1. Nicotinic Receptors and Anthelmintic Resistance Richard J. Martin, Adrian J. Wolstenholme, S. M. Williamson & Alan P. Robertson Department of Biomedical Sciences, Iowa State University, Ames, Iowa USA & Dept of Infectious Diseases, University of Georgia, Athens, Georgia

2. Nicotinic Anthelmintics 1.Imidazothiazoles: levamisole, agonist 2.Tetrahydropyrimidines: pyrantel, agonist 3.Quaternary Amines: bephenium, agonist 4.AADs: monepantel, agonist 5.Macfortines: paraherquamide & derquantel, antagonist

3. Complementary site on adjacent subunit Primary site on α subunit A B C D E F Y 93 W 86 Y 190 C 192 C 193 Y 198 W 149 Y 151 Y 111 Y 117 W 55/57 D 180 ABC Na +, Ca 2+ α Agonist binding site outside inside α

4. C. elegans nicotinic receptor subunits Jones et al., 2007: 29 subunits, 5 core structures AADs levamisole

5. Muscle nAChRs in C. elegans C. elegans levamisole sensitive receptor G= 30 pS (Qian et al, 2008) Analysis of the Brugia malayi (clade III) and Trichinella spiralis (clade I) genome sequences revealed only 9 and 8 clear nicotinic sequences respectively. In particular there were no sequences equivalent to the neuromuscular subunit genes lev-1, lev-8 and acr- 16, though unc-29, unc-38 and unc-63 were conserved. Cloning experiments using A. suum muscle preparations supported this analysis, and we obtained full-length cDNA clones encoding UNC- 29 and UNC-38, and a partial clone encoding UNC- 63. One nicotinic gene present in B. malayi (Bm1_48815 = unc-26) is not present in C. elegans. This gene is also present in A. suum and in clade V parasite species such as Haemonchus contortus. ACR 16 LEV 8 UNC 63 UNC 29 LEV 1 UNC 38 C. elegans nicotine sensitive receptor

6. Nicotinic anthelmintic 5 min 1g 10 nM 100 nM 300 nM 1  M 30  M 30 nM Control dose-response trace

7. 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

8. C O 1 O 2 O 3 L-type N-type 1.52.02.53.03.54.04.5 0 10 20 30 40 50 Number of observations (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

10. Asu-UNC38 Asu-UNC29

11. L-subtype receptor N-subtype receptor 1:5 Ascsu unc-38: unc-29 5:1 Ascsu unc-38: unc-29 levamisole nicotine

13. Kopp et al. 2008 IJP: qtPCR A. caninum pyrantel-sensitive vs -resistant. Expression of aar-38, aar-63 and aar-29 reduced Ref gene Isolateaar-38aar-63aar-29aar-19aar-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 ± 0.021 0.99 0.31 ± 0.018 0.89 0.41 ± 0.037 0.92 NT 0.37 ± 0.022 a a 1.2 ± 0.070 a a 1.5 ± 0. 035 a a 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

14. N-subtype nAChR subtypes and resistance L-subtype B-subtype levamisole resistance

15. Monepantel resistance: Hco-mptl-1; Hco-des - 2H; Hco-deg-3H; Rufener et al.,

16. Monepantel resistance: Hco-mptl-1, some lack splice acceptor (TTTCAG)

17. 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 codons 2. Altered expression levels of Hco-mptl-1 & Hco-des-2H and & Hco-deg-3H

18. rNA Inner nuclear membrane Putative L-type receptor subunits: UNC-38, UNC-29, UNC-63, LEV-1, ACR-13 LEV-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 ER UNC-68 C. elegans proteins involved in levamisole resistance

19. 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!

20. 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

21. UNC-38LEV-8 UNC-29 UNC-63 LEV-1 C. elegans levamisole- sensitive channel G= 30 pS