- Email: [email protected]
Definitive proof for a role of pfmdr 1 in quinoline resistance in Plasmodium falciparum
Ward and Bray
Definitive proof for a role of pfmdr 1 in quinoline resistance in Plasmodium falciparum S.A.Ward and P. G. Bray Department o Pharmacology & Therapeutics, University of Liverpool, Liverpool, UK
Key words: chloroquine, resistance, pfmdr 1, pgh 1
T
he idea that resistance to the 4 – aminoquinoline antimalarial chloroquine might involve an MDR type process was provided as early as 1987 by Martin et al.1 They were able to demonstrate convincingly the ability of verapamil to partially reverse chloroquine resistance in vitro, an observation that has been repeated by numerous groups using a range of chemosensitizers. This phenomenon coupled with the knowledge that chloroquine resistant parasites exhibited reduced drug accumulation compared to their sensitive counterparts stimulated the search for MDR homologues in plasmodium falciparum. These efforts led to the identification of pfmdr 12 and pfmdr2.3 Further investigations suggested that pfmdr 2 was unlikely to play any role in chloroquine resistance whereas pfmdr 1 was considered a good candidate drug resistance gene (for review see ref. 4). The pfmdr 1 gene encodes a 162 Kd protein (pgh1) with membrane topology typical of a P-glycoprotein which is localized primarily on the food vacuole and plasma membranes of the parasite.5 By analogy with MDR resistance in tumour cells, initial reports suggested that resistance to chloroquine and also to the related quinoline methanol mefloquine, was associated with amplification of pfmdr 12 with drug sensitive parasite isolates showing no evidence for amplification. However, subsequent studies provide compelling evidence that pfmdr 1 amplification is incompatible with chloroquine resistance, although it may be a factor in resistance to mefloquine and related drugs5 (reviewed in ref. 4). While amplification of pfmdr 1 failed to explain chloroquine resistance, evidence was presented to implicate mutations in pfmdr 1 as the basis for resistance. Foote et al.6 identified two mutant alleles of pfmdr 1, the K1 allele that codes for a single amino acid change from Asn86 to Tyr86 and the 7G8 allele that encodes four amino acid substitutions (Tyr184 to Phe184, Ser1034 to Cys1034,Asn1042 to Asp1042 and Asp1246 to Tyr1246). Based on these specific alleles, Foote et al.6 were able to predict correctly the chloroquine resistance status of 34 out of 36 parasite isolates in a blinded study. Despite this robust argument for a role of pfmdr 1 in quinoline susceptibility, several lines of evidence were reported which cast doubt on the universal applicability of this hypothesis. A number of studies, using both field and laboratory cultured isolates of P. falciparum have demonstrated the classical chloroquine resistance phenotype
80
Drug Resistance Updates (2000) 3, 80–81
2000 Harcourt Publishers Ltd
DOI: 10.1054/drup.2000.0136, available online at http://www.idealibrary.com on
(reduced energy dependent drug accumulation and the verapamil effect) without an association with either pfmdr 1 amplification or mutation (reviewed in ref. 4).These observations do not negate the potential role of pfmdr 1 in drug resistance but indicate that other genes must also be capable of contributing to the chloroquine resistance phenotype in a similar way. However, studies carried out by Wellems et al.7 seemed to completely rule out any role for pfmdr 1 in chloroquine resistance.These investigators carried out a genetic cross between the chloroquine sensitive parasite HB3 and the resistant isolate Dd2. Analysis of the progeny of this cross indicated that chloroquine resistance was inherited as a monogenic trait. The progeny exhibited only phenotypic characteristics of the sensitive parent (IC50s of 8 or 6 ng/ml and no verapamil effect) or the resistant parent (IC50s of 64 ng/ml and the demonstration of the verapamil effect). The authors were able to narrow down the single genetic locus to a segment on chromosome 78 and subsequent studies have identified a number of candidate resistance genes at this locus with Cg2 initially being proposed as the chloroquine resistance gene.9 pfmdr 1 is localized to chromosome 5 and therefore could not influence drug sensitivity in this genetic cross based on their analysis. Despite these observations, the group at the Walter and Elisa Hall Institute in Melbourne have continued to investigate pfmdr 1 as a potential drug resistance gene. This decision is vindicated by recent studies that indicate pfmdr 1 to be as predictive Cg2 in choroquine resistance.10 These investigations have led to the recent publication of an elegant study which has used allelic replacement to definitively prove a role for mutations in pfmdr 1 in resistance to chloroquine and a range of other commonly used antimalarials.11 Allelic exchange was performed (with appropriate controls) such that three of the known 7G8 mutations at 1034, 1042 and 1246 were introduced in place of the wild type pfmdr 1 sequence in the chloroquine sensitive isolate D10 (one transfected line incorporated only the 1246 mutation). Using a similar strategy, the mutations at 1034, 1042 and 1246 in the 7G8 chloroquine resistant isolate were replaced with wild type sequence. The resulting parasite lines were assessed for baseline sensitivity to chloroquine, mefloquine, quinine, halofantrine and artemisinin, the presence or absence of the verapamil effect and parasite accumulation of chloroquine and mefloquine. Considering the D10 isolate first, introduction of the 7G8 mutations at 1246 or at 1034, 1042 and 1246 had no effect on parasite sensitivity to chloroquine. In contrast sensitivity to mefloquine, halofantrine and artemisinin was increased and sensitivity to quinine was markedly reduced.The lack of any influence on chloroquine phenotype would confirm the view that there is a number of genes that operate in a coordinated manner to bring about chloroquine resistance. The introduction of a contributing resistance gene into a drug sensitive background, as in D10, therefore has no impact. Clearly these mutations alone are sufficient to reduce susceptibility to quinine. It would be interesting to see if the loss of quinine sensitivity is associated with the appearance of a verapamil effect, which has previously been demonstrated in some quinine resistant isolates. The increased
pfmdr 1 in quinoline resistance sensitivity to mefloquine, halofantrine and artemisinin is proof for a role of the gene in the control of parasite sensitivity to these drugs. This observation is in agreement with data showing that amplification of pfmdr 1 can reduce sensitivity to these drugs and confirms the view the mutations decrease the efficiency of pfmdr 1 in removing drug from its target site (either directly or indirectly) compared to wild type pfmdr 1. The replacement of the 7G8 mutations at 1034, 1042 and 1246 with wild type sequence, as would be predicted from the studies with the D10 lines, resulted in a decreased sensitivity to mefloquine, halofantrine and artemisinin and improved sensitivity to quinine.With respect to chloroquine, replacement with wild type pfmdr 1 sequence into a chloroquine resistant background resulted in a marked improvement in chloroquine sensitivity from a position of very high resistance (>350 nM) to a position moderate resistance (200 nM).This shift was associated with an improvement in high affinity chloroquine accumulation and a reduction in the size of the verapamil effect, although the effect was not abolished as seen in truly sensitive parasites.These data provide the first definitive proof that pfmdr 1 can contribute to chloroquine resistance and confirm the view that chloroquine resistance is multigenic. These studies must be considered a major step forward in our understanding of drug resistance in Plasmodium falciparum. It remains to be established whether pfmdr 1 can actually pumpout drug or whether the alterations in drug accumulation reflect an indirect effect, perhaps by altering transmembrane pH gradients and potential or by transporting the high affinity drug binding, heme or competitors of heme binding.12 Similarly, we await the identification of the other genes that are clearly essential to the chloroquine resistance phenotype and that are required for pfmdr 1 to exert influence on chloroquine sensitivity. Elucidation of the sequence of molecular events and the biochemical changes which accompany the move from fully chloroquine sensitive to highly resistant should provide a platform for the design of strategies to overcome chloroquine resistance. It is possible that Cg2 may be one of these other contributing genes and recent studies suggest that prediction of chloroquine resistance is improved if both pfmdr 1 and Cg2 are taken into consideration. Finally, the strategy adopted by the group in Melbourne emphasizes the power of this technology and provides the impetus to apply similar approaches to other crucial biological questions posed by P. falciparum.
Received and accepted 29 March 2000 Correspondence to: S.A.Ward Pharmacology Research Labs, 70 Pembroke Place, Liverpool L69 3GE, UK.Tel: +44(0) 151 794 8219; Fax: +44(0) 151 794 8217; E-mail: [email protected]
References 1. Martin SK, Oduola AMJ, Milhous WK. Reversal of chloroquine resistance in Plasmodium falciparum by verapamil. Science 1987; 235: 899–901. 2. Foote SJ,Thompson JK, Cowman AF, Kemp DJ.Amplification of the multidrug resistance gene in some chloroquine resistant isolates of Plasmodium falciparum. Cell 1989; 57: 921–930. 3. Zalis MG,Wilson CM, Zhang Y,Wirth WK. Characterisation of the pfmdr 2 gene for Plasmodium falciparum. Mol Biochem Parasitol 1993; 62: 83–93. 4. Bray PG,Ward SA.A comparison of the phenomenology and genetics of multi-drug resistance in cancer cells and quinoline resistance in Plasmodium falciparum. Pharmacol Ther 1998; 77: 1–28. 5. Cowman AF, Karcz S, Galatis D, Culvenor JG.A P-glycoprotein homologue of Plasmodium falciparum is localized on the digestive vaculoe. J Cell Biol 1991; 113: 1033–1045. 6. Foote SJ, Kyle DE, Martin RK, et al. Several alleles of the multidrug resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature 1990; 345: 255–258. 7. Wellems TE, Panton LJ, Gluzman IY, et al. Chloroquine resistance is not linked to mdr-like genes in Plasmodium Falciparum cross. Nature 1990; 345: 253–255. 8. Wellems TE,Walker-Jonah A, Panton LJ. Genetic mapping of the chloroquine resistance locus on Plasmodium falciparum chromosome 7. Proc Natl Acad Sci U.S.A. 1991; 88: 3382–3386. 9. Su XZ, Kirkman LA, Fujioka H,Wellems TE. Complex polymorphisms in a 330 kDa protein are linked to chloroquine resistance in Plasmodium falciparum in Southeast Asia and Africa. 1997; 91: 593–603. 10. Adagu IS and Warhurst DC.Association of cg2 and pfmdr 1 genotype with chloroquine resistance in field samples of Plasmodium falciparum from Nigeria. Parasitology 1999; 119: 343–348. 11. Reed MB, Saliba KJ, Caruana SR, Kirk K, Cowman AF. Pgh 1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 2000; 403: 906–909. 12. Bray PG, Janneh O, Raynes KJ, Mungthin M, Ginsburg H,Ward SA. Cellular uptake of chloroquine is dependent on binding to ferriprotoporphyrin IX and is independent of NHE activity in Plasmodium falciparum. J Cell Biol 1999; 145: 363–376.
2000 Harcourt Publishers Ltd Drug Resistance Updates (2000) 3, 80–81
81
Definitive proof for a role of pfmdr 1 in quinoline resistance in Plasmodium falciparum S.A.Ward and P. G. Bray Department o Pharmacology & Therapeutics, University of Liverpool, Liverpool, UK
Key words: chloroquine, resistance, pfmdr 1, pgh 1
T
he idea that resistance to the 4 – aminoquinoline antimalarial chloroquine might involve an MDR type process was provided as early as 1987 by Martin et al.1 They were able to demonstrate convincingly the ability of verapamil to partially reverse chloroquine resistance in vitro, an observation that has been repeated by numerous groups using a range of chemosensitizers. This phenomenon coupled with the knowledge that chloroquine resistant parasites exhibited reduced drug accumulation compared to their sensitive counterparts stimulated the search for MDR homologues in plasmodium falciparum. These efforts led to the identification of pfmdr 12 and pfmdr2.3 Further investigations suggested that pfmdr 2 was unlikely to play any role in chloroquine resistance whereas pfmdr 1 was considered a good candidate drug resistance gene (for review see ref. 4). The pfmdr 1 gene encodes a 162 Kd protein (pgh1) with membrane topology typical of a P-glycoprotein which is localized primarily on the food vacuole and plasma membranes of the parasite.5 By analogy with MDR resistance in tumour cells, initial reports suggested that resistance to chloroquine and also to the related quinoline methanol mefloquine, was associated with amplification of pfmdr 12 with drug sensitive parasite isolates showing no evidence for amplification. However, subsequent studies provide compelling evidence that pfmdr 1 amplification is incompatible with chloroquine resistance, although it may be a factor in resistance to mefloquine and related drugs5 (reviewed in ref. 4). While amplification of pfmdr 1 failed to explain chloroquine resistance, evidence was presented to implicate mutations in pfmdr 1 as the basis for resistance. Foote et al.6 identified two mutant alleles of pfmdr 1, the K1 allele that codes for a single amino acid change from Asn86 to Tyr86 and the 7G8 allele that encodes four amino acid substitutions (Tyr184 to Phe184, Ser1034 to Cys1034,Asn1042 to Asp1042 and Asp1246 to Tyr1246). Based on these specific alleles, Foote et al.6 were able to predict correctly the chloroquine resistance status of 34 out of 36 parasite isolates in a blinded study. Despite this robust argument for a role of pfmdr 1 in quinoline susceptibility, several lines of evidence were reported which cast doubt on the universal applicability of this hypothesis. A number of studies, using both field and laboratory cultured isolates of P. falciparum have demonstrated the classical chloroquine resistance phenotype
80
Drug Resistance Updates (2000) 3, 80–81
2000 Harcourt Publishers Ltd
DOI: 10.1054/drup.2000.0136, available online at http://www.idealibrary.com on
(reduced energy dependent drug accumulation and the verapamil effect) without an association with either pfmdr 1 amplification or mutation (reviewed in ref. 4).These observations do not negate the potential role of pfmdr 1 in drug resistance but indicate that other genes must also be capable of contributing to the chloroquine resistance phenotype in a similar way. However, studies carried out by Wellems et al.7 seemed to completely rule out any role for pfmdr 1 in chloroquine resistance.These investigators carried out a genetic cross between the chloroquine sensitive parasite HB3 and the resistant isolate Dd2. Analysis of the progeny of this cross indicated that chloroquine resistance was inherited as a monogenic trait. The progeny exhibited only phenotypic characteristics of the sensitive parent (IC50s of 8 or 6 ng/ml and no verapamil effect) or the resistant parent (IC50s of 64 ng/ml and the demonstration of the verapamil effect). The authors were able to narrow down the single genetic locus to a segment on chromosome 78 and subsequent studies have identified a number of candidate resistance genes at this locus with Cg2 initially being proposed as the chloroquine resistance gene.9 pfmdr 1 is localized to chromosome 5 and therefore could not influence drug sensitivity in this genetic cross based on their analysis. Despite these observations, the group at the Walter and Elisa Hall Institute in Melbourne have continued to investigate pfmdr 1 as a potential drug resistance gene. This decision is vindicated by recent studies that indicate pfmdr 1 to be as predictive Cg2 in choroquine resistance.10 These investigations have led to the recent publication of an elegant study which has used allelic replacement to definitively prove a role for mutations in pfmdr 1 in resistance to chloroquine and a range of other commonly used antimalarials.11 Allelic exchange was performed (with appropriate controls) such that three of the known 7G8 mutations at 1034, 1042 and 1246 were introduced in place of the wild type pfmdr 1 sequence in the chloroquine sensitive isolate D10 (one transfected line incorporated only the 1246 mutation). Using a similar strategy, the mutations at 1034, 1042 and 1246 in the 7G8 chloroquine resistant isolate were replaced with wild type sequence. The resulting parasite lines were assessed for baseline sensitivity to chloroquine, mefloquine, quinine, halofantrine and artemisinin, the presence or absence of the verapamil effect and parasite accumulation of chloroquine and mefloquine. Considering the D10 isolate first, introduction of the 7G8 mutations at 1246 or at 1034, 1042 and 1246 had no effect on parasite sensitivity to chloroquine. In contrast sensitivity to mefloquine, halofantrine and artemisinin was increased and sensitivity to quinine was markedly reduced.The lack of any influence on chloroquine phenotype would confirm the view that there is a number of genes that operate in a coordinated manner to bring about chloroquine resistance. The introduction of a contributing resistance gene into a drug sensitive background, as in D10, therefore has no impact. Clearly these mutations alone are sufficient to reduce susceptibility to quinine. It would be interesting to see if the loss of quinine sensitivity is associated with the appearance of a verapamil effect, which has previously been demonstrated in some quinine resistant isolates. The increased
pfmdr 1 in quinoline resistance sensitivity to mefloquine, halofantrine and artemisinin is proof for a role of the gene in the control of parasite sensitivity to these drugs. This observation is in agreement with data showing that amplification of pfmdr 1 can reduce sensitivity to these drugs and confirms the view the mutations decrease the efficiency of pfmdr 1 in removing drug from its target site (either directly or indirectly) compared to wild type pfmdr 1. The replacement of the 7G8 mutations at 1034, 1042 and 1246 with wild type sequence, as would be predicted from the studies with the D10 lines, resulted in a decreased sensitivity to mefloquine, halofantrine and artemisinin and improved sensitivity to quinine.With respect to chloroquine, replacement with wild type pfmdr 1 sequence into a chloroquine resistant background resulted in a marked improvement in chloroquine sensitivity from a position of very high resistance (>350 nM) to a position moderate resistance (200 nM).This shift was associated with an improvement in high affinity chloroquine accumulation and a reduction in the size of the verapamil effect, although the effect was not abolished as seen in truly sensitive parasites.These data provide the first definitive proof that pfmdr 1 can contribute to chloroquine resistance and confirm the view that chloroquine resistance is multigenic. These studies must be considered a major step forward in our understanding of drug resistance in Plasmodium falciparum. It remains to be established whether pfmdr 1 can actually pumpout drug or whether the alterations in drug accumulation reflect an indirect effect, perhaps by altering transmembrane pH gradients and potential or by transporting the high affinity drug binding, heme or competitors of heme binding.12 Similarly, we await the identification of the other genes that are clearly essential to the chloroquine resistance phenotype and that are required for pfmdr 1 to exert influence on chloroquine sensitivity. Elucidation of the sequence of molecular events and the biochemical changes which accompany the move from fully chloroquine sensitive to highly resistant should provide a platform for the design of strategies to overcome chloroquine resistance. It is possible that Cg2 may be one of these other contributing genes and recent studies suggest that prediction of chloroquine resistance is improved if both pfmdr 1 and Cg2 are taken into consideration. Finally, the strategy adopted by the group in Melbourne emphasizes the power of this technology and provides the impetus to apply similar approaches to other crucial biological questions posed by P. falciparum.
Received and accepted 29 March 2000 Correspondence to: S.A.Ward Pharmacology Research Labs, 70 Pembroke Place, Liverpool L69 3GE, UK.Tel: +44(0) 151 794 8219; Fax: +44(0) 151 794 8217; E-mail: [email protected]
References 1. Martin SK, Oduola AMJ, Milhous WK. Reversal of chloroquine resistance in Plasmodium falciparum by verapamil. Science 1987; 235: 899–901. 2. Foote SJ,Thompson JK, Cowman AF, Kemp DJ.Amplification of the multidrug resistance gene in some chloroquine resistant isolates of Plasmodium falciparum. Cell 1989; 57: 921–930. 3. Zalis MG,Wilson CM, Zhang Y,Wirth WK. Characterisation of the pfmdr 2 gene for Plasmodium falciparum. Mol Biochem Parasitol 1993; 62: 83–93. 4. Bray PG,Ward SA.A comparison of the phenomenology and genetics of multi-drug resistance in cancer cells and quinoline resistance in Plasmodium falciparum. Pharmacol Ther 1998; 77: 1–28. 5. Cowman AF, Karcz S, Galatis D, Culvenor JG.A P-glycoprotein homologue of Plasmodium falciparum is localized on the digestive vaculoe. J Cell Biol 1991; 113: 1033–1045. 6. Foote SJ, Kyle DE, Martin RK, et al. Several alleles of the multidrug resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature 1990; 345: 255–258. 7. Wellems TE, Panton LJ, Gluzman IY, et al. Chloroquine resistance is not linked to mdr-like genes in Plasmodium Falciparum cross. Nature 1990; 345: 253–255. 8. Wellems TE,Walker-Jonah A, Panton LJ. Genetic mapping of the chloroquine resistance locus on Plasmodium falciparum chromosome 7. Proc Natl Acad Sci U.S.A. 1991; 88: 3382–3386. 9. Su XZ, Kirkman LA, Fujioka H,Wellems TE. Complex polymorphisms in a 330 kDa protein are linked to chloroquine resistance in Plasmodium falciparum in Southeast Asia and Africa. 1997; 91: 593–603. 10. Adagu IS and Warhurst DC.Association of cg2 and pfmdr 1 genotype with chloroquine resistance in field samples of Plasmodium falciparum from Nigeria. Parasitology 1999; 119: 343–348. 11. Reed MB, Saliba KJ, Caruana SR, Kirk K, Cowman AF. Pgh 1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 2000; 403: 906–909. 12. Bray PG, Janneh O, Raynes KJ, Mungthin M, Ginsburg H,Ward SA. Cellular uptake of chloroquine is dependent on binding to ferriprotoporphyrin IX and is independent of NHE activity in Plasmodium falciparum. J Cell Biol 1999; 145: 363–376.
2000 Harcourt Publishers Ltd Drug Resistance Updates (2000) 3, 80–81
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