Forewarned is forearmed:
development of pyrethroid activity based protein
profiling probes (PyABPs) to predict insecticide
resistance liabilities
Mark J.I. Paine , Hanafy M. Ismail, Janet Hemingway , David Hong,
Paul M. O`Neill,
Drug Discovery: The first Commandment
Thou shalt determine if your drug is
metabolised by P450
drug
CPR
P450
d
drug
E.R.
O
2D6
3A4
OH
F
N
N
N
N
H
H
H
H
H
Cl
Most drugs
O
O
N
O
H
O
O
N
Tricyclics/ -blockers/ecstacy
O
O
O
O
O
O
O
O
O
O
N
N
O
O
O
© The Liverpool School of Tropical Medicine
71
Insecticide development: though shalt
determine if your insecticide is
metabolised by P450s
Insecticide
CPR
P450
Ins cticid
Insecticide
E.R.
6P3
6Z1
CH3 N
H3C
Br
Br
DDT
O
C
H
C
H
O
O
O
O
C
H
C
O
C
N
O
O
O
CH
O
O
O
H
C
C
OH
O
Pyrethroids
N
N
O
O
O
O
O
© The Liverpool School of Tropical Medicine
72
Malaria remains a global problem
R s
Reservoir
i
Pathogen
V ct
Vector
‘host’
host
•
•
•
•
•
400 – 900 m cases
3 m people die
Mainly children
Tropical Regions
Perpetuates poverty
Distribution of malaria
© The Liverpool School of Tropical Medicine
73
Bednets are frontline tools for malaria
control BUT only pyrethroids can be used
RESISTANCE MAP
• > 50 m nets
distributed in Africa
• Provides personal and
community
y protection
p
• long lasting, low
© toxicity,
The Liverpool School of fast
Tropical Medicine
acting
Source: Ranson et al. 2010
74
DDT Photo’s kindly provided by Dr Vijay Kumar, RMRI, Patna
MALARIA
CONTROL
RESTS
ON
TWO
TARGETS
© The Liverpool School of Tropical Medicine
75
Resistance monitoring is essential
© The Liverpool School of Tropical Medicine
76
WHO Resistance testing in
2014
By the time resistance is
spotted it is usually too late –
resistance g
genes are
widespread
© The Liverpool School of Tropical Medicine
77
P450’s play a major role in metabolic
resistance to pyrethroids: WHICH ONES?
Phenotype
Candidate
P450s key for:
1. Diagnostics
2. Insecticide
development
Challenges
• 100 – 200 P450s
• SNP frequency ~50 nt
Validate
Technology
Field test
© The Liverpool School of Tropical Medicine
78
AIM: fast track the identification of P450s
associated
i t d with
ith insecticide
i
ti id metabolism
t b li
Activity Based Protein Profiling Probes can target
P450s
Wright A. T. and Benjamin F. Cravatt (2007) Chemistry & Biology 14, 1043–1051
© The Liverpool School of Tropical Medicine
79
Pyrethroid Activity Based Probes
P ABP D
PyABP:
Design
i ffeatures
t
Warhead
Click Handle
Click Chemistry
P450
Rhod
Alexa
488
Label
Label Purify
Biotin
© The Liverpool School of Tropical Medicine
Purify
1 Deltamethrin backbone
1.
2. Warhead: attach to P450
3. Click Handle: attach to
reporter label and/or
affinity tag (biotin)
80
PyABP Suite
H.M. Ismail
Linker
Warhead
Cli k H
Click
Handle
dl
Controls
2EN
© The Liverpool School of Tropical Medicine
• Warhead and click
handle positioned at
major and minor sites of
attack
• Based on knowledge of
deltamethrin breakdown
by CYPs 6M2 and 6P3
81
PyABP validation - recombinant P450s
An. gambiae P450s +/
+/- NADPH
Con
Linker
Delta-ABP
Warhead
Cli k H
Click
Handle
dl
Controls
2EN
Screened against mosquito
P450s associated with Py
resistance – metabolisers
AND non metabolisers
© The Liverpool School of Tropical Medicine
Ismail et al PNAS 2013 110 (49) 19766-19771
82
PyABP validation - recombinant P450s
Screened An.
An gambiae P450s +/
+/- NADPH
Con
Linker
Delta-ABP
Warhead
Cli k H
Click
Handle
dl
Controls
2EN
Selective for deltamethrin metabolisers
Warhead best on major 4’ site of attack
SAR INFORMATIVE
© The Liverpool School of Tropical Medicine
Ismail et al PNAS 2013 110 (49) 19766-19771
83
Are Py-ABPs really activity based?
Validated with Dundee CPR and b5 KO
mice (Henderson, Finn, Wolf)
Linker
Warhead
Cli k H
Click
Handle
dl
b5 affects PyABP metabolism
Tag Free Click Chemistry ABP Probes Design
Control: 2EN-ABP
CPR needed for metabolism
© The Liverpool School of Tropical Medicine
Ismail et al PNAS 2013 110 (49) 19766-19771
84
Do PyABPs work?
Linker
Probe Validation:
identification of pyrethroid
py
metabolisers in rats (P7RS)
Warhead
Cli k H
Click
Handle
dl
Tag Free Click Chemistry ABP Probes Design
Cut Band
Control: 2EN-ABP
Tryptic Digest
LC/MS/MS
Peptide ID
© The Liverpool School of Tropical Medicine
85
Enzymes interacting with P7RS
Gel extraction
Protein
Mass
KDa
57.1
56
56.3
49.4
CYP2C11
CYP2C6
CYP2C23
Ugt2b37 similar to UDP-g2B5
precursor
UDP-g 2B5
60.6
CYP2C13
49.6
CYP2C7
56.2
CYP2D10
57
CYP2D1
57.1
CYP2D26
56.6
UDP g2B3
UDP-g2B3
60 5
60.5
FMO3
59.9
Epoxide hydrolase
52.5
UDP-g2B4
61
CYP2D3
56.9
Aldh3a2 Fatty aldehyde
54.0
dehydrogenase
CYP2C13 (male-specific)
55.8
CYP2B3
56.3
CYP4V3
60.5
CYP2C7
53.2
CYP2A2
56.3
CYP4F4
60.0
©CYP2C70
The Liverpool School of Tropical Medicine
56.1
CYP2A1
55 9
On-bead
On
bead digestion
b
emPAI
9.48
3.85
1.02
0.85
0.74
0.74
0.72
0.62
0.62
0.62
0 58
0.58
0.5
0.42
0.42
0.38
0.33
0.31
0.31
0.29
0.26
0.24
0.23
0.18
0 18
Protein
CYP2C11
CYP2D10
CYP2D1
CYP2D3
CYP2D26
UDP g 2B5
UDP-g
UDP-g 2B2
UDP-g 2B3
CYP2D18
CYP2C6
CYP2D4
Aldh3a2 Fatty aldehyde
dehydrogenase
Epoxide hydrolase
UDP-gg 2B4
UDP
CYP2B3
CYP2C23
CYP3A2
FMO3
CYP2C70
Protein disulfide-isomerase
CYP2C7
UDP-g 2B1
CYP4V3
Mass KDa Average
emPAI
(+NADPH)*
57.1
25.41
57
3.85
57.1
2.69
56.6
2.11
56.6
1.6
60 6
60.6
1 05
1.05
60.9
1.03
60.5
0.85
56.6
0.5
56
0.49
56.7
0.46
54
0.46
52.5
61
56.3
56.4
57.7
59.9
56.1
56.9
56.2
60.4
60.5
0.37
0 24
0.24
0.16
0.15
0.14
0.12
0.12
0.1
0.09
0.06
0.02
86
P7RS pulls down a network
of insecticide metabolising
enzymes
Enzyme
Py Activity
CYP2C11
KM 32 M,
Vmax 206
min-1
CYP2C6
and KM 22
M, Vmax
150 min-1
CYP2C13
Yes
CYP2D1
Yes
© The Liverpool School of Tropical Medicine
Ismail et al PNAS 2013 110 (49) 19766-19771
87
Interaction networks of CYP2C11 and UDP-g
2B5 according to STRING software
(h
(http://string-db.org/).
// i
db
/)
UDP-g 2B5
CYP2C11
Ugt2b
5
CYP2C
11
© The Liverpool School of Tropical Medicine
88
CONCLUSIONS
Synthesized
y
a new p
panel of ABPs,, PyABPs,
y
, directed towards
pyrethroid metabolizing P450s
• Interactions with mosquito P450s confirmed pyrethroid
specificity and provided useful SAR of Py-P450 interaction
• mouse liver KOs indicated modulation of activity by b5
• P7RS was capable of detecting deltamethrin metabolizing
P450s in a complex rat proteome, and other enzymes
associated with xenobiotic metabolism
• Suggests the probe has captured a ‘pyrethrome’, an
associated network of enzymes
y
involved in py
pyrethroid
metabolism.
• PyABPs useful for assessing P450-pyrethroid interactions in a
wide
id range off species
i and
d bi
biological
l i l systems,
t
© The Liverpool School of Tropical Medicine
89
Pyrethrome
Aldh
Pyrethroid
FMO
b5
Py -OH
P –OH
Py
OH
© The Liverpool School of Tropical Medicine
Py -OH
UGT
Glu-Py -OH
90
Can PyABPs be used for pre-emptive identification
of metabolic resistance markers?
Next step
p is to use P7RS against
g
An gambiae
Pre-affinity purification
Post-affinity purification
An gambiae
pyrethrome?
© The Liverpool School of Tropical Medicine
• mechanism
• diagnostics
• SAR
• new insecticide
?
91
ACKNOWLEDGEMENTS
LSTM
Hanafy Ismail
J
Janet
t Hemingway
H i
U of Liverpool
Pa l O’Neill
Paul
David Hong
Scripps
Benjamin F Cravatt
Pacific Northwest National Laboratory
Aaron T Wright
© The Liverpool School of Tropical Medicine
92