An explicitly designed paratope of amyloid-β prevents neuronal apoptosis in vitro and hippocampal damage in rat brain

Synthetic antibodies hold great promise in combating diseases, diagnosis, and a wide range of biomedical applications. However, designing a therapeutically amenable, synthetic antibody that can arrest the aggregation of amyloid-β (Aβ) remains challenging. Here, we report a flexible, hairpin-like synthetic paratope (SP1, ∼2 kDa), which prevents the aggregation of Aβ monomers and reverses the preformed amyloid fibril to a non-toxic species. Structural and biophysical studies further allowed dissecting the mode and affinity of molecular recognition events between SP1 and Aβ. Subsequently, SP1 reduces Aβ-induced neurotoxicity, neuronal apoptosis, and ROS-mediated oxidative damage in human neuroblastoma cells (SH-SY5Y). The non-toxic nature of SP1 and its ability to ameliorate hippocampal neurodegeneration in a rat model of AD demonstrate its therapeutic potential. This paratope engineering module could readily implement discoveries of cost-effective molecular probes to nurture the basic principles of protein misfolding, thus combating related diseases.


Introduction
The deposition of amyloid brils has consequences with numerous protein-misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's disease, Prion diseases, and type-2 diabetes. 1,2 The detailed molecular mechanism of Alzheimer's disease (AD) is not intelligible yet. However, growing shreds of evidence suggest that the aggregation of amyloidb peptide (Ab) from native non-toxic monomers to highly toxic amyloid brils in the extracellular space and formation of neurobrillary tangles (NFTs) in neurons are the principal hallmarks for the pathogenesis of AD. 3,4 In the past two decades, numerous strategies have been exercised to nd a cure for AD. 5 These strategies involve metal chelators, nanoparticles, the amyloidogenic core region (KLVFF) [6][7][8] or other fragments of the Ab peptide, 9,10 chemical chaperones, 11,12 peptide-based inhibitors, [13][14][15][16] small molecules, 5,17 and conformation-selective antibodies. [18][19][20][21] Antibody-based drug design is the most intriguing as antibodies engulf and eliminate the toxic Ab species. Besides, antibodies have demonstrated the scope and potential of immunotherapy. Nevertheless, they are associated with severe adverse effects such as Fc-mediated pro-inammatory immune responses. Recently, affibodies [22][23][24][25][26] have shown prevention of the self-aggregation of Ab by encapsulating the Ab peptide and reducing pro-inammatory immune responses, which led to a novel therapeutic approach against AD. [18][19][20][21][22][23][24][25][26][27] Among the mentioned strategies, a rationally designed, short peptide from a self-aggregation site of Ab showed promising results even in clinical trials with superior bioavailability and less toxicity. 5,28 Here, we aimed to construct an explicitly designed synthetic paratope inspired by a peptide fragment of Ab that could potentially be a clinical candidate for targeting Ab. A paratope is a part of an antibody known to recognize the epitope region of an antigen selectively. [18][19][20][21] The knowledge from prior investigations by our group and numerous reports has empowered us to construct a exible, parallel b-hairpin-like synthetic paratope (SP1, Fig. 1a and b). The size of the designed SP1 is smaller than that of any existing antibody and affibody. We explored its efficiency in binding to Ab using various spectroscopic techniques. The atomicscale mechanistic study by NMR dissected the recognition mechanism. We show that SP1 remarkably disaggregates the preformed Ab aggregates and potentially dissolves Ab plaques through different in vitro studies. Besides, SP1 reduces Ab 40 induced cytotoxicity, oxidative stress-mediated apoptotic events, and dysregulation of Ca 2+ homeostasis in human neuroblastoma SH-SY5Y cells. 29,30 SP1 also improves Ab 40 induced ROS generation and modulates apoptosis signalling in the cells. Notably, SP1 has therapeutic potential in vivo through less toxicity and ameliorating hippocampal neurodegeneration.

Results and discussion
Design and synthesis of the paratope The p / p stacking interactions play a central role in the selfassembly processes in most amyloidogenic proteins leading to their aggregation and disease progression. 31,32 The central core hydrophobic region of the Ab peptide (LVFFA), which acts as a selfrecognition unit, was chosen as a strand in the designed hairpinlike SP1. Two strands were connected in parallel through a exible unit (PEG) to construct the complete synthetic paratope molecule (Fig. 1a, b and ESI Scheme 1 †). We introduced N-methylation 33,34 in the alternate amino acids in each strand, preventing selfaggregation by blocking intermolecular H-bonding (Fig. 1a). In a similar principle, N-methylation should not allow further aggregation of the Ab peptide captured by SP1. Since the central core hydrophobic region (epitope) 6-8 of Ab is crucial for selfaggregation and senile plaque formation, we designed SP1 in such a way that it can selectively bind with the epitope and capture Ab from both sides with its two strands, as proposed in Fig. 1c. We introduced a control b-breaker peptide (CBp) with only one Nmethylated strand of SP1. Also, ve more peptides conjugated with suitable uorophores were hosted to investigate the mechanism of interaction between SP1 and Ab 40 (ESI Table 1 and Fig. 1-7 †). In the beginning, we conrmed that SP1 and CBp are nonamyloidogenic using combined CD, FTIR, TEM, and birefringence analyses (ESI Fig. 8 and 9 †).

Inhibition of Ab 40 amyloid formation
To investigate the inhibitory effect of SP1 on Ab 40 brillization, we performed various biophysical assays in the presence of different doses (0.5, 1, 2, and 5-fold molar excess of SP1), and CBp was used as a control. First, we monitored the kinetics of the amyloid formation of Ab 40 (50 mM) by thioavin T (ThT) assay ( Fig. 2a and b). The Ab 40 peptide alone aggregated with time, with a growth saturation point around 72 h, as evident from the surge in ThT uorescence intensity. However, in the presence of SP1, the intensity of uorescence decreased in a dose-dependent manner (Fig. 2a), and precisely, a 5-fold molar excess instigated $66% inhibition (Fig. 2b). Likewise, SP1 disaggregated the characteristic brillar aggregates of Ab 40 under TEM (Fig. 2c) in a dose-dependent manner. We inspected a few brillar aggregates at the lowest dose of SP1, demonstrating the essential requirement of an optimal concentration for Ab 40 disaggregation. Green-gold birefringence appears to be a standard result of Ab 40 aggregation under cross-polarized light post-staining with Congo red dye. Owing to amyloid formation, Ab 40 appears as green-gold birefringence (Fig. 2d) under cross-polarized light when stained with Congo red. Upon treatment with various doses of SP1, such type of birefringence ( Fig. 2d) disappeared, except at the lowest dose. In contrast, the control peptide (CBp) showed $45% inhibition of Ab 40 peptide aggregation with the experiments mentioned above in parallel (ESI Fig. 10a-c †). We noticed that a 5-fold dose is the minimal requirement for CBp to inhibit aggregation, whereas equimolar SP1 completely prevents it.

Disruption of the preformed Ab 40 aggregates
Understanding the kinetics of brillization led us to design an in vitro experiment of preformed bril disaggregation. In this experiment, the 60 h aged Ab 40 peptide was incubated further for 180 h (total 240 h) with SP1 and CBp separately at varying doses (0.5, 1, 2, and 5-fold molar excess). The high uorescence intensity of ThT shows that preformed Ab 40 brils were suppressed substantially with increased doses of SP1 and the control peptide ( Fig. 2e and ESI Fig. 11a †). Distinctly, we observed $57% (Fig. 2f) and $39% (Fig. 2f) disruption of the  We also performed TEM and Congo red staining experiments to examine the efficacy of SP1 disrupting the Ab 40 preformed brils. Equimolar or higher doses of SP1 disrupted the preformed brillar assembly of Ab 40 , as conrmed by TEM (Fig. 2g). In comparison, CBp disrupted the Ab 40 brils when treated with a 2-fold or higher molar excess. Upon incubation with an equimolar concentration of SP1, a remarkable disappearance of the preformed Ab 40 aggregates was evident in the Congo red birefringence staining experiment (Fig. 2h). However, we observed a signicant disappearance of birefringence only at a 5-fold molar excess of the control peptide (ESI Fig. 11b and c †). Notably, SP1 failed to demonstrate the efficacy of bril disruption of Ab 40 at a 0.5-fold molar concentration. Collectively, these results strongly demonstrate that SP1 is more efficient than CBp in disaggregating brils of Ab 40 .

Inhibition of Ab 42 amyloid formation
The aggregation of Ab 42 causes signicant neurotoxicity among all existing isoforms of Ab. 35 We, therefore, examined the inhibition efficacy of SP1 for Ab 42 aggregation. We performed similar biophysical experiments as described earlier for Ab 40 . The ThT assay showed that the aggregation rate of Ab 42 was much faster than that of Ab 40 at a 50 mM concentration (Fig. 3a), and aggregation started immediately reaching a plateau within 20 h. ThT uorescence intensity decreased in a dose-dependent manner by treating Ab 42 aggregates with SP1 ( Fig. 3a and b).
Around 84% inhibition of Ab 42 aggregation was observed when treated with a 5-fold molar excess of SP1 (Fig. 3b).
The aggregated Ab 42 in the absence of SP1 showed densely populated brillar structures under TEM (Fig. 3c), indicating  the amyloid signature, as previously reported. 36 With the assistance of SP1, the density of Ab 42 brils was reduced in a dose-dependent manner. Also, untreated Ab 42 aggregates exhibited green-gold birefringence upon staining with Congo red (Fig. 3d), and this signal was decreased in a dose-dependent manner by SP1. Collectively, these examinations indicate that the efficiency of SP1 for the inhibition of Ab 42 aggregates is comparable as observed with Ab 40 .

SP1 reduces Ab aggregate-induced dye leakage from LUVs
Smaller Ab oligomers or protobrils are more toxic than mature brils in AD progression due to their ability to disrupt membranes via pore formation. 37,38 Therefore, it is essential to examine whether SP1 can convert the toxic oligomeric species of Ab into a non-toxic one. To evaluate this, we performed a membrane leakage assay on carboxyuorescein-loaded large unilamellar vesicles (LUVs). 38 The time required for Ab 40 oligomer and mature bril formation is 12 h and 72 h, respectively (inferred from ThT assay, black curve, Fig. 2a), which directed us to set up the LUV leakage assay. Dye-loaded LUVs were incubated with the corresponding Ab 40 oligomers (12 h aged), mature brils (72 h), and freshly disaggregated Ab 40 brils in solution and SP1 or CBp (ESI Fig. 12b and c †). The uorescence intensity of complete dye release from LUVs by Triton X-100 served as a positive control (100% leakage), and untreated dye-loaded LUVs assisted as a negative control. The Ab 40 oligomers (12 h aged) caused rapid dye leakage of $40% until 100 min (ESI Fig. 12b and c †), whereas mature Ab 40 brils caused $15% leakage and the untreated LUVs showed a minimal leakage of $9% (ESI Fig. 12b and c †) during the same period. These results establish that the Ab 40 oligomers trigger more dye leakage than the mature brils, and hence are likely to be more toxic. 38 Membrane disruption by Ab 40 proceeds through a two-step mechanism. [39][40][41] In the rst step, Ab 40 monomers selfassemble to form soluble oligomers, which bind to the lipid membranes to form small ion-selective channel-like pores. During pore formation, the oligomers of Ab 40 further selfassemble and form larger aggregates that lead to the formation of mature brils which are released from the membrane. In the second step, the onset of Ab 40 aggregation and bril formation causes membrane disruption through a detergentlike mechanism. [39][40][41] Notably, freshly disaggregated Ab 40 brils by SP1 and CBp did not considerably damage the LUV membrane, evidenced by only $10% leakage from the LUVs, respectively, which is comparable to that from untreated LUVs. These results collectively affirm the potential ability of SP1 for disassembling preformed brils and other oligomers of Ab 40 to an innocuous species.

Monitoring early events by DLS and TEM
The inhibition of Ab 42 oligomer or bril formation by SP1 at different time intervals was further examined using DLS ( Fig. 4c and d) and TEM ( Fig. 4e and f)   $825 nm, and $940 nm, respectively (Fig. 4c), indicating the formation of oligomers (at 1 h or 5 h) and mature brils (at 10 h and 20 h), as observed in previous reports. 42,43 SP1 treated samples exhibited hydrodynamic diameters of $15 nm, $340 nm, $640 nm, and $730 nm, respectively (Fig. 4d). These results indicated that upon 5 h or more prolonged incubation of Ab 42 with SP1, it caused oligomer formation inhibition as smaller aggregates disappeared and were converted to larger, possibly amorphous aggregates. We further validated this phenomenon by TEM and observed that Ab 42 exhibited smaller aggregates at 1 h or 5 h. In contrast, dense brils appeared at 10 h or 20 h, suggesting oligomer formation at 1 h or 5 h, as observed in the DLS results. In the presence of SP1, we did not observe any brillar aggregates in all tested time intervals; instead, some amorphous aggregates were noted. These amorphous aggregates were non-toxic, as evident from the LUV experiments mentioned above and the cytotoxicity assay (vide infra). The formation of non-toxic amorphous species suggests that SP1 drives Ab 42 aggregation towards off-pathway aggregation in line with a previous report. 43

Prevention of Ab 40 induced cytotoxicity
Since the protobrils of Ab species induce cytotoxicity in neuronal cells, 44,45 we investigated the inhibition potential of SP1 against Ab 40 induced neurotoxicity in human neuroblastoma SH-SY5Y cells as a cellular model system of AD. 29,30 Initially, we explored the toxicity of SP1 and did not observe any discernible cytotoxicity even at the maximum concentrations (10 mM) used in the experiments (ESI Fig. 13a †). Further, the cells were incubated with 10 mM Ab 40 for 24 h in the absence or presence of graded concentrations of SP1 (0.5-10 mM). We observed a signicant reduction in the cell population treated only with Ab 40 compared to the negative control and in the absence of SP1. However, the incubation of SP1 ameliorated the toxic effect considerably at 5 mM ($82%), as determined by cell viability (ESI Fig. 13b †). Then, we explored the membrane damage induced by Ab 40 (ref. 45 and 46 ) using the lactate dehydrogenase (LDH) assay. Treatment of Ab 40 released a signicant amount of cytosolic LDH into the culture medium of SH-SY5Y cells. Co-incubation of Ab 40 with SP1 at the respective concentrations (5 mM and 10 mM) revealed a substantial reduction in LDH leakage into the cell culture medium as compared to only Ab 40 treated cells (ESI Fig. 13c †). These two ndings indicate that SP1 at a molar ratio of 1 : 2 (SP1 : Ab 40 ) is sufficient to demonstrate maximum inhibition of Ab 40 mediated cellular cytotoxicity. Interestingly, Ab 40 induced neuronal cell death was preserved for at least three days upon treatment with SP1 (5 mM) (ESI Fig. 13d and e †).

SP1 ameliorates oxidative stress injury, apoptosis, and Ca 2+ homeostasis
Condensed or fragmented nuclear bodies characterize the distinctive nature of apoptotic cells. To explore the anti-apoptotic and cytoprotective properties of SP1, we used Hoechst 33258 as a DNA staining dye. A signicant number of apoptotic cells were observed under a uorescence microscope when the cells were treated with Ab 40 (10 mM) for 24 h (Fig. 5a and b) compared to untreated cells. Upon co-incubation with SP1 (5 mM), the number of apoptotic cells holding damaged DNA was markedly reduced (Fig. 5b). These ndings illustrate the potency of SP1 in regulating Ab 40 induced DNA damage in SH-SY5Y cells. The underlying mechanism of this neuronal apoptosis and oxidative damage has been reported to be signicantly inuenced by ROS generation, followed by triggering mitochondrial apoptotic events. 47,48 In another experiment, we observed that the intensity of the ROS sensitive uorescent marker in SH-SY5Y cells increased in the presence of Ab 40 compared to untreated cells, and the co-incubation of cells with Ab 40 and SP1 (ratio 1 : 2) signicantly inhibited Ab 40 induced ROS production (Fig. 5c). Dyshomeostasis of Ca 2+ is also responsible for the increased production of Ab peptides, by which a degenerative feed-forward cycle is activated, resulting in accelerated apoptosis, synaptic dysfunction, and memory impairment. 49 To examine the effect of Ab 40 with or without SP1 on Ca 2+ homeostasis, we measured intracellular free Ca 2+ using a uorescent Ca 2+ indicator, Fura-2AM. Our results demonstrated that Ab 40 (10 mM) signicantly elevated intracellular Ca 2+ levels in SH-SY5Y cells as compared to untreated cells. Then co-incubation with a molar ratio of 1 : 2 (SP1 : Ab 40 ) reduced Ca 2+ levels to 98% compared to Ab 40 treated cells (Fig. 5d). The experiments showed that SP1 preserves Ca 2+ dyshomeostasis induced by Ab 40 via encumbering the oligomeric conversion of Ab 40 .

Effect of SP1 on Ab 40 induced apoptotic protein markers
Accumulation of Ab triggers the generation of intracellular free radicals and leads to the activation of caspases via releasing cytochrome-c from mitochondria. Bcl-2 family proteins, proapoptotic Bax proteins, and caspases are well known to be involved in the mitochondrial apoptotic pathway. 50 Western blot analyses of SH-SY5Y cells suggested that Ab 40 upregulates the level of Bax and causes a slight change in the Bcl-2 level, which is a signicant increase in the ratio of Bax/Bcl-2 expression ($3.2 fold) as compared to that in healthy cells. Interestingly, the expression of Bax protein was markedly downregulated by treatment with SP1 in SH-SY5Y cells for 24 h (Fig. 5e and f). Further, western blot analysis also revealed that the expression level of cleaved caspase-9 or caspase-3 signicantly decreased aer incubation with SP1 for 24 h (Fig. 5e, g  and h). However, treatment with Ab 40 alone leads to activation of caspase-3 directed DNA breakage, nuclear chromatin condensation, and neurocellular apoptosis. These outcomes conrm the active suppression of Ab 40 mediated mitochondrial apoptosis and cell death by SP1 by inhibiting Ab oligomer formation.

Evaluation of acute and sub-chronic toxicity of SP1 in vivo
We predicted the cytotoxicity of SP1 in Sprague-Dawley rats as per our previous report. 51 A total of 24 rats were used in this study (n ¼ 8/group) and divided into three groups: group1: control, no treatment; group 2: received 100 mg kg À1 of SP1 and group 3: received 500 mg kg À1 of SP1. The SP1 was administered into the tail vein for 42 days once in a day. We did not observe any cytoarchitectural changes in the liver and kidney tissue aer the injections of two different doses in the groups (Fig. 6a). Interestingly, SP1 causes neither mortality nor abnormal behavioral patterns in rats. Besides, we did not observe any signicant changes in the rats' body weight at two different doses (100 mg kg À1 and 500 mg kg À1 ) compared to their respective control groups on days 7, 21, and 42 by sub-chronic study (ESI Fig. 14 †). Notably, we did not nd any severe changes in the hematological and biochemical parameters in the group of rats treated with SP1 (100 mg kg À1 ) even aer 42 days. Almost similar observations in rats treated with SP1 (500 mg kg À1 ) were revealed, except for monocyte and SGOT levels (ESI Table 3 †). The tabulated biochemical prole (novel biomarkers of the liver and kidney) corroborates the safety charms of SP1 for further in vivo studies.

SP1 ameliorates hippocampal neurodegeneration in rat brain
The overproduction of Ab damages hippocampal neurons and causes cognitive impairments in AD. Previous data motivated us to explore the potential of SP1 in ameliorating hippocampal neurodegeneration. Cresyl violet staining was performed for identication of Nissl granules in neurons to reveal hippocampal neurodegeneration in this experiment. One-way ANOVA showed a signicant contrast in the intensity of granules in the hippocampus between the groups [F (4, 25) ¼ 102.4, P < 0.001].
Furthermore, Tukey's post hoc test suggested that the intrahippocampal microinjection of toxic Ab 40 in hippocampal neurons showed a signicant (P < 0.01) decrease in the intensity of Nissl granules (Fig. 6b) as compared to the control and sham groups, which indicates that neurons have degenerated. However, SP1 treatment at both the dosages (40 mM and 100 mM) in pre-Ab40 injected rats reduced the degeneration of hippocampal neurons signicantly (P < 0.01), demonstrated by the intensity of Nissl granules (Fig. 6b). Hence, we established that SP1 treatment exhibits neuroprotective function against Ab 40 induced neurotoxicity.

Investigation of the interaction between Ab and SP1
High-resolution 2D Heteronuclear Multiple Quantum Coherence (HMQC) NMR experiments were performed with 80 mM Ab40 with increasing concentrations of SP1 (titrated up to a molar ratio of 1 : 10). The Ab 40 backbone amide resonances resulted in concentration-dependent residue-specic chemical shi perturbations (CSPs) in the presence of SP1. At a molar ratio of 1 : 10, the molecular interaction resulted in notable CSPs, specically for the central hydrophobic-K 16 LVFFA 21 region (Fig. 7a). Similar observations were also made for the Cterminal region, particularly the I 31 IGL 34 stretch and the hydrophobic V36, V39, and V40 residues (Fig. 7b). These observations clearly indicated the specic involvement of these hydrophobic-rich segments in the molecular association with SP1. Recent studies have highlighted the K 16 LVFFA 21 segment to be essential for the Ab 40 brillation propensity. [52][53][54][55] Extensive reports have provided evidence for the segment to be closely  Table 2 †). (f-h) Densitometric analysis of changes in levels of the Bcl-2/Bax expression ratio, cleaved caspase-9, and cleaved caspase-3 (changes fold to control), respectively. The protein bands were quantified using smart view image analysis, and values are expressed as mean AE SEM (n ¼ 3 experiments per group) **p < 0.01, compared to the control group and ##p < 0.01 compared to Ab 40 treated group. associated with the dock-lock mechanism underlying Ab nucleation events. Thus, the SP1 mediated perturbation of this crucial domain suggests molecular interference in the docklock interactions of monomeric Ab, explaining the altered brillation. 55,56 Alternatively, the association of SP1 with the hydrophobic K 16 LVFFA 21 and the C-terminal segments also stands to explain the reduced membrane damage and subsequent toxicity. These hydrophobic segments have been shown to internalize within the hydrophobic acyl region of the lipid membranes, disrupting the membrane integrity. 57 Our recent studies have shown the crucial role played by the C-terminal residues in mediating cytotoxicity. Our mutation-based studies have suggested the role played by the GxxxG motifs from the C-terminal in aiding the helix-helix association and regulating the Ab brillation pathway. 58 Thus, a direct molecular association of SP1 with these segments indicates the inaccessibility of these segments necessary for wild-type Ab amyloidogenesis.
Interestingly, very similar observations were obtained for the residue-specic interaction studies between Ab 42 and SP1 (Fig. 7c). HMQC proles showed signicant CSPs, specically involving the central K 16 LVFF 19 segment and the C-terminal hydrophobic residues, including G29, G33, V36, I41, and A42 (Fig. 7d). The direct association of the terminal residues in Ab 42 is further reminiscent of the reduced cytotoxicity mediated upon SP1 interaction. Reports have found the increased Cterminal stability of Ab 42 to be entropically favorable for the cytotoxic brillation. 59 Thus, high CSPs for the C-terminal residues in Ab 42 corroborate well with the functional implication of SP1 in modulating Ab aggregation propensity.
Next, singular value decomposition (SVD) was used to obtain the residue-specic binding affinity of SP1 to Ab 40 . The CSPs for Ab 40 with SP1 were adjusted for both Dd N and Dd H to extract the dissociation constant (K D ) for each residue (see the NMR method and ESI Fig. 15-17 † for details). Comparatively lower K D values of $200 mM were obtained for the residues R5, L17, V24, K28, and G29 (ESI Table 4 †) of Ab 40 , indicating their functional unavailability in brillation. Once again, these data support the inhibition of the dock-lock mechanism of Ab 40 by SP1.
The lack of transferred NOE peaks (trNOEs) restricted us from determining the three-dimensional structure of SP1 bound to Ab 40 (data not shown). Although the designed paratope's (SP1) affinity is moderate, this is the rst example of a synthetic paratope to prevent Ab aggregation. However, we are working further to improve the molecular association.

†).
We considered the two most plausible structural alignments of SP1, hairpin-like or linear ( Fig. 8a and b). Since we observed that SP1 is non-amyloidogenic (ESI Fig. 8 †), self-aggregation with the hairpin-like conformation does not qualify. Now, if SP1 adopts a hairpin-like structure (Fig. 8a), it should exhibit intra-molecular FRET or intermolecular FRET through straightchain alignment (Fig. 8b). Interestingly, SP1C showed a unique FRET event in contrast to the mixture of equimolar SP1A and SP1B, which did not show a signicant change in emission. The time-resolved uorescence study further conrmed a similar observation (ESI Table 5 and Fig. 18 †). These combined pieces of information and the calculated Förster radius (R 0 ) 60-63 of the donor/acceptor system which was 27.9Å (Section 1.2.j in the ESI †) further corroborate the U-shaped or hairpin-like structural alignment of SP1 (Fig. 1b and 8a). Also, we obtained direct evidence of interaction between SP1 and the homologous sequence of the Ab 40 peptide, resulting in substantial FRET events and positive implication of time-resolved uorescence (ESI Table 6 and Fig. 19 †). The aforementioned studies were conducted to comprehend the interaction of SP1A to LP1B and that of SP1B to LP1A by incubating equimolar concentrations. Data, revealed through all the present studies, allowed us to propose two plausible modes of interaction, which demonstrate inhibition of amyloid formation and disruption of preformed aggregates of Ab 40 by SP1 (Fig. 8c and d). The proposed models were further validated through the FRET, time-resolved uorescence study, and Förster radius (R 0 ) calculations.
Briey, a signicant FRET event was observed when SP1C was mixed in pre-captured Ab 40 with SP1 solution, and no FRET events resulted in the mixture (SP1A + SP1B) added to the precaptured Ab 40 with SP1 solution (ESI Fig. 20 and Table 7 †).
We have noted earlier that the hairpin-shaped conformation of SP1 remains unaltered in the presence of Ab 40 aggregates. Therefore, these experiments together clarify that the U-shaped synthetic paratope prevents amyloid oligomer formation, most probably through the zipping action proposed in Fig. 8c.
To target a particular epitope of various amyloidogenic proteins including tau, Ab, a-synuclein, and b 2 -microglobulin, and to antagonize their aggregation, Nowick and co-workers previously developed rationally designed 54-membered cyclic peptides, amyloid b-sheet mimics (ABSMs). [64][65][66][67][68] The ABSM peptide was comprised of two strands, linked with two ornithine d-linkages. One of the two strands was selected for recognition of the target amyloidogenic protein, whereas another strand contained an unnatural amino acid, "Hao" used as a b-sheet breaker unit to inhibit the aggregation of the target disease protein. Due to the cyclic structure, these peptides were less exible yet effective in inhibiting the aggregation of various amyloidogenic proteins. In contrast, in the present study, we designed a hairpin-like exible synthetic paratope comprising two epitope-binding peptides connected through a PEG linker. The synthetic paratope can bind to the target epitope of the Ab peptide from both sides, and the presence of N-methylation on alternate amino acids does not allow Ab monomers to selfassemble to form amyloids.
The structural design of ABSM containing a "Hao" unit helped the cyclic peptide prevent ABSMs from aggregating in solution to form a larger aggregated network of b-sheets; instead, it dimerized and further self-assembled into oligomers. 64 In contrast, the synthetic paratope (SP1) did not selfassemble to form oligomers or larger aggregates due to the presence of N-methylation at alternate amino acids. In addition, the presence of PEG groups in the synthetic paratope, in contrast to the hydrophobic side chain of ornithine in ABSM, increases aqueous solubility, which is an essential factor from a therapeutic perspective. Moreover, due to the hairpin-like structure, the synthetic paratope exhibits more exibility and possibly can show a better efficacy to inhibit the aggregation of Ab or other amyloidogenic proteins than the existing peptide probe.

Conclusion
In the present study, we have demonstrated the design, synthesis, and characterization of a synthetic paratope (SP1) that selectively binds with the epitope LVFFA, a vital amyloidogenic part of the Ab peptide. A series of in vitro biophysical experiments, including NMR, support the inhibition of Ab 40 and Ab 42 aggregation by SP1 at an atomic resolution. SP1 was also equally efficient in disaggregating the preformed brillar assembly of the Ab40 peptide into non-toxic species. We speculate that the synthetic paratope may further enable for designing an affinity tag for AD diagnosis, a reporter of the Ab40 peptide, and a PROTAC type therapeutic against AD. The ability to ameliorate Ab 40 induced neurotoxicity, ROS generation, and apoptosis and maintain intracellular Ca 2+ homeostasis of SP1 is remarkable for the further construction of suitable antiapoptotic and anti-inammatory peptide probes. The designed, non-toxic synthetic paratope may gain considerable attention for ameliorating Ab-induced hippocampal neurodegeneration, corroborated by preliminary in vivo studies in a rat model of AD. In-depth investigations with animals may be carried out aer improving its binding affinity to the nanomolar level, which is at the micromolar range at present. Further improvement of solubility and enzymatic stability is also required.
We believe that the dissected zipping-mechanism for capturing the Ab 40 peptide by a synthetic paratope will signicantly facilitate the design of a great variety of paratopes. Such a smartly designed molecular construct may also nd applications in diverse directions spanning chemical biology, diagnostics, and therapeutics. Our ndings suggest that due to the structural exibility and moderate to weak affinity towards the target epitope, the synthetic paratope might lead to potential hit discoveries against Alzheimer's disease, extendable further to other amyloidoses.

Conflicts of interest
The authors declare no conict of interest.