Nicotinic Receptors and Anthelmintic ResistanceRichard J. Martin, Adrian J. Wolstenholme, S. M. Williamson & Alan P. RobertsonDepartment of Biomedical Sciences, Iowa State University, Ames, Iowa USA & Dept of Infectious Diseases, University of Georgia, Athens, Georgia
Nicotinic Anthelmintics
1. Imidazothiazoles: levamisole, agonist2. Tetrahydropyrimidines: pyrantel , agonist3. Quaternary Amines: bephenium , agonist4. AADs: monepantel , agonist5. Macfortines: paraherquamide &
derquantel, antagonist
Complementary site on adjacent subunit
Primary site
on α subunit
A
B
C
DE
F
Y93 W86
Y190 C192
C193 Y198
W149 Y151
Y111 Y117
W55/57
D180
A B C
Na+, Ca2+
α
Agonist binding site
outside
inside
α
C. elegans nicotinic receptor subunitsJones et al., 2007: 29 subunits, 5 core structures
AADs
levamisole
Muscle nAChRs in C. elegans
C. elegans levamisole sensitive receptor
G= 30 pS
(Qian et al, 2008)
ACR 16
ACR 16
ACR 16
ACR 16
ACR 16
LEV 8
UNC
63
UNC
29
LEV 1
UNC
38
C. elegans nicotine sensitive receptor
Nicotinic anthelmintic
5 min
1g
10 nM
100 nM300 nM
1 M30 M
30 nM
Control dose-response trace
Pharmacological Diversity in Ascaris
3 receptor types in Ascaris suum:
N-type(low sensitivity to paraherquaminde)
L-type(sensitive to paraherquamide
and 2 desoxo-paraherquamide: derquantel)
B-type(most sensitive to
2-desoxo-paraherquamide:
derquantel)
N-type
L-typeB-type
C
O1
O2
O3 B-type
L-type
N-type
1.5 2.0 2.5 3.0 3.5 4.0 4.5
0
10
20
30
40
50
Nu
mbe
r o
f obs
erv
atio
ns
(N)
Amplitude (pA)
N-type
L-type
B-type
3 channel currents & 3 nAChR subtypes
N-subtype
24pS
nicotine
L-subtype
35pS
levamisole
paraherquamide
B-subtype
45pS
bephenium
2-desoxoparaherquamide
(derquantel)
& paraherquamide
A B
C
mfglleallwlvvcgtsLTVGNEDAKRLYDDLMVNYNKHRRPAMSPHEPVTIKLKLRLSQIIDVHEIDQIMTYSVW LKQVWIDKKLSWDPKNYGGVSVLYVPYEMIWVPDIVLYNNADSNYNITISTKATLHYSGEVTWEPPAIFKSMCQID VRWFPFDEQKCHLKFGSWTYSEDLLVLELLDGEPHYELETNEFGEVDNITIVDDGIDLSDYYPSVEWDIMSRVAIR RTKNYPSCCPQSDAYIDIMYYLELRRKPLFYTVNLVFPCVGISFLTILVFYLPSDSGEKVTLCISILVALTVFFLL LTEIIPATSISLPLIGKYLLFTMVMVTLSVVVTVISLNLHFRTPTTHRMPEWVKWLFLKFLPKVLFMRRPLADTDD TYRRVSQRRGDNCEKVAINYHEHRVSRDIGRALSTSPVDERIQKLYYSPAVVKAFENVCFIAELLKKKDRDDKVDE DWKYVAMVLDRLFLLLFSFACFIGTVTILLQAPTLYDSREAIDLQYRPANISTPVTQ mhlpspaliivyyvlvallragvvlcSDDEERLMVDVFRGYNSLIQPVRNLSDTPIIVKIALQLVLLINVDERDQV MHTNVWLTLKWHDFQMKWNPVNYGEIQNIRVAPDKVWLPDIVFFNNADGNYEVSFMCNVVISHKGDMLWVPPAIYK SSCIIDVEFFPFDEQTCHLIFGSWTYNENEIKLEFEQAEWVDLSEYAPSSIWDVMDAPASLVNKRSRIEFQVRIRR KTLFYTVVLIIPTVLMAFLSMAVFFLPTDSGEKMTLTISVLLSIVVFLLLVSKILPPTSSTIPLMAKYLLLTFVLN VITILVTVIIINVYFRGPTTHRMPKWVRTTFLQVMPKLLCMQRPKQLLRKMAAHAAAQQANGAAVATALPGIGEFT LNPSAHHPFCPSADDSRFHPPSIELTHVDSVRQFERDPMTSAYYPLSADALRAIDAIEYITDHLKQDEEYKMYRDD WKYVAMIIDRLLLYVFFGITVGGTCGILFSAPYVFQGVDQRAELQRLIHLYKNGGEI
Asu-UNC38
Asu-UNC29
L-subtype receptor
UNC29
UNC29
UNC29
UNC38
UNC38
UNC29
UNC29
UNC29
UNC38
UNC38
N-subtype receptor
UNC29
UNC38
UNC29
UNC38
UNC38
UNC29
UNC38
UNC29
UNC38
UNC38
1:5 Ascsu unc-38: unc-29
5:1 Ascsu unc-38: unc-29
levamisole
levamisole
nicotine
nicotine
Kopp et al. 2008 IJP: qtPCR A. caninum pyrantel-sensitive vs -resistant. Expression of aar-38, aar-63 and aar-29 reduced
Ref gene Isolate aar-38 aar-63 aar-29 aar-19 aar-15
aar-8 PR/NT
60S PR 0.11 ± 0.013 0.29
0.66 ± 0.0090
0.55 0.94 ± 0.046 0.62 0.54 ±
0.0210.99
0.31 ± 0.018
0.890.41 ± 0.037
0.92
NT0.37 ± 0.022a
1.2 ± 0.070a
1.5 ± 0.035a
0.55 ± 0.0082
0.35 ± 0.091
0.45 ± 0.018
Also no changes in E153 and Q57 – pyrantel-specific sites Rayes et al. (2006)
1.aar-29 -I50L, S206T,I309L (adults and larvae), I422L in both isolates and L466M in the NT isolate,
2.aar-63-L393I(adults and larvae)
3.No 38 amino-acid changes
4.15-46 nucleotide changes
N-subtype
nAChR subtypes and resistance
L-subtype
B-subtype
levamisole resistance
Monepantel resistance: Hco-mptl-1; Hco-des -2H; Hco-deg-3H; Rufener et al.,
Monepantel resistance: Hco-mptl-1, some lack splice acceptor (TTTCAG)
Molecular detection of resistance: levamisole & monepantel
• Kopp et al., Did not find specific SNPs associated with resistance
• E153 & Q57 not associated with pyrantel resistance• Expression levels of UNC-38, UNC-29 and UNC-63 are
reduced in pyrantel resistance • Rufener et al., Hco-mptl-1 & Hco-des-2H, 1. a panel of mutations (null mutants) including deletions
at intron-exon boundaries giving mis-spliced transients & premature stop codons2. Altered expression levels of Hco-mptl-1 & Hco-des-2H and & Hco-deg-3H
rNAInner nuclear
membrane
Putative L-type receptor subunits:
UNC-38, UNC-29, UNC-63, LEV-1, ACR-13LEV-10
NRA-1
SOC-1 TPA-1
TAX-6
UNC-22
LEV-11
Muscle contraction
UNC-50 RIC-3
Translation
UNC-74
Processing & assembly
Ca CaER
UNC-68
C. elegans proteins involved in levamisole resistance
Resistance may be due to a panel of functional null mutant genes with deletions and insertions and reduced expression of the target receptor genes + other sites!
Acknowledgments• Alan Robertson• Sally Williamson• Sreekanth Puttachary• Sam Buxton• Cheryl Clark• Adrian Wolstenholme• Steve Koppe• Andrew Kotze• Supported by NIH: R01 R 01 AI 047194
UNC-38 LEV-8
UNC-29
UNC-63 LEV-1
C. elegans levamisole-sensitive channel
G= 30 pS