the negative control, no hemoglobin was released in case of ENPs and CNPs treated RBCs. Having biocompatible CLC loaded nanoparticles; we further checked the efficacy of CNPs to modulate Httex1 aggregation in HD-150Q cells expressing tNhtt-EGFP-150Q.

Figure 7. CLC loaded nanoparticles prevents aggregate formation in HD-150Q cells.


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(a) Effect of CLC loaded nanoparticles on cell viability. Cell toxicity was determined using MTT assay by incubating CNPs with HD-150Q. n = 4, ns- not significant, comparison was done using T-test. (b) Hemocompatibility of BSA nanoparticles was determined using hemolysis assay. 1% Triton-X 100 was used as a positive control and PBS as a negative control. The ENPs and CNPs were suspended at 800 µg mL-1 (n = 3) and RBC lysis was determined by absorbance at 540 nm. An inset shows the image of samples after centrifugation with complete hemolysis in case of Triton-X 100 while RBC pellet in PBS, ENPs, and CNPs. *p < 0.05, ns- not significant, comparison with control was done using one-way ANOVA with Holm-Sidak post-hoc test. (c) Representative fluorescent images of Neuro-2a cells expressing tNhtt-EGFP-150Q. Aggregates are shown by green fluorescence (GFP) and nucleus by blue fluorescence (DAPI). Untreated cells were considered as control (c). (d) ENP treated cells. (e) CNP (250 µg mL-1 CLC concentration) treated cells. Scale bar for c, d and e is 50 µm. (f) Effect of BSA nanoparticles treatment on Neuro-2a cells expressing tNhtt-EGFP-150Q. Percentage of cells containing aggregates in the absence and presence of nanoparticles. n = 15, *p < 0.05, pairwise comparison was done using one-way ANOVA with Holm-Sidak post-hoc test. (g) Schematic representation of CLC action on Httex1 aggregation. (1) CLC loaded BSA nanoparticle formulation on digestion with trypsin (2) releases CLC to prevent Httex1 aggregation. (3) BSA-CLC complex form ternary complex with NT17 of Httex1 to inhibit its aggregation. Htt-150Q expression results in the formation of green fluorescent aggregates when induced with PNST (Figure 7c). Both ENPs and CNPs were incubated with HD-150Q cells for 24 h after inducing expression. Cells treated with ENPs (Figure 7d) and CNPs (Figure 7e) for 24 h postinduction showed fewer green fluorescent puncta in comparison to control. Quantification of GFP puncta per cell revealed an inhibitory effect of both ENPs and CNPs, with CNPs showing


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better efficiency in aggregation inhibition (Figure 7f). Effect shown by ENPs is in accordance with the published literature and our in vitro result where BSA alone also showed an inhibitory effect (Figure 5a).50-52 CNPs treatment of HD-150Q cells replicated the anti-aggregation effect of CLC observed in vitro. CNPs could now be tested in rodent HD model for in vivo efficacy of CLC as a part of preclinical study for this therapeutic formulation against HD. Based on similar concept of hydrophobic drug binding to serum albumin, nanoparticle formulation of paclitaxel (Abraxane) was approved for oncotherapy.60 4

Conclusions To summarize, a hydrophobic molecule calmidazolium chloride was tested for aggregation

modulation of Httex1 as a potential therapeutic molecule for Huntington’s disease. CLC targets the initial step of Httex1 aggregation, which is a potential therapeutic target in HD. We speculate the involvement of aromatic interactions of CLC with a hydrophobic face of NT17 domain of Httex1 to prevent its aggregation. Since CLC adsorbs to the vial surface during aggregation reaction, we have developed a serum albumin-based assay to prevent adsorption of CLC while simultaneously monitoring aggregation kinetics (Figure 7g). The BSA-CLC complex formation and characterization were done with the help of tryptophan fluorescence, FCS, DLS, NMR and molecular docking studies. The increased inhibitory potential of serum albumin-CLC complex was achieved due to a ternary complex formation with NT17, which is involved in oligomer formation during aggregation (Figure 7g). The serum albumin based nanoparticle formulation of CLC was developed to overcome the limitations of poor aqueous solubility of CLC, DMSO cytotoxicity and CLC adsorption to the culture dish. CLC loaded BSA nanoparticles with size < 150 nm were synthesized and characterized. CNPs treatment of HD-150Q cells resulted in lower aggregate load. A hydrophobic molecule CLC showed potential as a HD therapeutic molecule


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both in vitro and in HD-150Q cells and can now be tested for toxicity and efficacy in rodent HD model. It is now possible to develop a similar formulation (BSA-CLC) platform for other hydrophobic aggregation inhibitors against aggregation-prone proteins or even different targets in other diseases. Though the BSA-CLC complex is specific for HD, the usage of it could be extended to proteins following similar aggregation mechanism. With further improvement in the encapsulation efficiency, CNPs could provide a promising therapeutic formulation for preclinical studies. Although, use of high dose of CLC could pose a potential toxic risk as it blocks calcium release form TRPV1 channels and calcium homeostasis is important for cell survival.24 Considering existing calcium imbalance in neurons affected by Httex1 aggregation in HD62, targeting neurons with CLC could be a good multitarget strategy by simultaneously alleviating protein aggregation and blocking excess calcium release. To minimize risk to benefit ratio, we propose to use targeted delivery approach by tagging nanoparticles with angiopep-2 peptide that assist in cargo delivery across BBB, which will provide a platform for testing of multitarget strategy in HD mouse model.63 ASSOCIATED CONTENT Supporting Information. Full details of supporting methods, details Proton NMR spectroscopy, HSA tryptophan fluorescence quenching measurements, fluorescence correlation spectroscopy (FCS), HSA labeling and sample preparation, FCS data analysis, computational docking of NT17 peptide to BSA, RP-HPLC analysis of CNPs to determine encapsulation efficiency, Characterization of BSA nanoparticles, fourier transform infrared spectroscopy (FTIR), circular dichroism (CD) spectroscopy.


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Figure S1. Effect of CLC on aggregation kinetics of Q35 peptide. Figure S2. CLC vial surface adsorption kinetics. Figure S3. Effect of salt on the vial surface adsorption. Figure S4. 1H NMR spectra of CLC. Figure S5. CLC vial surface adsorption in the presence of serum albumin. Figure S6. Effect of BSA concentration on CLC vial surface adsorption. Figure S7. 1H NMR spectra of BSA in the presence of CLC. Figure S8. Three-dimensional structures of serum albumin, Figure S9. Tryptophan fluorescence quenching assay. Figure S10. Change in relative abundance of serum albumin complex in FCS. Figure S11. Docking of BSA-NT17 and BSA-CLC-NT17. Figure S12. CLC loaded BSA nanoparticles (CNPs). Figure S13. Effect of cross-linking and CLC loading on BSA structure in nanoparticles. Table S1. Particle size, polydispersity index and zeta potential of BSA nanoparticles. Table S2. Encapsulation efficiency (EE) of BSA nanoparticles. The supporting information file is available free of charge. AUTHOR INFORMATION Corresponding Author *Email: [email protected] Department of Biological sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur-208016, India. Tel. +91-512-259-1077. Author Contributions This work is planned and executed by VS and AKT. VS performed most of the experiments. BS and PS performed and analyzed FCS experiments. ARJ performed mammalian cell culture experiments. RNVKD carried out the docking studies overseen by HF. VS and AKT analyzed and interpret all the data. The manuscript was written through contributions of all authors. All


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authors have given approval to the final version of the manuscript. The work is supervised and directed by AKT. Conflict of Interest Disclosure The authors declare competing interest: An Indian patent application number 1182/DEL/2015 titled ‘A drug screening process and a drug formulation for Huntington’s disease’' has been submitted by AKT and VS and IIT Kanpur based on these results.

ACKNOWLEDGMENT AKT acknowledges Indian Council of Medical Research (ICMR/BSBE/2016489) for funding the project. VS acknowledge CSIR, IITK and ICMR for the fellowship. This work is part of PhD thesis of VS. RNVKD and HF acknowledge Biomedical Research Council, A*STAR for their support. We thank Dr. Nihar Ranjan Jana (NBRC) for providing Neuro-2a HD-150Q cells. We also acknowledge contribution of Mangesh Bawankar towards initial studies with CLC, Avinash for help with hemolysis assay and CMBR Lucknow for providing access to NMR facility. REFERENCES 1. Hung, C.-W.; Chen, Y.-C.; Hsieh, W.-L.; Chiou, S.-H.; Kao, C.-L. Ageing and Neurodegenerative Diseases. Ageing Res. Rev. 2010, 9, S36-S46. 2. Gitler, A. D.; Dhillon, P.; Shorter, J. Neurodegenerative Disease: Models, Mechanisms, and a New Hope. Dis. Models & Mech. 2017, 10, 499-502. 3. Bates, G. Huntingtin Aggregation and Toxicity in Huntington's Disease. Lancet 2003, 361, 1642-1644. 4. Mangiarini, L.; Sathasivam, K.; Seller, M.; Cozens, B.; Harper, A.; Hetherington, C.; Lawton, M.; Trottier, Y.; Lehrach, H.; Davies, S. W.; Bates, G. P. Exon 1 of the HD Gene with an Expanded CAG Repeat is Sufficient to Cause a Progressive Neurological Phenotype in Transgenic Mice. Cell 1996, 87, 493-506. 5. Jayaraman, M.; Mishra, R.; Kodali, R.; Thakur, A. K.; Koharudin, L. M.; Gronenborn, A. M.; Wetzel, R. Kinetically Competing Huntingtin Aggregation Pathways Control Amyloid Polymorphism and Properties. Biochemistry 2012, 51, 2706-2716.


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6. Mishra, R.; Jayaraman, M.; Roland, B. P.; Landrum, E.; Fullam, T.; Kodali, R.; Thakur, A. K.; Arduini, I.; Wetzel, R. Inhibiting Nucleation of Amyloid Structure in a Huntingtin Fragment by Targeting α-Helix Rich Oligomeric Intermediates. J. Mol. Biol. 2012, 415, 900-917. 7. Thakur, A. K.; Jayaraman, M.; Mishra, R.; Thakur, M.; Chellgren, V. M.; L Byeon, I.-J.; Anjum, D. H.; Kodali, R.; Creamer, T. P.; Conway, J. F.; M Gronenborn, A.; Wetzel, R. Polyglutamine Disruption of the Huntingtin Exon 1 N Terminus Triggers a Complex Aggregation Mechanism. Nat. Struct. Mol. Biol. 2009, 16, 380-389. 8. Thakur, A. K.; Yang, W.; Wetzel, R. Inhibition of Polyglutamine Aggregate Cytotoxicity by a Structure-Based Elongation Inhibitor. FASEB J. 2004, 18, 923-925. 9. Nagai, Y.; Fujikake, N.; Ohno, K.; Higashiyama, H.; Popiel, H. A.; Rahadian, J.; Yamaguchi, M.; Strittmatter, W. J.; Burke, J. R.; Toda, T. Prevention of Polyglutamine Oligomerization and Neurodegeneration by the Peptide Inhibitor QBP1 in Drosophila. Hum. Mol. Genet. 2003, 12, 1253-1259. 10. Popiel, H. A.; Burke, J. R.; Strittmatter, W. J.; Oishi, S.; Fujii, N.; Takeuchi, T.; Toda, T.; Wada, K.; Nagai, Y. The Aggregation Inhibitor Peptide QBP1 as a Therapeutic Molecule for the Polyglutamine Neurodegenerative Diseases. J. Amino Acids 2011, 2011, 1-10. 11. Marelli, C.; Maschat, F. The P42 Peptide and Peptide-Based Therapies for Huntington’s Disease. Orphanet J. Rare Dis. 2016, 11, 24. 12. Escalona-Rayo, O.; Fuentes-Vázquez, P.; Leyva-Gómez, G.; Cisneros, B.; Villalobos, R.; Magaña, J. J.; Quintanar-Guerrero, D. Nanoparticulate Strategies for the Treatment of Polyglutamine Diseases by Halting the Protein Aggregation Process. Drug Dev. Ind. Pharm. 2017, 43, 871-888. 13. Büning, S.; Sharma, A.; Vachharajani, S.; Newcombe, E.; Ormsby, A.; Gao, M.; Gnutt, D.; Vopel, T.; Hatters, D. M.; Ebbinghaus, S. Conformational Dynamics and Self-Association of Intrinsically Disordered Huntingtin Exon 1 in Cells. Phys. Chem. Chem. Phys. 2017, 19, 1073810747. 14. Yu, C.; Niu, X.; Jin, F.; Liu, Z.; Jin, C.; Lai, L. Structure-Based Inhibitor Design for the Intrinsically Disordered Protein C-Myc. Sci. Rep. 2016, 6, 22298. 15. Levine, Z. A.; Larini, L.; LaPointe, N. E.; Feinstein, S. C.; Shea, J.-E. Regulation and Aggregation of Intrinsically Disordered Peptides. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 2758-2763. 16. Lamberto, G. R.; Binolfi, A.; Orcellet, M. L.; Bertoncini, C. W.; Zweckstetter, M.; Griesinger, C.; Fernández, C. O. Structural and Mechanistic Basis Behind the Inhibitory Interaction of Pcts on α-Synuclein Amyloid Fibril Formation. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 21057-21062. 17. Porat, Y.; Abramowitz, A.; Gazit, E. Inhibition of Amyloid Fibril Formation by Polyphenols: Structural Similarity and Aromatic Interactions as a Common Inhibition Mechanism. Chem. Biol. Drug Des. 2006, 67, 27-37. 18. Xie, H.; Peng, J.; Liu, C.; Fang, X.; Duan, H.; Zou, Y.; Yang, Y.; Wang, C. AromaticInteraction-Mediated Inhibition of Beta-Amyloid Assembly Structures and Cytotoxicity. J. Pept. Sci. 2017, 23, 679-684. 19. Gazit, E. Mechanisms of Amyloid Fibril Self-Assembly and Inhibition. FEBS J. 2005, 272, 5971-5978. 20. Arosio, P.; Vendruscolo, M.; Dobson, C. M.; Knowles, T. P. J. Chemical Kinetics for Drug Discovery to Combat Protein Aggregation Diseases. Trends Pharmacol. Sci. 2014, 35, 127-135.


34

Page 35 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


21. Williams, A. D.; Sega, M.; Chen, M.; Kheterpal, I.; Geva, M.; Berthelier, V.; Kaleta, D. T.; Cook, K. D.; Wetzel, R. Structural Properties of Aβ Protofibrils Stabilized by a Small Molecule. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 7115-7120. 22. Sunagawa, M.; Kosugi, T.; Nakamura, M.; Sperelakis, N. Pharmacological Actions of Calmidazolium, a Calmodulin Antagonist, in Cardiovascular System. Cardiovasc. Drug Rev. 2000, 18, 211-221. 23. Sunagawa, M.; Yokoshiki, H.; Seki, T.; Nakamura, M.; Laber, P.; Sperelakis, N. Direct Block of Ca2+ Channels by Calmidazolium in Cultured Vascular Smooth Muscle Cells. J. Cardiovasc. Pharmacol. Ther. 1999, 34, 488-496. 24. Oláh, Z.; Jósvay, K.; Pecze, L.; Letoha, T.; Babai, N.; Budai, D.; Ötvös, F.; Szalma, S.; Vizler, C. Anti-Calmodulins and Tricyclic Adjuvants in Pain Therapy Block the TRPV1 Channel. PLoS One 2007, 2, e545. 25. Dudek, N. L.; Dai, Y.; Muma, N. A. Protective Effects of Interrupting the Binding of Calmodulin to Mutant Huntingtin. J. Neuropathol. Exp. Neurol. 2008, 67, 355-365. 26. Zainelli, G. M.; Ross, C. A.; Troncoso, J. C.; Fitzgerald, J. K.; Muma, N. A. Calmodulin Regulates Transglutaminase 2 Cross-Linking of Huntingtin. J. Neurosci. 2004, 24, 1954-1961. 27. Page, T. A.; Kraut, N. D.; Page, P. M.; Baker, G. A.; Bright, F. V. Dynamics of Loop 1 of Domain I in Human Serum Albumin When Dissolved in Ionic Liquids. J. Phys. Chem. B 2009, 113, 12825-12830. 28. Sengupta, B.; Acharyya, A.; Sen, P. Elucidation of the Local Dynamics of Domain-III of Human Serum Albumin over the ps-µs Time Regime Using a New Fluorescent Label. Phys. Chem. Chem. Phys. 2016, 18, 28548-28555. 29. Sengupta, B.; Das, N.; Sen, P. Elucidation of µs Dynamics of Domain-III of Human Serum Albumin During the Chemical and Thermal Unfolding: A Fluorescence Correlation Spectroscopic Investigation. Biophys. Chem. 2017, 221, 17-25. 30. Sengupta, B.; Yadav, R.; Sen, P. Startling Temperature Effect on Proteins When Confined: Single Molecular Level Behaviour of Human Serum Albumin in a Reverse Micelle. Phys. Chem. Chem. Phys. 2016, 18, 14350-14358. 31. Irwin, J. J.; Shoichet, B. K. Zinc − a Free Database of Commercially Available Compounds for Virtual Screening. J. Chem. Inf. Model. 2005, 45, 177-182. 32. Irwin, J. J.; Sterling, T.; Mysinger, M. M.; Bolstad, E. S.; Coleman, R. G. Zinc: A Free Tool to Discover Chemistry for Biology. J. Chem. Inf. Model. 2012, 52, 1757-1768. 33. Bujacz, A. Structures of Bovine, Equine and Leporine Serum Albumin. Acta Crystallogr. D 2012, 68, 1278-1289. 34. Grosdidier, A.; Zoete, V.; Michielin, O. Fast Docking Using the Charmm Force Field with Eadock DSS. J. Comput. Chem. 2011, 32, 2149-2159. 35. Grosdidier, A.; Zoete, V.; Michielin, O. Eadock: Docking of Small Molecules into Protein Active Sites with a Multiobjective Evolutionary Optimization. Proteins: Struct., Funct., Bioinf. 2007, 67, 1010-1025. 36. Grosdidier, A.; Zoete, V.; Michielin, O. Swissdock, a Protein-Small Molecule Docking Web Service Based on Eadock DSS. Nucleic Acids Res. 2011, 39, W270-W277. 37. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. Development and Validation of a Genetic Algorithm for Flexible Docking. J. Mol. Biol. 1997, 267, 727-748. 38. Langer, K.; Balthasar, S.; Vogel, V.; Dinauer, N.; von Briesen, H.; Schubert, D. Optimization of the Preparation Process for Human Serum Albumin (HSA) Nanoparticles. Int. J. Pharm. 2003, 257, 169-180.


35


Page 36 of 38

39. Goswami, A.; Dikshit, P.; Mishra, A.; Nukina, N.; Jana, N. R. Expression of Expanded Polyglutamine Proteins Suppresses the Activation of Transcription Factor Nfκb. J. Biol. Chem. 2006, 281, 37017-37024. 40. Vöpel, T.; Bravo-Rodriguez, K.; Mittal, S.; Vachharajani, S.; Gnutt, D.; Sharma, A.; Steinhof, A.; Fatoba, O.; Ellrichmann, G.; Nshanian, M.; Heid, C.; Loo, J. A.; Klärner, F.-G.; Schrader, T.; Bitan, G.; Wanker, E. E.; Ebbinghaus, S.; Sanchez-Garcia, E. Inhibition of Huntingtin Exon-1 Aggregation by the Molecular Tweezer CLR01. J. Am. Chem. Soc. 2017, 139, 5640-5643. 41. Fukazawa, T.; Yamazaki, Y.; Miyamoto, Y. Reduction of Non-Specific Adsorption of Drugs to Plastic Containers Used in Bioassays or Analyses. J. Pharmacol. Toxicol. Methods 2010, 61, 329-333. 42. Song, D.; Hsu, L.-F.; Au, J. L. S. Binding of Taxol to Plastic and Glass Containers and Protein under in Vitro Conditions. J. Pharm. Sci. 1996, 85, 29-31. 43. Ilevbare, G. A.; Taylor, L. S. Liquid–Liquid Phase Separation in Highly Supersaturated Aqueous Solutions of Poorly Water-Soluble Drugs: Implications for Solubility Enhancing Formulations. Cryst. Growth Des. 2013, 13, 1497-1509. 44. Glisoni, R. J.; Chiappetta, D. A.; Finkielsztein, L. M.; Moglioni, A. G.; Sosnik, A. SelfAggregation Behaviour of Novel Thiosemicarbazone Drug Candidates with Potential Antiviral Activity. New J. Chem. 2010, 34, 2047-2058. 45. Kirstein, S.; Daehne, S. J-Aggregates of Amphiphilic Cyanine Dyes: Self-Organization of Artificial Light Harvesting Complexes. Int. J. Photoenergy 2006, 2006, 1-21. 46. LaPlante, S. R.; Aubry, N.; Bolger, G.; Bonneau, P.; Carson, R.; Coulombe, R.; Sturino, C.; Beaulieu, P. L. Monitoring Drug Self-Aggregation and Potential for Promiscuity in OffTarget in Vitro Pharmacology Screens by a Practical NMR Strategy. J. Med. Chem. 2013, 56, 7073-7083. 47. Meti, M. D.; Nandibewoor, S. T.; Joshi, S. D.; More, U. A.; Chimatadar, S. A. MultiSpectroscopic Investigation of the Binding Interaction of Fosfomycin with Bovine Serum Albumin. J. Pharm. Anal. 2015, 5, 249-255. 48. Möller, M.; Denicola, A. Protein Tryptophan Accessibility Studied by Fluorescence Quenching. Biochem. Mol. Biol. Educ. 2002, 30, 175-178. 49. Nusrat, S.; Siddiqi, M. K.; Zaman, M.; Zaidi, N.; Ajmal, M. R.; Alam, P.; Qadeer, A.; Abdelhameed, A. S.; Khan, R. H. A Comprehensive Spectroscopic and Computational Investigation to Probe the Interaction of Antineoplastic Drug Nordihydroguaiaretic Acid with Serum Albumins. PLoS One 2016, 11, e0158833. 50. Algamal, M.; Ahmed, R.; Jafari, N.; Ahsan, B.; Ortega, J.; Melacini, G. AtomicResolution Map of the Interactions between an Amyloid Inhibitor Protein and Amyloid Beta (Aβ) Peptides in the Monomer and Protofibril States. J. Biol. Chem. 2017, 292, 17158-17168. 51. Algamal, M.; Milojevic, J.; Jafari, N.; Zhang, W.; Melacini, G. Mapping the Interactions between the Alzheimer’s Aβ-Peptide and Human Serum Albumin Beyond Domain Resolution. Biophys. J. 2013, 105, 1700-1709. 52. Milojevic, J.; Costa, M.; Ortiz, A. M.; Jorquera, J. I.; Melacini, G. In Vitro Amyloid-Beta Binding and Inhibition of Amyloid-Beta Self-Association by Therapeutic Albumin. J Alzheimers Dis 2014, 38, 753-765. 53. Tam, S.; Spiess, C.; Auyeung, W.; Joachimiak, L.; Chen, B.; Poirier, M. A.; Frydman, J. The Chaperonin TRiC Blocks a Huntingtin Sequence Element that Promotes the Conformational Switch to Aggregation. Nat. Struct. Mol. Biol. 2009, 16, 1279-1285.


36

Page 37 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


54. Thévenet, P.; Shen, Y.; Maupetit, J.; Guyon, F.; Derreumaux, P.; Tufféry, P. PEP-FOLD: an Updated De Novo Structure Prediction Server for Both Linear and Disulfide Bonded Cyclic Peptides. Nucleic Acids Res. 2012, 40, W288-W293. 55. Arndt, J. R.; Kondalaji, S. G.; Maurer, M. M.; Parker, A.; Legleiter, J.; Valentine, S. J. Huntingtin N-Terminal Monomeric and Multimeric Structures Destabilized by Covalent Modification of Heteroatomic Residues. Biochemistry 2015, 54, 4285-4296. 56. Schneidman-Duhovny, D.; Inbar, Y.; Nussinov, R.; Wolfson, H. J. Patchdock and Symmdock: Servers for Rigid and Symmetric Docking. Nucleic Acids Res. 2005, 33, W363W367. 57. Mashiach, E.; Nussinov, R.; Wolfson, H. J. Fiberdock: Flexible Induced-Fit Backbone Refinement in Molecular Docking. Proteins: Struct., Funct., Bioinf. 2010, 78, 1503-1519. 58. Monsellier, E.; Redeker, V.; Ruiz-Arlandis, G.; Bousset, L.; Melki, R. Molecular Interaction between the Chaperone Hsc70 and the N-Terminal Flank of Huntingtin Exon 1 Modulates Aggregation. J. Biol. Chem. 2015, 290, 2560-2576. 59. Yuan, C.; Gao, J.; Guo, J.; Bai, L.; Marshall, C.; Cai, Z.; Wang, L.; Xiao, M. Dimethyl Sulfoxide Damages Mitochondrial Integrity and Membrane Potential in Cultured Astrocytes. PLoS One 2014, 9, e107447. 60. Ron, N.; Cordia, J.; Yang, A.; Ci, S.; Nguyen, P.; Hughs, M.; Desai, N. Comparison of Physicochemical Characteristics and Stability of Three Novel Formulations of Paclitaxel: Abraxane, Nanoxel, and Genexol PM. Cancer Res. 2008, 68, 5622-5622. 61. Ma, X.; Sun, X.; Hargrove, D.; Chen, J.; Song, D.; Dong, Q.; Lu, X.; Fan, T.-H.; Fu, Y.; Lei, Y. A Biocompatible and Biodegradable Protein Hydrogel with Green and Red Autofluorescence: Preparation, Characterization and in Vivo Biodegradation Tracking and Modeling. Sci. Rep. 2016, 6, 19370. 62. Giacomello, M.; Oliveros, J. C.; Naranjo, J. R.; Carafoli, E. Neuronal Ca(2+) Dyshomeostasis in Huntington Disease. Prion 2013, 7, 76-84. 63. Xin, H.; Sha, X.; Jiang, X.; Chen, L.; Law, K.; Gu, J.; Chen, Y.; Wang, X.; Fang, X. The Brain Targeting Mechanism of Angiopep-Conjugated Poly(Ethylene Glycol)-Co-Poly(ƐCaprolactone) Nanoparticles. Biomaterials 2012, 33, 1673-1681.


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For Table of Contents Only Calmidazolium chloride and its complex with serum albumin prevent Huntingtin exon1 aggregation Virender Singh†, R. N.V. Krishna Deepak‡, Bhaswati Sengupta€, Abhayraj S. Joshi†, Hao Fan‡§, Pratik Sen€ and Ashwani Kumar Thakur†*


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Calmidazolium Chloride and Its Complex with Serum Albumin Prevent

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