April 26th (Sat.), 2014
Takuya Matsumoto (D2)

~ Case Study of Peptidream ~How was an artificial ribozyme
developed into bio-venture.

0. peptidream

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

1

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PeptidreamX

Too small for inhibiting
protein-protein interaction

OX

Antibody

0. peptidream

High selectivity and high binding affinity

High permeability of membrane

OOHOOHN-methylHN
MeR

D-configurations

H2N

R

Potential immunogen
Low permeability of membrane

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Inhibitive capacity of proteinprotein interaction
High selectivity (low side-effects)‘Non-Standard’ Peptide

OHigh permeability of intestinalor cell membrane (oral dose)

Small Molecule

Why Peptide?

0. peptidream

Limited to canonical amino acids(> 10 peptide bond formation / sec)

Ribosomal synthesis3 the lack of a suitable screening methodology from the huge peptide libraries

Insufficient library size
(adaptability to any amino acids)

Chemical synthesis

 the lack of a suitable synthetic methodology for producing the diverse
compound libraries required for drug discovery

A major obstacle to the development of peptide drugs

What is Peptidream doing?

Professional career
1994-1997:
Postdoctoral fellow, Massachusetts General Hospital,
Harvard Medical School (Prof. Jack W. Szostak)
1997-2002:
Assistant professor, University at Buffalo,
The State University of New York
2002-2003:
Associate professor (tenured), University at Buffalo,
The State University of New York
2003-2005:
Associate professor, Research Center for Advanced Science and Technology,
The University of Tokyo
2005-2010:
Professor, Research Center for Advanced Science and Technology,
The University of Tokyo
2010-present:
Professor, Graduate School of Science, The University of Tokyo

Education
1986:
B.Sc., Okayama University (Prof. Sigeru Torii)
1987:
University of Lausanne (Prof. Manfred Schlossor)
1989:
M.Sc., Okayama University (Prof. Sigeru Torii)
1994:
Ph.D., Massachusetts Institute of Technology (Prof. Satoru Masamune)

Prof. Hiroaki Suga

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0. peptidreamScreening

3. RAndom Peptide Integrated Discovery (RAPID) system

2. Flexible In-vitro Translation (FIT) system

Catalyst

1. Flexizyme (an RNA-based artificial aminoacyl-tRNA synthetase)

Peptide Discovery Platform System

Peptidream’s Technology (Today’s Contents)

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Library(Synthesis)

0. peptidream~ Acc. Chem. Res., 2011, 44 (12), pp 1359–1368. ~1. Flexizyme

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1. Flexizyme

aa = amino acid

Charging amino acids onto tRNAs

Transcription & Translation

Overview

5’3’

anti-codon loop

Flexizyme:
an RNA-based artificial ARSs (rybozyme),
which has broad substrate scope of bothamino acids and tRNAs.

vs7* discriminating base

A 76
C 75
C 74
N 73*

3’

Aminoacyl - tRNA synthetases (ARSs):a family of protein enzymes which chargeamino acids onto the 3’-terminus of tRNAtRNA

1. Flexizyme

Storage of genetic information = DNACatalyst for chemical reactions = Protein

Current life

In order to prove the hypothesis, many
artificial ribozymes that resemble various
functions of current protein enzymes
have been developed.

RNAEarly life on earth

81. Flexizyme

hypothesis that self-replicating RNA molecules were precursors to current life, which
is based on DNA, RNA and proteins.

The RNA world hypothesis:

Historical Background (RNA World Hypothesis)evolutioncontrols

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1. Flexizyme

Original reaction (Peptidyl Transfer)

Lohse, P. A.; Szostak, J. Nature 1996, 381, 442.

The pool RNA was constructed fromcompletely random-sequence RNA, withoutbias toward any known sequence or structure.

Methods (In vitro selection) & Results

Target reaction

The first artificial ribozyme scoping of tRNA-like molecule

Peptidyl Transferase

(4)Acyl transferfrom catalyst to tRNA“ping-pong”
process

Acyl transferfrom reagent to catalyst

(3)

(2)

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1. Flexizyme

Lee, N.; Bessho, Y.; Wei, K.; Szostak, J. W.; Suga, H. Nat. Struct. Biol. 2000, 7, 28.

After 8 roundstRNAacylating reagent

catalyst(1)

The First Artificial Aminoacyl-tRNA Synthetase (step1)

2.

1.

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

Biotin-(L)-Gln-CME

Evolution pressure

11Retention of theoriginal
oligonucleotideribozyme acyl-transfer
reaction

Selectivity toward
side chain of Gln

Lee, N.; Bessho, Y.; Wei, K.; Szostak, J. W.; Suga, H. Nat. Struct. Biol. 2000, 7, 28.

Amino acidrecognizing region

New design

Ribozyme in RNA world (?) → selectivity toward specific amino acidvsPrevious artificial ribozyme (ATRib) → no selectivity toward amino acid

The First Artificial Aminoacyl-tRNA Synthetase (step2) 1 1. Flexizyme

suspended

1. Biotin-(L)-Gln-CME
2. Biotin-(L)-Phe-3'-ACCAAC-5'
3. Biotin-(L)-Phe-CME
4. Biotin-(L)-Leu-CME
5. Biotin-(L)-Val-CME

Lee, N.; Bessho, Y.; Wei, K.; Szostak, J. W.; Suga, H. Nat. Struct. Biol. 2000, 7, 28.

the complex mechanisms involving
the equilibrium shift of acyl-transfer chemistry

Ribozyme-catalyzed aminoacylation of tRNA

Amino acids selectivity of evolved ribozyme

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The First Artificial Aminoacyl-tRNA Synthetase (step2) 2 1. Flexizyme

This time Strategy

Previous Strategy

1. Flexizyme

A possible early world

Catalytic activity ?

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 catalyst domain and recognition domain wereevolved at the same time.
5’ leader catalytic domain can be removed by M1like ribozyme to yield mature aminoacyl-tRNAs.

 cis-acting rybozyme

 catalyst domain and recognition domain wereevolved separately.

 trans-acting rybozyme

5’ leader domain has no function
In the “modern world”.

The current world

aa-tRNA maturation pathway

How to charge specific amino acid onto tRNA ?

Next Design (Catalysis by 5’-Leader)Results

Methods

controls

1. Flexizyme

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for detail discussion,
J. Am. Chem. Soc. 2001, 123, 7178.

3’-OH of 3’-terminal
adenosine was selectivelyaminoacylated !

Saito, H.; Kourouklis, D.; Suga, H. EMBO J. 2001, 20, 1797.

evolution

Aminoacylation by 5’-Leader Sequence Domain 1

Trans-aminoacylation !

Saito, H.; Kourouklis, D.; Suga, H. EMBO J. 2001, 20, 1797.

RNase P RNA cleavage of pre-tRNA

1. Flexizyme

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The critical recognition element is
the phenylalanyl (benzyl) side chain.

(L)-Tyr 0.55; (D)-Phe 0.18

After aminoacylation, the biotinylation was performed.

Amino acids selectivity

Aminoacylation by 5’-Leader Sequence Domain 2

Specificity
Versatility

Applied Science16

1. FlexizymeScienceBasicExpanding the Scope of Catalyst

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1. Flexizyme

Modification Design

Murakami, H.; Saito, H.; Suga, H. Chem. Biol. 2003, 10, 655.

Nucleic Acids Res. 2002, 30, 5151.

RNA 2001, 7, 1867.

for biochemical structural studyies,

Original Ribozyme

Expanding the Scope of tRNA 1

Evolved ribozyme (Flexizyme)

1. Flexizyme

Murakami, H.; Saito, H.; Suga, H. Chem. Biol. 2003, 10, 655.

Flexizyme (Fx3) shows greater activity toward various tRNAs !
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Aminoacylation activities of Fx3 and r24mini
toward various tRNAs.

Just Deleting
N46 ~ N56

Comparison of the the ribozyme activities
between Flexizymes (Fx2–5) and r24mini.

Original ribozyme (r24mini)

Expanding the Scope of tRNA 2

Benzyl group is embedded
in leaving groups.

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1. Flexizyme

Murakami, H.; Ohta, A.; Ashigai, H.; Suga, H. Nat. Methods 2006, 3, 357.

Modification Design

Fx3 only recognize
the benzyl group.

Hypothesis

Expanding the Scope of Amino Acids

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1. Flexizyme(A) L-Amino acid with nonproteinogenic side chain. (B) D-Amino acid. (C) R-N-acylamino acid.
(D) R-N-Alkyl-amino acid. (E) β-Amino acid. (F) R-Hydroxy acid.
(G) R-N-Peptidyl-amino acid. (H) γ-Aminoacyl-amino acid.

Scope of side chains

P2J2/1aP1aP1U1A

J1a/3

J1a/2

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Interaction model with full-length tRNA

1. Flexizyme

Xiao, H.; Murakami, H.; Suga, H.; Ferre-D'Amare, A. R. Nature 2008, 454, 358.

The secondary structure

Flexizyme Structure (Crystal Structural Studies) 1

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1. Flexizyme

Xiao, H.; Murakami, H.; Suga, H.; Ferre-D'Amare, A. R. Nature 2008, 454, 358.

critically import base-pairs

Flexizyme Structure (Crystal Structural Studies) 2

Xiao, H.; Murakami, H.; Suga, H.; Ferre-D'Amare, A. R. Nature 2008, 454, 358.

tRNA Acceptor End Recognition:(1) a partial cross-strand stack of the base of tA76 with tC75-G55 pair(2) the base of tA76 makes van der Waals contact with the ribose of G24(3) a hydrogen bond between N1 of tA76 and the 2’-OH of A23(4) a hydrogen bond between the 2’-OH of tA76 and N2 of G24

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1. Flexizyme

Amino Acid Recognition:
Interaction with O6 of G24 by its partial positive charge at the center of the phenyl ringFlexizyme Structure (Crystal Structural Studies) 3Amino Acid Recognition: Bn of LG

aa = amino acid

Charging amino acids onto tRNAsSmall summary

5’3’anti-codon loop

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* discriminating base

A 76
C 75
C 74
N 73*tRNA Recognition: N73-C75 (+A76)

1. Flexizyme

~ Nat. Protoc. 2011, 6, pp779–790. ~2. FIT system25

2. FlT system

Flexizyme

aa = amino acid+

Reconstituted cell-free translation system

Overview= FIT system
26(from GeneFrontier Corporation)

2. FlT systemHecht, S.M.; Alford B.L.; Kuroda Y.; Kitano S. J. Biol. Chem. 1978, 253, 4517.
Schultz, P.G. et al. J. Am. Chem. Soc. 1991, 113, 2722.

2. Chemical Synthesis

Szostak J. W. et al. PLoS One 2 2007, e972.

2. FlT system

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Tirrell, D. A. et al. Methods, 2005, 36, 291.

Engineered ARSs

Szostak J. W. et al. Proc. Natl. Acad. Sci. USA 2006, 103, 4356.

Miss-acylation with natural ARSs

1. Aminoacyl - tRNA Synthetases (ARSs)

Non-Canonical AAs onto tRNA (the Other Ways)

spontaneouslyGoto, Y.; Katoh, T.; Suga, H. Nat. Protoc. 2011, 6, 779.

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2. FlT system

Peptoid:
artificially designed peptides composed of N-substituted glycine building blocks

SN2 Reaction

Michael Addition

Huisgen Cycloaddition

Application

~ Chemistry & Biology 2011, 18, 1562. ~3. RAPID system

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3. RAPID system

Phenotype molecule

Genotype molecule

Screening peptides one by one ???

In vitro selection of RNA and DNA

Information Flow

How to Select Bioactive-Peptide ?

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3. RAPID systemKeefe, A. D.; Szostak, J. W. Nature 2001, 410, 715.

Roberts, R. W.; Szostak, J. W. Proc. Natl. Acad. Sci. USA 1997, 94, 12297.

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3. RAPID system

Nemoto, N.; Miyamoto-Sato, E.; Husimi, Y.; Yanagawa, H. FEBS Lett. 1997, 414, 405.

mRNA Display (In Vitro Virus)

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3. RAPID system

Yamagishi, Y.; Shoji, I.; Miyagawa, S.; Kawakami, T.; Katoh, T.; Goto, Y.; Suga, Y.
Chemistry & Biology 2011, 18, 1562.

FIT System + mRNA Display = RAPID SystemOverview

3. RAPID system

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Selected Macrocyclic N-methyl-peptide

Yamagishi, Y.; Shoji, I.; Miyagawa, S.; Kawakami, T.; Katoh, T.; Goto, Y.; Suga, Y.
Chemistry & Biology 2011, 18, 1562.

Reprogrammed genetic code

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

E6AP

Application to the selective inhibitor against E6AP

N-H amino acids (no N-Me)
Kd > 1000 nM

Yamagishi, Y.; Shoji, I.; Miyagawa, S.; Kawakami, T.; Katoh, T.; Goto, Y.; Suga, Y.
Chemistry & Biology 2011, 18, 1562.

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3. RAPID system

Both thioester linkage & N-Me modification are essential !

Linear (no thioeter linkage)
Kd = 180 nM

Kd = 0.60 nM

Application to the selective inhibitor against E6AP

4. Summary

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4. Summary

FIT system+Ribosomal Synthesis of Unnatural Peptides
Josephson, K.; Hartman, M. C. T.; Szostak, J. W.
J. Am. Chem. Soc., 2005, 127, 11727.

Flexizyme

The RNA world hypothesismRNA Display+36

Peptidream Inc.

RAPID system

Reconstituted cell-freetranslation system

4. Summary

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4. Summary

Even though flexizymes facilitate the preparation of tRNAs charged with Damino acids or β-amino acids,some of these amino acids could not be consecutively elongated becauseof poor compatibility of naturally occurring ribosomes.
(It should be noted that the initiation event turned out to be more amenable
to a wide variety of amino acids.)

Limitation