2007 – 2009 publications

Macrocyclic lactone resistance:1. Understanding resistance mechanisms to

different MLs2. Progress with possible markers for ML

resistances

Roger PrichardInstitute of Parasitology

McGill University, Montreal, Canada

Ligand-gated ion channels

Fig. 4. Dose-response curves for L-glutamate at mutant H. contortusGluClα3B channels. , wild-type; , V235A; , ✖

E114G; , L256F. McCaverna et al. Mol. Pharmacol. 2009

H. contortus GluClα3B expressed in Xenopus oocytes

The effects of ivermectin resistance-associated mutations on the ability of glutamate to activate H. contortus GluCl3B channels (mean SEM)

Mutation EC50 for L-Glutamate Hill Number

Wild type 27.6 ±2.7 1.89 ± 0.35 E114G 31.5 ± 3.2 1.48 ± 0.31V235A 26.2 ± 2.5 1.97 ± 0.35L256F 92.2 ± 3.5*** 1.09 ± 0.16**T300S No channels

** p 0.01, *** p 0.001

Radioligand binding assays using[3H]ivermectin and membranepreparations from COS-7 cells transfected with wild-type and mutant H. contortus GluClα3B. The insets show the Scatchard plots for each mutant.McCaverna et al. Mol. Pharmacol. 2009

The effects of mutations in H. contortus GluClα3B on binding of 3[H]ivermectin to membrane preps from transfected COS-7 cells. Mean± SEM

Mutation Kd 3[H]Ivermectin Binding, nM

Wild type 0.35 ± 0.1E114G 0.39 ± 0.07V235A 0.32 ± 0.09L256F 2.26 ± 0.78***L256W 2.51 ± 0.7***L256Y 1.84 ± 0.49***L256V 0.79 ± 0.24*T300S 0.76 ± 0.25

* p< 0.05; *** p< 0.001

Annelies Van Zeveren in her Ph.D. studies looked for the L256F SNP, in GluClα3

homolog, in an ivermectin resistant strain of Ostertagia ostertagi that she and her

colleagues had experimentally selected, but did not find the L256F mutation.

L256F, in GluClα3, may be quite rare in different nematode isolates. However, if it does occur it does seem to produce an

‘IVM-resistance phenotype’

A dopamine-gated ion channel (HcGGR3*) from Haemonchus contortus is expressed in the

cervical papillae and is associated with macrocyclic lactone resistance

Rao VT, Siddiqui SZ, Prichard RK, Forrester SG.Mol Biochem Parasitol. 2009 Jul;166(1):54-61. Epub

2009 Mar 4.

Electrophysiology of HcGGR3 in Xenopus laevis oocytes

Response to dopamine

Response to other amines

HcGGR3 forms a homomeric channel and is primarily gated by dopamineRao et al. Mol. Biochem. Parasitol. 2009

Expression of HcGGR3 in lab macrocyclic lactone (ML)-selected strains

of Haemonchus contortus (♀)

qPCR: Standard curve relative quantification methodFold change as compared to expression in PF23 strain

(normalized with 18srRNA)

anova: P<0.0001

HcGGR3 is down regulated in lab strains of ML- selected worms

Level of expression in PF23 strain

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Macrocyclic lactone selected lab strains

PF23: ML sensitive strainIVF23 & MOF23: Lab selected strains

Rao et al. Mol. Biochem. Parasitol. 2009

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PF23 IVF23 MOF23

Allel

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Strains of H. Contortus

Genotyping of Hcggr3 for the region encoding 3’ UTR(single ♂s)

N = 30

Selection of HcGGR3 allele in Macrocyclic lactone resistance

GGTGGGTAAACGATAGATCAACTATATCGCACATAAAAAATACAATGCAAACACATATTTTGTAAACGTTAATTCCACCGTAGCCAATTAACCGTATAAATATGCAATTCAACCTAATTGAGTTGCATTATCACATTCGTTGATTTCTCTAAATGTAGAGAGTTGAGGAG

PF23: ML sensitive strainIVF23 & MOF23: Lab selected strains

Heterozygous: T/CHomozygous: T/T

Rao et al. Mol. Biochem. Parasitol. 2009

Pharyngeal pump rates of wild-type C. elegans after 2.5 h exposure to 2-fold serial dilutions of IVM and MOX (mean ± SD). A circle ( ) represents pumping rate in

non-treated worms, a triangle ( ) indicates pumping rate in IVM exposed worms and a square ( ) indicates pumping rate in MOX exposed worms.

Ardelli et al. 2009 Vet Parasitol

Motility phenotype of wild-type C. elegans after exposure to 2.5 nM of drug (mean ± SD). A circle ( ) represents velocity in non-treated worms, a triangle ( ) indicates velocity in IVM exposed worms and a square ( )

indicates velocity in MOX exposed wormsArdelli et al. 2009 Vet Parasitol

Transcriptional profiles of GluCl genes in wild-type C. elegans after exposure to 2.5 nM of IVM or MOX for 2h (mean ± SD). The black bars represent gene transcription after exposure to IVM and the grey bars

represent gene transcription after exposure to MOX. The y-axis represents the fold change in transcription relative to non-treated controls and listed

along the x-axis is the C. elegans gene. Changes that are statistically different between the drug treatments (p < 0.05) following IVM or MOX

exposure are indicated with .Ardelli et al. 2009 Vet Parasitol

ABC transporters

Effect of the ivermectin (IVE) and selamectin (SEL)] on mitoxantrone (MXR) accumulation mediated by human ABCG2 and murine Abcg2. Transduced MDCKII cells were preincubated with or without Ko143 (1 µM) or the other tested compounds (50 µM). Mean MXR fluorescence is shown in terms of relative arbitrary units. The bars indicate the means ± S.D. ** p< 0.01, comparing the difference between human ABCG2- and murine Abcg2- transduced MDCKII cells. §§, p< 0.01, comparing the difference between IVE and SEL in murine Abcg2-transduced cells. §§§, p< 0.001, comparing the difference between IVE and SEL in human ABCG2-transduced cells.

Merino et al. Drug Met. Disp. 2009

Natural Allelic Variants of Bovine ATP-Binding Cassette Transporter ABCG2: Increased Activity of the Ser581 Variant and Development of Tools for the Discovery of New ABCG2 Inhibitors

Selamectin was a significantly more potent inhibitor of the Tyr581 variant compared with the wild-type Ser581

Half-transporters

Cross-resistance to anthelmintics. C. elegans L1s in M9 buffer were incubated in serial dilutions of moxidectin (MOX), levamisole (LEV), albendazole (ALB) or pyrantel (PYR), and their fold-resistance determined in IVR6 (solid) and IVR10 (hatched) relative to Bristol N2 strain (1; dotted line). James & Davey, Int. J. Parasitol. 2008

IVM selection on C. elegans

ABC transporter gene expression in ivermectin-resistant Caenorhabditis elegans. pgp-1, pgp-2, mrp-1, mrp-2, mrp-5 and mrp-6 were amplified from cDNA using gene-specific primers in IVR6 (solid) and IVR10 (hatched) strains cultured with ivermectin. James & Davey, Int. J. Parasitol. 2008.

‘Natural’ IVM resistance in C. elegans results in overexpression of a number of ABC transporter genes

(Pgps & mrps)

The expression of gcs-1 and gstp-1 (B) was examined in IVR6 (solid) and IVR10 (hatched) strains cultured on ivermectin. Bars show means ± SD of the fold change in gene expression relative to the Bristol N2 strain from 2 independent experiments. * indicates P≤0.05; ** indicates P≤0.01. James & Davey, Int. J. Parasitol. 2008.

c-glutamylcysteinesynthetase (gcs-1), the rate-limiting enzyme for glutathione synthesis was overexpressed in IVR10,

while a glutathione transferase π (gstp-1) was overexpressed in IVR6

Allelic variation in an Onchocerca volvulus ABC transporter was reduced in IVM-treated human populations in Ghana in 1999

Ardelli & Prichard, Trans R Soc Trop Med Hyg. 2007, 101:1223

05

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Frequency

P=0.0077

P=0.048 P=0.048

P=0.029

P=0.054

BEFORE IVM

AFTER IVM 13x

PLP (half-transporter) Genotype before IVM and after 13X IVM: Onchocerca

volvulus

Fisher’s exact test Pre IVM= 66Post IVM=35

Female worms

Allelic selection after 13 three-monthly doses of IVM in female wormsLoss of polymorphism. Bourguinat et al. 2008 Mol Biochem Parasitol

Position 1 =AA/AG/GG Position 2 =CC/CG/GGPosition 4 =AA/AT/TT

Mrp expression in C. elegans following exposure to 2.5 nM IVM, compared with wild-type worms not exposed to drug

Mrp expression in C. elegans following expsure to 2.5 nM MOX, compared with wild-type worms not exposed to drug

B Ardelli & Prichard J. Helminthol. in press

Analysis of H. contortus showed that expression of both

thioredoxin 12-kDa (HcTrx1) and the 16-kDa (HcTrx3)

genes were increased in an IVM-resistant strain relative to

a sensitive strain.

Sotirchos, Hudson , Ellis & Davey. Free Radic Biol

Med. 2008

Thioredoxins and IVM resistance

β-tubulin and ML resistance

H. contortus: Comparison of the frequency of 200TTC-phenylalanine, 200TTC/TAC-phenylalanine/tyrosine and 200TAC-tyrosine in unselected

(PF), IVM selected (IVF) and MOX selected (MOF) strains

Tyr at amino acid 167 or 200: PF = 11.1%; IVF = 48.1%; MOF = 51.9%

Mottier & Prichard 2008 Pharmacogenetics & Genomics

β-tubulin

BEFORE TXAFTER TX

Onchocerca volvulus: β- tubulin Female worms after 13 x IVM

N before=183N after 4 doses=59N after 13 doses=39

IVM selection caused a significant change in β-tubulin genotype

Bourguinat et al. 2007 PLoS NTD

Before and after 13 three-monthly doses

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Fisher’s exact test

P=1e-6

P=1e-8

TAC/TTC in H. contortus β-tubulin in Swedish flocks which had been under BZ or ML treatment

“An allele frequency of ≥65% was detected in one of the two flocks in 13 (29%) of the 45 farms examined. On many farms (24, 25, 33, 36, 37, 39, 42, 43 and 44) the allele frequency was similar in both the BZ and ML treated flocks”

Höglund et al. Vet Parasitol, 2009

Conclusions• ML resistances appear to be multigenic

• Phenotypic effects of IVM & MOX markedly different

• Differences in genes implicated in IVM & MOX resistances

• Possibly GluCls involved in ML resistances, but data not conclusive

• ABC transporters induced & overexpressed in ML resistances, but not same between IVM & MOX

• MLs select on β-tubulin

• Thioredoxins may also be overexpressed in ML resistances