Peptidomimetic-Based Multidomain Targeting Offers Critical

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Peptidomimetic-based Multi-Domain Targeting Offers Critical Evaluation of A# Structure and Toxic Function Sunil Kumar, Anja Henning, Mazin Magzoub, and Andrew D. Hamilton J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b13401 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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Journal of the American Chemical Society

Peptidomimetic-based Multi-Domain Targeting Offers Critical Evaluation of Aβ Structure and Toxic Function

Sunil Kumar1*, Anja Henning-Knechtel2, Mazin Magzoub2*, and Andrew D. Hamilton1*

1

2

Department of Chemistry, New York University Biology Program, New York University Abu Dhabi

*Correspondence: [email protected], [email protected], [email protected] Lead Contact: [email protected] Address: 1Department of Chemistry, New York University, New York, NY 10003, USA. 2

Biology Program, New York University Abu Dhabi, P.O. Box 129188, Saadiyat Island Campus,

Abu Dhabi, United Arab Emirates. 1 ACS Paragon Plus Environment

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ABSTRACT The prevailing hypothesis stipulates that the pre-amyloid oligomers of Aβ are the main culprits associated with the onset and progression of Alzheimer’s disease (AD), which has prompted efforts to search for therapeutic agents with the ability to inhibit Aβ oligomerization and amyloidogenesis. However, the lack of clinical progress is impeded by the limited structural information about the neurotoxic oligomers. To address this issue, we have adopted a synthetic approach, where a library of oligopyridylamide-based small molecules was tested against various microscopic events implicated in the self-assembly of Aβ. Two oligopyridylamides bind to different domains of Aβ and affect distinct microscopic events in Aβ self-assembly. The study lays the foundations for a dual recognition strategy to simultaneously target different domains of Aβ for further improvement in anti-amyloidogenic activity. The data demonstrate that one of the most effective oligopyridylamides forms a high affinity complex with Aβ, which sustains the compound’s activity in cellular milieu. The oligopyridylamide was able to rescue cells when introduced 24 h after the incubation of Aβ. The rescue of Aβ toxicity is potentially a consequence of the colocalization of the oligopyridylamide with Aβ. The synthetic tools utilized here provide a straightforward strategic framework to identify a range of potent antagonists of Aβ-mediated toxic functions. This approach could be a powerful route to the design of candidate drugs for various amyloid diseases which have so far proven to be ‘untargetable'.

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INTRODUCTION The conversion of intrinsically disordered peptides and proteins into intractable amyloids via a sequence of intermediate structures is a pathological hallmark of many neurodegenerative disorders, including Alzheimer’s disease (AD), Parkinson’s disease, and Huntington’s disease; non-neuropathic organ localized amyloidosis such as type 2 diabetes; dialysis related amyloidosis; HIV-AIDS; and cancer1-11. AD is the most prevalent progressive neurodegenerative disorder, accounting for ~60-80% of all the cases of dementia12,13. The number of patients suffering from AD is ~47 million, which is expected to increase to 75 million by 2030, representing a massive economic and social burden on society. The amyloid β peptide (Aβ) is generated by successive cleavage of an amyloid precursor transmembrane protein by β- and δ-secretases13,14. Among several isoforms, Aβ42 and Aβ40 are the most pathophysiologically relevant species, with Aβ42 being more aggressive in terms of its amyloidogenic and toxic nature. The process of Aβ42 aggregation, via sampling of a series of intermediate conformations, is associated with its neurotoxic nature. Recent advances in the field underpin the link between soluble oligomeric intermediate structures of Aβ42 and neuronal cell toxicity which is associated with the progression of AD4, 15. Therefore, approaches that facilitate the inhibition or the disruption of the oligomeric intermediates of Aβ could in principle set a launching pad for the treatment of AD. There exists a large body of work focused on identifying antagonists of Aβ aggregation using various methods, including screening, computational modeling, electrospray ionization-ion mobility spectroscopy-mass spectrometry, and an in vivo periplasmic platform16-18. These have resulted in the identification of various antagonists of Aβ self-assembly, including natural products such as polyphenols19,20 and resveratrol,21 dyes,22,23 cucurbit[7]uril,24 curcumin,25 protein antibodies,26-30 molecular chaperones,31-35 β-sheet mimetics,36-38 N-methylated peptides,39 cyclic-KLVFF and its analogues,40-41 ligand D-737 and its derivatives,42 molecular tweezers (MTs),43 β-cyclodextrins,44 cyclic α-peptides,45 trimeric aminopyrazole derivatives,46 rationally designed foldamers.47,16 Most of these efforts have involved inhibiting/delaying Aβ fibrillation or disintegrating Aβ fibers. However, in recent years, oligomer structures have been established as 3 ACS Paragon Plus Environment


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the key molecular species associated with the onset of AD4,15. In a different approach, ligands were developed to stabilize Aβ in an α-helical48-50 or a β-hairpin conformation51 (Figure 1b, c). The strategy was very effective as the stabilized non de novo Aβ structures did not mediate cytotoxicity or facilitate aggregation. Recently, we have designed a series of α-helix mimetics that act by constraining the Aβ peptide into a helical structure thereby preventing its oligomerization and fibrillation (Figure 1a, d). Alpha-helix mimetics are structured scaffolds that harbor properties of an α-helix as they present surface functionalities in a well-defined order to mimic the side chain residues of the helix surface at positions i, i+3/i+4, and i+752-54. Alpha-helix mimetics have been shown to disrupt various disease relevant protein-protein interactions55-62. Here, we present a study of the effects of these α-helix mimetics on Aβ mediated toxicity in mouse neuroblastoma (N2a) cells. The study illuminates the role of Aβ oligomerization in inducing toxicity and demonstrates the targeting of Aβ by two oligopyridyalmides equipped with contrasting functional groups. The oligopyridylamides were very effective in inhibiting fibrillation and ameliorating Aβ mediated cytotoxicity in N2a cells. NMR and the chemical nature of the oligopyridylamides suggests that they are potentially binding to two different subdomains of Aβ. This is useful from a mechanistic and therapeutic viewpoint as it provides important insights into the role of different domains in Aβ self-assembly processes and the desired chemical nature for efficient antagonists of Aβ toxic functions. More importantly, the study underscores a strategy for dual recognition as an approach towards more potent inhibitors of Aβ aggregation and cytotoxicity.


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RESULTS AND DISCUSSION We have recently reported the design and synthesis of a library of oligopyridylamides to inhibit the aggregation of the Aβ peptide.49 From the library, a tripyridylamide, ADH-41 (Figure 1a, d) was found to be one of the most potent antagonists of Aβ fibrillation.49 Extensive in vitro characterization of the interaction of ADH-41 with Aβ was carried out using a battery of techniques, including immunoassays, spectroscopy (NMR and CD), ITC, and fluorescence titration.49 These studies revealed that ADH-41 effectively inhibited the oligomerization, and primary and secondary nucleation mediated fibrillation of Aβ. Here, we have assessed the effect of ADH-41 and its analogs on Aβ mediated cytotoxicity in neuroblastoma cells. Oligopyridylamides inhibit Aβ oligomerization, fibrillation, and cytotoxicity The self-assembly and oligomerization of Aβ are associated with the pathology of AD and ADH41 potently inhibits these processes. Therefore, we investigated the effects of ADH-41 on Aβ mediated toxicity. The cell-based experiments were conducted using mouse neuroblastoma cells (N2a), and cell viability was quantified using the CellTiter 96 Aqueous One Solution (MTS) assay. Treatment with 5 µΜ Aβ42 reduced the viability of N2a cells to 73±3% and 48±2% after 24 and 72 h, respectively (Figure 2a and Figure S1). At an equimolar ratio of ADH-41, the cell viability increased to 99±2% and 98±2% at 24 h and 72 h, respectively (Figure 2a, Figure S1). Importantly, ADH-41 was equally effective in protecting cells from Aβ42 toxicity at substoichiometric ratios and rescues cytotoxicity in a dose dependent manner with an IC50 of 1.4±0.1 µM (Figure 2a, b). ADH-41 alone was not toxic to N2a cells under the conditions used for the cell viability assays (Figure S2). The antagonist activity of ADH-41 towards Aβ42 mediated cytotoxicity and aggregation is dependent on the side chain functionalities projected from the surface of the oligopyridylamide. A structure activity relationship study was employed where analogs of ADH-41 were designed by varying the hydrophobicity of the side chains (Figure 2c, d). The rank order for the antagonist activity of the oligopyridylamides is ADH41>ADH46>ADM-40≥ADM-45A for Aβ42 mediated cytotoxicity and aggregation (Figure 2d), which follows the order of hydrophobicity of the molecules. Thus, a decrease in hydrophobicity is detrimental to the antagonist activity of the oligopyridylamides against Aβ42 mediated cytotoxicity. The spatial arrangement of the surface 5 ACS Paragon Plus Environment


functionalities of oligopyridylamides is also essential for their antagonist activity. Compounds ADH-41 and ADH-37 are similar in chemical composition, but differ in the spatial arrangement of their surface functionalities (Figure 2e). In marked contrast to ADH-41, ADH-37 did not have a noticeable effect on the cytotoxicity incurred in N2a cells by Aβ42 (Figure 2f). ADH-37 was also a weak inhibitor of Aβ42 aggregation as it only decreases the ThT fluorescence intensity of the amyloid reaction from 100% to 74% (Figure 2f). Clearly, the functionalities and their spatial location projected on the surface of the oligopyridylamides are essential in exerting antagonist activities. ADH-41 exhibits specific interactions with Aβ42. To assess the binding specificity of the oligopyridylamides toward their target protein, the antagonist activity of ADH-41 was tested against Aβ42 and islet amyloid polypeptide (IAPP) mediated cytotoxicity and aggregation. IAPP is a hormonal peptide co-secreted with insulin by pancreatic β-cells, and its misfolding is associated with the pathology of type 2 diabetes63. IAPP and Aβ42 share ~50% sequence similarity64,65. Notably, the Aβ(15-21) and Aβ(26-32) sequences share a high degree of sequence commonality with the IAPP(10-16) and IAPP(21-27) domains, respectively, which have also been proposed as core nucleation sites for amyloidogenesis (Figure 2g)64,65. These similarities likely account for the observation that many Aβ antagonists also inhibit IAPP amyloid formation and vice versa. Although ADH-41 completely inhibited Aβ42 mediated toxicity and fibrillation, it did not have any effect on IAPP fibrillation or toxicity in rat INS-1 cells (Figure 2h). Taken together, these studies suggest that the binding interaction between ADH-41 and Aβ is protein specific and sensitive to the functionalities presented on the surface of the inhibitor. From the oligopyridylamide library screen, a second set of molecules was identified as potent inhibitors of Aβ aggregation and, in contrast to ADH-41, they are negatively charged. Among anionic oligopyridylamides, ADH-31, a dianionic tetrapyridylamide, was the most effective antagonist of Aβ aggregation as measured by ThT-based amyloid kinetic assays66 (Figure 3a). The kinetic profile of 5 µM Aβ42 showed a sigmoidal response, which yielded a t50 (time required to reach 50% ThT fluorescence) of 2.1±0.2 h (Figure 3b, Figure S3). The aggregation of Aβ42 was wholly suppressed, with very little formation of ThT positive fibers, in the presence of ADH-31 at an equimolar ratio (Figure 3b, c). Immunoassays and TEM image analysis revealed that ADH-31 inhibited the formation of the neurotoxic Aβ oligomers. TEM images showed that 6 ACS Paragon Plus Environment

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5 µM Aβ42 forms fibers in 12 h (Figure 3d); however, no fibers were detected in the presence of ADH-31 after 12 h at an equimolar ratio (Figure 3e). An ELISA assay67,68 was employed to assess the effect of ADH-31 on the oligomerization of Aβ42 (Figure 3f). Samples of 2 µM Aβ42 were incubated in the absence and presence of ADH-31 at an equimolar ratio for 0 h, 3 h and 6 h and then detected using an Aβ oligomer-specific monoclonal antibody (OMAB)67,68 (Figure 3f). The absorbance increased gradually from 0 to 6 h indicating an increase in the amount of soluble oligomers of Aβ42. In marked contrast, the absorbance of the ADH-31-Aβ42 complex was significantly lower at all time points (Figure 3f). An orthogonal dot blot assay was utilized to examine the effect of ADH-31 on Aβ oligomer formation. Briefly, 2 µM Aβ42 was incubated in the absence and presence of ADH-31 at an equimolar ratio for various durations, and the samples were applied to a nitrocellulose membrane and detected using a polyclonal antibody (A11) specific for Aβ oligomers (Figure 3g). A time-dependent increase in the amount of the A11sensitive Aβ42 oligomeric structures was reflected in the progressive enhancement in the chemiluminescence intensity of the dots, which reached a maximum intensity around 6 h (Figure 3g). The intensity decreased after 12 h, due to formation of fibers that are not sensitive to the A11 antibody. In the presence of ADH-31 at an equimolar ratio, under matched conditions, weak intensities of the dots were observed during the whole time course of the amyloid reaction (Figure 3g). Results from the dot blot assay strongly corroborate the ELISA assay and indicate that ADH-31 inhibits Aβ42 oligomerization. The antagonist activity of ADH-31 towards Aβ fibrillation could be attributed to stabilization of a secondary structure in Aβ. The far UV-CD spectrum of a sample of 20 µM Aβ42 transitioned from that of a random coil to a β-sheet conformation over 24 h (Figure 3h). However, no β-sheet formation was observed in the presence of ADH-31. Instead, Aβ42 adopts an α-helical conformation in the presence of ADH-31 at an equimolar ratio, characterized by two minima at ~208 nm and ~222 nm, which remained stable even after 24 h (Figure 3h). We also employed 2D HSQC NMR to investigate the structural changes in Aβ induced by ADH31. To minimize the aggregation and precipitation of the complex, the concentrations of Aβ42 and ADH-31 were restricted to 40 and 80 µM, respectively. The assignments for chemical shifts of Aβ42 residues were carried out using published reports69,70. A perturbation was observed in the structure of Aβ42 in the presence of ADH-31 reflected by the change in the chemical shifts 7 ACS Paragon Plus Environment


related to various residues (Figure 3i). The highest change in the chemical shifts was localized to residues spanning from Glu11 to Phe20 (Figure 3i, j). The data indicate that this region is a potential binding site of ADH-31. This suggests that the two negatively charged carboxylate functional groups of ADH-31 interact and form salt bridges with the positively charged domain of Aβ42 (His13-Lys16), while the hydrophobic side chains of ADH-31 stabilize the hydrophobic domain of Aβ42 (Ile17 to Phe20). CD data also suggest a conformation transition from random coil to an α-helix in the presence of ADH-31. We have shown that Aβ40 has the tendency to undergo a transition from a weakly folded state to an α-helix conformation from residues 13-24 and 28-36 in the presence of a cationic oligopyridylamide49. However, binding site of the cationic oligopyridylamide differs from that of the anionic oligopyridylamide. To our knowledge, this is the first report of two molecules with a similar scaffold interacting with different domains of Aβ and effectively inhibiting aggregation and oligomerization. ADH-31 rescues Aβ42 induced cytotoxicity in N2a cells. The viability of N2a cells decreases to 44±1% upon exposure to 5 µM Aβ42, but was rescued to 97%±1% when ADH-31 was added at an equimolar ratio (Figure 3i), with a dose dependent profile and an IC50 of 2.5±0.1 µM (Figure 3i, j). In contrast to ADH-41, ADH-31 was very effective in inhibiting IAPP mediated fibrillation and cytotoxicity. ThT-based amyloid assay for the aggregation of IAPP yielded a t50 of 3.5±0.3 h (Figure S5). In the presence of ADH-31 at an equimolar ratio, IAPP aggregation was completely suppressed (Figure 3k), and IAPP induced toxicity in rat insulinoma cells (INS1) was rescued from 65±6% to 98±4% (Figure 3k). We have previously reported that certain negatively charged oligopyridylamides inhibit IAPP mediated toxicity and fibrillation59-62. The binding site for the negatively charged oligopyridylamides on IAPP was suggested to involve residues R11 to H18, which is a common domain of IAPP and Aβ and linked to amyloidogenesis (Figure 2g, orange line). From NMR, ThT amyloid and cytotoxicity assays we hypothesize that the binding site of ADH-31 is in the vicinity of this common domain of Aβ and IAPP as it contains dominant positively charged (His13, His14, and Lys16) and hydrophobic (Leu17, Val18, Phe19, and Phe20) regions that complement the surface functionalities of ADH-31. “Since both the oligopyridylamides target different domains of Aβ, we sought to investigate whether ADH-31 and ADH-41 can work in tandem against Aβ aggregation. ThT-based amyloid and ELISA assays were utilized to probe the cooperative antagonist activity of the 8 ACS Paragon Plus Environment

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oligopyridylamides against Aβ aggregation (Figure S6). In the presence of either of the oligopyridylamides alone (ADH-31 or ADH-41), the fibril mass of Aβ42 was reduced from 100 % to ~20% (Figure S6a) at a stoichiometric ratio of 1:0.1 (Aβ42:oligopyridylamide). No additional decrease in the fibril mass was observed when the amyloid reaction of Aβ42 was treated with a combination of ADH-31 and ADH-41 at a stoichiometric ratio of 1:0.1 [Aβ42:ADH-31 (0.05)+ADH-41 (0.05)] as the fibril mass decreased from 100% to ~20%. An ELISA assay was also employed to assess the cooperative effect of ADH-31 and ADH-41 on the oligomerization of Aβ42 (Figure S6b). Samples of 2 µM Aβ42 were incubated in the absence and presence of ADH-31 or ADH-41 alone for 0 h, 3 h and 6 h and then detected using an Aβ oligomer-specific monoclonal antibody (OMAB) (Figure S6b). The absorbance of Aβ42 alone increased gradually from 0 to 6 h indicating an increase in the amount of soluble oligomers. In marked contrast, the absorbance of the ADH-31-Aβ42 (or ADH-41-Aβ42) complex was significantly lower at 3 h and 6 h (Figure S6b) at a stoichiometric ratio of 1:0.5 (Aβ42:oligopyridylamide, 2 µM: 1 µM). Under matched conditions, in the presence of a combination of ADH-31 and ADH-41 (0.5 µM each), the inhibition of Aβ42 oligomerization was comparable to the individual ligand (ADH-31 or ADH-41 alone), reflected by the absorbance values (Figure S6b). The assays were conducted at lower concentrations to monitor the cooperative effect of the oligopyridylamides because at higher concentrations their antagonist activity was complete against Aβ42 oligomerization or fibrillation. The HSQC-NMR data suggest that the both the molecules, ADH-31 and ADH-41 share a common binding motif on Aβ, which is the hydrophobic region spanning from Ile17 to Phe20. Therefore, we speculate that instead of showing a cooperative effect, both the molecules will compete in anti-amyloidogenic activity against Aβ, as we have observed in the amyloid kinetic and ELISA assays. The data precludes the possibility of positive cooperativity of the oligopyridylamides in inhibiting Aβ42 fibrillation.” Oligopyridylamides

eliminate

preformed

oligomers

and

suppress

seed-catalyzed

aggregation of Aβ. Aβ oligomers have been proposed as the primary neurotoxins associated with the pathogenesis of AD4; therefore, we sought to assess the effects of the most potent oligopyridylamides, (ADH-31 and ADH-41) on preformed oligomers and seed-catalyzed processes. ADH-31 and ADH-41 were added during the growth phase of an Aβ42 (5 µΜ) amyloid reaction (Figure 6a). Both 9 ACS Paragon Plus Environment


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oligopyridylamides were effective in inhibiting Aβ42 when added during the lag period (black arrow, 2 h, Figure 4a). Interestingly, ADH-31 was more effective in inhibiting the growth phase of Aβ42 amyloidogenesis than ADH-41. Image analysis of Aβ42 fibrillation supports the ThT amyloid assay results. TEM images of Aβ42 after 2 h revealed a nearly homogenous distribution of round particles confirming the formation of Aβ42 oligomers (Figure 4b, and zoom in region), as has been reported by others71,72. No formation of Aβ42 oligomers was observed when ADH-31 or ADH-41 were added to the preformed Aβ42 oligomers at an equimolar ratio (Figure 4c, d). ELISA assay confirms the formation of Aβ42 (2 µΜ) oligomers after 3 h and 6 h reflected by a gradual increase in the absorbance of the solution (Figure 4e). In marked contrast, no evidence of oligomers was observed when the oligopyridylamides were added at an equimolar ratio at 3 h and 6 h after the start of the Aβ42 amyloid reaction (Figure 4e). It is interesting to note that ADH31 was more effective than ADH-41 in inhibiting Aβ42 oligomerization under matched conditions (Figure 4e). The oligopyridylamides were also tested for their effect on the cytotoxicity induced by the preformed oligomers in N2a cells. Aβ42 (5 µΜ) was incubated in buffer for 0, 1, 2, and 3 h and then introduced to the cells, which reduced the cell viability to 48%, 46%, 57%, and 62%, respectively (Figure 5f). Both oligopyridylamides were effective at rescuing Aβ42 mediated toxicity when incubated with the preformed Aβ42 oligomers. Whereas ADH-41 was a better antagonist than ADH-31 in rescuing toxicity when they were preincubated with Aβ42 (t=0 h, Figure 4f). , ADH-31 was more effective in inhibiting toxicity of the preformed oligomers. ADH-41 was able to increase cell viability from 46% to 72%, 57% to 68%, and 62% to 67% when added to Aβ42 after 1, 2, and 3 h from the start of the amyloid reaction, respectively. Under matched conditions, ADH-31 rescued cell viability from 46% to 90%, 57% to 89%, and 62% to 95% when added to Aβ42 after 1, 2, and 3 h from the start of the amyloid reaction, respectively (Figure 4f). We also compared the effect of both oligopyridylamides on the secondary nucleation processes of Aβ42 aggregation. Preformed fibers (seeds) catalyze Aβ aggregation via secondary nucleation processes and generate the key neurotoxic oligomers of Aβ4273,74. Thus, from a therapeutic point of view, it is important to assess the effect of oligopyridylamides on Aβ42 aggregation processes predominantly driven by secondary nucleation. The aggregation of 5 µM Aβ42 bypasses the lag phase in the presence of preformed fibers (10% v/v) and decreases the t50 10 ACS Paragon Plus Environment

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from 2.7±0.2 h to 0.3±0.1 h (Figure 4g). In the presence of ADH-31 and ADH-41, the seed catalyzed fibrillation of 5 µΜ Aβ42 was completely suppressed at an equimolar ratio (Figure 4g, h) as reflected in the small change in the fluorescence intensity in comparison to the control reaction (Aβ42+seeds, Figure 4g, h). The effect of the oligopyridylamides on seed-induced Aβ42 cytotoxicity in N2a cells was also assessed. The cell viability decreased from 47% to 31% in the presence of 5 µΜ Aβ42 and 5 µΜ Aβ42+seeds (10% v/v), respectively (Figure 5h), an observation consistent with earlier reported work73. Under identical conditions, the cell viability was rescued from 31% to 69% and 63% in the presence of ADH-31 and ADH-41 (5 µΜ each), respectively. There is a possibility that the oligopyridylamides disintegrate Aβ42 fibers and consequently affect secondary nucleation. We conducted a ThT amyloid assay and TEM image analysis to monitor the effect of ADH-31 and ADH-41 on the preformed fibers of Aβ42. No significant change in the ThT fluorescence intensity was observed for the preformed fibers of Aβ42 in the presence of ADH-31 and ADH-41 at an equimolar ratio or at higher doses (Figure S7a). The ThT-based results were also validated by TEM images (Figure S7b-S7d). The study suggests that ADH-31 and ADH-41 inhibit Aβ42 fibrillation either by acting on Aβ42 oligomers or the monomers. ADH-41 is a better antagonist than ADH-31 of Aβ42 primary nucleation processes, including aggregation and cytotoxicity. In contrast, ADH-31 is a better antagonist than ADH-41 of Aβ oligomerization and secondary nucleation processes. These results suggest that ADH-31 interacts with an Aβ subdomain that may be required to initiate the secondary nucleation processes. ADH41 is a moderate inhibitor of secondary nucleation and oligomerization, indicating that the binding domain of ADH-41 (Ile17 to Glu22) on Aβ is only partially associated with the secondary nucleation processes. It has been suggested that during Aβ fibrillation, the N-terminal β-strand extends from Glu11 to Glu22 and Ile31 to Ala41, with a turn stabilized by a salt bridge between Asp23 and Lys2872. We hypothesize that the dianionic ADH-31 interacts with the positively charged (His13 to Lys16) and hydrophobic (Leu17 to Phe20) domains of Aβ and blocks the secondary nucleation processes, whereas ADH-41 only interacts with the hydrophobic domain (Leu17 to Phe20) of Aβ and, therefore, only partially affects these processes. Based on our results, we propose a couple of possible mechanisms for the mode of action of the oligopyridylamides against Aβ amyloidogenesis. When the oligopyridylamides are added to the monomeric form of Aβ peptide (at time t=0), the oligopyridylamides presumably sequester and 11 ACS Paragon Plus Environment


modulate Aβ conformation into an α-helical state. The oligopyridylamide-mediated conformational switch (helical state) in Aβ leads to off-pathway structures, which are non-toxic and amyloid incompetent. However, when the oligopyridylamides are added to preformed oligomers of Aβ, they disrupt the oligomers and inhibit the associated toxic functions. It should be noted that while the 2D NMR, cytotoxicity, and ELISA assays do not provide direct evidence, it is possible that the oligpyridylamides interact with Aβ oligomers and disintegrate them. Another possibility is that the oligopyridylamides sequester Aβ monomer which are in rapid exchange with Aβ oligomers (in the heterogeneous mixture) and consequently decrease the concentration of Aβ oligomers in the solution. Oligopyridylamides retain antagonist activity in cellular milieu Despite the identification of several small molecule ligands with promising anti-amyloid properties in solution studies, whether their activity is retained in extra- and intra-cellular milieu is often unknown16. There are a number of limitations associated with the investigation of small molecule-Aβ interaction in cellular milieu: (1) confocal fluorescence imaging is one of the most widely used techniques to probe binding interactions in cellular media, which requires fluorescent tags on the molecules. Ligands identified as inhibitors of Aβ aggregation are often difficult to modify synthetically in order to append a fluorescent tag; (2) the fluorescent tag might affect the binding of the ligand to Aβ; (3) the molecule might not be stable in the cellular milieu (e.g. due to proteolytic cleavage) and thus does not retain the binding with Aβ; (4) the binding interaction between the ligand and Aβ might be weaker in the cellular environment. To address these questions, we have utilized confocal imaging to monitor the interactions between Aβ42 and ADH-41 in cellular milieu. ADH-41 and Aβ42 were labeled with fluorescein (ADH41F) and Texas-Red (AβTR), respectively. Confocal fluorescence imaging confirmed the cellular uptake of both Aβ42 peptide (4 µM Aβ42 + 1 µM AβTR) and ADH-41 (4 µM ADH-41 + 1 µM ADH-41F) by N2a cells within 24 h of incubation (Figure 5a). The observation that Aβ42 is readily taken by the cells is consistent with reports that suggest Aβ partially exerts its neurotoxic effects via disruption of mitochondrial function75,76. The cell permeability of ADH-41 is not surprising as it is a di-cation with a calculated partition coefficient (log P) of 1.8 (theoretical = 1.4). The cell viability decreased from 100% to 65% when N2a cells were incubated with 4 µM Aβ42 (+1 µM AβTR) for 24 h (Figure 5a). There was no inherent toxicity associated with ADH-41 12 ACS Paragon Plus Environment

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as the cell viability was 98% in the presence of ADH-41 alone (4 µM ADH-41 + 1 µM ADH41F) (Figure 5b). To monitor the effect of ADH-41 on Aβ42-mediated cytotoxicity, a solution of Aβ42 (4 µM Aβ42 + 1 µM AβTR) was premixed with ADH-41 (4 µM ADH-41 + 1 µM ADH-41F) and introduced to the cells. Under matched conditions, the cell viability was completely restored in the presence of ADH-41 at an equimolar ratio (Aβ42:ADH-41 1:1, 5 µM each). The Aβ42ADH-41 complex was readily taken up by N2a cells, indicating that ADH-41 did not affect the cell permeability of Aβ42 (Figure 5c). Moreover, the observation that the Aβ42–ADH-41 complex permeates through the cell membrane and localizes intracellularly indicates a tight binding event which is not weakened by the extra-and intra-cellular milieu (Figure 5c). We further sought to compare the cell permeability and intracellular localization of the Aβ42-ADH-41 complex with that of Aβ42 or ADH-41 alone (Figure S8). A solution of Aβ42 (4 µM Aβ42 + 1 µM AβTR) was premixed with 5 µM unlabeled ADH-41 and introduced to the cells (Figure S8a). As observed with the peptide and compound alone, the labeled Aβ42-unlabled ADH-41 complex was partially localized at the mitochondria. Similarly, the complex of 5 µM unlabeled Aβ42 with ADH-41 (4 µM ADH-41 + 1 µM ADH-41F) partially colocalized with mitochondria (Figure S8b). The rescue of cytotoxicity in N2a cells is a consequence of the colocalization of protein and small molecule. We hypothesize that ADH-41 rescues toxicity by modulating the toxic structures of Aβ42 into non-toxic off-pathway structures without compromising the peptide’s cellpermeability. One of the likely contributors to cytotoxicity has been shown to be the intracellular accumulation of Aβ4277-79. We therefore sought to investigate the rescue of intracellular cytotoxicity induced by Aβ42. N2a cells were treated with a toxic dose of 5 µM Aβ42 for 24 h which decreased the cell viability from 100±2% to 64±1% (Figure 5d). A solution of 5 µM ADH-41 was added to N2a cells pretreated with with 5 µM Aβ42 for 12 h, and the cytotoxicity was measured after 12 h (total 24 h). The cell viability was increased from 64±1% to 93±2% when ADH-41 was added 12 h after the addition of Aβ42 to N2a cells (Figure 5d). ADH-41 was very effective even at higher toxic insult induced by Aβ42. The cell viability is attenuated to 48±2% for N2a cells when incubated with 5 µM Aβ42 for 72 h. The delayed addition of ADH-41 after 24 and 48 h of exposure to Aβ42 led to the restoration of the cell viability to 79±2 and 102±2, respectively (Figure S9). The rescue of toxicity is associated with the colocalization of ADH-41 with Aβ42 13 ACS Paragon Plus Environment


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(Figure 5d). ADH-41 rescues N2a cells from Aβ42 mediated cytotoxicity in both scenarios, either coincubated with Aβ42 or added after a delay of 12-48 h. The data suggest that the cytotoxicity induced by Aβ occurs partially due to intracellular mechanisms. ADH-41 potentially disaggregates the intracellular neurotoxic oligomers of Aβ and rescues cytotoxicity, a result that is corroborated by our immunoassays and ThT amyloid assays. Overall, ADH-41 binds and modulates the toxic structures of Aβ42 into non-toxic conformations with a remarkable selectivity. A proposed model for the binding mode of oligopyridylamides to Aβ Based on the solution and cellular data, we propose a model for the binding interaction of the oligopyridylamides with various facets of Aβ conformation (Figure 6). We expect that the model will aid in developing a better understanding of Aβ-small molecule interactions for future generation of antagonists of toxic functions of the peptide. Both oligopyridylamides, cationic (ADH-41) and anionic (ADH-31), inhibit primary nucleation by inducing an oligomerizationand fibrillation-incompetent α-helix conformation in Aβ. ADH-31 is more effective in disrupting preformed toxic oligomers and inhibiting seed catalyzed secondary nucleation. NMR suggests that ADH-31 interacts with residues span from His13 to Phe20. The conformation of the monomers in Aβ oligomers is such that residues from His13 to Phe20 are partially structured into β-strands and these residues are partially exposed to the solvent (Figure 6). Therefore, ADH-31 potentially caps these residues using salt bridge and hydrophobic interactions and inhibits further oligomerization of Aβ. ADH-41 interacts with residues from Leu17 to Asp23, which are completely structured into a β-strand; therefore, ADH-41 does not have access to its binding domain in the oligomer as it is completely buried within the β-sheet structure. ADH-41 is able to inhibit Aβ oligomerization, albeit with moderate activity, probably by interacting with a small population of unstructured Aβ present in the solution.


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CONCLUSIONS Aberrant protein self-assembly is the hallmark of an array of amyloid-related debilitating medical conditions, including AD, PD, Huntington’s disease, T2D, cancer, and HIV-AIDS.1-11 AD, the most common type of dementia, is a rapidly growing social and economic burden.3,12 The limitations of current therapies are attributed to poor diagnosis and weak mechanistic understanding of the factors responsible for the onset and progression of AD. The Aβ protein is a precursor whose self-assembly processes are considered to be the causal agent responsible for the onset and progression of AD.4,15 The discovery of therapeutic agents for AD is dwarfed by the highly elusive, heterogeneous, and dynamic nature of the toxic Aβ oligomers associated with AD. In the past few years, a panoply of small molecule antagonists of Aβ aggregation have been reported, which target various facets of Aβ peptide and inhibit its fibrillation.19-50 Some of these molecules were very effective agents in inhibiting Aβ-mediated toxic functions; however, they suffered from a number of limitations which preclude them from being considered as potential lead therapeutics for AD16: (1) most ligands harbor planar aromatic character and the main mode of their antagonist activity is attributed to hydrophobic interactions, which make them nonspecific antagonists of Aβ aggregation;81-84 (2) some ligands self-aggregate and their mode of action is the sequestration of Aβ, which is non-specific;81-84 (3) some ligands delay Aβ aggregation and therefore enhance the population of toxic oligomers; (4) the antagonist activity of most of the ligands against Aβ aggregation within complex cellular milieu has been demonstrated, which is a key prerequisite for ligands to be potential therapeutic agents16.

In the present study we demonstrate a strategy for the design of oligopyridylamide-based compounds to modulate the structure and toxic functions of Aβ, a peptide whose aggregation is associated with AD. The strategy has been adapted from an already established protocol where affibodies51 and peptoids48 have been employed to manipulate the structure and toxic functions of Aβ. These approaches were novel as they focused on targeting the pre-amyloid structures rather than the amyloid fibers. However, they were limited in their ability to be optimized because the manipulation of side chain residues potentially alters the conformation and consequently compromises the activities of these molecules. In contrast, the chemical properties 15 ACS Paragon Plus Environment


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of the oligopyridylamide scaffold can be modified using a convenient synthetic approach without perturbing their conformation. We propose that various domains of Aβ can be targeted using the same scaffold but with different chemical functional groups. The antagonist activity of the oligopyridylamides towards Aβ aggregation is sensitive to the spatial arrangement and chemical nature of the functionalities projected from the surface. The oligopyridylamides affect distinct pathological facets of Aβ via disparate mechanisms. We have also shown that an oligopyridylamide, ADH-41, forms a complex with Aβ intracellularly and retains its antagonist activity in cellular milieu. The approach of targeting proteins using structured scaffolds has been employed in a number of areas; however, manipulating the structure and function of amyloidogenic proteins using these protein-like small molecules is a recent development. Using oligopyridylamides, we have outlined a chemical landscape that illuminates a pathway to design potent antagonists of Aβ aggregation. The strategy is versatile owing to the diversity in functional groups that can be utilized on different scaffolds with distinct architectures. Overall, we have devised a simple, yet powerful, approach that can be conveniently manipulated based on the target protein, which harbors potent anti-amyloid properties both in solution and cellular milieu, and functions as a tool to gain insight into the mechanistic details of protein-ligand interactions in living cells. Such understanding will aid in illuminating the toxic pathways of various amyloidogenic proteins. We believe that this approach will have a broader impact as similar structures are sampled by many intrinsically disordered proteins, such as α-synuclein in PD, IAPP in T2D, mutant p53 in cancer, and prostatic acid phosphatase in HIV-AIDS.


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MATERIALS AND METHODS Materials. All peptides used in this study (Aβ40, Aβ42, and IAPP) were purchased at >98% purity from Anaspec (Fremont, CA, USA) and used without further purification.

15

N- labeled

wild-type human Aβ40 was purchased from rPeptide (Bogart, GA, USA) and used without further purification. ThT was purchased from Acros Organics (NJ, USA). The 96-well plates (black, flat bottom) were purchased from Corning Coster (Corning, NY, USA). All chemicals were purchased from commercial suppliers and used without further purification. Silica plates (with UV254, aluminum backed, 200 µm) and silica gel (standard grade, particle size = 40–63 µm, 230-400 mesh) for flash column chromatography were purchased from Sorbent Technologies (Norcross, GA, USA). Dry solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,6-Dichloro-3-nitropyridine, alkyl iodides, alkyl alcohols, anhydrous triethylamine, 2-chloro-1methylpyridinium iodide, tert-butyl bromoacetate, HPLC grade trifluoroacetic acid, and triethylsilane (TES) were purchased from Sigma Aldrich (St. Louis, MO, USA). Peptide Preparation. Aβ40, Aβ42, and IAPP were dissolved in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) (1 mg/mL) and kept at r.t. for 1 h with occasional vortexing to maintain them in monomeric state. Peptides were then aliquoted into small fractions and lyophilized overnight and stored at -80 °C until use. Peptide concentration was checked by dissolving one of the aliquots in water and measuring absorbance at 280 nm. For solution-based assays, lyophilized samples were allowed to equilibrate at r.t. for 20 min. and then dissolved in pure DMSO (Amresco, Solon, OH, USA). The concentration of the stock solution for all the biophysical assays was 0.5-1.0 mM. Synthesis of Oligopyridylamide based α-Helix Mimetics. The synthetic protocol and the characterization of the relevant compounds are presented in detail in the supplementary information. ThT-based Kinetic Assay. Kinetic assays were conducted on a FlexStation 3 Multi-Mode Microplate reader from Molecular Devices (Sunnyvale, CA, USA). Experiments were conducted in triplicate in a 96-well plate with a final volume of 200 µL per well. Every measurement was an average of 50 readings. The aggregation of amyloidogenic peptide (Aβ40, Aβ42, and IAPP) was initiated by addition of peptide from a stock solution (in DMSO, 0.5-1 mM) to phosphate buffer. The final concentration of each peptide was different based on their aggregation (see 17 ACS Paragon Plus Environment


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main text). The stoichiometry of ThT dye for each peptide was 0.5:1 (ThT:peptide). Peptide aggregation were monitored by ThT fluorescence (λex = 445 nm and λem = 485 nm). The blank sample contained everything except peptide. The sample data were processed by subtracting the blank and renormalizing the fluorescence intensity by setting the maximum value to one. Kinetic assays in the presence of small molecules were conducted under the same conditions except that the small molecules were added from a stock solution (1 mM or 10 mM in DMSO) to keep the final concentration of DMSO less than 1.0% (v/v). Small molecules were added to the wells with ThT and buffer and mixed gently with a pipette before adding the peptide. To keep the conditions identical, an equal amount of DMSO was added to the wells with the peptide only reactions. Kinetic profiles were processed using Origin (version 9.1). Kinetic curves were fit using the built-in sigmoidal fit. Each run was fit independently to extract the t50 (time required to reach 50% of the maximum fluorescence intensity). Error bars represent standard deviations from the mean of at least three independent experiments. Seed-Catalyzed Kinetic Assay. Seeds of Aβ40 / Aβ42 were prepared by incubating 200 µM of Aβ40 / Aβ42 in phosphate buffer at r.t. The samples were aged for 48 h and the formation of fibers was confirmed by TEM and ThT before storage at -20 °C until use. For the aggregation of Aβ40, 10% (based on the monomeric Aβ40, v/v) seeds were added along with ThT in phosphate buffer to the 96-well plate. The aggregation was initiated by the addition of monomeric Aβ40 followed by gentle mixing. The process was similar in the case of Aβ42 except that the seed concentration was 5% (based on the monomeric Aβ42, v/v). Transmission Electron Microscopy (TEM) Analysis. Aβ42 (5 µM) was incubated in phosphate buffer in the absence and presence of oligopyridyalmides at various time intervals and stoichiometric ratios. Aliquots of these samples were then applied to glow-discharged carboncoated 300-mesh copper grids for 2 min and dried. Grids were negatively stained with uranyl acetate (2%, w/v) and dried. Micrographs of grids were examined on a Phillips CM12 Cryoelectron Microscope equipped with Gatan 4k × 2.7k CCD camera at 120-kV accelerating voltage. 18 ACS Paragon Plus Environment

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Circular Dichroism (CD) Spectroscopy. A freshly prepared stock solution of Aβ42 (500 µM) was diluted to 15 µM in phosphate buffer for CD measurements. The spectra of Aβ42 were recorded at 0.5 nm intervals from 190 to 260 nm with an averaging time of 10 sec. and an average of three repeats on a Aviv Stopped Flow CD Spectropolarimeter (Model 202SF). Spectra were recorded using the identical method as described above, except Aβ42 was diluted in the solution of oligopyridyalmides in phosphate buffer at an equimolar ratio. Buffer conditions: 150 mM KCl, 50 mM NaPi, pH 7.4. ELISA. The ELISA was performed according to a previously published method39. A NuncImmuno MaxiSorp plate (Sigma Aldrich, St. Louis, MO, USA) was incubated with 2 µg mL-1 Aβ oligomer-specific antibody (OMAB, Agrisera, Sweden) in PBS buffer overnight at 4 °C (200 µl/ well). Wells were blocked with 5% fat-free milk in PBS buffer with 0.1% Tween 20 (PBST) for 1 h at 4 °C and washed with PBST buffer (× 3). Wells were then treated with Aβ42 samples overnight at 4 °C. Aβ42 samples were prepared by incubating 30 µM Aβ42 in the absence and presence of oligopyridylamides at an equimolar ratio at various time intervals at r.t. Samples were diluted by 1:30 in PBS buffer before adding to a 96-well plate. After adding Aβ42 samples, the wells were washed (× 3) with PBST buffer followed by the addition of 6E10 antibody (1/1000 dilution in 5% nonfat free milk in PBST buffer) for 1 h at r.t. Wells were washed (× 5) with PBST buffer and treated with an anti-mouse HRP-conjugated IgG (1/10,000 dilution in 5% nonfat free milk in PBST buffer). Wells were washed (× 5) with PBST buffer and treated with TMB Peroxidase EIA Substrate Kit (Biorad, Hercules, CA, USA). The plates were developed until the color of the solution turned blue. The reaction was stopped by adding 100 µL of 1N H2SO4 to each well. The color of the solution changed to yellow from blue. The absorbance was recorded at 450 nm on a FlexStation 3 Multi-Mode Microplate reader from Molecular Devices (Sunnyvale, CA, USA). Each sample was repeated in triplicate. Dot Blot Assay. Samples of 2 µM Aβ42 were incubated at various time intervals (see main text for detail) in the absence and presence of oligopyridylamide at an equimolar ratio. The samples were then applied to a nitrocellulose membrane and dried at r.t. for 1 h or overnight at 4 °C. The membranes were then blocked with 5% nonfat milk in Tris buffer (20 mM Tris, pH 7.4) for 1 h at r.t. The nitrocellulose membranes were then washed (× 3) with 20 mM Tris, pH 7.4 supplemented with 0.01% Tween-20 (TBST) and incubated with polyclonal A11 antibody 19 ACS Paragon Plus Environment


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(1/1000 dilution in 5% nonfat milk in TBST, Life Technologies Corp., Grand Island, NY, USA) overnight at 4 °C. Samples were then washed (× 3) with TBST buffer and incubated with horseradish peroxidase (HRP) conjugated anti-rabbit IgG (1/500 dilution in 5% nonfat free milk in TBST) at r.t. for 1 h. The dot blots were then washed with TBST buffer (× 3), developed using the ECL reagent kit (Amersham, Piscataway, NJ, USA), and imaged using a Typhoon FLA 9000 instrument (GE Healthcare Life Sciences, Pittsburgh, PA, USA) using chemiluminescence settings. A similar experiment was repeated using 6E10 antibody (1/1000 dilution in 5% nonfat free milk in TBST, Biolegend, San Diego, CA, USA) for comparison. Two-dimensional HSQC NMR Spectroscopy. Two-dimensional

1

H-15N HSQC NMR

experiments were performed on a 600 MHz Bruker instrument. Uniformly labeled 15N-Aβ40 was purchased from rpeptide (Bogart, GA, USA). The stock solution of 1 mg mL-1 was dissolved in 10 mM NaOH, aliquoted into small fractions, lyophilized, and stored at -80 °C until use. The concentration of each aliquot was determined spectroscopically at 280 nm using an extinction coefficient of 5690 M-1cm-1. Experiments were carried out in 20 mM NaPi, pH 7.4 by maintaining a solution ratio of 90:10 (H2O:D2O) according to a previously published method to ensure that Aβ40 is in the monomeric state. A stock solution of 20 mM oligopyridylamide was prepared in DMSO-d6 (pure, HPLC grade). For each NMR experiment, a freshly prepared aliquot of

15

N-Aβ40 was used to avoid potential complication from amyloid formation. NMR

spectra were recorded using fresh sample of 60 µM

15

N-Aβ40 in 20 mM NaPi, pD 7.4 in the

absence and presence of oligopyridylamide at a stoichiometric ratio of 1:2 (Aβ40: oligopyridylamide) at 7 °C on a 600 MHz Bruker instrument equipped with a triple resonance HCN cryoprobe. The maximum dilution of the

15

N-Aβ40 sample with the titration of

oligopyridylamide was

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