Nicotinic Receptors and Anthelmintic Resistance

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