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Biomimetics

Chemomimetics How?

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USING EVOLUTIONARY DESIGN TO EXPAND

GENETICALLY ENCODED CHEMISTRY

FAPESP SEMINAR

São Paulo, April 15th 2014

WWW.PROVIVI.COM

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Who We Are

Our breakthrough technology was developed in the Arnold lab at Caltech

― Pedro Coelho, Ph.D.

• Co-founder and CEO

• Winner of Demetriades and McCoy awards for best PhD thesis in biotechnology and chemistry at Caltech

― Prof. Frances Arnold, Ph.D.

• Co-founder and board member

• Pioneer of “directed evolution”

• Elected to all three National Academies in the U.S.A. (Sciences, Engineering, Medicine)

• Co-founder of Gevo, Inc. (NASDAQ:GEVO)

― Peter Meinhold, Ph.D.

• Co-founder and CTO

• Named one of world’s top innovators under the age of 35 by MIT’s Technology Review magazine

• Co-founder of Gevo, Inc. (NASDAQ:GEVO)

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Provivi

We engineer biocatalysts to perform synthetic chemical functions

Unique Solutions in Agchem Mission

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“To use the power of synthetic

biology to make products with

improved performance”

Strategy

End-user solutions driven

Initial Focus: Agchem

1. Synthetic pyrethrum,

currently sourced from

flowers in Africa

2. Cyclopropyl isosteres to

create products with

improved performance

3. High efficacy enantiopure

pesticides

First Nature Identical Pyrethrum

Advantages:

• Very safe broad spectrum insecticide

• Non-persistent. Fast photolysis

• Few cases of resistance reported

• Ideal for household and post-harvest crop

protection

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chrysanthemate

pryrethrolone

Challenges:

• Unstable supply chain. Chrysanthemum

farmed mainly in East Africa and Australia

• Complex mixture of six esters

• Biosynthesis in plant not fully elucidated

• Significantly more expensive than

pyrethroids and allethrin

Cyclopropyl Isosteres for Improved Products

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Bioorg. Med. Chem. Lett. 18, 4118 (2008) (Bristol-Myers Squibb) Bioorg. Med. Chem. Lett. 17, 828 (2007) (Merck)

Bioorg. Med. Chem. Lett. 19, 1575 (2009) (Japan Tobacco)

O

NC

O N

N

COOH

COOH

COOH

t1/2 (rat) = 0.7 h

trans t1/2 (rat) = 5.9 h

cis t1/2 (rat) = 7.4 h

Inreased metabolic stability

ATR

AZIN

E

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• 1950–1993 non-biodegradable; accummulated in soil

• From 1993 onwards, fast degradation was observed

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?

ATR

AZIN

E CH

LOR

OH

YDR

OLA

SE

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J. L. Seffernick et al., Rapid Evolution of Bacterial Catabolic Enzymes: A Case Study with Atrazine Chlorohydrolase. Biochemistry 40, 12747 (2001)

9 amino acid mutations (98% sequence identity)

The introduction of synthetic compounds drives the evolution of novel catabolic activities

ATR

AZIN

E CH

LOR

OH

YDR

OLA

SE

“NA

TUR

A NO

N FA

CIT SA

LTUS”

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DN

A

NO

NN

ATU

RA

L CA

TALYSIS

Computational design

Mechanism-based modeling

e.g. diels-alder, retro-aldol, Kemp

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Hybrid Bio-TM catalysts

Artificial co-factor in a host protein

e.g. hydrogenation, C-H activation

D. Baker et al., PLoS One 6, e19230 (2011) T. R. Ward, Acc. Chem. Res. 44, 47 (2011)

Do Novel Reactions Require New Active Sites?

DN

A

NO

NN

ATU

RA

L CA

TALYSIS

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novel reactions

novel enzymes old enzymes

?

1. “enzymes evolve because they have evolved” 2. high performance in vivo

DN

A

NO

NN

ATU

RA

L CA

TALYSIS

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nonnatural reactions

old scaffold (e.g. P450s)

natural reactions

C–H C–OH

C=C epoxide

R2S R2SO R2SO2

RCOH RH + CO2

RCH2CH2R’ RHC=CHR’

natural selection + biological reagents

directed evolution + synthetic reagents

C=C cyclopropanes

C–H C–NHR

(por.+)FeIV=O

Fe=X (X = O, NR, CR2)

Isoelectronic

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O, NR, and CR2 Transfers Share Mechanistic Features

1. Do P450s show promiscuous cyclopropanation activity?

2. Is there an evolutionary pathway for improving the novel activity?

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Heme Proteins Show Promiscuous Activity

for Olefin Cyclopropanation

16 P. S. Coelho, E. M. Brustad, A. Kannan, F. H. Arnold, Science 339, 307 (2013)

* Diastereomeric ratios and enantiomeric excess were determined by GC analysis. † (R,S) – (S,R). ‡ (R,R) – (S,S). § Bioconversion conducted at 0.1 M citrate buffer pH = 4.0.

catalyst axial ligand cat. loading

(% mol eq) TTN cis:trans*

%ee

cis†

%ee

trans‡

catalase O-Tyr 0.16 0 - - -

CPO§ S-Cys 0.40 0 - - -

HRP N-His 1.00 9 7:93 8 -7

cyt c N-His, S-Met 1.00 19 6:94 0 12

Mb N-His 1.00 43 6:94 -1 2

P450BM3 S-Cys 0.20 5 37:63 -27 -2

hemin - 0.20 73 6:94 -1 0

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Engineered P450BM3 Variants Are Stereoselective

Cyclopropanation Catalysts

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Screened 92 P450BM3 variants for activity and altered stereoselectivity. Top 10:

P450 % yield* TTN cis:trans† %ee cis %ee trans

WT 1 5 37:63 -10 -9

WTF87A 1.2 6 37:63 26 -6

H2A10 33.4 167 60:40 -95 -78

H2-4-D4 41.2 206 53:47 -79 -33

H2-5-F10 58.8 294 16:84 -41 -63

C2C12R1 1.6 8 36:64 45 1

C3E4R1 1.6 8 43:57 51 -7

X7R1 2.4 12 33:67 23 -4

12 R1 6.2 31 17:83 9 -2

C2E6 R1 4.6 23 27:73 25 -6

C2G9 R1 48 240 9:91 10 -2

7-11D 32 160 35:65 -22 -18 * based on EDA. † Diastereomeric ratios and enantiomeric excess were determined by GC analysis.

P. S. Coelho, E. M. Brustad, A. Kannan, F. H. Arnold, Science 339, 307 (2013)

Related to 9-10A TS F87V (inactive)

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BM3-T268A is Highly Active

18 P. S. Coelho, E. M. Brustad, A. Kannan, F. H. Arnold, Science 339, 307 (2013)

P450 (Holo) V78 F87 T268 I263 TTN cis:trans %ee cis %ee trans

WT - - - - 5 37:63 -10 -9

WT-F87A - A - - 6 38:62 26 -6

WT-F87V - V - - 9 30:70 -33 -26

WT-T268A - - A - 323 1:99 -15 -96

WT-F87V T268A - V A - 274 32:68 -77 -99

WT-V78A F87V T268A A V A - 190 32:68 -70 -20

A single active site mutation is sufficient

Mutations outside the active-site also influence the stereochemical outcome

still trans selective!

Stereoselective Biocatalytic Cyclopropanation

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P. S. Coelho, E. M. Brustad, A. Kannan, F. H. Arnold, Science 339, 307 (2013)

15.0 17.5 20.0 22.5 25.0 27.5

-4.0

-3.0

-2.0

-1.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

uV(x1,000)

Chromatogram

Hemin

P450BM3

-T268A

15.0 17.5 20.0 22.5 25.0 27.5

-0.50

-0.25

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

4.00

uV(x10,000)

BM3-CIS

BM3-CIS-T438S

15.0 17.5 20.0 22.5 25.0 27.5-0.50

-0.25

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

4.00

4.25uV(x10,000)

Ph COOEt

SS

Ph COOEt

S R

COOEt

R R

Ph

R S

COOEtPh

OEt

O

N

N

+

cis trans

15.0 17.5 20.0 22.5 25.0 27.5-0.50

-0.25

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

4.00

4.25uV(x10,000)

catalyst (0.2 mol%)

styrene EDAChemomimetics

Non-Natural Reactions Catalyzed in vivo

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• No protein purification

• Novel metabolic pathways

• Biobased production of

chemicals currently made

with synthetic methods

• Expand the chemical

toolbox for in vivo studies of

cellular function

Motivation Challenges

• Biocatalyst assembly in

functional form in vivo

• Biocompatibility and

permeability of the

synthetic reagents

• Need to drive the reaction

with NAD(P)H

• Is EDA stable in the

presence of cells?

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Redox “Gating” of the P450 Cycle

Redox diagram from: Daff et al., Redox control of the catalytic cycle of flavocytochrome P-450 BM3. Biochemistry 36, 13816 (1997)

NADPH

Need decent substrate binding

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Rationale for Axial Serine Ligation

1) Increase the FeIII/FeII redox

potential and therefore facilitate

NAD(P)H driven reduction

1) Reported to abolish

monooxygenation activity when

introduced in a mammalian P450.1

1 K. P. Vatsis, H. M. Peng, M. J. Coon, J. Inorg. Biochem. 91, 542 (2002) P. S. Coelho et al. Nature Chemical Biology 9, 485–487 (2013)

TEC

HN

OLO

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EVELO

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C400S Does NOT Alter the Crystal Structure

X-ray structure confirms serine-heme ligation

TEC

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P. S. Coelho et al. Nature Chemical Biology 9, 485–487 (2013)

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P411 Does NOT Look Like a P450

FeII-CO peak at 411 nm; no longer a “P-450”

BM3-CIS ABC-CIS

P. S. Coelho et al. Nature Chemical Biology 9, 485–487 (2013)

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Reduction Potential: Resting P411 > NADPH

ΔE° = +130 mV similar to BM3 – BM3(palmitate)

Maraia Ener

Na2S2O4 −660

−420

−290

−320

BM3

NAD(P)H

ABC

+130 mV

E°’ (mV vs NHE)

TEC

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P. S. Coelho et al. Nature Chemical Biology 9, 485–487 (2013)

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NADPH Driven Cyclopropanation in vitro T

ECH

NO

LOG

Y DEV

ELOP

MEN

T

P. S. Coelho et al. Nature Chemical Biology 9, 485–487 (2013)

In vivo Cyclopropanation: The New State-of-the-Art

• P450 wild-type: 5 total turnovers

• P411 in vivo: 67,000 total turnovers

• Previous state-of-the-art (Rh): 45,000

total turnovers

• High product titer: ~ 30 g L-1

• High productivity: > 1 g L-1 h-1

• High enantioselectivity: 99% eecis

• Enzyme is active inside intact bacterial

cells. No enzyme purification is

required

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P. S. Coelho et al. Nature Chemical Biology 9, 485–487 (2013)

• Lyophilized whole cells,

buffer, substrates and

nothing else

Readily Applied to Commercial Product Synthesis

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• Levomilnacipran (Fetzima) is a selective serotonin and

norephineprine reuptake inhibitor

• Recently approved by FDA for treatment of clinical depression

• This enzymatic route presents an attractive green alternative that

compares favorably to current synthesis of levomilnacipran

Z. J. Wang et al. Angew. Chemie Int. Ed. In press

Proprietary Technology: Novel Reactions Beyond Cyclopropanation

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LATFO

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$10Bn Opportunity in Chiral Crop Protection Chemicals

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NA

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ESTICID

ES Non-chiral 70%

Single isomer 8%

Mixed Isomers

22% Chiral 30%

Note: 73% of all chiral pesticides are still sold as mixed isomers

All pesticides

Single enatiomers reduce application

rates and undesired side effects

Syngenta’s asymmetric synthesis of (S)-metolachlor

Provivi’s chiral synthesis has the potential

to enable the launch of single

enantiomers on a cost competitive unit

activity basis

Conclusions

Science:

• Old scaffolds, new reactions

• Small changes in structure, big

changes in function

• Fe can go quite far…

• Why no Ser-Heme proteins in

nature?

Provivi’s technology:

• Novel proprietary technology with

broad synthetic applications

• Provivi established a new benchmark

for olefin cyclopropanation

• Has been demonstrated on a first

commercial target

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Acknowledgements

• Frances Arnold

• Caltech: Jared Lewis, Eric Brustad, John

McIntosh, Jane Wang, Chris Farwel

• Provivi: Peter Meinhold, Mike Chen