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Nicotinic Receptors and Anthelmintic Resistance

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.

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Nicotinic Receptors and Anthelmintic Resistance

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  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 • Imidazothiazoles: levamisole, agonist • Tetrahydropyrimidines: pyrantel , agonist • Quaternary Amines: bephenium , agonist • AADs: monepantel , agonist • Macfortines: paraherquamide & derquantel, antagonist

  3. A B C Na+, Ca2+ Complementary site on adjacent subunit Agonist binding site D E Y111 Y117 outside W55/57 α α W149 Y151 C D180 F Y190 C192 C193 Y198 Y93 W86 B A inside Primary site on α subunit

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

  5. 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. 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. Muscle nAChRs in C. elegans G= 30 pS (Qian et al, 2008) ACR 16 LEV 8 UNC 63 ACR 16 ACR 16 LEV 1 UNC 29 UNC 38 ACR 16 ACR 16 C. elegans levamisole sensitive receptor C. elegans nicotine sensitive receptor

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

  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 B-type L-type

  8. 50 L-type B-type 40 B-type O 3 30 L-type O Number of observations (N) 20 2 N-type N-type B-subtype 45pS bephenium 10 O 1 L-subtype 35pS levamisole 0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 N-subtype 24pS nicotine Amplitude (pA) C 2-desoxoparaherquamide (derquantel) & paraherquamide paraherquamide 3 channel currents & 3 nAChR subtypes B A C

  9. Asu-UNC38 Asu-UNC29

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

  11. Kopp et al. 2008 IJP: qtPCR A. caninum pyrantel-sensitive vs -resistant. Expression of aar-38, aar-63 and aar-29 reduced Also no changes in E153 and Q57 – pyrantel-specific sites Rayes et al. (2006) aar-29 -I50L, S206T,I309L (adults and larvae), I422L in both isolates and L466M in the NT isolate, aar-63-L393I(adults and larvae) No 38 amino-acid changes 15-46 nucleotide changes

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

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

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

  15. 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

  16. 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-68 Ca Ca ER Processing & assembly UNC-22 UNC-74 Translation UNC-50 RIC-3 LEV-11 Inner nuclear membrane Muscle contraction rNA C. elegans proteins involved in levamisole resistance

  17. 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!

  18. 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

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

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