Lit. Seminar (M1)

'10. 7. 28. (Wed.)
Shogo HASHIZUME (M1)

Chemistry and Biology of FK506
S. L. Schreiber's pioneering work of the "chemical biology"

Stuart L. Screiber, Ph.D. was born in 1956. He received B.A. degree at theUniversity of Virginia in 1977, and Ph.D. at Harvard University under the supervisionof R. B. Woodward and Y. Kishi in 1981. After that, he joined the faculty at Yale
University and was promoted to Associate Professor in 1984 and Full Professor in1986. In 1988, he returned to Harvard. Now, he is Professor of Chemistry andChemical Biology at Harvard University and Director of Chemical Biology of theBroad Institute of Harvard and MIT.
He is well-known for his strategy that synthesized molecules can be utilized asprobe of biological molecule and suggested one field of science, "chemical biology".
As the pioneer in this field, his first famous work is synthesis of FK506 andbiological study using synthesized FK506 and its analogues.

T oday's theme
Contents
1. Introduction and Background2. Total Synthesis of FK506 and 13C-labelled FK506
3. Exploring of Molecular Target of FK506 and Its Binding Mechanism
4. Elucidation of Signal Tr ansduction Pathway Inhibited by FK506
5. Summary and Outlook

1. Intr oduction and Backgr ound
1-1. FK506 and other immunosupressants
MeOMeMeO

9H

MeJ. Am. Chem. Soc. 1987, 109, 5031
J. Antibiotics 1987, 40, 1249
Immun. Today 1989, 10, 6O

MeFK506 was isolated from Streptomyces tsukubaensis
by research group of Fujisawa Pharma. Co. in 1987.

OOH 8ONHOH O

FK506 shows more potent immunosurpressive activity
than cyclosporins, both in vivo and in vitro.

1OTacrolimus has been used for immunosupressant afterorgan transplant, myasthenia gravis, rheumatism, etc.
since authorized in 1993.

OMeO
Me MeOHToday, Astellas Pharma Inc. is selling Tacrolimus asPrograf and Graceptor .

FK506 (Tacrolimus)

MeCsA and rapamycin are similar immunosupressants in view of acting mechanism.
They inhibit signal transduction pathway in T lymphocyte selectively.
MeMeNOO

Me N

H

MeN

MeO MeMeO

H MeNNMeHOOH OMeONMeMeO

MeN

MeOOMeONMeMeMeMe H
MeN

H

MeO
OHOOMeMeN

MeO
Mecyclosporin A (CsA)

OOO
NH

NHO

MeMeOMe

MeMeHOMeOHMe

rapamycin
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1-2. Immune system as background of this workref),D.Male ,The immune system(31995)

1995

Innate immunity
Adaptive immunity

Humoral immunity : served by B-lymphocyte
Cellular immunity : served by T-lymphocyte
Helper T-lymphocyte (HTL)
Cytotoxic T-lymphocyte (CTL)

Network of lymphocytes

B-lymphocyte

Cytokines accelates
immunity actions

HTLCTL

AntigenPr esenting

Producing
antibodies(=Humoral immunity)

Antigen- Presenting
Cell (APC)

Cytotoxity(=Cellular immunity)

This figure shows a very simplified immune system.
Actual system is far more complicated.

Interleukin-2 (IL-2) : one of the cytokines
Secreted by helper T-lymphocyte when HTL was stimulated by antigen via T-cell receptor (TCR)
Activates grouwth and differentiation of HTL, CTL, etc.

It was already clear when Schreiber set out this work that FK506's biological activity has
relationship with IL-2.
M. J. Tocci et al. had showed that FK506 apperantly inhibits the accumulation of IL-2
gene mRNA.
M. J. Tocci et al.
J. Immunology 1989, 143, 718

But FK506's inhibition mechanism at molecular level was still unclear...
Schreiber's work made it clear star ting f r om total synthesis of FK506.

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2. T otal Synthesis of FK506 and 13C-labelled FK506

MeO
MeO

Me9O
H HOO

MeMeO

S. L. Schreiber et al.
J. Org. Chem. 1989, 54, 9J. Org. Chem. 1989, 54, 15
J. Org. Chem. 1989, 54, 17J. Org. Chem. 1989, 54, 4267
J. Am. Chem. Soc. 1990, 112, 5583

These two carbonyl are involved in binding to the target molecule.

8(The reason will be stated later.)NH

Labelling these carbon (C8 and C9) would reveal the details ofbinding mechanism ??

1OH OOOMeO
Me MeThe key is a route enabling tointroduce this C-2 unit independently.

OHFK506
considering
this strategy....
Retrosynthesis

Synthesis of C27-C34 unit

MeO2COMe

CH2N243

Synthesis of C20-C26 unit followed by coupling with C27-C34 unit and C1-N7 unit

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Synthesis of C10-C19 unit

Completion of the synthesis
6 is easily obtained from-bromo acetic acid.
(13C2- -Br AcOH is alsocommercially available.)

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3. Exploring of Molecular T ar get of FK506 and Its Binding Mechanism
3-1. Identif ication of FK506-binding protein
In case of related immunosupressant cyclosporin A (CsA)...
MeMeN
O

H

MeN

MeH MeNN
MeH

O

Me N

MeO

OH O

MeMeNN
MeMe

MeMe HMeNO

OMebinding
cyclophilin

ONHO

MeO

MeN

MeN
MeO MeG. Fischer et al. Nature 1989, 337, 476
N. Takahashi et al. Nature 1989, 337, 473

OMetarget protein of CsA andcis-trans peptidyl-prolyl isomerase

Mecyclosporin A (CsA)

also binding ???
At first, FK506's target seems to be also cyclopilinbecause the two agents showed similar bioactivity.

FK506

But these have little structual similarity..
Schreiber and co-workers tried to identify the FK506-binding protein (FKBP).
S. L. Schreiber et al. Nature 1989, 341, 758
MeO

MeMeO
H

MeMeO

OMeMeOOOHO

1. L-Selectride
2. N3(CH2)2COCl
3. HS(CH2)3SHNHOH OMeOOMeO
Me MeHMeOOOHOMeNHOOH O

4. affigel-10

OMeO

HOMeMeOH

FK506
calf thymusorhuman spleen

OO
N
H

affigel-10

FK506-affigel
homogenize

centrifuge

clear supernatant(= mixture of proteins)

FK506
solution
FK506-affigel

FKBP binds
to affinity gel

washing out FKBPFKBP selectively
sticks to affigel
get FKBP
They succeeded in isolating FKBP by this method.
(lane 1 and 5 in figure)
And this isolated FKBP was anti-cyclophiline IgE.
The result showed cyclophilin and FKBP are
antigenically different. (lane 3 and 7)
a: from bovine thymus
b: from human spleen
immunoblot: react with anti-cyclophilin IgE

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They tested this speculation by anotherexperiment.
FK506 was 14C-labelled and exposed
assey with FKBP.

MeO

MeMeO
H

MeO

MeMeO

OHO

N14O

Me

C-benzoyl chloride

HOOOHO

N
H

OH O
OMeO
Me MeOOMe OOMe MeOH

FK506

14OC

Ph

32-[1-14C]-benzoyl-FK506

14C-FK506

FKBPH

MeOH O

MeMeO

O

unlabelled FK506or CsA
measure 14Cstrength of FKBP(The results in left graph)
14C-FK506
bound to FKBP

measure 14Cstrength of FKBP
(FK506: right graph
CsA: data not shown)

Unlabelled FK506 displaced 14C-labelled
FK506, but contrarily, CsA didn't.

395nm absorbance means activityof FKBP-mediated isomerization

Isomerization activity of FKBP was also tested (right figure).
Suc-Ala-Ala-Pro-Phe-4-nitroanilide as model peptide-substrate.
(This experiment was developed by Fischer et al. (Natur e 1989, 337, 476))

cis-trans isomerization activity was increased with FKBP (A),compared with exp. without FKBP (E).
And this FKBP activity was inhibited by FK506 relatively to the conc..
Furthermore, CsA had no effect about FKBP inhibition. (data not shown)

A: FKBP
B: FKBP with 27nM of FK506
C: FKBP with 54nM of FK506
D: FKBP with 270nM of FK506
E: control

In addition to these experiments, FKBP's amino acid sequence didn't match cyclophilin's one.
And FKBP's sequence matched none of the sequence from gene database.
(= This isolated FKBP was unknown and new enzyme.)

This series of experiments showed...
CsAcyclophilin

FK506

FKBP

commonenzymatic property(cis-tr ans peptidylprolyl isomeraze)

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3-2. Mechanism of FK506-FKBP binding
MeOMeO

Me9O
H HOO

MeS. L. Schreiber et al.
Science 1990, 248, 863

MeConsidering FKBP is cis-trans peptidyl-prolyl isomeraze,
this group may be involved in the binding.

O8N
H

OH O

Two hypotheticalmechanism of rotamease

OOMeO
Me MeOH

FK506
FK506 binds to FKBP in tetrahedral-adduct mechanismor twisted-amide mechanism ??

FK506 binds as a mimic oftransition state of peptide substrate??
[8,9-13C] FK506 was used to determine the binding mechanism.
(by 13C-NMR measurement of labelled FK506 with or without recombinant-FKBP(rFKBP))
13C-FK506 only

figure A: 13C spectrum of [8,9-13C]FK506
This two pairs coupled doublet peaks means cis- and transamide rotamers of FK506 in solution state.

13C-FK506 with rFKBP
figure B: 13C spectrum of complex between [8,9-13C]FK506 and FKBP
FK506 takes a single rotamer in binding-state with FKBP.

Peak of tetrahedral carbon
is expected in this region...

This NMR study reveals that FK506 binds to FKBP not covalently but by taking stabilized twistedamide state as mimic of the peptidyl-prolyl-cis-trans isomerization of its peptide-substrate.
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4. Elucidation of Signal T ransduction Pathway Inhibited by FK506
4-1. FKBP-mediated isomerization has a role f or T -cell activation??
It was proved by the experiments above that FK506 binds to FKBP selectively.
And FKBP is identical to peptidyl-prolyl-cis-trans isomerase.
But does FKBP really have a crucial role for T cell activation pathway ???
stimulant

T cell activation? ?

FK506 inihibits
the role of FKBPFKBP promotes
one of these steps

This hypothetical signaling pathway involving FKBP is doubtful
because of several experimental results below.

Schreiber and co-workers conducted molecular cloning and overexpression of human-FKBP.
(It is for making recombinant FKBP for assay with some imunnosupressants and thier analogues.)
S. L. Schreiber et al. Nature 1990, 346, 671
And Sigal et al. reported FK506 inhibits calcium-dependent lyphokine gene transcription.
N. H. Sigal et al. J. Immun. 1990, 144, 251
Judging from FKBP's amino acids sequence, however, FKBP is probably neither a direct activator of genetranscription nor a calcium-dependent protein (it has no calcium-binding motif).

Schreiber synthesized a FK506's analogue, 506BD, and tested its binding activity against FKBP.
S. L. Schreiber et al.
Science 1990, 250, 556
MeO
MeO
H

MeMeO

MeO

MeO

O
OHO

MeMeO
HN

MeO

MeOMeOMe MeFK506

OHOHOHOH OO

putative FKBPbinding region

NO HOOOMeOMeMe MeOH506BD

X-ray structure shows 506BD adopts a geometry similar
to that of the putative binding domain in FK506.

506BD actually bound to FKBP and inhibited its rotamase activity.
And 506BD could effectively displace FK506 by FKBP. (506BD's Ki value was 5 nM)
However, 506BD showed different biological effects from those of FK506.
506BD didn't prevent IL-2 production from T-cell.
Furthermore, 506BD reversed FK506's inhibition of IL-2 production. (figure A)
Although 506BD has no effect on inihibition of T cell activation by CsA, it reversed inhibiton byrapamycin. (figure C)(Experimental data in the next page)506BD works as antagonist for FK506 and rapamycin, not for CsA.

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- 506BD+ 506BD

without 506BD
1 M 506BD
2 M 506BD

control

Schreiber also demonstrated that complexes between FKBP and FK506 or rapamycin might inhibit two
distinct signaling-pathway in T lympocytes.
S. L. Schreiber and G. R. Crabtree et al.
Proc. Natl. Acad. Sci. USA 1990, 87, 9231
IL-2 production of T-cell was inhibited by FK506 and CsA
but not by rapamycin. (figure A and B)
And 100-fold excess of rapamycin reversed the inhibition
by FK506. (figure C and D)

Crabtree et al. had reported that a T-cell specific transcription factor (NF-AT) is controlling IL-2 gene.
And NF-AT is activated after stimulation of antigen receptor of T cells.
(G. R. Crabtree et al. Science 1988, 241, 202)
Schreiber shows that FK506 inhibits a factor of pathway activating
NF-AT by making model system. (figure A on right side)

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B: A ribonuclease assay of transcripted mRNAs
C: Effects of FK506 & Rap. on the NF-AT activity
measured as galactosidase activity
D: Electrophoreic mobility shift assay using theNF-AT binding site as probe

All results suggest that NF-AT activity is inhibited by FK506
and rapamycin reverses this FK506's inhibitory actions.
Considering these three series of experimental facts...
Either FK506 or rapamycin (Rap) binds to FKBP at the same binding site and works as antagonist
against each other. This is supported by the fact that Rap has very a very similar structure to theFK506's putative binding site to FKBP.
FK506 apperently inhibits a T-cell activation pathway involving NF-AT, but Rap didn't. This factsuggests that Rap inhibits another step of T-cell activation.
FKBP bound to FK506 or Rap interact with different target moleculein separable pathways in T-cell activation ??

binds to common
binding-site
FK506
acting on each target
in different signaling pathway
concerning the T-cell activation

FK506-FKBP
complex
FKBP

Rap
Rap-FKBP
complex4-2. Genuine FK506's target

S. L. Schreiber et al.
Cell 1991, 66, 807

The next Schreiber's attempt is identification of the target of FK506-FKBP complex.
He used a complex between FK506 and a modified FKBP, glutathione S-transferase-FKBP12 fusion
protein (GFK).
(This modified protein was prepared in an artificial genetic way and glutathione S-transferase-cyclophilin
fusion protein (GCyP) was also made in the same way.)
Assay to identify the target protein was conducted by immobilizing this GFK.

immobilizedglutathione

GFKFK506

assay with
mixture of proteinsglutathione binding-site and FK506 binding-site
had been proved to be different.
10/ 14GFK
GFK + extract
GFK-FK506 + extract
GFK-rap. + extract
GCyP
GCyP + extract
GCyP-CsA + extract

Only when immobilized GFK or GCyP was assayed with proteinsin presence of FK506 or CsA, resectively, 4 bands (61kD, 57kD,17kD, 15kD) appeared.
These proteins might be the target proteinsof FKBP-FK506 complex.

Experiment of Fig.2 is whether solution of immunosupressant and/or
immunophilin can wash out the target protein from immmobilizedglutathione-bound GFK-FK506 complex.
This result shows only FKBP-FK506 and CyP-CsA can bind to thetarget proteins (=wash them out from immobilized system).
(And EGTA (Ca2+ ion chelator) also dissociates binding between thetarget protein and FKBP-FK506 complex.)
By Ca2+ dependency of target proteins to bind to immunophiline-immunosupressant complex, theyspeculate one of them is calmodulin.
1: Marker
And other three proteins may be subunits of
2: auth. calmodulin + Ca2+
calmodulin-binding protein.
2+
calcineurin A 3: EGTA elute + Ca
Comparison in SDS-page between EGTA
4: auth. calcineulin + Ca2+eluate from immobilized GFK-FK506 and
5: auth. calmodulin + EGTA
6: EGTA elute + EGTA
authentic samples revealed the four proteins
7: auth. calcineulin + EGTA
as described in figure 3.
calmodulincalcineurin B

Western blotting analysis with anti-calcineurin antibodies (fig.4) and 45Ca2+ ligand blotting(fig.5) also identify these proteins.

The experiments showed that calcineurin, a Ca2+- and calmodulin-dependent serine/threonine
phosphatase is a common target of FKBP-FK506 and CyP-CsA complex.
So, it's natural to consider that calcineurin regulates phosphorylation state of some downstream
target, which might be a component of signaling pathway.
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Schreiber got insights about further details of binding between FK506-FKBP complex and calcineurin.
S. L. Schreiber et al.
Biochemistry 1992, 31, 3896
FK506-FKBP complex inhibits calcineurin by binding in 1:1 ratio.
And calmodulin enhances the affinity between FK506-FKBP complex
and calcineurin. (Table I)
It was also revealed that FK506-FKBP and CsA-CsP are specificinhibitors for calcineurin among some protein phosphatases. (Table III)

This graph shows inhibition rate is saturated when FKBP-FK506
complex become equal to its target, calcineulin.

4-3. T he whole picture of the signaling pathway
The "upstream" signaling pathway of calcineurin emanating from T cell receptor had been disclosed
by other reaserchers.
And Schreiber revealed that calcineurin has a critical role for T cell activation and inhibition ofcalcineurin deactivates NF-AT-mediated gene transcription.
So the remaining disputable point is how calcineurin is related to NF-AT activities.
It was demonstrated by a research of A. Rao et al.
A. Rao et al.
J. Biol. Chem. 1993, 268, 3747
Calcineurin, a Ca2+-regulated protein, has an ability of dephosphorylating NF-AT.
NF-AT takes an inactivated state and is localized in the cytoplasm when phosphorylated.
Contrarily, it takes an activated state and is localized in the nucleus when dephosphorylated.
Briefly, calcineurin activates NF-AT directly.

So Schreiber's and other researchers' studies disclosed the whole picture of the signal-transduction
pathway of TCR-initiated T-cell activation. (Figure below)

T cell activation

inactiveNF-AT

activeNF-AT

IL-2 genetranscription

Signal transduction pathway f r om TCR to transcription of IL-2 gene in T-cell
12/ 144-4. Structural studies of FK506-FKBP-calcineurin-calmodulin complex
Schreiber gave some insights about FK506-FKBP's binding to calcineurin from the standpoint of structure.
The FK506 binding site of FKBP was identified by X-ray crystallography and NMR spectroscopy.
S. L. Schreiber et al. Angew. Chem. Int. Ed. Eng. 1992, 31, 384
They reported some mutagenesis experiments of FKBP.
The residues which have a critical role to bind to calcineurin
was identified.
(These residues was showed in the left fig. on the top ofthe next page.)
S. L. Schreiber et al.
J. Am. Chem. Soc. 1993, 115, 819

Some analogues of FK506 had been tested for binding with FKBP and calcineurin.
The results showed C21-allyl group and C15-methoxy group are important to bind to calcineurin.
(These group also showed in the fig. as well.)S. L. Schreiber et al. Biochemistry 1992, 31, 3896

FKBP-FK506 complex

FK506 with the binding region of FKBPThe residues in FKBP and the groups in FK506
influencing the binding to calcineurin are displayed.

FK506-FKBP complex make a composite surface for binding to calcineurin.
And FK506 might act as "molecular glue" between FKBP and calcineurin.
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5. Summary and Outlook
Schreiber et al. disclosed inside the "black box" of FK506-inhibited pathway in T cell.
TCR stimulant?

!!!

!!!

?
FK506
inhibition
T cell activation
responce
Their work started from organic synthesis, then was expanded to molecular-biological studies.
Since then, a series of studies like this work has attracted many chemists' attention, and became
one field of science, "chemical biology", which so many scientists participate in nowadays.

What distinguished him from other chemists is that he was thinking "what the total synthesis is for".
Using the synthesized molecule for testing its biological effects was a brand-new science in thosedays.

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