Amyloid Probe-Insights into Its Binding Sites and SelectivityLiterature Seminar #2
2021/10/14
Wataru Atsumi (B5)1

Table of contents
lIntroduction
Background of amyloid-selective fluorescent probes

lBinding sites of probes to Aβ
lSelectivity of probes
1. Selectivity to Aβ oligomers
2. Selectivity to tau aggregates

lSummary2

Amyloids and human diseases

The aggregation of peptides intoamyloid fibrils is the hallmark of
misfolding diseases known as
amyloidosis.
e.g.
• Alzheimer‘s disease• Perkinson’s disease• Huntington’s diseaseZaida L. Almeida and Rui M. M. Brito, Molecules, 2020, 25(5), 1195.

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The process of amyloid formation

Iadanza, M.G., Jackson, M.P., Hewitt, E.W. et al. Nat Rev. Mol. Cell Biol. 2018, 19, 755–773.

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Cytotoxicity of amyloids

Not only fibrils but oligomers represent cytotoxicity.
Iadanza, M.G., Jackson, M.P., Hewitt, E.W. et al. Nat Rev. Mol. Cell Biol. 2018, 19, 755–773.

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Fluorescent probe for amyloid
FSO

NFB

O

R1

N

Thioflavin T (ThT)

R2

R3X

NH2X

R4CRANAD

NSO3NaNN
N

NaO3S

H 2N

Congo Red (CR)

NF

B

N
F

BODIPYThe strengths of fluorescent probeü Real-time monitoring of self-assembly in vitroü Recognition of amyloids in vivo
ü To identify aggregation inhibitor
ü Lower cost than PET (Positron Emission Tomography)
ü No exposure to radioactivity
Especially, ThT has been used to elucidate the process of aggregate formation.
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The mechanism of amyloid recognitionn W/O amyloid aggregatesn W/ amyloid aggregates**Rotation

amyloid probeRotation*Excitation*ExcitationNo emission

Non-radiation
decayEmission

Non-radiation
decay

Ø In the unbound state, intramolecular rotation causes rapid self-quenching.
→ No light emission
Ø The presence of amyloid aggregates restricts the intramolecular rotation.
→ The enhancement of fluorescence intensity
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Problems• It is still unclear how amyloid probes bind tofibrils.
• Heterogeneous structures of amyloids in vivo
make it difficult to elucidate theirpharmacophore.
• For accurate diagnosis, protein-selective
amyloid probes need to be developed.

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Table of contents
lIntroductionBackground of amyloid-selective fluorescent probes

lBinding sites of probes to AβlSelectivity of probes
1. Selectivity to Aβ oligomers
2. Selectivity to tau aggregates

lSummary
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The structure of Aβ fibrilsn Fibril structure of Aβ determined by cryo-EM
Hydrophobic clusters

salt bridges

Ø Two twisted protofilaments composed of Aβ(1–42) molecules stacked in a parallel.
Ø A single Aβ subunit forms an LS-shaped structure.
Lother Gremer, et al. Science, 2017, 358, 116–119.

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Binding sites of ThTn Multiple binding sites (BSs) of ThTSNNAβ fibril
Thioflavin T (ThT)Ø BS1 & BS2
・Relatively abundant (approximately one site per 4-35 monomers)
・BS1 and BS2 are thought to be in close proximity (∵FRET).
・Composed of surface grooves created by aligned side chainsØ BS3・Less abundant than BS1 and BS2 (approximately one site for
300 monomers)
Ashley A. Reinke and Jason E. Gestwicki, Chem. Biol. Drug Des. 2011, 77, 399–411.

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Binding sites of ThT
n Two binding channels (molecular docking study)

At least five, spatially consecutive, hydrophobic (Phe only or Phe and Val)side chains are important.
→ Hydrophobic interactions and π-π stacking(Similar findings have been reported by Dr. Koide and Dr. Makabe)
Ashley A. Reinke and Jason E. Gestwicki, Chem. Biol. Drug Des. 2011, 77, 399–411.

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Binding sites of ThT
n Two binding channels (molecular docking study)

SNN
Thioflavin T (ThT)
positively chargedSNH
N
BTA-1
neutralNeutral BTA-1 has much better affinity to Aβ fibrils→ Neutral molecules can bind deeper into the hydrophobic binding grooves.
e.g. Phe only site (left) is deeper and narrower than Phe/Val site (right).
Ashley A. Reinke and Jason E. Gestwicki, Chem. Biol. Drug Des. 2011, 77, 399–411.

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Binding sites of Congo Red (CR)n Multiple binding sites of ThTNH2
NSO3NaNNN

NaO3S

Aβ fibril

H 2N
Congo Red (CR)Ø BS2One site per 3 monomers
Shared by ThT (The competition between ThT and CR was reported.)Ø BS4Unique binding site for CR (Discrete binding sites of ThT and CR was alsoobserved.)
Lower density
BS4 may be at the face of the growing fibril.
Ashley A. Reinke and Jason E. Gestwicki, Chem. Biol. Drug Des. 2011, 77, 399–411.

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Binding sites of Congo Red (CR)n The binding channel of CR (molecular docking study)Ø The CR-binding channel is longer and narrower than that of ThT.
Ø The aligned residues are largely polar and non-aromatic (such as Asn and Gly).
→Ionic or polar interactions may be important.

Ashley A. Reinke and Jason E. Gestwicki, Chem. Biol. Drug Des. 2011, 77, 399–411.

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Binding sites of Congo Red (CR)
Fibril axis

BS2BS4

BS2 → Approximately 78 % of the total CR binding clusters
BS4 → 11 % of the total binding↓

An explanation for the difference between CR binding affinity(Kd high nM ~ low μM) and its inhibition capacity (Kd mid-high μM)
CRCR

CR

CR
CR

CR

CR

CR

saturation of BS2

CRCR

CR

CR
CR

CR

CR

CR

Inhibition ofthe fibril growing

Ashley A. Reinke and Jason E. Gestwicki, Chem. Biol. Drug Des. 2011, 77, 399–411.

CRCR16

Another example of binding modesn Binding sites of ThT and CR (molecular dynamic simulation)n ThT・ThT binds across multiple Aβ peptides, forming π-π stacking with F20.
→ It recognizes only Aβ fibrils (not single Aβ).

n CR・CR binds to the groove between Y10 and V12 forming edge-to-face aromatic andhydrophobic interactions.
・Carbonyl oxygen → Forming hydrogen bond with E11・Sulfonate group → Exposed to the solvent

Frieg, B, Lother Gremer, et al. Chem. Commun. 2020, 56 (55), 7589– 7592.

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Table of contents
lIntroduction
Background of amyloid-selective fluorescent probes

lBinding sites of probes to Aβ
lSelectivity of probes
1. Selectivity to Aβ oligomers
2. Selectivity to tau aggregates

lSummary18

Selective detection for Aβ oligomers

Aβ oligomer
Probe for Aβ fibrils(e.g. ThT)Aβ oligomer
oligomer-selective probe

Aβ fibrils

Aβ fibrils

Ø Oligomeric soluble Aβ is responsible for the pathogenesis ofAlzheimer’s disease.
Ø There are a number of fibril-specific dyes but few dyes which
preferentially recognize Aβ oligomers.
Ø It is necessary to develop oligomer-selective small fluorescentmolecular probes.
Chai Lean Teoh et al. J. Am. Chem. Soc. 2015, 137, 13503−13509.

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Selective detection for Aβ oligomers

The highest fluorescence enhancement with Aβ oligomers↓BD-Oligo preferentially recognizes Aβ oligomers
over monomers or fibrils
Chai Lean Teoh et al. J. Am. Chem. Soc. 2015, 137, 13503−13509.

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Selective detection for Aβ oligomers
n ThT & BD-Oligon Pelleting assay

SNN

plateauThioflavin T (ThT)Aβ aggregates selective
Medecrease

NFOHB

NFOO
CCl3OEt

BD-OligoAβ oligomer selective

The majority of Aβ
is still in solutionn TEM images

Aβ starts to form a large
segmenting materials

Day 0 and Day 1: No sign of fibrils
Day 4: Fibril formation was observed.
The presence of β-sheet structure alone does not suffice
to explain the binding specificity of BD-Oligo.
↓What is the structural feature of Aβ oligomers?

Chai Lean Teoh et al. J. Am. Chem. Soc. 2015, 137, 13503−13509.

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A working model of Aβ oligomers
n The structure of the mimic of Aβ17-36 trimer

Structural features of Aβ trimer

ü The trimer consists of three twisted β-hairpins.
ü Phe19 and Val36 form hydrophobic patchesat the center of Aβ trimer.

Importantly, these hydrophobic
patches are exposed only in Aβ oligomers
but not in Aβ fibrils (oligomer-specific).

Ryan K. Spencer, Hao Li, and James S. Nowick, J. Am. Chem. Soc. 2014, 136, 5595−5598.

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A working model of Aβ oligomers
n The structure of the mimic of Aβ17-36 trimer

The side chains of Leu17, Phe19, Ala21,Asp23, Ile32, Leu34 and Val36 form
extensive hydrophobic clusters.

Ryan K. Spencer, Hao Li, and James S. Nowick, J. Am. Chem. Soc. 2014, 136, 5595−5598.

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Molecular docking using working model

Site-directed thermodynamics analysis ofthe BD-Oligo complex with Aβ oligomer (Aβ17-36)

BD-oligo recognizes F19 and V36 in Aβ oligomers.

Ø Hydrophobic patches (F19)π-π stacking with aromatic ring od BD-oligoØ V36
Hydrophobic interaction / CO---H bonding with carbonyl group of BD-oligo
Chai Lean Teoh et al. J. Am. Chem. Soc. 2015, 137, 13503−13509.

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Ex vivo binding of BD-Oligo in AD mouse brain
Intraperitonealinjectionwith BD-Oligo

24 hr.

Ex vivo imaging
BD-Oligo labeling not only appeared in the central core of Aβ plaques
but was also present in the periphery of plaques.

↓This result reflects the hypothesis that Aβ plaque may function
as a reservoir for soluble oligomers.
Chai Lean Teoh et al. J. Am. Chem. Soc. 2015, 137, 13503−13509.

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Other examples of Aβ oligomer probes
MeOO

BOONMeNN

MeMeCNN

MeO

MeAN

NO2

AD-1

*AD-1 also recognizes Aβ monomers and aggregates

The main binding mode is the π-π stacking interactions
between Phe 19 and aromatic rings of probes.
Their twisted structures may affect the binding affinity to oligomers ??

left: Yiran Ge et al. ACS Chem. Neurosci. 2021, 12, 19, 3683–3689.
right: Guanglei Lv et al. Chem. Commun., 2016, 52, 8865-8868.

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Table of contents
lIntroduction
Background of amyloid-selective fluorescent probes

lBinding sites of probes to Aβ
lSelectivity of probes
1. Selectivity to Aβ oligomers
2. Selectivity to tau aggregates

lSummary27

Difficulties in developing tau-selective probes• Common β-sheet structure• The lack of clear pharmacophore• The structural diversity of tau proteins• PTMs (Post-Translational Modification)↓
Rational design of tau-selective probes is difficult…

However, there are two hypotheses to guidethe molecular design of tau binders.
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Two hypotheses
1. A distance of 13 to 19Å between the donor and acceptor
parts benefits tau selectivity.
~13Å

13~19ÅA

D→ Tau selective detection

A

D→ Shorter distance favors
Aβ plaques

2. Dyes containing fused ring system tend to have tau
selectivity over Aβ fibrils.
SHNONNN

N

Examples of fused ring system
Ariza,M. et al. J. Med. Chem. 2015, 58, 4365−4382.
Maruyama M. et al. Neuron, 2013, 79, 1094−1108.
Peter Verwilst et al. J. Am. Chem. Soc. 2017, 139, 13393−13403.

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New tau-selective probes
Tau 1 & Tau 2: Rationally designed probesü About 14Å between donor and acceptor
ü Fused ring systemü→ More likely to interact with the β-sheet fibrillar aggregatespresent in PHF-tau

ü Short synthetic routeü Tau selective detection in vitro and in vivo
Peter Verwilst et al. J. Am. Chem. Soc. 2017, 139, 13393−13403.

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Selective responses to Tau aggregates
Tauw/ tau aggregatesü No significant fluorescencein heparin onlyü Time-dependent fluorescentenhancement

Aβw/ Aβ aggregatesü Trivial responses tothe presence of Aβ aggregates

Tau 1&2 are highly selective to Tau protein aggregatesover Aβ aggregatesPeter Verwilst et al. J. Am. Chem. Soc. 2017, 139, 13393−13403.

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In cell assay*OA: Okadaic acid(an inhibitor of phosphatases) → In vitro tau hyperphosphorylation model

Both of two probes (especially Tau 1) are capable of detecting
hyperphosphorylated tau aggregates selectively over Aβ aggregateseven in cellular environments.
Peter Verwilst et al. J. Am. Chem. Soc. 2017, 139, 13393−13403.

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Ex vivo imaging of Tau 1

Intracranially injected with Tau 1

ü Anti tau and Tau 1→a very high degree of overlap
ü Anti Aβ / APP and Tau 1→virtually no overlap
Tau 1 co-localized with tau
but not with Aβ
under these conditionsPeter Verwilst et al. J. Am. Chem. Soc. 2017, 139, 13393−13403.

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In vivo imaging of Tau 1
n Protocol

Excitation at 670 nm

Detection at 699 nmTail vein injection
with Tau 1
Tau P301L model mouse / WTn Result

P301L mutation → Aggregation-prone tau protein

A significantly enhanced fluorescencein the transgenic mouse model↓

Tau 1 can detect the presence oftau tangles in mice !!

Peter Verwilst et al. J. Am. Chem. Soc. 2017, 139, 13393−13403.

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PHF fragment (306VQIVYK311)
Q
V
YKI

IVVVKYQ
Dry and tight interface•PHF sequence has been demonstrated to play a pivotal rolein the aggregation of tau protein.

•The crystal structures of VQIVYK peptide consists the steric zipper structure.

•Co-crystallization with some compounds reveals a propensity togenerate tunnels along the fibril axis (See next page).

M. von Bergen et al. PNAS, 2000, 97 (10), 5129-5134.
Meytal Landau et al. PLoS Biol. 2011, 9, 1–13.

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Co-crystallization with dyes

OO

MeO

OMeHOOH

curcuminNC

CNMeMeN
MeDDNP

These dyes bind to the β-sheets with their long axes parallel to the fiber axis.
Meytal Landau et al. PLoS Biol. 2011, 9, 1–13.

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Co-crystallization with dyesn Crystal structure (DDNP)

apolar cavities
apolar cavities

The atomic groups lining the tunnel are about half apolar (colored in white)and half polar (colored in red and bleu).
↓This cylindrical, partially apolar structure favors
various apolar or aromatic compounds.
These cavities also provide biding sites of apolar drugs (benzodiazepines and anesthetics etc.)→ An explanation for the altered pharmacokinetic properties and increased sensitivity in elderly patients ??
Meytal Landau et al. PLoS Biol. 2011, 9, 1–13.

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Molecular docking studies (Tau 1)

Tau 1 demonstrated a tight fit in the tunnel.
The main binding mode is the hydrophobic interactions
between aromatic rings of Tau1 and side chains of Val .

Peter Verwilst et al. J. Am. Chem. Soc. 2017, 139, 13393−13403.
Ahmed A. Elbatrawy et al. ACS Sens. 2021, 6, 2281-2289.

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Molecular docking studies
n Binding affinity of tau-selective probes

Ø Lower binding affinity of BAP-1→ Due to its smaller surface area ?
Ø F-ATPZ-38 & astemizole → Favorable to interact with other topologies ?

Peter Verwilst et al. J. Am. Chem. Soc. 2017, 139, 13393−13403.

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Table of contents
lIntroduction
Background of amyloid-selective fluorescent probes

lBinding sites of probes to Aβ
lSelectivity of probes
1. Selectivity to Aβ oligomers
2. Selectivity to tau aggregates

lSummary40

SummaryØThe binding mode of amyloid probes has been wellstudied by utilizing molecular docking simulation ormolecular dynamics simulation.
ØThese studies provide insights into the pharmacophoreand keys to the rational design of amyloid probes.
ØHowever, these findings need to be interpreted withcaution as the exact structures of amyloid fibrils(or oligomers) in vivo are still unclear.
Thank you for your listening !!
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APPENDIX

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Molecular docking simulation*結合部位があらかじめ定められている場合に有効(今回取り扱った各⽂献では、結合部位を想定した上でMDが⾏われている)
OISTのウェブサイトより: https://www.oist.jp/ja/news-center/photos/14053

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Molecular dynamics simulation

http://guillefix.me/cosmos/static/Molecular%2520dynamics.html

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Detailed structure of Aβ fibril

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Three distinct binding sites

Kd1 : プローブ濃度を固定し、Aβモノマー濃度を変動させた時の結合定数
Kd2 : Aβモノマー濃度を固定し、プローブ濃度を変動させた時の結合定数
Kd1 /Kd2 : モノマーいくつ分に対してそのプローブの結合部位があるかを⽰す値
ThT, IMPY-Hb, IMPY-Me→ 30~40モノマーあたり⼀つの結合部位 (BS1)
TZDM, TZPI, BF1→ 3~5モノマーあたり⼀つの結合部位 (BS2)→２つの結合部位があることの⽰唆する
前者(BS1)より後者(BS2)の⽅がより多くAβ上に存在するということがわかる
その他、300モノマーあたりに⼀つ存在するBSもあるという結果が→BS3とした
Lockhart A. et al. J. Biol. Chem., 2005, 280(9), 7677-84.

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FRET (Fluorescence Resonance Energy Transfer)

Distance-dependent interaction

This figure shows CFP (donor) andYFP (acceptor) absorption andemission spectra. Overlap between
CFP emission and YFP absorption(shaded region) leads to efficient FRETinteraction.
https://www.semrock.com/fret.aspx

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Works of Dr. Koide and Dr. Makaben PSAM as a model of β-sheet structure

Matthew Biancalana, Koki Makabe, Akiko Koide and Shohei Koide, J. Mol. Biol. 2009, 385, 1052–1063

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Works of Dr. Koide and Dr. Makaben Tyrosine is the key to the binding mode of ThTAt least 4~6 tyrosines are needed.
Matthew Biancalana, Koki Makabe, Akiko Koide and Shohei Koide, J. Mol. Biol. 2009, 385, 1052–1063

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Works of Dr. Koide and Dr. Makabe→ 5-YY/LL is the most favorable.

Ø Aromatic side chains of Tyr is important.
Ø The Leu ladder seems to have a secondary rolein forming ThT-binding site.
Matthew Biancalana, Koki Makabe, Akiko Koide and Shohei Koide, J. Mol. Biol. 2009, 385, 1052–1063

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Works of Dr. Koide and Dr. Makabebis-ANS seems to share
the binding sites with ThT.

No evidence for binding of Congo Redto 5-YY/LL→ Binding site of 5-YY/LL is too small??
(CR: ~26 Å, ThT: ~15 Å)Matthew Biancalana, Koki Makabe, Akiko Koide and Shohei Koide, J. Mol. Biol. 2009, 385, 1052–1063

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Co-crystallization with Orange G
16KLVFFA21OH
N

NSO3Naco-crystallization
SO3Na

Orange G
Meytal Landau et al. PLoS Biol. 2011, 9, 1–13.

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Co-crystallization with Orange GThis crystal structure may provide a pharmacophoreof Aβ-selective probes??
Meytal Landau et al. PLoS Biol. 2011, 9, 1–13.

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A working model of Aβ oligomers
n peptide 1a: as a mimic of Aβ17-36

The structure of trimers or higher-order oligomers were not known.

↓ü Crystallization was achieved by modifying side chains of Aβ17-36.
ü The crystal structure of Aβ trimer provides a working model.

Ryan K. Spencer, Hao Li, and James S. Nowick, J. Am. Chem. Soc. 2014, 136, 5595−5598.

54Tau selectivity

13-19 Å between D & A↓
Well detected!

Maruyama M. et al. Neuron, 2013, 79, 1094−1108.

*AT8: Anti PHF-tau antibody

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TICT of Tau 1 and Tau 2
Tau 1

Tau 2

HOMO

LUMO

HOMO

LUMO

Electron redistribution from the aniline / aminonaphthalene in the HOMO
to the BODIPY core in the LUMO↓Involvement of TICT process (large Stokes shift)(Large stokes shift contributes to better signal over noise ratio)Peter Verwilst et al. J. Am. Chem. Soc. 2017, 139, 13393−13403.

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Molecular docking studies (Tau 2)

Tau 2 also demonstrated a tight fit in the tunnel.

Peter Verwilst et al. J. Am. Chem. Soc. 2017, 139, 13393−13403.

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Check the selectivity of ThT & BAP-1

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Another example of tau-selective probe

The design of this probe also follows two hypotheses of tau-selectivity.
Molecular docking study demonstrated the high affinity to 306VQIVYK311 tunnel as well.
Ahmed A. Elbatrawy et al. ACS Sens. 2021, 6, 2281-2289.

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