Lactacystin©

1. Discovery, producing organism and structure1-4)
Lactacystin was originally isolated from a broth of Streptomyces lactacystinicus strain OM-6519T
while screening for neurite outgrowth inducers in Neuro 2a, a cell line of murine neuroblastoma
cells. The absolute configuration of the structure was confirmed by X-ray crystallographic analysis2).

HOOC
O

H SNO

H3C

HN

CH3
O

CH3CH3HO
Lactacystin

HO

Streptomyces lactacystinicus OM-6519T

2. Physical data
Colorless crystal. C15H24N2O7S; mol wt 376.13. Sol. in H2O, MeOH, DMSO, pyridine. Insol. inbenzene, EtOAc, CHCl3.
3. Biological activity5-9)1) Lactacystin inhibits cell cycle progression5) and induces neurite outgrowth in Neuro 2a cells1).

Control (day 4)
Lactacystin (1.3 µM, day 4)
Photomicrographs of morphological changes induced by lactacystin in Neuro 2a cells2) Lactacystin inhibits proteasome activity6). Proteasome exhibits three protease activities, of thethree, lactacystin inhibits chymotrypsin-like activity most potently, followed by tyrpsin-like activity, but its inhibitory activity against peptidylgultamylpeptide hydrolytic activity is relativelyweak.

Ubiquitin conjugation

Proteolysis
Ubiquitin

Ubiquitin
ATPATP

E1, E2, E3

26S proteasome

Protein(Substrate)

Amino acids

PeptidesAntigenpresentation

19S complex
Lactacystin
Omuralide

20S proteasome
19S complex

The molecular structure of
rat liver 26Sproteasome
based on electron microscopy6)

Model of intracellular protein degradation mediated by
the ubiquitin-proteasome system
Lactacystin is specifically bound to the N-terminal threonine residue of certain β subunits (subunit
X for mammalian cells6) and subunit β5 for yeast8)). Recently, it has been proposed that lactacystin is converted non-enzymatically to the β-lactone derivative (Omuralide), which reacts directlywith the threonine active site.

HNHOOCO
H3C

H SNOOCH3HOO
H3C

CH3Proteasome

OH CH3αH3C
CH3HNOHOOOmuralide

CH3 OHONH2ThrX(β5)βCH3 OOH3C

βH ONHOHOO NH2CH3CH3X(β5)α4. Structure–activity study3)
Lactacystin derivatives were evaluated in neurite outgrowth of Neuro 2a cells and growth inhibition of osteosarcoma cells. The same derivatives were found to elicit both activities, suggestingthat they are based on the same mechanism. The presence of a hydroxyisobutyl group on C-5 andthe 9S configuration are important, as is the 6S,7R configuration of the γ-lactam ring, revealing
specific structural requirements for activity. The most significant Discovery was that clasto-lactacystin β-lactone, later designated omuralide by Prof. Corey, shows almost the same activity aslactacystin10).
(A)

OH RN

H3C RSHS
69HOHOEssential
7(B)Replaceablewith R1, R 2
but not H

Essential

Essential

Replaceablewith Et, i-Pr, n-Bu, Bn
but not H

O

H3C

O
CH37SH

CH3Replaceablewith CH3O

R1CH3OS

HOOC

NH

R2OH

O

O

Essential

Essential

Essential

H
N

CH3CH3H
N S

NH2H
N

COOH
OS

Summary of Structure-Activity Studies of Lactacystin (A) and Omuralide (B)5. Biosynthesis11,12)
COOH

COOH

NH2
L-Valine

IsoButyryl-CoA

COSCoA

Propionyl-CoA

HN

HOOC

Acetyl-CoA

HO COOH

COSCoA

COOH
OHC
Methylmalonicsemialdehyde

COSCoAOCOSCoAOHS

COSCoA

COOH

Acetyl-CoA
HOOC
O

H2N

H S
NO

H 3C

COOH

COSCoA

H
N

CH3O

HOOC

CH3HS

CH3HO
Lactacystin

HOOC

NH2HO
L-Serine

COOH
CHO

O
Pyruvate

NH2

L-CysteineHO

L-Leucine

Acetyl-CoA

COOH

HOOC NH2COOH
HO(HO)

O

HO

H
N

HOOC
COOH

NH2

S
L,L-Cysteine

(HO)

2

6. Total synthesis
The total synthesis of lactacystin has been reported by several groups. The following scheme isŌmura’s approach13-15).
OH

Ph

H2N

1) HCl (gas)
(97%)

HO2C

2) PhC(OMe)3(82%)

O

(56%)

CO2Me

H
N

H
N CO H

O2(82%)

CO2Me

H SNO

1) O3, DMS
2) NaClO2NHO

1) Pd, HCO2–NH4+AcOH
2) 0.1N NaOHNO

CO2MeOCO2MeO

AllylO2C

HO

MeO2C

(2 steps, 70%)

Ph

HO(85%)(+)-Ipc2B

N
OHC

O
N

PhO

Benzene

HO2C

O
N

Ph

DCC, DMSO,TFA, Pyridine

Ph

LHMDS,HCHO

HO

H
NO

Pd(PPh3)4,HCO2H, Et3N

BOPCl, Et3N
(79%)

HO

HO2C
O

H SN
O

HOHOSH
CO2AllylHNO(81%)

HOHO

Lactacystin
Overall yield 14 % in 11 steps

7. Application
Lactacystin is recognized as the most specific inhibitor of proteasome due to its mode of action.
Therefore, lactacystin has been used to reveal and support the cellular proteasome functions.
1) Cell cycle: [571], [642], [667], [719], [735]2) Apoptosis: [599], [676], [691], [692], [713], [716], [739]3) Immunoantigen processing: [666], [667]4) Protein degradation on ER: [593]5) Receptor degradation: [594], [606], [653]
6) Transcription factor degradation: [643], [674], [769],
7) Other regulatory protein degradation: [618], [627], [641], [654], [670], [678], [698],[704], [752], [756], [770], [771], [780], [783], [818]8) Anti-tumor: [750],
9) Anti-malaria: [830]10) Anti-angiogenesis: [697]
The above brackets [ ] show references for the applications (See Publications).

8. Lactacystin is commercially available from Kyowa Medex.
9. References
1. [455] S. Ōmura et al., J. Antibiot. 44, 113-116 (1991)
2. [456] S. Ōmura et al., J. Antibiot. 44, 117-118 (1991)
3. [760] H. Tomoda et al.,Yakugaku Zasshi 120, 935-949 (2000)
4. [1178] A. Take et al., J. Antibiot. 68, 322-327 (2015)
5. [571] M. Katagiri et al., J. Antibiot. 48, 344-346 (1995)
6.		
G. Fenteany et al., Science 268, 726-731 (1995)
7.		
K. Tanaka et al. J. Leukocyte Biol. 56, 571 (1994)
8.		
M. Groll et al., Nature 386, 463-471 (1997)
9.		
L. R. Dick et al., J. Biol. Chem. 272, 182-188 (1997)
10.		
E. J. Corey et al., Chem. Pharm. Bull. 47, 1-10 (1999)
11. [546] A. Nakagawa et al., Tetrahedron Lett. 35, 5009-5012 (1994)
12. [589] S. Takahashi et al., J. Antibiot. 48, 1015-1020 (1995)
13. [507] T. Sunazuka et al., J. Am. Chem. Soc. 115, 5302 (1993)
14. [510] T. Sunazuka et al., Tetrahedron Lett. 34, 4447-4448 (1993)
15. [628] T. Nagamitsu et al., J. Am. Chem. Soc. 118, 3584-3590 (1996)
16.		
L. Kozlowski et al., Tumur Biol. 22, 211-215 (2001)