Design, Synthesis and Enzymatic Inhibition of Novel Unusual Amino Acids as a Transition State Analogue of Amyloid Precursor Protein Peptide

Article abstract of DOI:10.1007/s10989-020-10015-9

Abstract: β-secretase 1 (BACE1) plays a pivotal role in the pathology of Alzheimer^?s disease via accumulation beta amyloid in the brain. In this context, identifying new scaffolds that block BACE1 is of great importance despite all pharmacokine

Full text of DOI:10.1007/s10989-020-10015-9

International Journal of Peptide Research and Therapeutics
https://doi.org/10.1007/s10989-020-10015-9
Design, Synthesis and Enzymatic Inhibition of Novel Unusual Amino
Acids as a Transition State Analogue of Amyloid Precursor Protein
Peptide
Atefe Ghodrati1 · Loghman Firoozpour2 · Saeed Balalaie3 · Faezeh Sadat Hosseini1 · Sorour Ramezanpour3 ·
Najme Edraki4 · Naser Mohtavinejad5 · Massoud Amanlou1,6
Accepted: 2 January 2020
© Springer Nature B.V. 2020
Abstract
׳
β-secretase 1 (BACE1) plays a pivotal role in the pathology of Alzheimers disease via accumulation beta amyloid in the brain.
In this context, identifying new scafolds that block BACE1 is of great importance despite all pharmacokinetic drawbacks
that peptide-like structures have. Here, we report a new core structure based on novel unusual amino acids by substituting
phenyl amide group in the P1 position and small alkyl groups in the P1′ site that results in the formation of new biological
active peptides in micromolar level. Three diferent scafolds were designed based on docking studies to efciently interact
with critical Asp32 residue in the active site of BACE1 and incorporated in peptides synthesis by Fmoc solid-phase peptide
synthesis (SPPS) methodology to achieve desired compounds in good yield. The inhibitory activity of all synthesized peptides
was examined by FRET-based enzymatic assay. The peptide 7 showed the best inhibitory activity with IC50=98.14 µM.
Results of this investigation revealed that utilizing unusual amino acids as building blocks for the synthesis of peptidomimet-
ics would be an option for the development and optimization of pharmaceutical structures.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10989-020-10015-9) contains
supplementary material, which is available to authorized users.
* Massoud Amanlou
amanlou@tums.ac.ir
1
Department of Medicinal Chemistry, Faculty of Pharmacy,
Tehran University of Medical Sciences, Tehran, Iran
2
Drug Design, Development Research Center, The Institute
of Pharmaceutical Sciences (TIPS), Tehran University
of Medical Sciences, Tehran, Iran
3
Peptide Chemistry Research Center, K. N. Toosi University
of Technology, Tehran, Iran
4
Medicinal and Natural Products Chemistry Research Center,
Shiraz University of Medical Sciences, Shiraz, Iran
5
Department Radiopharmacy, Faculty of Pharmacy, Tehran
University of Medical Sciences, Tehran, Iran
6
Experimental Medicine Research Center, Tehran University
of Medical Sciences, Tehran, Iran
Vol.:(0123456789)
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International Journal of Peptide Research and Therapeutics
Graphic Abstract
O
OH
OH
O
H
N
O
N
O
O
H
N
H2N
O
O
N
H
O
HN
H
O
OH
OH
O
O
OH
OM00-3
Rational Design
BACE-1 inhibition assessment
Docking Methodoloy
H
N
O
O
O
O
H
N
O
OH
HO
N
H
N
O
O
H
N
H
NH2
O
NH
NH
O
OH
O
O
OH
Most Potent
(7)
The inhibition of β-secretase 1 (BACE1) is potentially important approach to treatment of Alzheimer disease (AD). Novel
series of peptides coupled to the new unusual amino acids scafold were investigated as BACE1 inhibitors in this study.
The design of these peptides was mostly afected by OM00-3. Based on observations, in good agreement between BACE-1
inhibition assessment and docking methodology, peptide 7 was the most potent compound between all and could be the
basis of future studies.
Keywords BACE-1 · Synthesis · Peptidomimetic · Unusual amino acids · OM00-3
In recent years, the design of non-peptidic β-secretase
Introduction
׳
1 (BACE1) inhibitors as anti- Alzheimer s agents has been
׳
considered, in this regard, several BACE1 inhibitors have
been introduced to clinical trial, unfortunately, until now,
none of them can successfully pass the trial phase (Volloch
and Rits 2018; Moussa 2017). So, these challenges of non-
peptidic BACE1 inhibitors might drown keen attention of
medicinal chemists to modify and develop the rational
design of peptidic inhibitors to reach acceptable results.
The majority of peptidomimetic BACE1 inhibitors are
merged into a non-cleavable transition-state isostere motif,
which is the key binding element. Some of these essential
motifs include hydroxyethylene (HE), hydroxyethylamine
(HEA), statin and reduced amide pseudopeptide backbones
(Ghosh and Osswald 2014). Many eforts have been done
to obtain efcient inhibitors of BACE1 via designing new
scafolds that could be replacing the non-cleavable isostere
intermediate (Verdie et al. 2007).
Among the many types of dementia, Alzheimer s disease
(AD) is considered one of the most common diseases that
mainly arises in the elderly. It deteriorates the nervous sys-
tem, negatively afects memory, cognitive abilities and even-
tually leads to death (Nussbaum and Ellis 2003; Cummings
׳
2004). In a person’s brain with Alzheimer s diseases, the
presence of amyloid plaques and neurofbrillary tangles are
regarded as the main markers of the diseases. The former is
formed by the accumulation of amyloid beta (Aβ), the pep-
tides produced as a result of successive proteolysis of amy-
loid beta precursor protein (APP), and the latter is formed
by tau protein flaments aggregated through a hyperphos-
phorylation mechanism (Nguyen et al. 2006; Medeiros et al.
2011). Consecutive cleavage of APP by beta and gamma
secretase enzymes ends in the production of amyloid plaques
therefore synthesis of any compounds that could inhibit such
enzymes would help to slow the progression of the disease.
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International Journal of Peptide Research and Therapeutics
Recently, synthesis of unusual amino acids (UAAs) and
utilizing them as building blocks have attracted consider-
able attention in the feld of drug discovery that would
beneft in function of therapeutic agents and characteris-
tics improvement (Cardillo et al. 2006; Stevenazzi et al.
2014). In this way, a range of approved and clinical stage
drugs that contain unusual amino acids have been reported
(Blaskovich 2016). Peptides containing UAAs show more
conformation limitation and enhanced rigidity of peptide
that result in more resistance to proteasome-mediated
degradation, more selectivity and afnity to receptor in
contrast to peptides containing usual amino acids.
Materials and Methods
General Chemistry
Solvents that have been used during peptide synthesis
including trifuoroacetic acid (TFA), methanol, dichlo-
romethane (DCM), acetonitrile (ACN) and dimethylfor-
mamide (DMF) were provided from Merck (Germany).
Trietheylsilane (TES), (1-[Bis(dimethylamino)methylene]-
1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafuoro-
phosphate (HATU), N,N-diisopropylethylamine (DIPEA),
and benzotriazole-1-yloxytris(pyrrolidino)phosphonium
hexafuorphosphate (PyBOP) and N-(9H-Fluoren-9-yl-
methoxycarbonyloxy)succinimide (Fmoc-OSu) were pur-
chased from Sigma-Aldrich (USA). Besides, 2-chlorotri-
tylchloride (2-CTC) resin and 9-fuorenylmethoxycarbonyl
(Fmoc)-protected amino acids were bought from Bachem
(Switzerland), Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH,
Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH, Fmoc-Phe-OH are
the amino acids in this work. All other reagents were in
analytical grade or higher purity from Sigma-Aldrich or
Merck.
The peptide OM00-3 (Fig. 1) ofers high efciency as
a peptidomimetic BACE1 inhibitor (Hong et al. 2002).
This inhibitor contains a non-cleavable hydroxyethylene
isostere at the cleavage site. The Ki of OM00-3 was deter-
mined 0.3 nM and it is mainly used as a lead compound
for designing other efective inhibitors.
Here, we report the synthesis of the new structural
analog of OM00-3 containing novel unusual amino acids
(Fig. 2) as a substitution of Leu-Ala hydroxyethylene isos-
tere in the OM00-3 backbone (Fig. 3).
Melting points were measured by a Kofer hot stage
1
apparatus. H and 13C NMR spectra were obtained by
Bruker FT-500 and Bruker FT-300 using tetramethylsilane
(TMS) as the internal standard. IR spectra were acquired
using a Nicolet Magna FT-IR 550 spectrophotometer (KBr
disks) and Mass-ESI-POS (Apex Qe-FT-ICR instrument)
spectrometer was used to gain high-resolution mass spec-
tra of the components. The compounds’ purity was deter-
mined by HPLC (Column C-18, Eurospher 100, 7 µm).
Fig. 1 Structure of OM00-3
Fig. 2 Structures of synthesized
UAAs 2–4
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International Journal of Peptide Research and Therapeutics
ºC IR (KBr, cm−1): 3326, 2970, 1729, 1699, 1665 1H-NMR
(500 MHz, DMSO-d6): 3.32 (s, 2H, CH2), 4.30 (t, j=6.5 Hz,
1H, CH), 4.46 (d, j=6.2 Hz, 2H, CH2), 7.33–7.47 (m, 8H,
Ar–H), 7.75 (d, j=7.2 Hz, 2H, Ar–H), 7.90 (d, j=7.6 Hz,
2H, Ar–H), 9.64 (s, 1H, NH), 10.03 (s, 1H, NH). 13CNMR
(125 MHz, DMSOd6): 43.8, 46.6, 65.5, 118.7, 119.6, 120.2,
125.1, 127.1, 127.7, 133.8, 140.8, 143.8, 153.4, 164.2,
169.3.
Synthesis of Protected UAA3: (11b)
Fmoc-protected diamine (1 mmol, 0.31 g) and succinic
anhydride (1 mmol, 0.11 g) were dissolved in dry Acetone
(5 mL). The mixture was stirred overnight. After completion
of the reaction (checked by TLC), the mixture was fltered
and washed with ethyl acetate to give pure product. White
powder. Yield:>80%; M.P. 240 ºC. IR (KBr, cm−1): 3312,
3054, 2936, 1700, 1663, 1H-NMR (500 MHz, DMSO-d6):
2.49–2.52 (m, 4H, CH2), 4.29 (t, j=6.5 Hz, 1H, CH), 4.46
(d, j=6.0 Hz, 2H, CH2), 7.31–7.49 (m, 10H, Ar–H), 7.73
(d, j=7.2, 2H, Ar–H), 7.88 (d, j=7.35, 2H, Ar–H), 9.60(s,
1H, NH), 9.86 (S, 1H, NH), 11.9 (brs, 1H, COOH). 13CNMR
(125 MHz, DMSO-d6): 28.9, 30.9, 46.7, 65.5, 118.7, 119.4,
120.2, 125.1, 127.1, 127.7, 134.1, 134.3, 140.8, 143.9,
153.5, 169.7, 173.9.
Fig. 3 BACE1 peptide inhibitor containing the proposed UAAs 2–4
Synthesis of Protected UAA4: (11c)
Protected unusual 11c was synthesized in more than 80%
yield according to the synthesis method of compound 11b
using glutamic anhydride instead of succinic anhydride.
White powder. Yield:>80%; M.P. 247 ºC. IR (KBr, cm−1):
3321, 3021, 2938, 1701, 1665 1H-NMR (500 MHz, DMSO-
d6):1.72–1.84 (m, 2H, CH2), 2.24–2.34 (m, 4H, CH2),
4.27 (t, j=6.9 Hz, 1H, CH), 4.45 (d, j=6.7 Hz, 2H, CH2),
7.31–7.48 (m, 10H, Ar–H), 7.73 (d, j=7.1 Hz, 2H, Ar–H),
7.89 (d, j = 7.3 Hz, 2H, Ar–H), 9.59 (s, 1H, NH), 9.79 (s,
1H, NH). 13CNMR (125 MHz, DMSO-d6): 20.6, 33.1, 35.3
46.7, 65.6, 118.7, 119.7, 119.9, 120.3, 125.2, 127.2, 127.8,
134.3, 140.9, 143.9, 144, 153.5, 170.5, 174.3.
Synthesis of Fmoc‑Diamine (10)
To a solution of 1, 4-phenylenediamine (1 mmol, 0.1 g) in
ACN (5 ml), fmoc-OSu (1 mmol, 0.33 g) was added drop-
wise. The mixture was stirred at room temperature for 1 h.
The progress of the reaction was monitored using TLC.
After completion the reaction, the resultant precipitate was
fltered and washed with ACN. White powder; Yield:>90%;
M.P. 201 ºC; IR (KBr, cm−1): 3391, 3311, 3204, 3042, 2947,
1688. 1H-NMR (500 MHz, DMSO-d6): 4.29 (t, j=6.5 Hz,
1H, CH), 4.41 (d, j=6.2, 2H, CH2), 5.1 (brs, 2H, NH2), 6.53
(d, j=6.2 Hz, 2H, Ar–H), 7.1 (d, j=6.2 Hz, 2H, Ar–H), 7.36
(t, j = 7.0 Hz, 2H, Ar–H), 7.44 (t, j = 7.2 Hz, 2H, Ar–H),
7.76 (d, j=7.0, 2H, Ar–H), 7.92 (d, j=7.4 Hz, 2H, Ar–H),
9.6 (s, 1H, NH). 13C NMR (125 MHz, DMSO-d6): 46.7,
65.3, 114.4, 120.2, 120.3, 125.2, 127.1, 127.7, 140.8, 143.9,
154.0.
Procedure for the Synthesis of Peptide Sequence
The details of the practical procedure for the synthesis
of peptides (H-Glu-Leu-Asp-UAAs-Val-Glu-Phe-OH) is
described here as an example. The frst amino acid Fmoc-
Phe-OH (1 mmol) was attached to the 2-CTC resin (1.0 g)
using DIPEA (8 mmol) in DMF (10 ml). After incubation
for 2 h at room temperature, the mixture was fltered and
washed with DMF (3×10 ml), and the remaining trityl chlo-
ride groups of the resin were capped during the reaction
with solution of DCM/MeOH/DIPEA (20:2.4:1.2 ml) in a
30-min shake. The Fmoc group in each step of coupling
Synthesis of Protected UAA2: (11a)
The mixture of Fmoc-protected diamine (1 mmol, 0.31 g)
and Meldrum’s acid (1 mmol, 0.1 g) were refuxed overnight
in THF/DCM (1:1 mL). The mixture was concentrated and
the residual was washed with ethyl acetate and fltered to
give pure product. White powder, Yield: > 80%; M.P. 231
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International Journal of Peptide Research and Therapeutics
was removed by adding 25% piperidine in DMF (14 ml) to
the vessel and shaking the mixture for 30 min. A solution
of Fmoc-Glu-OH (3 mmol), TBTU (3 mmol), and DIPEA
(3.5 mmol) in 10 ml of DMF were added to the vessel to
attach the second amino acid (Glu) to the peptide chain.
After 2 h, the presence of free primary amino groups was
checked by Kaiser testing, other amino acids were coupled
to the chain in the same procedure respectively. For the
coupling of aromatic amine Fmoc-UAAs-OH the coupling
reagent PyBOP (3 mmol) was used with DIPEA (8 mmol)
in anhydrous DCM/DMF (10 ml, 1:1).
1,4-phenylendiamine solution in dry ACN results in the
immediate formation of a white solid with more than 90%
yield (10) that despite its simple structure its synthesis has
not yet been reported. Subsequently, nucleophilic addition of
the primary amine group in Fmoc-protected diamine (10) in
the presence of Meldrum’s acid, succinic anhydride or glu-
tamic anhydride provided a series of novel unusual protected
amino acids (11a), (11b) and (11c) in high yields.
The fnal peptidic compounds (Fig. 3) containing unusual
amino acids were synthesized through solid-phase peptide
synthesis (SPPS) methodology on the surface of 2-CTC
resin (Golmohammadi et al. 2018). All Fmoc-L-amino acids
were coupled to the peptide chain using TBTU and DIPEA
in DMF. PyBOP was the coupling reagent that has been used
for the coupling aromatic amine of Fmoc-UAAs-OH to the
peptide sequence. Deprotection of Fmoc groups in each step
was done in the presence of piperidine in DMF. TFA was
used for cleavage of the side chain-protected peptides from
the resin and all protecting groups were removed with a mix-
ture of TFA/(TES)/H2O/MeOH. The crude peptides were
precipitated in diethyl ether. The purifcation and identif-
cation of each peptide chain were analyzed via preparative
HPLC and ESI–MS as shown in Table 1. All the peptides
were purifed to achieve a purity>90%.
Chloranil is used to test for if there is any free aromatic
amine in the mixture. For cleavage of peptide from solid
phase, solution of 1% TFA in DCM (99 ml) was used which
was neutralized by 4% pyridine in MeOH (46 ml). After
solvent evaporation under reduced pressure in a rotary
evaporator, the peptide was precipitated in distilled water
and was fltered. Finally, cocktail TFA/TES/H2O/MeOH
(90:5:2.5:2.5) (10 ml) was used to remove all the protecting
groups. In this step, after the evaporation of the solvent, the
fnal peptide was precipitated in cold diethyl ether. Only for
peptide 8, cleavage and fnal deprotection were performed
simultaneously in a cocktail container followed by the cou-
pling steps.
It is worth mentioning that in the present study, in syn-
thesizing the UAAs 2–4, acylation of amine groups was
performed with high yields through free coupling reagent
reaction with no needs for recrystallization or chromato-
graphic methods to obtain purifed compounds. In the next
step, the substitution was replaced on the modifed Leu-Ala
scafold (OM00-3) and all peptides 5−8 were synthesized
by Fmoc solid-phase peptide synthesis as described in pre-
vious sections. To establish the optimal coupling condition
of Fmoc-UAAs–OH with Fmoc-Asp –OH, we investigated
Results and Discussion
Chemistry
General procedure for the synthesis of N‐protected UAAs
2–4 is summarized in Scheme 1. 1,4-phenylendiamine (9)
was used as the starting material for the synthesis of a novel
scafold in this study. Dropwise addition of Fmoc-OSu to
Scheme 1 Reagents and condi-
tions: (i) Fmoc-OSu, ACN, rt,
1 h, 90–95%; (ii) Meldrum’s
acid, THF/ DCM, refux,
overnight,>80% (iii) Succinic
anhydride, dry acetone, RT,
overnight,>80%; (iv) Glutamic
anhydride, dry acetone, rt,
overnight,>80%
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International Journal of Peptide Research and Therapeutics
Table 1 Physico-chemical
parameters of synthesized
peptide
Peptide
Peptide sequence
Yield (%)
MW
RT (min)
5
6
7
8
NH2-Glu-Leu-Asp-UAA6-Val-Glu-Phe-OH
NH2-Glu-Leu-Asp-UAA7-Val-Glu-Phe-OH
NH2-Glu-Leu-Asp-UAA8-Val-Glu-Phe-OH
NH2-Leu-Asp-UAA8-Val-OH
67
64
62
65
926.97
940.99
955.02
549.28
18.427
26.328
26.003
15.983
Table 2 Coupling reagents for coupling aromatic amines of UAA 2–4
Table 3 BACE1inhibitory activity peptides 5–8
Entry
Coupling reagents
Time (h)
Yield (%)
Peptide Inhibition (%)
IC50 (µM)
(200 µM)
(100 µM)
(50 µM)
1
2
3
4
HATU
TBTU
DIC
24
24
24
2
–
–
–
5
6
7
8
22.98 1.99 16.32 2.38 7.64 7.28 >200
84.00 18.44 46.11 6.49 28.29 6.13 111.80 20.51
97.06 4.58 53.87 6.73 40.57 3.06 98.14 2.12
62.73 3.92 44.49 7.69 21.78 3.54 167.00 32.90
PyBOP
60–70
OM99-2
–
–
–
14.70 nM
using diferent coupling reagents such as HATU, DIC, and
TBTU in the DCM/DMF (1:1) as a solvent.
Data are expressed as Mean SE (three independent experiments)
OM99-2 was tested at 10, 1 and 0.1 nM
Aryl amine group of proposed UAAs are weakly nucleo-
philic amine and our result indicated that N-acylation had
to be done in the presence of 8 equivalents of DIPEA when
PyBOP was used as coupling reagent in manual coupling
for 2 h (Entry 4 in Table 2). On the other hand, the reaction
totally failed when HATU or TBTU or DIC were used as
activating agents.
of each), BACE1 enzyme (10 µl) and BACE1 substrate
(10 µl) and then stopped with 2.5 M sodium acetate. Spec-
trophotometer recorded fuorescence at 545 nm for excita-
tion and 585 nm for emission into 96-well plate to monitor
the hydrolysis of the substrate (BMG Labtech). OM99-2
(H-Glu-Val-Asn-Leu-Ψ-Ala-Glu-Phe–OH) was employed
as a reference inhibitor. Hence in each concentration of test
compounds the percentages of enzyme inhibition were cal-
culated regarding maximum enzyme activity wells (contain-
ing substrate plus enzyme) and baseline wells (containing
substrate). IC5o concentrations were determined by Expert
software version 1.34 for Windows. Each experiment was
repeated three times.
As sites, located near the transition-state isostere (P1 and
P1′), on both P and P’ sides are more efective than the distal
sites (P4 and P4′) Therefore, we also presented the design
and synthesis of short peptide 8 as an analog of OM00-3 by
′
removing positions P3, P4, and P4 to determine if P1 and
P1′ new scafolds are selected properly or the chain length
is responsible for the seen efects. The achievement of the
least diference in comparison with peptide 7 revealed the
designed structures for S1–S1′ enzymatic sites has been cor-
rectly selected that could have relatively efective interac-
tions with the enzyme.
Biological Activity
BACE1 inhibitory activity of peptides containing UAAs
2–4 was examined in vitro using FERT assay. Positive con-
trols were performed using a known potent peptidomimetic
inhibitor (OM99-2) and the BACE1 kit employed contained
a particular APP-based peptide substrate (Rh-EVNLDAEFK
quencher) and a BACE1 enzyme. The results are shown in
Table 3. All the peptides synthesized exhibited fair to good
BACE1 inhibitory activities. The highest potency was rep-
resented by the peptide 7, which had the largest group in
P1´site and the most fexible core.
BACE‑1 Inhibition Assessment
The BACE1 fluorescence resonance energy trans-
fer (FRET) assay was carried out according to
manufacturers’instructoins (Invitrogen. https://tools.invit
rogen.com/content/sfs/manuals/L0724.pdf).
To obtain 3 × solutions, both BACE-1(provided by
Baculovirus expression) and Rh-EVNLDAEFK-Quencher
as substrate were diluted by BACE-1 assay bufer (50 mM
sodium acetate, pH 4.5). Besides, 3×solutions of the difer-
ent concentrations of test compounds were acquired through
diluting stock solutions of inhibitors in DMSO using assay
bufer (fnal concentration of DMSO in the test plate was
6%). Reaction was incubated at room temperature for 90 min
under dark condition with a mixture of test samples (10 µl
Docking Study
To explore the mode of interactions of synthesized peptides
with critical Asp32 residue in active site of BACE1, dock-
ing simulations were performed using the AutoDock Vina
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International Journal of Peptide Research and Therapeutics
(Ver. 1.1.0) (Trott and Olson 2010). The ability of software
to predict the best docking pose and docking accuracy was
determined with re-docking of co-crystallized inhibitor
(OM00-3) with pdb ID: 1M4H. For this purpose, the ligand
has been removed from crystallographic structure and then,
has been docked in the active site of the protein. Calculated
RMSD value was less than 2 Å. The result of this proto-
col and superimposition of docked structure and crystallo-
graphic ligand shown in Fig. 4 and revealed that the binding
orientation and interaction of both structures are similar.
The structures of all compounds have sketched by Mar-
vinSketch applet (Marvin package, ChemAxon Company).
AutoDockTools (ADT, Ver. 1.5.6) (Morris et al. 2009) was
used for preparing input fles and analyzing docking results.
Polar hydrogens and rotatable bonds were assigned by ADT.
Docking with a maximum number of 25×106 energy eval-
uations and 50 runs per simulation using the Lamarckian
Genetic Algorithm (LGA) were performed. All other param-
eters were set to their default values. The docked conforma-
tions of each ligand were ranked into clusters based on the
binding energy. After clustering analysis, conformation with
the most favorable binding energy was selected.
was performed on all designed peptides. Due to the high
fexibility of peptides, several docking programs have been
used with diferent levels of prediction accuracy to predict
protein–peptide mode of interaction (Ciemny et al. 2018).
Between these applications, AutoDock Vina shows the best
docking performance and scoring power on short peptides
up to four residues (Rentzsch and Renard 2015). The results
of our work showed that AutoDock Vina is still suitable for
peptides up to seven residues (Figs. 4, 5, 6).
Investigating Binding Model of Synthesized
Peptides Over BACE1
Docking results indicated that all the designed peptides bind
to the active site with negative binding energy in the range
of−9.0 kcal/mol. As shown in Fig. 5 the new scafold rep-
resented identical binding modes with other transition-state
analogs of BACE1 inhibitors. The nitrogen atom of phenyl
amide substitution in the novel synthesized scafold plays the
same role as the hydroxyl group in OM00-3 does. The fap
residues Thr72 binds with another nitrogen in the new scaf-
fold. The key H-bonding interaction between the new scaf-
fold and Asp 32 was observed during the docking and the
amide backbone of the inhibitor made hydrogen bonds with
Arg128, Tyr198, Thr232, Thr72 and Gly230 while the side
chain Glu and Asp formed hydrogen bonds with Thr231,
Thr72, Thr329 and Gly34. Docking pose of peptide 7 was
shown in Fig. 6
Reliability of the Docking Protocol
The main approach to achieve new peptidomimetic
β-secretase inhibitors is to create structures that mimic
transition-state isostere. The S1 site of BACE1 is large
hydrophobic pocket and groups like cyclohexyl and benzyl
at the P1 position can efciently fll the S1 site, so phenyl
amide was used as benzyl mimic in the P1 position. In addi-
tion, because the S1′ position accommodates hydrophobic
groups properly, replacement of small alkyl groups in the
Conclusion
In this study, an efcient approach is described for the syn-
thesis of unusual amino acids as new scafolds that could
replace the HE isostere of OM00-3 forming analogs of
′
P1 site is considered to be efcient (Ghosh and Osswald
2014; Silvestri 2009) to confrm design rationale, docking
Fig. 4 Superimpose of redocked
pose (orange) with crystallo-
graphic structure (green) (Color
fgure online)
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International Journal of Peptide Research and Therapeutics
Fig. 5 Representation of bind-
ing modes of the most active
peptide 7 in the active sites of
BACE1
Fig. 6 Docking pose of peptide
7 in the active sites of BACE1
tetra and heptapeptides. The key component of new scaf-
folds consists of a cyclic amide group that interacts with
catalytic Asp side chain. This novel scafold also includes
Compliances with Ethical Standards
Conflict of interest The authors declare that they have no confict of
interest.
′
an alkyl moiety which easily fts hydrophobic S1 pocket.
The most potent peptide 7 exhibited 98 µM BACE-1
inhibitory activity using FRET assay. SAR study of pre-
sented peptidic BACE-1 inhibitors emphasizes the impor-
tant role of amide NH of unusual amino acids in key
H-bonds with Asp32 and Gly230 residues. PyBOP was
used in the presence of 8 eq of DIPEA to couple aromatic
amine group of UAAs to peptide sequences that aforded
the product in good yield and could be utilized in ami-
dation of other weakly nucleophilic aromatic amines.
Besides, the same connections and bonds between peptide
7 and OM003 that reported above, may well justify the
in vitro inhibitory ability of peptide 7.
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