Ho

w w

as

an

art

ific

ial

rib

oz

ym

e

de

ve

lop

ed

in

to b

io-v

en

ture

. ~

Ca

se S

tud

y o

f P

ep

tid

ream

~

Ap

ril

26

th (

Sat.

), 2

014

Tak

uy

a M

ats

um

oto

(D

2)

2013

年06月11日東大発ＶＢのペプチドリーム、東証マザーズ上場－自社創薬に本腰

http://www.peptidream.com/index.htm

l

0. peptidream 1

Peptidream

0. peptidream 2

Why Peptide?

Small Molecule

Antibody

‘Non-Standard’ Peptide

O X

Hig

h p

erm

eab

ilit

y o

f in

testi

nal

or

cell

mem

bra

ne (

ora

l d

ose)

To

o s

mall

fo

r in

hib

itin

g

pro

tein

-pro

tein

in

tera

cti

on

O X

Inh

ibit

ive c

ap

acit

y o

f p

rote

in-

pro

tein

in

tera

cti

on

Hig

h s

ele

cti

vit

y (

low

sid

e-e

ffects

)

Po

ten

tial im

mu

no

gen

Lo

w p

erm

eab

ilit

y o

f m

em

bra

ne

Hig

h p

erm

eab

ilit

y o

f m

em

bra

ne

Hig

h s

ele

cti

vit

y a

nd

hig

h b

ind

ing

aff

init

y

H2N

R

OH

O

D-c

on

fig

ura

tio

ns

HN

R

OH

OM

e

N-m

eth

yl

0. peptidream 3

What is Peptidreamdoing?

�th

e l

ack o

f a s

uit

ab

le s

yn

theti

c m

eth

od

olo

gy f

or

pro

du

cin

g t

he d

ivers

e

co

mp

ou

nd

lib

rari

es r

eq

uir

ed

fo

r d

rug

dis

co

very

A m

ajor obstacle to the development of peptide drugs

Ch

em

ical

syn

thesis

Rib

oso

mal syn

thesis

Lim

ite

d t

o c

an

on

ica

l a

min

o a

cid

s

(> 1

0 p

ep

tid

e b

on

d f

orm

ati

on

/ s

ec

)

�th

e l

ack o

f a s

uit

ab

le s

cre

en

ing

meth

od

olo

gy f

rom

th

e h

ug

e p

ep

tid

e lib

rari

es

Ins

uff

icie

nt

lib

rary

siz

e

(ad

ap

tab

ilit

y t

o a

ny a

min

o a

cid

s)

0. peptidream 4

Prof. Hiroaki Suga

Ed

uc

ati

on

1986:

B.Sc., O

kayama U

niversity (Prof. Sigeru

Torii)

1987:

University of Lausanne (Prof. M

anfred Schlossor)

1989:

M.Sc., O

kayama U

niversity (Prof. Sigeru

Torii)

1994:

Ph.D., M

assachusetts Institute of Technology (Prof. Satoru M

asamune)

Pro

fes

sio

na

l c

are

er

1994-1997:

Postdoctoral fello

w, Massachusetts G

eneral Hospital,

Harvard M

edical School ( Prof. Jack W

. Szostak)

1997-2002:

Assistant professor, U

niversity at Buffalo,

The State U

niversity of New York

2002-2003:

Associate professor (tenured), U

niversity at Buffalo,

The State U

niversity of New York

2003-2005:

Associate professor, R

esearch C

enter for Advanced Science and Technology,

The University of Tokyo

2005-2010:

Professor, R

esearch C

enter for Advanced Science and Technology,

The University of Tokyo

2010-present:

Professor, G

raduate School of Science, The U

niversity of Tokyo

1.

Fle

xiz

ym

e(an RNA-based artificial aminoacyl-tRNAsynthetase)

2.

Fle

xib

le In

-vit

ro T

ran

sla

tio

n (

FIT

) syste

m

3.

RA

nd

om

Pep

tid

e I

nte

gra

ted

Dis

co

very

(R

AP

ID)

syste

m

Peptide Discovery Platform

System

0. peptidream 5

Peptidream’sTechnology (Today’s Contents)

Catalyst

Library

(Synthesis)

Screening

1. F

lexiz

ym

e

1. Flexizyme 6

~ A

cc

. C

he

m.

Re

s.,

20

11,

44(12), pp 1359–1368. ~

1. Flexizyme

Overview

7

Transcription & Translation

an

ti-c

od

on

lo

op

tRNA

5’

3’

A C C N

3’

Charging amino acids onto tRNAs

Fle

xiz

ym

e:

an RNA-based artificial A

RSs (rybozyme),

which has broad substrate scope of both

amino acids and tRNAs.

Am

ino

ac

yl

-tR

NA

syn

theta

ses

(AR

Ss):

a family of protein enzymes which charge

amino acids onto the 3’-term

inus of tRNA

aa= amino acid

vs

76

75

74

73

*

* d

isc

rim

ina

tin

g b

as

e

Historical Background(RNA World Hypothesis)

Th

e R

NA

wo

rld

hyp

oth

es

is:

Current life

Early life on earth

hypothesis that self-replicating R

NA m

olecules were precursors to current life, which

is based on DNA, RNA and proteins.

Catalyst for chemical reactions = P

rote

in

Storage of genetic inform

ation = D

NA

RN

A

1. Flexizyme 8

In order to prove the hypothesis, many

artificial ribozymes that resemble various

functions of current protein enzymes

have been developed.

PeptidylTransferase

1. Flexizyme 9

Lohse, P. A.; Szostak, J.

Na

ture

19

96,

38

1, 442.

Th

e f

irs

t a

rtif

icia

l ri

bo

zym

e s

co

pin

g o

f tR

NA

-lik

e m

ole

cu

le

Target reaction

Original reaction (PeptidylTransfer)

Methods (In vitro selection) & Results

Th

e p

oo

l R

NA

wa

s c

on

str

uc

ted

fro

m

co

mp

lete

ly r

an

do

m-s

eq

ue

nc

e R

NA

, w

ith

ou

t

bia

s t

ow

ard

an

y k

no

wn

se

qu

en

ce

or

str

uc

ture

.

controls

evolution

The First Artificial Aminoacyl-tRNASynthetase(step1)

1. Flexizyme 10

Lee, N.; Bessho, Y.; W

ei, K.; Szostak, J. W.; Suga, H.

Na

t. S

tru

ct.

Bio

l. 2

00

0,

7, 28.

Acyl transfer

from reagent to catalyst

acyla

tin

gre

ag

en

t

tRN

A

cata

lyst

(1)

(2)

(3)

(4)

Acyl transfer

from catalyst to tRNA

“ping-pong”

process

Aft

er

8 r

ou

nd

s

The First Artificial Aminoacyl-tRNASynthetase(step2) 1

1. Flexizyme 11

Lee, N.; Bessho, Y.; W

ei, K.; Szostak, J. W.; Suga, H.

Na

t. S

tru

ct.

Bio

l. 2

00

0,

7, 28.

Pre

vio

us a

rtif

icia

l ri

bo

zym

e (

AT

Rib

) →

no

sele

cti

vit

y t

ow

ard

am

ino

acid

Rib

ozym

e i

n R

NA

wo

rld

(?

) →

sele

cti

vit

y t

ow

ard

sp

ecif

ic a

min

o a

cid

vs

Amino acid

recognizing region

New design

Evo

luti

on

pre

ss

ure

1.

Se

lec

tivit

y t

ow

ard

sid

e c

ha

in o

f G

ln

2.

Re

ten

tio

n o

f th

e

ori

gin

al

oli

go

nu

cle

oti

de

-

rib

ozym

e a

cyl-

tra

ns

fer

rea

cti

on

Biotin-(L)-Gln-C

ME

Biotin-L-Phe-3'-ACCAAC-5'

The First Artificial Aminoacyl-tRNASynthetase(step2) 2

1. Flexizyme 12

Lee, N.; Bessho, Y.; W

ei, K.; Szostak, J. W.; Suga, H.

Na

t. S

tru

ct.

Bio

l. 2

00

0,

7, 28.

Amino acids selectivity of evolved ribozyme

1. Biotin-(L)-

Gln

-CME

2. Biotin-(L)-

Ph

e-3

'-A

CC

AA

C-5

'

3. Biotin-(L)-

Ph

e-C

ME

4. Biotin-(L)-

Le

u-C

ME

5. Biotin-(L)-

Va

l-CME

Ribozyme-catalyzed aminoacylationof tRNA

the

co

mp

lex

me

ch

an

ism

s i

nvo

lvin

g

the

eq

uil

ibri

um

sh

ift

of

ac

yl-

tra

ns

fer

ch

em

istr

ysu

sp

en

ded

Next Design (Catalysis by 5’-Leader)

1. Flexizyme 13

Ho

w t

o c

ha

rge

sp

ec

ific

am

ino

ac

id o

nto

tR

NA

?

aa-tRNAmaturation pathway

5’ le

ad

er

do

main

has n

o f

un

cti

on

In t

he “

mo

dern

wo

rld

”.

Cata

lyti

c a

cti

vit

y ?

Pre

vio

us

Str

ate

gy

�trans

-ac

tin

g r

yb

ozym

e

�c

ata

lys

t d

om

ain

an

d r

ec

og

nit

ion

do

ma

in w

ere

evo

lve

d separately.

Th

is t

ime

Str

ate

gy

�cis

-ac

tin

g r

yb

ozym

e

�c

ata

lys

t d

om

ain

an

d r

ec

og

nit

ion

do

ma

in w

ere

evo

lve

d at the same tim

e.

5’ le

ad

er

ca

taly

tic

do

ma

in c

an

be

re

mo

ve

d b

y M

1-

lik

e r

ibo

zym

e t

o y

ield

ma

ture

am

ino

ac

yl-

tRN

As

.

Th

e c

urr

en

t w

orl

dA

po

ss

ible

ea

rly w

orl

d

Aminoacylationby 5’-Leader Sequence Domain 1

1. Flexizyme 14

Saito, H.; Kourouklis, D.; Suga, H.

EM

BO

J.

20

01,

20, 1797.

controls

evolution

3’-

OH

of

3’-

term

ina

l

ad

en

os

ine

wa

s s

ele

cti

ve

ly

am

ino

ac

yla

ted

!

for detail discussion,

J.

Am

. C

he

m.

So

c. 2

00

1,

12

3, 7178.

Methods

Results

Aminoacylationby 5’-Leader Sequence Domain 2

1. Flexizyme 15

Saito, H.; Kourouklis, D.; Suga, H.

EM

BO

J.

20

01,

20, 1797.

(L)-

Tyr

0.5

5;

(D)-

Ph

e0

.18

Aft

er

am

ino

acyla

tio

n, th

e b

ioti

nyla

tio

nw

as p

erf

orm

ed

.

Amino acids selectivity

RNaseP RNA cleavage of pre-tRNA

Trans-aminoacylation!

Th

e c

riti

cal

reco

gn

itio

n e

lem

en

t is

the p

hen

yla

lan

yl

(ben

zyl)

sid

e c

hain

.

Expanding the Scope of Catalyst

1. Flexizyme

Basic

S

cie

nce

Ap

plied

Scie

nce

Sp

ecif

icit

y

Vers

ati

lity

16

Expanding the Scope of tRNA1

17

1. Flexizyme

Murakami, H

.; Saito, H.; Suga, H.

Ch

em

. B

iol.

20

03,

10, 655.

RN

A2

00

1,

7, 1867.

Nu

cle

ic A

cid

s R

es

. 2

00

2,

30

, 5151.

for biochemical structural studyies,

Original Ribozyme

Modification Design

Expanding the Scope of tRNA2

18

1. Flexizyme

Murakami, H

.; Saito, H.; Suga, H.

Ch

em

. B

iol.

20

03,

10, 655.

Original ribozyme (r24mini)

Evolved ribozyme (Flexizyme)

Just Deleting

N46~ N

56

Comparison of the theribozyme activities

between Flexizymes(Fx2–5) and r24mini.

Aminoacylationactivities of Fx3 and r24mini

toward various tRNAs.

Fle

xiz

ym

e(F

x3

) s

ho

ws

gre

ate

r a

cti

vit

y t

ow

ard

va

rio

us

tR

NA

s!

Expanding the Scope of Amino Acids

1. Flexizyme

Murakami, H

.; O

hta, A.; Ashigai, H.; Suga, H.

Na

t. M

eth

od

s 2

00

6,

3, 357.

19

Hypothesis

Fx

3 o

nly

re

co

gn

ize

the

be

nzyl

gro

up

.

Be

nzyl

gro

up

is

em

be

dd

ed

in l

ea

vin

g g

rou

ps

.

Modification Design

Scope of side chains

(A)

L-A

min

o a

cid

wit

h n

on

pro

tein

og

en

ics

ide

ch

ain

. (B

) D

-Am

ino

ac

id.

(C)

R-N

-ac

yl-

am

ino

ac

id.

(D)

R-N

-Alk

yl-

am

ino

ac

id.

(E)

β-A

min

o a

cid

. (F

) R

-Hyd

rox

ya

cid

.

(G)

R-N

-Pe

pti

dyl-

am

ino

ac

id.

(H)

γ-A

min

oa

cy

l-a

min

o a

cid

.

1. Flexizyme 20

Xiao, H.; M

urakami, H

.; Suga, H.; Ferre-D

'Amare, A. R.

Na

ture

20

08,

45

4, 358.

1. Flexizyme

FlexizymeStructure (Crystal Structural Studies) 1

U1

A

P1

P1

a

J2/1

a

J1

a/3

P2

J1

a/2

The secondary structure

Interaction m

odel with full-length tRNA

21

Xiao, H.; M

urakami, H

.; Suga, H.; Ferre-D

'Amare, A. R.

Na

ture

20

08,

45

4, 358.

1. Flexizyme

FlexizymeStructure (Crystal Structural Studies) 2

22

cri

ticall

y i

mp

ort

base-p

air

s

Xiao, H.; M

urakami, H

.; Suga, H.; Ferre-D

'Amare, A. R.

Na

ture

20

08,

45

4, 358.

1. Flexizyme

FlexizymeStructure (Crystal Structural Studies) 3

23

Am

ino

Ac

id R

ec

og

nit

ion

:

Interaction with O

6 of G24 by its partial positive charge at the center of the phenyl ring

tRN

AA

cc

ep

tor

En

d R

ec

og

nit

ion

:

(1)a partial cross-strand stack of the base of tA76 w

ith tC75-G

55 pair

(2)the base of tA76 m

akes van der Waals contact with the ribose of G24

(3)a hydrogen bond between N

1 of tA76 and the 2’-OH of A23

(4)a hydrogen bond between the 2’-OH of tA76 and N2 of G24

Small summary

an

ti-c

od

on

lo

op

5’

3’

A C C N

Charging amino acids onto tRNAs

tRNARecognition: N73-C75 (+A76)

Amino Acid Recognition: Bnof LG

aa= amino acid

76

75

74

73

*

* d

iscri

min

ati

ng

base

1. Flexizyme 24

2. F

IT s

yste

m

2. FlTsystem 25

~ N

at.

Pro

toc

. 2

011

, 6

, p

p7

79

–7

90

.~

Overview

26

2. FlTsystem

Reconstituted cell-free translation system

(from G

eneFrontierCorporation)

aa= amino acid

Flexizyme

+

= F

IT s

yste

m

27

2. FlTsystem

Non-Canonical AAs onto tRNA(the Other Ways)

1. Aminoacyl-tRNASynthetases(ARSs)

Mis

s-a

cyla

tio

n w

ith

natu

ral A

RS

s

SzostakJ. W.

et

al.

Pro

c.

Na

tl.

Ac

ad

. S

ci.

US

A2

00

6,

10

3, 4356.

SzostakJ. W.

et

al.

PL

oS

On

e 2

20

07

, e972.

Tirrell, D. A.

et

al.

Me

tho

ds,

20

05,

36, 291.

En

gin

eere

d A

RS

s

2. Chemical Synthesis

Hecht, S.M

.; Alford B.L.; Kuroda Y.; Kitano S.

J.

Bio

l. C

he

m.

19

78,

25

3, 4517.

Schultz, P.G

. e

t a

l. J

. A

m.

Ch

em

. S

oc

. 1

99

1,

11

3, 2722.

2. FlTsystem

Application

28

HuisgenCycloaddition

Michael Addition

spontaneously

SN2 Reaction

Pep

toid:

artificially designed peptides composed of N-substituted glycine building blocks

Goto, Y.; Katoh, T.; Suga, H.

Na

t. P

roto

c.

20

11,

6, 779.

3. R

AP

ID s

yste

m

29

3. RAPID system

~ C

he

mis

try

& B

iolo

gy

20

11,

18, 1562. ~

30

In vitro selection of RNA and DNA

3. RAPID system

How to Select Bioactive-Peptide ?

Inform

ation Flow

Sc

ree

nin

g p

ep

tid

es

on

e b

y o

ne

??

?

Ph

en

oty

pe

mo

lec

ule

Ge

no

typ

e m

ole

cu

le

31

3. RAPID system

mRNA Display (In Vitro Virus)

Nemoto, N.; M

iyamoto-Sato, E.; H

usim

i, Y.; Yanagawa, H.

FE

BS

Le

tt.

19

97,

41

4, 405.

Roberts, R. W.; Szostak, J. W.

Pro

c.

Na

tl.

Ac

ad

. S

ci.

US

A1

99

7,

94, 12297.

Keefe, A. D.; Szostak, J. W.

Na

ture

20

01,

41

0, 715.

3. RAPID system 32

Overview

Yamagishi, Y.; Shoji, I.; M

iyagawa, S.; Kawakami, T.; Katoh, T.; G

oto, Y.; Suga, Y.

Ch

em

istr

y &

Bio

log

y 2

011,

18, 1562.

FIT System + m

RNA Display = RAPID System

3. RAPID system 33

Application to the selective inhibitor against E6AP

Yamagishi, Y.; Shoji, I.; M

iyagawa, S.; Kawakami, T.; Katoh, T.; G

oto, Y.; Suga, Y.

Ch

em

istr

y &

Bio

log

y 2

011,

18, 1562.

Reprogrammed genetic code

E6 protein originates from the high-risk types 16 and 18 human papillomavirus (HPV).

Selected M

acrocyclicN-m

ethyl-peptide

E6AP

3. RAPID system 34

Application to the selective inhibitor against E6AP

Yamagishi, Y.; Shoji, I.; M

iyagawa, S.; Kawakami, T.; Katoh, T.; G

oto, Y.; Suga, Y.

Ch

em

istr

y &

Bio

log

y 2

011,

18, 1562.

Kd

= 0

.60

nM

Lin

ea

r (n

o t

hio

ete

rli

nk

ag

e)

Kd

= 1

80

nM

N-H

am

ino

ac

ids

(n

o N

-Me

)

Kd

> 1

00

0 n

M

Bo

th t

hio

es

ter

lin

ka

ge

& N

-Me

mo

dif

ica

tio

n a

re e

ss

en

tia

l !

4. S

um

mary

35

4. Summary

4. Summary

The RNA world hypothesis

Flexizyme

Reconstituted cell-free

translation system

+

FIT system

+

mRNA Display

RAPID system

36

Peptidream

Inc.

Ribosomal Synthesis of Unnatural Peptides

Josephson, K.; Hartman, M. C. T.; Szostak, J. W.

J.

Am

. C

he

m.

So

c., 2

005,

12

7, 11727.

Limitation

4. Summary 37

Even

th

ou

gh

fle

xiz

ym

es

facil

itate

th

e p

rep

ara

tio

n o

f tR

NA

sch

arg

ed

wit

h D

-

am

ino

acid

s o

r β

-am

ino

acid

s,

so

me o

f th

ese a

min

o a

cid

s c

ou

ld n

ot

be c

on

secu

tively

elo

ng

ate

d b

ecau

se

of

po

or

co

mp

ati

bil

ity o

f n

atu

rall

y o

ccu

rrin

g r

ibo

so

mes.

(It

sh

ou

ld b

e n

ote

d t

hat

the i

nit

iati

on

even

t tu

rned

ou

t to

be m

ore

am

en

ab

le

to a

wid

e v

ari

ety

of

am

ino

acid

s.)