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Protective effects of a neurohypophyseal hormone analogue on prion aggregation, cellular internalization and toxicity Nikita Admane, Ankit Srivastava, Salma Jamal, Bishwajit Kundu, and Abhinav Grover ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00299 • Publication Date (Web): 13 Aug 2019 Downloaded from pubs.acs.org on August 13, 2019

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ACS Chemical Neuroscience

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Protective effects of a neurohypophyseal hormone analogue on

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prion aggregation, cellular internalization and toxicity

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Nikita Admane1, Ankit Srivastava2, Salma Jamal1, Bishwajit Kundu2*, Abhinav Grover1*

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1School

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2Kusuma

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Delhi, India – 110016

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*Corresponding authors

of Biotechnology, Jawaharlal Nehru University, New Delhi, India - 110067 School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New

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ABSTRACT

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Herein, we report novel neuroprotective activity of a neurohypophyseal hormone analogue

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desmopressin (DDAVP) against toxic conformations of human prion protein. Systematic analysis

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using biophysical techniques in conjunction with surface plasmon resonance, high-end

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microscopy, conformational antibodies and cell-based assays demonstrated DDAVP’s specific

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binding and potent anti-aggregating effects on prion protein (rPrPres). Besides subjugating

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conformational conversion of rPrPres into oligomeric forms, DDAVP also exhibits potent fibril

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modulatory effects. It eventually ameliorated neuronal toxicity of rPrPres oligomers by

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significantly reducing their cellular internalization. Molecular dynamics simulations showed that

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DDAVP prevents β-sheet transitions in the N-terminal amyloidogenic region of prion and induces

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antagonistic mobilities in the α2-α3 and β2-α2 loop regions. Collectively, our data proposes

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DDAVP as a new structural motif for rational drug discovery against prion diseases.

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KEYWORDS: Prion, Amyloid, Desmopressin, Oligomer, Microscopy, Toxicity

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Currently there are no clinically proven therapeutics available for transmissible spongiform

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encephalopathies (TSEs) which include Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-

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Scheinker (GSS) and fatal familial insomnia (FFI) in humans, bovine spongiform encephalopathy

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(BSE) in cattle and chronic wasting disease (CWD) in deer and elks[1]. Etiology of these rapidly

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progressing fatal dementias commonly involve conversion of glycosyl phosphatidylinositol (GPI)

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anchored normal prion protein PrPC (Cellular form) into an aggregation-prone and infectious form

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PrPSc (Scrapie form)[2]. Unfortunately, none of the previously reported anti-prion compounds

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including trehalose, rapamycin, tamoxifen, FK506, IU-1 provided any firm evidence for their

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efficacy against human prion diseases and eventually failed in the clinical trials[3-5]. In fact,

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clinical efficacy of several small molecules reported against other neurodegenerative diseases

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(NDs) remain arguable and require rigorous in vivo testing [6-9]. In this manuscript, we report

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repurposing of desmopressin (DDAVP; 1-deamino-8-D-arginine vasopressin) as a new

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pharmacologically relevant scaffold that binds strongly to the native PrPC repertoire and inhibits

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its conversion into neurotoxic, PrPSc-like form (Figure S1A). DDAVP is an octapeptide, synthetic

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analogue of neurohypophyseal hormone vasopressin (anti-diuretic hormone) that acts as an agonist

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for the vasopressin type 2 receptors (V2r) and is widely used for treating diabetes insipidus,

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hemophilia A, von Willebrand disease and high blood urea levels[10]. A previous in vivo report

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showed that the plasma levels of PrPC decreased following DDAVP therapy in patients with von

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Willebrand disease[11]. Unfortunately, no discrete evidences discerning the mechanism of

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interaction between PrPC and DDAVP were reported in the recent past. In this study, using a battery

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of biophysical and cell-based techniques, we characterized the hetero-molecular interaction

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between DDAVP and protease-resistant prion fragment (residues 90-231; rPrPres, Figure S1B).

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Figure 1. Binding interactions and effect on prion aggregation. (A) SPR sensorgrams for evaluating interactions of rPrPres with DDAVP. Various concentrations of DDAVP were injected at a rate of 10 μl/min and the association kinetics was monitored for 300 s, followed by dissociation for next 200 s. (B) Dose-response curve for interaction of rPrPres with DDAVP. The equilibrium response values were extrapolated from the fitted data calculated using the BIA evaluation 4.1 software. (C) Thermal denaturation of rPrPres (black)and rPrPres with DDAVP (red; 1:1 molar ratio) measured as change in secondary structure (θ222) with increasing temperature. First derivatives (dθ/dT) of normalized melting curves are shown, where the maxima indicate melting temperature (Tm)for each system. (D) Molecular representation of docking pose of DDAVP at the interface of β2-α2 loop and C-terminal of prion protein structure (PDB:2KUN). The zoomed section shows residues involved in molecular interactions. (E) Non-covalent bonding interactions of DDAVP at the binding pocket. (F) Change in ThT fluorescence plotted as a function of time to represent aggregation kinetics of rPrPres alone (black traces) and in the presence of varying concentrations of DDAVP (colored traces). The aggregation kinetics of rPrPres followed a sigmoidal transition and a dose-dependent abrogation in ThT fluorescence was observed in presence of DDAVP. (G) The relative subduing of ThT intensity upon increasing concentration of DDAVP. (H) The kinetic inhibition of rPrPres aggregation in presence of DDAVP represented by dose-dependent decrease in rate of aggregation. 3 ACS Paragon Plus Environment


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This protease-resistant, 142 residues fragment acts as a PrPSc repertoire designated as PrP 27-30

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that conveys prion infectivity[12, 13]. Thus, a construct comprising residues 90 to 231 has been

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widely utilized to study self-replicating conformational conversion, infectivity, and the strain

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phenomenon in prions[14, 15]. First, we carried out surface plasmon resonance (SPR) experiments

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to affirm the hetero-molecular interaction between rPrPres and DDAVP. Analysis of dose-response

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sensorgrams suggested the existence of a favorable DDAVP binding pocket in rPrPres structure

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with a dissociation constant (KD) of 4.6 ± 0.2 μM (Figure 1A-B). Interestingly, individual

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association (kon) and dissociation (koff) rates indicated fast association and a significantly slow

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dissociation rate for DDAVP (Figure 1A-B, Table S1). Far-UV CD based thermal melting

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experiments suggested that rPrPres begins to lose its native secondary structure above 50 °C with

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a distended transition phase (Tm=68.3oC; Figure 1C). Evidently, DDAVP binding increased the

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thermal stability of prion protein as compared to the unbound protein (ΔTm=2.3oC; Tm=70.6 oC).

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Interestingly, we did not observe any significant change in the tertiary structure of rPrPres upon

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titrating with DDAVP, as assessed by Tryptophan/Tyrosine fluorescence (Figure S2A). Although

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gradual decrement of fluorescence was noted, but any appreciable shift in the spectra (indicating

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conformational change) was not observed (Figure S2B). Next, we performed acrylamide

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quenching experiments for studying changes in the local environment of tryptophan residue and

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to evaluate any effect on compaction of prion structure. Interestingly, in presence of DDAVP,

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rPrPres exhibited >50% reduction in dynamic quenching constant (Ksv; 17.3 M-1 to 8.7 M-1),

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indicating lower penetration of quencher and higher protein compaction (Figure S3). This also

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indicated that DDAVP perhaps subdues hydrophobic surfaces of rPrPres by inducing compaction

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in its structure. This notion was confirmed by a gradual decrement in ANS fluorescence with

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increasing DDAVP concentrations with >2-fold lowering at higher molar concentrations of


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DDAVP (Figure S4). These observations showed that DDAVP binds robustly to rPrPres with

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moderate affinity and stabilizes its structure but with a still unclear mechanism. For deducing the

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plausible binding mechanism, flexible molecular docking was performed. The top 3 docking

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clusters indicated that DDAVP engages in a binding pocket encompassing the β2-α2 loop and

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theα3helix in the rPrPres structure (Figure 1D; Table S2). Analysis of the most favorable binding

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pose showed the presence of several H-bonding and hydrophobic interactions corroborating the

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enthalpy driven, stabilizing interactions between DDAVP and rPrPres. DDAVP formed five H-

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bonds with residues, Gln98, Met166, Ser222 and Gln217, from the crucial β2-α2 loop and α3 helix.

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The peptide-scaffold of DDAVP was further stabilized by hydrophobic interactions with Gly93,

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Gly94, Ser97, Trp99, Asn100, Ala116, Met129, Tyr163, Arg164, Pro165, Tyr169, Phe175,

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Tyr218, Glu221, Gln223 and Gln227 (Figure 1E; Table S3). Also, DDAVP showed higher non-

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covalent interactions as compared to Epigallocatechin gallate (EGCG) which is a known prion

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binding molecule that affects its transition into a non-scrapie like conformation [16, 17].

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Interestingly, both DDAVP and EGCG induced the formation of stabilizing salt-bridge

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interactions upon binding in the β2-α2 loop and α3 helix regions of prion protein (PrP) (Table S3

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and S4). These results motivated us to study the effect of DDAVP on rPrPres aggregation by

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various biophysical and dye-based probes. To examine the effects of DDAVP on prion

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aggregation, Thioflavin T (ThT) fluorescence assay was carried out. Aggregating rPrPres species

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followed a typical sigmoidal transition with a distinct lag phase of ~28 h and an exponential phase

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up to 90 h that begins to saturate after 100 h (black trace, Figure 1F). The inhibition efficiency

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of DDAVP was monitored by analyzing ThT based kinetics with respect to untreated rPrPres

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(100%).



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Figure 2. Effects on fibrillation, size distribution and secondary structure. Electron micrographs of rPrPres aggregates formed (A) alone and (C) in presence of DDAVP. Corresponding AFM images of rPrPres aggregates formed (B) alone and (D) in presence of DDAVP after 140 h incubation. Three-dimensional view of each AFM images is also shown for clarity. Scale bars represent 100 nm for TEM and 200 nm for AFM images. Particle size distribution of aggregating species monitored at different time points (0h, 80h and 140h) represented as color bars for (E) rPrPres alone and (F) in presence of DDAVP. (G)Far-UV CD spectra of native rPrPres (red) and aggregates formed after 140 h incubation in absence (red) and presence of DDAVP (blue). CD spectra of native, unaggregated rPrPres (black) is also shown. (H) Second derivative ATR FTIR spectra of aggregates depicting the amide I region. Component peaks in spectra of rPrPres (black) alone and DDAVP (red) incubated samples are also shown. All data were collected after 140 h incubation. In all experiments a 1:3 molar ratio of rPrPres and DDAVP was used.


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The inhibitory potential of DDAVP was evident even at 5-fold and 2-fold low sub-stoichiometric

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concentrations with ~11% and 18% reduction in ThT maxima respectively (Figure 1G).

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Interestingly, at higher molar ratios, the effect was more dramatic with >50% reduction in ThT

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maxima at 3-fold or higher concentrations of DDAVP. The interaction plausibly affects the

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kinetics of primary nucleation event which is evident by >25% lowering in aggregation rate and

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>2-fold enhancement in the lag-time (~66 h) at these concentrations (Figure 1H; Table S5). Any

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possible quenching effect was ruled out after we verified no spectral overlaps or subduing of ThT

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fluorescence in presence of DDAVP in buffer solutions as well as with actual amyloid aggregates

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(Figure S5). The inhibitory effects of DDAVP on rPrPres aggregation was also reverified using

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another amyloid specific dye Nile red that corroborated our ThT data (Figure S6). Additionally,

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Rayleigh scattering also showed similar kinetic attenuation of higher order aggregate formation in

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presence of DDAVP (Figure S7). Moreover, to ascertain the specificity of DDAVP, we tested its

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effect on an unrelated protein lysozyme which is implicated in senile amyloidosis leading to multi-

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organ dysfunction[18]. ThT-based aggregation assay showed that addition of increasing molar

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ratios of DDAVP did not significantly alter lysozyme’s aggregation kinetics (Figure S8). These

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evidences demonstrated that DDAVP specifically binds to and modulates fibrillation of prion

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protein.

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Transmission electron microscopy (TEM) of end-stage aggregates (~150 h) showed distinct inter-

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twined fibrillar amyloids in control rPrPres samples (Figure 2A). In contrast, the DDAVP

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incubated samples revealed mostly amorphous, aggregates with sporadic structures resembling

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short, disrupted fibers (Figure 2C). Further, analysis using atomic force microscopy (AFM)

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showed that the untreated rPrPres fibrils were stacked laterally (average width ~6 nm) and attained

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20-25 nm heights (Figure 2B). Conversely, topographical analysis in case of DDAVP incubated 7 ACS Paragon Plus Environment


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samples confirmed their heterogenous and mostly amorphous nature. Eventually, the sparsely

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observed short fibrils were thin (1-2 nm width), unstacked and displayed low height (3-6 nm)

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profiles suggesting lower protein content (Figure 2D). Next, the modulatory effect of DDAVP on

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rPrPres was studied using time-matched particle sizing based on dynamic light scattering (DLS)

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measurements. The data showed clear difference in size distribution at mid-exponential phase (80

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h) and saturation phase (140 h). At 80 h, rPrPres showed a size distribution of150-550 nm species

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that gradually transitioned into large aggregates with 800-2000 nm hydrodynamic radii at 140 h

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(Figure 2E). On the contrary, the DDAVP incubated aggregates showed comparatively

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heterogenous size distribution profiles at both time points. Evidently, the aggregates in mid-

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exponential phase and saturation phase showed a size distribution of 30-450 nm and 120-650 nm

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respectively, indicating an altered aggregation pathway (Figure 2F). Apparently, gradual

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appearance of higher-order particle sizes during rPrPres aggregation corroborated their on-pathway

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nature that plausibly gets averted in presence of DDAVP. Absence of dense, fibrillar aggregates

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in presence of DDAVP even after an extended incubation (200 h) further substantiated an aversion

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from the fibrillogenic pathway (Figure S9).

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Besides, these results also reaffirmed the microscopic observations of heterogenous aggregates

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formed in presence of DDAVP. Perhaps, these heterogenous aggregates were a resultant of altered

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aggregation kinetics due to stabilization of rPrPres native conformation by DDAVP that eventually

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hinders its conversion into a misfolded and aggregation-prone state. It has been previously shown

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that scrapie-like conversion of prion protein involves conversion of its majorly helical architecture

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(α1-α3) into a β-sheet structure [19]. Thus, to ascertain these secondary structural changes, we

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carried out far-UV circular dichroism (CD) and ATR-FTIR analysis of both type of aggregates.

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The far-UV CD signal of unaggregated, monomeric rPrPres exhibited a typical double minimum 8 ACS Paragon Plus Environment

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at 210 and 222 nm confirming its majorly α-helical architecture[20]. On the other hand, saturation

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phase rPrPres aggregates (140 h) showed a single minimum at 218 nm indicating their β-sheet rich

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conformation (Figure 2G). Interestingly, DDAVP incubated samples showed considerable loss of

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the 222 peak but still showed significant 210 nm minima peak that indicated the presence of

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random coil structure along with residual helical content. These observations were supported by

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the FTIR data of rPrPres aggregates that showed a major peak at 1625 cm-1corresponding to cross-

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β-sheets along with a subsidiary random coil content (1665 cm-1). Similarly, DDAVP samples

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comprised mainly of a disordered (1643 cm-1) structure with some helical (1663 cm-1) content

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reaffirming the CD data (Figure 2H). Overall, these evidences strongly indicated that the hetero-

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molecular interactions of rPrPres and DDAVP impedes the higher order, β-sheet conversion of the

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former. Additionally, the presence of residual helical content in DDAVP samples also corroborate

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its structure stabilizing effects on rPrPres that eventually affects its fibrillogenic pathway. It has

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been reported that infectivity and seeding activity peaks markedly in oligomeric prion conformers.

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Our TEM and AFM based analysis of rPrPres oligomeric intermediates formed at early growth

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phase (~45 h) showed spheroid morphology (4-6 nm width). Albeit, it must be noted that the AFM

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images of oligomeric aggregates showed more clustered and less spherical morphologies owing to

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samples being dried before imaging. On the other hand, aggregates formed in presence of DDAVP,

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only showed sparse and morphologically irregular shaped aggregates (1-2 nm width; DDAVP

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aggregates) (Figures 3A-B). This intrigued us to further probe into DDAVP’s ability to modulate

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rPrPres fibrillogenic pathway by studying its direct effect on oligomeric and fibrillar aggregates.



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Figure 3. Effect on prion oligomerization and toxicity. (A) Electron micrographs of oligomeric aggregates formed by (A) untreated and (B) DDAVP treated rPrPres. Corresponding AFM height images are shown in inset. In each case, three-dimensional AFM images of aggregates are also shown for clarity. Scale bars represent 100 nm for TEM and 200 nm for AFM images. (C) Antibody dot blot analysis showing comparative A11 and OC immunoreactivities for untreated rPrPres oligomers and fibrils alongside corresponding time matched DDAVP aggregates. Loading controls using prion-specific (SC-15312) antibody are also depicted. (D) Analysis of the variation in the intensities of dot blots between different aggregate types. Asterisks represent statistical significance as p-value< 0.001.(E) MTT based toxicity assessment of different concentrations of rPrPres oligomers and DDAVP aggregates on Neuro-2a neuroblastoma cells. (F) DIC images of Neuro-2a cells after the addition of PBS buffer, rPrPres oligomers and DDAVP aggregates for 24 hours.

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Eventually, addition of DDAVP to rPrPres oligomers formed at early growth phase, ensued a

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dramatic reduction in ThT fluorescence in less than 5 h that later subjugated into 6-fold less ThT

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responsive aggregates (compared to control) (Figure S10A). End-stage morphology assessed by

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TEM showed only clustered, unstructured aggregates as compared to typical fibrils found in 10 ACS Paragon Plus Environment

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control samples (Figure S10 B-C). Moreover, incubation of mature rPrPres fibrils (150 h; late

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saturation phase) with DDAVP emanated a time-dependent decline in ThT fluorescence (Figure

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S11A). One-week incubation under these conditions resulted in ~50% reduction of ThT response

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with electron micrographs showing smaller, detached fibril fragments as compared to untreated

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fibrils (Figure S11 B-C). Additionally, fibril depolymerization was also confirmed by DLS

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experiments that showed redistribution of higher order (>1000 nm) rPrPres aggregates into

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significantly lower sized (30% cellular uptake within 4 h of rPrPres oligomer

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addition that augmented gradually in a time-dependent manner (Figures S13-S15). 13 ACS Paragon Plus Environment


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In contrast, cellular uptake of DDVAP treated oligomers remained very limited and accounted

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only for ~ 5-10% even after 16 h incubation (Figures S14-S15). At this stage, we also examined

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if apoptotic effects entail cellular internalization of rPrPres oligomers in Neuro-2a cells. We

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observed cell rounding, detachment and membrane-blebbing mostly in rPrPres oligomers treated

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cells. We quantified these features in 16 h post-treated cells and found a 5-fold increase in cell

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numbers showing apoptotic features with a major decline in cell viability (Figure S16).

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Interestingly, DDAVP treated oligomers continue to show remarkably high healthy cell counts

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(>80% compared to buffer/mock treated cells). These results strongly suggested that DDAVP

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essentially affects the membrane interacting surfaces of rPrPres oligomers which in turn subdue

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internalization. Overall, these observations also corroborated our initial hypothesis that DDAVP

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alters toxic, oligomeric conformation of prions. Hence, to investigate the mechanism of

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conformational alterations in prion structure resulting from its hetero-molecular association with

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DDAVP, molecular dynamics (MD) simulations were carried out. Firstly, the prion structure and

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docked DDAVP ensemble were analyzed for any changes in secondary structural elements.

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Interestingly, DDAVP ensemble showed lowering in variations of both backbone RMSD and

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radius of gyration (Rg) suggesting stabilization of structural elements and relatively compact prion

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structure (Figure S17). Nonetheless, the stable RMSD of both systems suggested that their

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trajectories were suitable for further analysis on bonding, residue interactions and sampling of

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conformations. We next analyzed the binding pocket encompassing the β2-α2 loop (residues

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Tyr162, Met166, Tyr169, Asp173, Phe175) and the C-terminal region of the α3 helix (residues

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Gln217, Tyr218, Gln221, Ala224, Tyr225, Arg228). As a major contribution to specific binding,

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the five H-bonds formed by residues Gln98, Met166, Ser222 and Gln217 with DDAVP remained

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stable during entire simulation trajectory (Figure S18).


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Figure 5. Effects on prion conformation and structural variations. (A) Residue-wise RMSF plots of prion alone (black) and prion-DDAVP complex (red). Two regions exhibiting highest variability at the N-terminal (a) and C-terminal (b) are highlighted by boxes. (B) Distribution of beta-sheets content (%) in prion structure (black) and prion-DDAVP complex (red). (C) The probability distribution of solvent assessible surface area (SASA)for prion structure (black) and prion-DDAVP complex (red). A stereo-view of superposed structures sampled during simulation of (D)prion structure alone and (E) prion-DDAVP complex. Regions (a) and (b) are marked for structural comparison of both ensembles. Free energy landscapes (FELs) depicting Gibbs free energy surfaces in kcal/mol projected as a function of RMSD and gyration radius (Rg) for (F) prion alone and (G) prion-DDAVP complex. FELs showing free energy surfaces projected as function of beta-sheet content (%) and RMSD for (H) prion alone and (I) prion-DDAVP complex. Black arrows in FEL plots indicate lowest free energy basins in each case.

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Apparently, the stable inter-molecular H-bonding between DDAVP and prion binding pocket

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residues reaffirmed a strong association. Intriguingly, the two H-bonds formed by Met 166 residue

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with terminal D-Arg residue of DDAVP affected the conformational orientation of crucial Tyr 169

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residue of β2-α2 loop. The hydroxy-phenyl ring of Tyr169 moved 0.5Å closer towards Phe175

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which in turn moved inward too, indicating a plausible alignment of opposing electrostatic

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potentials of both the rings through a T-shaped stacking interaction (bi-directional arrows, Figure

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S19A). The π-stacking between Tyr169 and Phe175 in prion structure has been earlier reported to

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stabilize the β2-α2 loop [27]. This was corroborated by the formation of an extra helical component

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restricting the mobility of β2-α2 loop in DDAVP complex (black arrow, Figure S19A). Together,

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this indicated that engagement of DDAVP incurs similar stabilization mechanism where structural

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rigidity to β2-α2 loop due to constrained mobility of conserved Tyr 169 prevents pathogenic

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conversion of prions [28, 29]. Additionally, this hetero-molecular association also altered the

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positions of hydrophobic residues in the binding pocket (Figure S19B). Next, a comparative

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analysis of residue-wise RMSF was done that showed two major structural variabilities in both

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prion ensembles (dashed boxes); one incorporating the N-terminal amyloidogenic region (a;

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residues 100-130) and the other involving α2-α3 region (b; residues 180-200) (Figure 5A).

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Structural ensembles sampled during the simulation trajectory for both systems were superposed

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to analyze structural differences in both these regions. We found that the N-terminal

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amyloidogenic region of PrP transmuted into β-sheets during the course of simulation indicating

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the attainment of a pro-amyloidogenic conformation (region ‘a’; Figure 5D)[30]. However, the

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DDAVP ensembles lacked similar structural transition with the corresponding N-terminal region

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remained largely unstructured (Figure 5E). Previous NMR based studies demonstrated the

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presence of a rigid α2-α3 region with a relatively flexible inter-helix loop in native prion


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structure[31, 32]. Based on this, a ‘banana-peeling’ mechanism was proposed that attributed loss

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of loop dynamicity for transforming α2-α3 region into the aggregation seed that promote

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conversion into a scrapie-like conformation [33]. Incidentally, the crucial loop connecting α2-α3

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helices showed relatively higher mobility and disorder in DDAVP ensembles suggesting a stable

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native-like architecture (region ‘b’; Figure 5E). Further, on assessing the variations in secondary

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structural content, we observed ~2-fold decrease in β-sheet content in prion structure in presence

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of DDAVP (Figure 5B). Interestingly, DDAVP ensembles also showed relatively higher (~15%)

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disordered elements (turns and coils; Figure S20). On probing more, two stretches encompassing

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residues 100-110 and 112-120 at the N-terminal of un-liganded prion structure were found

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showing highest probability of transforming into β-sheets (Figure S21). Interestingly,

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crystallographic studies have shown that the N-terminal residues 113-120 incorporate a conserved

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palindromic motif AGAAAAGA that adopts a β-sheet fold (termed ‘β0’) and may mediate β-

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enrichment of native prion during aggregation [34]. This further substantiated the N-terminal β-

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sheets observed in prion ensembles sampled during the simulations (region ‘a’; Figure 5D).

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Apparently, β-sheets were also evident through the formation of new local contacts in the N-

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terminal region of the prion ensembles (Figure S22). In addition, an overall increase in solvent

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accessibility (SASA) of prion structure indicated solvation of hydrophobic core corroborating a

356

possible unfolding event impelling a destabilized conformation (Figure 5C). Subsequently,

357

DDAVP ensembles showed lower SASA distribution indicating their relatively stable structural

358

fold. Finally, RMSD, β-sheet content and gyration radius (Rg) were used as reaction coordinates

359

to construct the two-dimensional free-energy landscape (FEL) for each system. Free-energy

360

landscapes (FELs) show the entire conformational space and aid in identifying preferred low

361

energy conformations during the course of simulations. The FEL with RMSD and Rg as reaction



362

coordinates for PrP alone showed the presence of two local basins suggestive of an altered

363

conformational space (black arrows, Figure 5F). On the contrary, prion structure complexed with

364

DDAVP displayed a single low energy basin indicating the overall stability of PrP structure during

365

the entire simulation (Figure 5G). Similarly, FELs projecting β-sheet content and RMSD showed

366

large, dispersive conformational space for PrP alone indicating the presence of more diverse, β-

367

sheet rich species as compared to DDAVP ensembles that clustered into a single basin harboring

368

relatively more compact and lower β-sheet conformations (Figure 5H-I). Overall, structural

369

insights obtained here substantiated stabilization of the prion fold architecture due to its hetero-

370

molecular interaction with DDAVP molecule.

371

In summary, our study demonstrated that the hetero-molecular interaction of DDAVP with rPrPres

372

essentially affect latter’s conformational transition into toxic intermediates. We propose that owing

373

to its strong H-bonding based enthalpy driven binding, DDAVP mediates it’s effect by interacting

374

with multiple aggregating species. Most importantly, it not just averts fibrillation in oligomeric

375

intermediates by converting them into off-pathway species but also represses their toxic epitopes.

376

This eventually affects membrane associations and neuronal cell uptake of toxic oligomers that is

377

reportedly associated with accumulation and amplification of prion infectivity. Since most

378

previously identified molecules against NDs failed to demonstrate efficacy, or displayed high

379

toxicity, alternative pharmacotherapies including drug repurposing are now considered. Drug

380

repurposing eventually is most advantageous, owing to established pharmacokinetic and

381

pharmacodynamic profiles of tested molecules that subsequently abbreviates drug discovery

382

investments. In fact, several drugs are currently repurposed for treatment of Alzheimer’s disease

383

(Sildenafil, Amphotericin B), Parkinson’s disease (Nilotinib, Inosine and Isradipine),

384

Huntington’s disease (Olanzapine, Tetrabenazine) and Amyotrophic Lateral Sclerosis (Triumeq, 18 ACS Paragon Plus Environment

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385

Ibudilast) [35, 36]. Unfortunately, there has been a dearth of similar repurposing efforts against

386

prion diseases in recent years. Through the presented work, we propose that repurposing DDAVP

387

could be the newest alternative in counteracting prion diseases. Our work warrants further in vivo

388

testing using animal models for ascertaining its inhibitory potential against different prion strains

389

and their infectivity. Moreover, newer DDAVP analogues could serve as a starting avenue for

390

developing better therapeutics against prion diseases and other similar neuropathies.

391 392 393

ASSOCIATED CONTENT

394

Publications website at DOI:

395

Detailed experimental methods and materials; Structures of PrP and DDAVP; Docking score of

396

clusters; ThT Kinetics of lysozyme; Light scattering data; SPR equilibrium parameters; Fibril and

397

oligomer modulation data; Supplementary Molecular dynamics (MD) data.

398 399 400 401

Supporting Information. The Supporting Information is available free of charge on the ACS

AUTHOR INFORMATION Corresponding Authors

402

Dr. Abhinav Grover, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India -

403

110067, Telephone: +91-11-26738728; FAX: +91-11-26702040; E-mail: [email protected]

404

[email protected]

405

Dr. Bishwajit Kundu, Kusuma School of Biological Sciences, Indian Institute of Technology Delhi

406

(IIT Delhi), New Delhi, India - 110016, Telephone: +91-11-26591037; FAX: +91-11- 2658 2282;

407

E-mail: [email protected]

408

Author Contributions

409

A.G. and B.K. coordinated the study. A.S. and N.A. designed experiments and analyzed the data;

410

N.A. conducted most of the biophysical and cell-based experiments; S.J. conducted computational

411

experiments; A.S. and N.A. compiled and wrote the manuscript. All authors reviewed the

412

manuscript.



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414

Acknowledgements

415

The authors thank the Jawaharlal Nehru University (JNU) and the Indian Institute of Technology

416

Delhi (IIT Delhi) for infrastructural support. N.A. and S.J. acknowledge scholarship support from

417

University Grant Commission (UGC) and Indian Council of Medical Research (ICMR)

418

respectively. The authors also acknowledge Advanced Instrumentation Research Facility (AIRF),

419

JNU, New Delhi for technical assistance in performing TEM and Confocal experiments. Dr Shalini

420

Gupta and Dr Shruti Khanna at the Department of Chemical Engineering and Central Research

421

Facility (CRF) IIT Delhi are deeply acknowledged for assistance in using Biacore 3000 SPR

422

facility.

423

Notes

424

The authors declare no competing financial interest.

425

ABBREVIATIONS USED

426

DDAVP,

427

isothiocyanate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SASA,

428

Solvent accessible surface area; SPR, Surface plasmon resonance; TEM, Transmission electron

429

microscopy; AFM; Atomic force microscopy; CD, Circular dichroism; MD, Molecular dynamics.

430

REFERENCES

431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446

1. 2. 3. 4. 5. 6. 7.

Desmopressin;

DIC,

Differential

interference

contrast;

FITC,

Fluorescein

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Figure 1. Binding interactions and effect on prion aggregation.

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Figure 2. Effects on fibrillation, size distribution and secondary structure.


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Figure 3. Effect on prion oligomerization and toxicity.



Figure 4. Effects on neuronal cell apoptosis and internalization of toxic oligomers.


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Figure 5. Effects on prion conformation and structural variations.


Protective effects of a neurohypophyseal hormone ... - ACS Publications

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