Amyloid-Î² Peptide Targeting Peptidomimetics for Prevention of

Subscriber access provided by OPEN UNIV OF HONG KONG

Article

Amyloid-# Peptide Targeting Peptidomimetics for Prevention of Neurotoxicity Dmytro Honcharenko, Alok Juneja, Firoz Roshan, Jyotirmoy Maity, Lorena Galan-Acosta, Henrik Biverstal, Erik Hjort, Jan Johansson, Andre Fisahn, Lennart Nilsson, and Roger Stromberg ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00485 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 26, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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

ACS Chemical Neuroscience

Amyloid-β Peptide Targeting Peptidomimetics for Prevention of Neurotoxicity

Dmytro Honcharenkoa,*, Alok Junejaa, Firoz Roshanb, Jyotirmoy Maitya, Lorena GalánAcostac, Henrik Biverstålc,d, Erik Hjorthc, Jan Johanssonc, André Fisahnb, Lennart Nilssona, and Roger Strömberga,* a Department b

of Biosciences and Nutrition, Karolinska Institutet, 14183 Huddinge, Sweden

Neuronal Oscillations Laboratory; Neurogeriatrics Division; Center for Alzheimer Research

Department of Neurobiology, Care Sciences and Society; Karolinska Institutet, 17164 Solna, Sweden c

Division for Neurogeriatrics, Department of Neurobiology, Care Sciences and Society,

Karolinska Institutet, 14183 Stockholm, Sweden d

Department of Physical Organic Chemistry, Latvian Institute of Organic Synthesis, Riga

LV-1006, Latvia

1 ACS Paragon Plus Environment

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

Page 2 of 39

Abstract A new generation of ligands designed to interact with the α-helix/-strand discordant region of the amyloid- peptide (Aβ) and to counteract its oligomerization is presented. These ligands are designed to interact with and stabilize the Aβ central helix (residues 13-26) in an α-helical conformation with increased interaction by combining properties of several firstgeneration ligands. The new peptide-like ligands aim at extended hydrophobic and polar contacts across the central part of the Aβ, i.e. “clamping” the target. Molecular dynamics (MD) simulations of the stability of the Aβ central helix in the presence of a set of secondgeneration ligands were performed and revealed further stabilization of the Aβ α-helical conformation, with larger number of polar and non-polar contacts between ligand and Aβ, compared to first-generation ligands. The synthesis of selected novel Aβ–targeting ligands was performed in solution via an active ester coupling approach or on solid-phase using an Fmoc chemistry protocol. This included incorporation of aliphatic hydrocarbon moieties, a branched triamino acid with an aliphatic hydrocarbon tail and an amino acid with a 4′-N,Ndimethylamino-1,8-naphthalimido group in the side chain. The ability of the ligands to reduce Aβ1–42 neurotoxicity was evaluated by gamma oscillation experiments in hippocampal slice preparations. The “clamping” second-generation ligands were found to be effective antineurotoxicity agents and strongly prevented the degradation of gamma oscillations by physiological concentration of monomeric Aβ1–42 at a stoichiometric ratio.

Keywords: Alzheimer’s disease, amyloid-β peptide, synthetic ligands, α-helical conformation, molecular dynamics, gamma oscillations


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


Introduction Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia, accounting for about 60% of all cases.1,

2

AD belongs to the group of

conformational diseases, which arise from protein misfolding and aggregation,3,

4

and is

associated with cerebral extracellular deposits, called plaques, which are mainly composed of amyloid fibrils formed from the amyloid- peptide (A).5, 6 According to the amyloid cascade hypothesis the aggregation of soluble Aβ and formation of insoluble fibrillar A in plaques is the central pathogenic event in AD.7-9 However, numerous studies indicate that the prefibrillar soluble oligomers, which are produced early in the aggregation pathway, possess a toxic nature.10-12 Currently available treatments of AD are only symptomatic, do not stop the neurodegenerative process and only delays the development of symptoms.13 However, substantial efforts are being focused on the development of methods to prevent the occurrence or progression of AD.14 Several strategies13, 15 have been proposed to achieve inhibition of fibrillization/oligomerization of Aβ which also include targeting of Aβ in an elongated, βstrand-like conformation with a range of small organic molecules or peptide-based inhibitors.16 The main problems of these approaches are lack of specificity and potential accumulation of cytotoxic soluble, non-fibrillar A aggregates.17 Other strategies such as inhibition of the secretase enzymes18, 19 and passive or active immunization20 may have their limitations through interference with important physiological functions or development of undesirable side effects.21 The toxic oligomeric species are predominantly formed from monomeric Aβ1-42 involving a fibril-catalyzed secondary nucleation mechanism22 and alteration of this pathway was presumed to be one of the approaches to suppress neurotoxicity of Aβ1-42. The use of the chaperone-like protein domain, BRICHOS, which interferes with secondary nucleation in Aβ1-42 by binding to the surfaces of fibrils and redirecting the aggregation reaction to a pathway involving primary nucleation with fewer cytotoxic oligomers, is an eminent example of this strategy.23-26 Alternatively, formation of Aβ oligomers and fibrils could be delayed or prevented by masking the binding site of monomeric Aβ to inhibit its oligomerization through primary as well as secondary nucleation pathways. Initially, when generated from the amyloid precursor protein (APP), A harbors a discordant α-helix that is composed of residues that strongly favor β-strand formation.27 This region of Aβ (K16LVFFAED23) serves as a binding 3 ACS Paragon Plus Environment


Page 4 of 39

sequence during Aβ polymerization,28, 29 and structural models for amyloid fibrils30-32 support that the residues 17–24 in the discordant helix of Aβ1–42 are crucial for fibril formation.33 It has been shown that the removal of Aβ positions 14–23 prevents fibril formation and their replacement results in the reduction of fibrillization.28, 34, 35 This suggests that stabilization of the discordant α-helix in monomeric Aβ could inhibit its oligomerization by preventing sheet structure formation as well as hampering monomeric Aβ from oligomerization through secondary nucleation. Previously, we have reported on inhibition of Aβ aggregation by means of ligands that are designed to bind and stabilize the 13–26 region of Aβ in an α-helical conformation, in a state similar to its native structure.36-38 Oral administration of these inhibitors in Drosophila melanogaster expressing human Aβ1-42 in the central nervous system resulted in prolonged lifespan, decrease of locomotor dysfunction and reduction of neuronal damage.36 The ligands also strongly reduced the Aβ1-42-induced reduction of gamma oscillations in hippocampal slices, an activity which plays an important role in higher cerebral processes and is known to be considerably reduced in patients diagnosed with AD.39 Our studies revealed that low molecular weight compounds, designed to target and stabilize the discordant α-helix, inhibit Aβ aggregation and prevent accumulation of cytotoxic species. This concept was also supported by molecular dynamics simulation studies that uncover details of the mechanism of unfolding of the Aβ central helix and retardation of the folding in presence of amyloid targeting ligands.40-42 In this study we describe novel second-generation “clamping” ligands designed to enhance the interaction with α-helical Aβ through the extended combination of hydrophobic and polar contacts across the central part of the Aβ in order to reduce its aggregation and associated neurotoxicity. For the preparation of new ligands, either solution or solid-phase synthesis protocols were used. Interactions between the ligands and Aβ and their impact on the helix stability of Aβ were studied by molecular dynamics (MD). The efficiency of secondgeneration ligands to reduce or prevent Aβ1-42 neurotoxicity was evaluated using gamma oscillation experiments in hippocampal slice preparations.

Results and Discussion Design of second-generation ligands First-generation Aβ targeting ligands36 were based on two different classes of compounds, the zwitterionic peptide-like compound Pep1b (Figure 1A) and the fatty acylpolyamine Dec4 ACS Paragon Plus Environment

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


Figure 1. Chemical structures of the first-generation ligands Pep1b (A) and Dec-DETA (B) and second-generation ligand DAEPep (C) designed to bind the α-helical form of the Aβ13–26. The naming and numbering of ligands: carbon (black), nitrogen (blue), and oxygen (red) atoms. The naming and numbering of Aβ13–26 residues are shown in black. Arrows indicate the interactions between Aβ13–26 residues and different groups of the ligands. (D) Molecular graphics of stabilization of α-helical Aβ by second-generation ligand DAEPep. The ligand DAEPep (colored yellow) designed to “straddle” or “clamp” the central helix of the Aβ by formation of hydrophobic contacts in two regions and polar contacts in two regions. The Aβ13–26 is rendered as stick presentations with overlayed ribbon (α-helical secondary structure is shown in red). DETA (Figure 1B). These ligands were designed to interact with two polar regions and one hydrophobic region (e.g., Pep 1b) or with one polar region and another hydrophobic region (e.g., Dec-DETA) in order to stabilize the helical conformation of the central region (residues 13-26; H13HQKLVFFAEDVGS26) of Aβ. The contact maps from simulations of both Aβ– ligand complexes showed distinct distribution of contacts for each Aβ–ligand complex.40, 41 The novel ligands presented here are designed to exploit the contact surfaces of both DecDETA and Pep1b by structurally including elements allowing for this and with the aim to increase interaction between ligand and Aβ surface. The idea is that the ligands would act like a “clamp” for the Aβ by interaction with more regions, i.e., to interact with two hydrophobic regions as well as two or three polar areas. An example of potential interactions of a designed ligand, DAEPep, is shown in Figure 1C. The second-generation ligand DAEPep retains potential cationic interactions with Lys16 and a partially protonated His13 via the carboxylate groups and enhances potential hydrophobic interactions with Leu17, Val18 and Ala21 by addition of hydrocarbon chain. To keep interaction with the negatively charged residues 5 ACS Paragon Plus Environment


Page 6 of 39

Glu22 and Asp23 the structure includes a triamino acid moiety, which mimics the cationic function in Dec-DETA and the positively charged arginine residue in Pep1b. Incorporation of such triamino acids into an Aβ targeting ligand has been shown to be an effective replacement of the arginine in Pep1b.37 The “clamping” or “straddling” of the central helix of the Aβ by the designed ligands is visualized in Figure 1D. The properties of such ligands could also be advantageous for blood-brain barrier permeability. Their size, moderate lipophilicity and amphipathic nature could facilitate passive transmembrane diffusion.43 Effect of ligands on the helix stability of Aβ13–26 Initially, a larger number of new ligands (Table S1, SI) were designed and evaluated for the ability to stabilize the central helix of the Aβ using implicit solvent model GBMV II44 MD simulations and compared with first-generation ligands Pep1b and Dec-DETA. Numerous variations in the structure were tested in order to explore Aβ‒ligand interactions by modulation of ionic and hydrophobic functions of the ligand. During the implicit solvent simulations the Aβ13–26 unfolded in absence of ligands as backbone heavy atoms average RMSD was ~ 4 Å and the average number of α-helical backbone hydrogen bonds (αHBs) < 2. On the other hand Aβ13-26 with most of the ligands was still in a helical or partially helical state with an average RMSD < 2 Å and αHBs 3‒6 (Table S1, SI). A number of the novel ligands designed to cover several surfaces of the Aβ displayed substantially better stabilization of Aβ13-26 compared to the first-generation ligand Pep1b as it is seen in average values of αHBs count and backbone RMSD (Table S1, SI). Based on the results from the implicit solvent model GBMV II MD simulations two second-generation ligands DAEPep and CUAEPep (ligands 8 and 10 in Table S1, SI) that were among the top performers, and also feasible synthesis targets, therefore, were selected for MD simulations using explicit solvent model. During explicit solvent MD simulations, using the TIP3P water model, the Aβ13–26 unfolded in the absence of ligands with an average RMSD of conformers of 3.9 Å and αHBs of 1.6 (Figure 2A) as also reported in our recently performed explicit solvent simulation studies.40,

41

The selected ligands enhanced the stability of Aβ13–26 in a

helical state as is seen from values of αHBs count and backbone RMSDs (Figure 2A). The first-generation ligands, Pep1b and Dec-DETA displayed significantly higher RMSD values and lower αHBs counts than the new DAEPep and CUAEPep ligands. The average number of polar and non-polar contacts with Aβ13–26 was also more beneficial for the new ligands (Figure 2A), especially for DAEPep. Aβ13–26 has a higher average number of


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


Figure 2. (A) Average αHBs count, backbone RMSD of Aβ13–26 in the absence and presence of ligands Pep1b, Dec-DETA, DAEPep and CUAEPep and average number of polar and nonpolar contacts between Aβ13–26 and the corresponding ligands. Parameters from complexes of Aβ13–26 with ligands after ten independent 20 ns long molecular dynamics simulations at 360 K in explicit solvent. (B) Distribution of the backbone heavy atoms RMSD and (C) number of αHBs of Aβ13–26 middle region (15–24) at 360 K in Aβ13–26 alone (black), Aβ13–26 with DecDETA (red), Aβ13–26 with Pep1b (green), Aβ13–26 with DAEPep (blue) and Aβ13–26 with CUAEPep (brown). (D) Distribution of the number of polar, i.e., the number of hydrogen



Page 8 of 39

bonds (HB), and (E) non-polar contacts between Aβ13–26 and Dec-DETA (red), Pep1b (green), DAEPep (blue) and CUAEPep (brown) at 360 K. polar and non-polar contacts with DAEPep than with CUAEPep which may suggest that the extra acetamide functional group in CUAEPep in comparison to DAEPep brings some modulation in the interaction between ligands and Aβ13–26 and leads to a decrease of polar and non-polar contacts with Aβ13–26. The distributions of the backbone heavy atoms RMSD and number of αHBs of Aβ13–26 are summarized in Figure 2B and 2C. The majority of conformations of Aβ13–26 have RMSD < 3 Å and 1–4 αHBs with Dec-DETA, while with Pep1b, conformers of Aβ13–26 have RMSD < 4 Å and αHBs is in the range 2–5. For ligands DAEPep and CUAEPep the Aβ13–26 the RMSD is < 2 Å and the αHBs is > 3 (Figure 2B and 2C). The polar and non-polar contact distributions in the presence of ligands are summarized in Figure 2D and 2E. Since second-generation ligands DAEPep and CUAEPep are extended from the scaffolds of the first generation ligands Pep1b and Dec-DETA, they make a larger total number of polar and non-polar contacts to Aβ13–26 as compared to the first-generation ligands. Interactions between the ligands and Aβ13–26 To examine whether the ligands displayed contacts to Aβ in the way they were designed to do, the contact maps between Aβ13-26 and ligands (Figure 3) were analyzed. Visual inspection of the trajectories revealed that both the Aβ and the ligands are quite flexible and that the ligands sometimes detached from Aβ but bound to Aβ again. During the simulations of Aβ1326

with first-generation ligands Pep1b and Dec-DETA the contact probabilities were found to

be lower than 0.6, while with second-generation ligands DAEPep and CUAEPep the contact probabilities are as high as 0.8, indicating that the second-generation ligands have more extensive interaction with Aβ13-26 than the first-generation ligands. Considering contacts between the acidic residues (E22 and D23) of Aβ13-26 and the basic functional groups of ligands, contact probabilities are higher for the basic functional groups (N5, N6, and N7) of ligands DAEPep and CUAEPep than for the basic functional groups (N5, N7′, and N8′) of Pep1b and comparable with (N5′ and N6) of Dec-DETA. Basic functional groups of DAEPep have higher preference to bind to D23, while basic functional groups of CUAEPep show binding preference to E22 (Figure 3). The hydrocarbon tails of secondgeneration ligands (C27–C35 of DAEPep and C27–C36 of CUAEPep) have higher contact probabilities for residues V18 (for CUAEPep) and F19 (for DAEPep) of the Aβ13-26 middle 8 ACS Paragon Plus Environment

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


non-polar part. In contrast, low contact probabilities are found between the hydrocarbon tail (C27–C35) of Dec-DETA and residues from L17 to A21 of Aβ13-26. The amide functionality at



Page 10 of 39

Figure 3 Contact maps between Aβ13-26 and ligands using explicit solvent at 360 K. The contact probability between the center of geometry of side chain heavy atoms of each residue of Aβ13-26 and each heavy atom of ligands (A) Pep1b, (B) DAEPep, (C) CUAEPep and (D) Dec-DETA, which ranges from 0 (white) representing no contacts to 1 (blue) representing all contacts throughout simulation. Numbers in boxes on the X axis represent basic (blue), acidic (red), nonpolar (green), and uncharged-polar (black) residues of Aβ13-26. On Y axis are the respective ligand atoms. The chemical structures of ligands Pep1b (E), Dec-DETA (F), DAEPep (G) and CUAEPep (H) are shown. The naming and numbering on ligands: carbon (gray), nitrogen (blue), and oxygen (red) atoms. The naming and numbering of Aβ13–26 residues is basic (blue), acidic (red), nonpolar (green), and uncharged-polar (black). Arrows indicate the interactions between mentioned residues of Aβ13–26 (head) and different groups of the ligands (tail). the end of the hydrocarbon chain in CUAEPep showed some contact probability with the basic H14 residue of Aβ13-26. Interestingly, both second-generation ligands, DAEPep and CUAEPep, displayed weaker contact probability between acidic residues of the ligands (O1, O2, O4, and O5) and basic residue of Aβ13-26 (H13 and K16) compared to Pep1b, possibly due 10 ACS Paragon Plus Environment

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


to imperfect positioning when long hydrocarbon chains interact with hydrophobic residues in Aβ13-26. However, since the hydrocarbon chain in DAEPep and CUAEPep makes strong nonpolar contacts with L17, V18, F19 and A21 of Aβ13-26, the overall contact probability of DAEPep and CUAEPep is increased compared to first-generation ligands Pep1b and DecDETA. Overall, ligand DAEPep displayed the highest contact probability and, in comparison to CUAEPep, also a higher average number of polar and non-polar contacts (Figure 2A). The above results indicate that the second-generation ligands DAEPep and CUAEPep can interact with the central helix of the Aβ peptide and by “clamping” Aβ13-26 the new ligands display stronger non-polar interactions through the hydrocarbon tail while still keeping specific polar contacts. Based on MD simulations data and feasibility of synthesis we chose several promising second-generation ligands for evaluation of their anti Aβ1-42 neurotoxicity properties. This set of ligands included the model ligand DAEPep, its derivative CUAEPep, ligand DAEDmnPep (corresponding to ligand 32 in Table S1, SI), which resembles DAEPep and

contains

(R)-2-amino-4-(4′-N,N-dimethylamino-1,8-naphthalimido)

[(R)-2-amino-4-

DMNA] butanoic acid unit instead of D-Trp residue, and ligand 8AEDmnPep (corresponding ligand 31 in Table S1, SI), containing 4-DMNA functionality and a hydrocarbon branched triamino acid. Preparation of Aβ targeting ligands DAEPep and CUAEPep The DAEPep and CUAEPep ligands were synthesized via an active ester coupling approach in solution followed by attachment of the aliphatic hydrocarbon moiety. The synthesis started from the protected dimer 1 (Scheme 1) which was prepared according to the previously reported procedure.36 After selective deprotection of the tert-butoxycarbonyl (Boc) group at the α-position of the L-diaminobutyric acid (L-Dab) residue using 50% trifluoroacetic acid (TFA) in dichloromethane (DCM) containing 2% of 1,2-ethanedithiol–water (1:1 v/v) the intermediate 2 was reacted with an excess of glutaric anhydride in DCM–pyridine (4:1 v/v) mixture to give compound 3 in 96% yield. Removal of the Fmoc (fluorenylmethyloxycarbonyl) protecting group from the D-Trp residue was achieved by means of 20% piperidine in dimethylforamide (DMF). For the attachment of the Nβ-(2-aminoethyl)-2,3-diaminopropionic acid (AE-L-Dap) triamino acid building block45 to the α-amino function of D-Trp the free carboxylic acid group of the protected AE-L-Dap (5) was converted to an active ester. The corresponding pentafluorophenyl (5a) and p-nitrophenyl (5b) esters were synthesized via a carbodiimide mediated method46 (see SI, Scheme S1). Coupling of the pentafluorophenyl ester 5a to intermediate 4 in DCM–pyridine (2:1 v/v) 11 ACS Paragon Plus Environment


Page 12 of 39

Scheme 1. Synthesis of ligands DAEPep and CUAEPep. O

O

OBn

O

NHFmoc

N H

BocHN

TFA-DCM HS(CH2)2SH, H2O

O

NHFmoc

N H

H2N

94%

HN

2

HN

1

OBn

O

O

O

96% DCM, pyridine

O

O

OBn

O

HO

O

Piperidine, DMF

HO

97%

OR

CbzHN

5: R = H 5a: R = Pfp 5b: R = Pnp Cbz N NHBoc

HO

O

HO

O N H

N H

NHCbz Cbz N

H N

NHFmoc

N H 3

HN

H N

NHCbz Cbz N

NHBoc

O

HN

6

OBn

O

O N H

N H

62% for a; 73% for b

O

OBn

O

a) 5a, DCM, pyridine; b) 5b, DCM, pyridine, DIPEA

O

N H

O

O

OBn

O

HN

4

O

NH2

N H

N H

O

O

TFA-DCM, TIS, H2O

95%

NH2

O

HN

7

8

O

NH2

HO O

a)

b)

O2N

O

O

HO

NH2

O

Pyridine

O

9

O

Cl

O

DIPEA, pyridine

OBn

O N H

N H

NHCbz Cbz N

H N O

O

O R

N H

a) 10: R = 4

CH3 , 83%

HN

NH2 , 92%

b) 11: R = 5O

a) H2 (g), Pd/C AcOH, methanol

O HO

O

O N H

OH

b) H2 (g), Pd/C AcOH

O N H HN

NH2

H N O

H N

O N H

R

a) 12 (DAEPep, Ligand 8): R = 4

CH3 , 35% NH2 , 41%

b) 13 (CUAEPep, Ligand 10): R = 5O


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


mixture afforded derivative 6 in 62% yield (Scheme 1). The pentafluorophenyl ester 5a was found to be very reactive and somewhat unstable during purification. However, reaction of 4 with the less reactive p-nitrophenyl ester 5b in DCM–pyridine (2:1 v/v) and N,N′diisopropylethylamine (DIPEA) (1.5 eq.) gave higher yield (73%) of compound 6. The Boc protecting group on the side chain of the AE-L-Dap residue was removed using 25% TFA in DCM containing 5% of triisopropylsilane (TIS)–water (1:1 v/v) to provide key precursor 7 in 95% yield. Attachment of the 10-carbon aliphatic tail to the primary amino function of 7 was performed using an excess of decanoyl chloride in pyridine to obtain protected compound 10 with a yield of 83%. Synthesis of ligand CUAEPep with the longer 12-carbon chain with a terminal amide functional group was achieved using an active ester of 12-amino-12oxododecanoic acid. This derivative was made by first converting monoethyl dodecanedioate to 8 by treatment with ammonia in methanol and then was reacted with p-nitrophenol in pyridine in the presence of N,N′-diisopropylcarbodiimide (DIC) to afford p-nitrophenyl ester 9 (see SI, Scheme S2). The reaction of active ester 9 with the precursor 7 afforded protected compound 11 in 92% yield (Scheme 1). Deprotections of 10 and 11 were accomplished by catalytic hydrogenation on Pd/C in acetic acid or acetic acid–methanol (1:1 v/v) mixture to give corresponding final compounds 12 (DAEPep) and 13 (CUAEPep). Preparation of the Aβ targeting ligands DAEDmnPep and 8AEDmnPep The Aβ targeting ligands DAEDmnPep and 8AEDmnPep were prepared using an Fmoc chemistry based solid-phase approach and several in house developed amino acid derivatives. The synthesis of DAEDmnPep comprises of attachment of 2,3-diaminopropanic acid derivative 1547 to the Wang-supported intermediate 1448 at the α-amino position of a DMNAbutanoic acid unit and subsequent coupling of decanoic acid after Fmoc removal to afford solid supported compound 18 (Scheme 2). For the 8AEDmnPep ligand the recently developed hydrocarbonbranched triamino acid 1647 was directly attached at the α-amino position of DMNA-butanoic acid residue of 14 and after Fmoc removal the solid supported intermediate 20 was obtained (Scheme 2). Removal of the Boc protecting groups and cleavage from the solid support was achieved by the corresponding treatment of the 18 and 20 with a cleavage cocktail (TFA‒H2O‒TIS). After the hydrolysis of the methyl ester of the incorporated D-DabOMe residue with 0.05 M aqueous methanolic LiOH solution the final ligands DAEDmnPep (19) and 8AEDmnPep (21) (Scheme 2) were isolated using RP-HPLC.



Page 14 of 39

Scheme 2. Solid-phase synthesis of ligands DAEDmnPep and 8AEDmnPep. N

O

O

O OCH3

14

FmocHN BocN

BocHN

N H

N H

O

O N

O

NHFmoc

O

NBoc

NHFmoc BocHN

a) 40% piperidine/ DMF b) HOBt, DIC, DMF, 6 h, rt

16

COOH N

15

40% piperidine/ DMF for 20

COOH

O N

O

17 O

O

O OCH3 N H

N H

O

N

O

N H

N H

O

O

O OCH3

O NHBoc Boc N

HN

NHFmoc

O

O N

O

O NHBoc

HN

20

a) 40% piperidine/ DMF b) Decanoic acid, HOBt, DIC, DMF, 6 h, rt N Boc

O

NH2 N

a) TFA, H2O, TIS, 4 h, rt b) LiOH, MeOH, H2O, 1 h, rt O

HO

O

O

O N H

O

O OH N H

O NHBoc Boc N

HN

18 O

N

O

N H

N H

O

N

O

O OCH3

O

N

O N H

O

a) TFA, H2O, TIS, 4 h, rt b) LiOH, MeOH, H2O, 1 h, rt

NH2 HN O

21 (8AEDmnPep, Ligand 31)

N H

NH2 N

O HO

O

O

O OH N H

N

O N H

NH2 HN

19 (DAEDmnPep, Ligand 32)

O

O H N

O N H

Prevention of gamma oscillation degradation by interference with Aβ folding Recently we have shown that ligands designed to target the Aβ α-helix and interfere with its folding can retard the Aβ-induced degradation of gamma oscillations.36-38 We then investigated to see if neuroprotection would also be obtained with the new “clamping ligands”. Selected second-generation ligands were investigated for the effect on pharmacologically induced rhythmic network activity in the gamma-frequency range (30–80 Hz, gamma oscillations49, 50) in hippocampal slice preparations. Gamma oscillations are vital 14 ACS Paragon Plus Environment

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


Figure 4. Inhibitory effect of the second-generation ligands DAEPep and CUAEPep, and first-generation ligands Dec-DETA and Pep1b on the Aβ-induced reduction of gamma oscillation power in hippocampal slices. (A) Traces of kainate-induced gamma oscillations in area CA3 of naїve slices (Control). (B) Reduction of gamma oscillation power after 15 min incubation with 50 nM Aβ1–42 before kainate superfusion. (C) Traces of kainate-induced gamma oscillations after incubation with 50 nM Aβ1–42 in the presence of 250 nM DAEPep and (D) 250 nM CUAEPep. (E) Power spectra of gamma oscillations in a naїve slices (black), after incubation with Aβ1–42 alone (red) and in the presence of 250 nM DAEPep (green) and (F) 250 nM CUAEPep (green). (G) Summary histogram of gamma oscillation power in naїve slices (Control), Aβ1–42 incubated slices (Aβ), Aβ1–42 incubated slices in the presence of second-generation ligands DAEPep (Aβ + DAEPep) and CUAEPep (Aβ + CUAEPep), and Aβ1–42 incubated slices in the presence of first-generation ligands Pep1b (Aβ + Pep1b) and Dec-DETA (Aβ + Dec-DETA). component of brain function and play an essential role in its higher processes, such as learning, memory and cognition.51 This rhythmic electrical activity is known to be



Page 16 of 39

significantly reduced in patients diagnosed with AD, who suffer from deficiencies in their cognitive faculties.39, 52, 53 Initially we performed studies with 5-fold excess of DAEPep and CUAEPep ligands with respect to Aβ1-42 concentration and also compared their performance with the first-generation ligands Pep1b and Dec-DETA. Gamma oscillations were induced by superfusing horizontal hippocampal slices from C57BL/6 mice with 100 nM kainate. Local field potential (LFP) recordings in area CA3 revealed control gamma oscillations of 5.58 10-09 ± 3.98 10-10 V2 power (n=16; Figure 4A). Incubation of slices at physiological concentration of Aβ1–42 (50 nM) for 15 min prior to kainate superfusion led to a significant reduction of gamma oscillation power (1.97 10-09 ± 2.98 10-10 V2; n=12; U=188.0, n1=16, n2=12, p

Amyloid-Î² Peptide Targeting Peptidomimetics for Prevention of

Recommend Documents