Download Essentials of Glycobiology Lecture 42 June 9, 1998 Jeff Esko

Essentials of Glycobiology
Lecture 34
May 28, 2002
Jeff Esko
Glycosylation Inhibitors
Overview
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Indirect inhibitors and metabolic poisons
Tunicamycin - Inhibition of dolichol-PP-GlcNAc
assembly
Plant Alkaloids - Natural inhibitors of glycosidases
Substrate analogs - Directed synthesis of
inhibitors
Glycoside primers - Mimicking what already works
Inhibitors of glycolipids and GPI anchors
Neuraminidase inhibitors - Rational design from Xray crystallography
Introduction
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Need to perturb
structure to
understand
function
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Inhibitors can
lead to
therapeutics
Indirect Inhibitors and Metabolic Poisons
In vitro inhibitors
- nucleotide sugar analogs, acceptor
analogs
Sugar analogs
- deoxyglucose, fluorinated sugars,
mannosamine analogs
Compounds that interfere with transport
- Brefeldin A, ionophores, ammonium
chloride
Compounds that block intermediary
metabolism
- 6-diazo-5-oxo-L-norleucine (DON),
chlorate
Tunicamycin
O
HN
O
RCHN
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Blocks the transfer of
GlcNAc-1-P from UDPGlcNAc to dolichyl-P
(GPT)
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Resistant mutants
overproduce GPT
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Km for UDP-GlcNAc is ~3
x 10-6 M, whereas the Ki
value for tunicamycin is
~5 x 10-8M
OH
HO
O
O
O
NHAc
HO
CH2
N
O
H
H
H
OH
H
OH
O
OH
HO
OH
Plant Alkaloids - Natural Inhibitors of Glycosidases
a2
±
a3
a3
a3
a3
a2
a2
a2
a3
a3
a2
a2
a2
a6
a6
Asn
a2
a2
a3
a3
b4
b4
a2
a2
a6
a6
b4
Asn
a2
a3
a3
b4
a-glucosidase I
a2
a6
a6
a3
b4
a-glucosidase II
b4
Asn
a3
a6
a6
b4
Asn
Australine
Kifunensin
Deoxynojirimycin
Castanospermine
a3
b4
a-mannosidase I
Deoxymannojirimycin
a3
b2
a6
a3
a6
GlcNAc TI
a6
b4
b4
a-mannosidase II
b4
Asn
b4
Asn
Swainsonine
Plant Alkaloids
HO
H
OH
CH2OH
HO
N
OH
H
N
OH
OH
HO
N
OH
HO
H
C H 2OH
OH
Australine
a-Glucosidase I
N
OH
HO
OH
Castanospermine
Deoxymannojirimycin
a-Glucosidase I and II
a-Mannosidase I
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Swainsonine
a-Mannosidase II
Alkaloids contain polyhydroxylated ring
systems that mimic the orientation of
hydroxyl groups in the natural substrates
Protonation of the ring nitrogen may mimic
the positive charge developed on the ring
oxygen during the hydrolytic reaction
Substrate Analogs - Directed Synthesis of Inhibitors
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Modify the acceptor or
nearby hydroxyl group in a
substrate
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Exhibit Ki values in the
range of the Km for the
parent substrate
Hindgaul and coworkers
Glycosidase Inhibitors
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PUGNAC
Galactonolactone
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Many inhibitors of
lysoomal and bacterial
hydrolases are
available
Generally they mimic a
proposed transition
state since C1 takes
on a trigonal
orientation
Synthetic Substrate Analogs
Glycoside Primers - Substrate Mimicry
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Prepare compounds that
resemble biosynthetic
HO
intermediates
HO
Conjugate to a
hydrophobic aglycone to
enhance uptake and
activity
Alkylation or acylation also
works
H
H
O
O
H
OH
H
H
H
H
O
OH
O
O
O
H
O
H
O
O
H
Sarkar et al (1995) Proc. Natl. Acad. Sci. USA 92: 3323
Xyloside Primers Block Proteoglycan Glycosylation
Proteoglycan
6S
6S
6S
2S NS
O
NS
6S
O
NS
NS 2 S
6S
6S
O
NS
NS 2S
Cell
O
6S
Xylose
6S
Hydrophobic
Aglycone
O
NS
NS 2S
Xyloside
Fritz & Esko (2001) Methods Mol Biol. 171:309
Types of Primers
OAc
OAc
OAc
O
O
O
AcO
OAc
Gal
AcO
O
NHAc
GlcNAc
Naphthalene
methanol
Properties of Glycoside Primers
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Priming usually exceeds normal levels of
synthesis on endogenous conjugate
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The enhanced rate of synthesis of
oligosaccharides on primers may alter the
assembly of other classes of glycoconjugates
than those generated on the primer
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Inhibition is rarely complete
Properties of Glycoside Primers
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Primers can reveal alternate glycosylation
pathways
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Primers represent starting points for making
membrane permeable analogs that might have
inhibitory activity without acting as a primer
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Glycosides can be absorbed through the gut
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Many glycosides occur naturally, especially in
plants
Natural Inhibitors of Glycosylation
Alkaloids
Flavonoids
Carotenoids
Steroids
and others
Glucosides
Arabinosides
Xylosides
Ribosides
Rhamnosides
and others
Natural Glycoside Primers
Psittacanthus
cucullaris
1
1 mg/ml
0.33
0.11
.037
.012
2
3
Alchornea
triplinervia
4
1
2
3
Maieta
guianensis
4
1
2
3
4
Plant extracts
were prepared
by graded
solvent
extraction (1-4)
and assayed for
GAG priming
activity
.004
0
Taylor et al. (1998) J. Biol. Chem. 273:22260
Natural GAG primers
O
4"
HO
O
5"
O
4"
2" O H 1"
HO
3"
HO
4
MeO
O
6
1
7'
O
H
O
3"'
5"'
HO
4"'
OH
O Me
OH
3,3'-Di-O-methyl ellagic acid4-O-b-D-xylopyranoside
H
3
5
2
6
O
7'
1'
O
O
2'
6'
5'
H
O
7
1
OH
3'
4'
2"'
6"'
4
MeO
1"'
O
2'
6'
5'
2" O H 1"
O
7" C = O
7
1'
O
3"
H
5
3
2
5"
3'
4'
OH
OMe
2EAGX)
3,3'-Di-O-methyl ellagic acid
4-O-(3"-galloyl)-b-D-xylopyranoside
Glycolysis
Gal
Glc
ATP
Fuc
Man
GlcNAc
ATP
ATP
Pi
Gal-1-P
Glycogen
Glc-6-P
Fuc-1-P
-NH3 Glutamine
UTP UDP-Glc
UDP-Gal
Man-6-P
Fru-6-P
NAD
UDP-Glc
GlcN-6-P
Glc-1-P
Man-l-P
AcCoA
NAD+
GTP
Dol-P
ATP
GDP-Man
GlcNAc-6-P
UDP-GlcA
GTP
Dol-P-Glc
NADP
-CO2
GlcNAc-l-P
GalNAc
GalNAc-1-P
UTP
GDP-Fuc
UTP
UDP-Xyl
ATP
Dol-P
Dol-P-Man
UDP-GalNAc
UDP- GlcNAc
ManNAc
ATP
CMP-Neu5Gc
ManNAc-6-P
PEP
NADP
CMP-Neu5Ac
CTP
Neu5Ac
Neu5Ac-9-P
Mannosamine
derivatives (ManNR)
can be used by this
pathway
Mannosamine Analogs
Mahal et al. (1997) Science 276:1125
Metabolism of ManAzide
results in display of cell
surface azides.
Reaction with
exogenously delivered
phosphine via the
Staudinger ligation
creates an amide-linked
cell surface conjugate.
Saxon & Bertozzi (2001) Annu. Rev. Cell Dev. Biol. 17:1
Inhibitors of Glycolipid Assembly
Xylosides have a mild effect
on glycolipid formation, due
to the assembly of a GM3
like compound
a3
b4
b -O-R
b-O-Cer
Glucosylceramide acts like a
primer; an analog containing an
exocyclic epoxide inhibits
glycolipid formation (IC50 ~8 µM)
b-O-Cer
O
Inhibitors of Glycolipid Assembly
HO
N-butyldeoxynojirimycin
inhibits Glc-Cer synthesis
CH2OH
N
HO
HO
The best inhibitors are
analogs of the long chain
base (sphingosine
analogs)
O
N
HO
O
N
H
DL-threo-PDMP
Inhibitors of GPI anchors
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Mannosamine inhibits GPI anchor formation:
ManNH2-Man-GlcN-PI is a poor substrate for the
a2mannosyltransferase
Trypanosomes selectively take up and exchange
fatty acid analogs (10-(propoxy)decanoic acid) for
acyl chains on glycosylphosphatidylinositol
O
O
HO
Neuraminidase Inhibitors
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A neuraminidase inhibitor
was deduced by assuming
a carbocation arises at C1
in the transition state,
which would cause C1 to
adopt a trigonal (sp2) planar
configuration
Compounds mimicking this
geometry had inhibitory
activity (µM Ki value)
Rational design from X-ray crystallography
Potent Sialic Acid Analogs
A sialic acid analog
containing a positively
charged guanidinium
group instead of O4 had a
Ki of 10-11 M due to an
additional salt bridge
Non-carbohydrate analogs
based on of benzoic acid
mimic the partially planar
ring of the proposed
intermediate
OH
OH
HO
O
HNOCH3C
H 2N C
COOH
CH2
NH
NH
4-guanido-Neu5Ac-2-ene
Therapeutics
Relenza and Tamiflu are drugs based on these
sialic acid analogs
N-butylated alkaloids are under development as
anti-viral and anti-tumor agents
Other pharmaceutical applications of
glycosylation and glycosidase inhibitors will be
discussed later