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Chapter 7 Chloroquine and Hemozoin
CHAPTER
7 Chloroquine and Hemozoin
Drug accumulation by lysosomes had first been noticed in mammalian cells by workers at the NIMR and applying this concept to CQ accumulation, Homewood, Warhurst, Peters and Baggaley (1972) were able to explain how CQ could accumulate in the acidic FVs of the Plasmodium. Warhurst and co-workers also found that quinine and WR142,490 competitively inhibited the CQ-induced morphological changes in P. berghei; later, WR142,490 would be developed as mefloquine (Warhurst and Thomas, 1975). In 1963, David Warhurst (1938– ) who received his doctor of philosophy (PhD) at Leicester University, United Kingdom (1964) took a position at National Institute for Medical Research (NIMR, London, United Kingdom) in the Division of Parasitology headed by Frank Hawking. Working under a grant from the World Health Organization (WHO, Geneva, Switzerland) he was drawn to the problem of the mode of action of chloroquine (CQ) by its morphological effects on Plasmodium bergheiinfected red cells, that is, food vacuole (FV) swelling and microscopic clumping of hemozoin due to engulfment of vacuoles in an autophagic vacuole (Warhurst and Hockley, 1967a,b). The clumping effect was seen with other species of malaria (P. cynomolgi but not with P. falciparum where digestion already occurs in a large single vacuole) and with other 4-aminoquinolines related to CQ, and this correlated with anti-plasmodial activity. In addition, a proportionality was found between anti-malarial activity for 4-aminoquinolines and quinine and the inhibition of b-hematin formation (Warhurst, 1987; Warhurst et al., 2003).
Advances in Parasitology, Volume 67 ISSN 0065-308X, DOI: 10.1016/S0065-308X(08)00407-7
#
2008 Elsevier Ltd. All rights reserved.
59
60
Irwin W. Sherman
In the early 1960s the mode of action of chloroquine was generally believed to be through binding to deoxyribonucleic acid (DNA), and the evidence, particularly from Hahn’s group at the Walter Reed Army Institute of Research (WRAIR, Washington), was thought to be conclusive (Ciak and Hahn, 1966). However, as discussed above, subsequent studies by Warhurst and colleagues demonstrated that the mechanism was incorrect. Since that time various other theories have been postulated as to the mechanism of action of CQ (Fitch, 2004; Jiang et al., 2006; Sullivan, 2002b). Chou and Fitch (1980) and Orjih et al. (1981) found that ferriprotoporphyrin (FP) and its complex with CQ were toxic for erythrocytes and malaria parasites; since CQ treatment reduced FP dimerization in vivo in CQ-sensitive P. berghei-infected red cells, Chou and Fitch (1993) claimed this explains the accumulation of toxic, undimerized FP after CQ treatment, and that the chemotherapeutic effect is due to toxic, undimerized FP that binds CQ. Sullivan (2002b) suggests that CQ binds to the FP and caps the growing crystal thereby preventing further FP incorporation; in this theory FP binding to the growing face of the hemozoin crystal is the crucial feature for the action of CQ, and this is only seen when the crystal is growing (proven by incorporation of radiolabelled drug into the hemozoin). Non-covalent binding of CQ to hemozoin would remove CQ from solution equilibrium and lead to its accumulation in the FV. Although this notion was very convincing when the polymer structure of hemozoin was believed, it is presently less so with the varied new 4-aminoquinolines that require a range of possible sites on the hemozoin (Warhurst, personal communication). The worldwide spread of CQ resistance has led to a significant resurgence of malarial morbidity and mortality concomitant with interest in the manner by which resistance develops. Investigations into the mechanisms of CQ resistance have generated several different models, including reduced influx of CQ, increased efflux of CQ, pH effects on drug accumulation and/or receptor availability, glutathione (GSH) degradation of hematin or formation of CQ-hematin complexes. It is generally accepted that CQ enters the acidic FV by passive diffusion as an uncharged species and becomes trapped in the vacuole in its di-protonated, membraneimpermeable form; the di-protonated CQ is retained in the FV as a hematin-CQ complex. If resistance involves restricted access of CQ to hematin such that there are reduced drug levels in the digestive vacuole, then possible models for how this might be achieved are: (1) efflux of CQ from the digestive vacuole via an energy-coupled mechanism, (2) leakage of CQ out of the digestive vacuole down its concentration gradient with energy driving the vacuolar proton pump such that a concentration gradient of protonated CQ is maintained (rather than energy being coupled to drug movement per se), (3) a pH-dependent reduction in CQ accumulation and (4) passive outwards movement of protonated
Chloroquine and Hemozoin
61
CQ through a gated aqueous pore (O’Neill et al., 2006). It is generally thought that differences in the digestive vacuole pH are not primarily responsible for CQ resistance, therefore, other models have received much more attention and all likely involve the P. falciparum chloroquine transporter (PfCRT; Valderramos and Fidock, 2006). The PfCRT gene was identified through the analysis of a genetic cross between a CQ-resistant and a CQ-sensitive clone (Fidock et al., 2000). The 45-kDa PfCRT protein has been localized to the digestive vacuolar membrane and it shows extraordinary amino acid sequence diversity among geographic isolates involving as many as 15 residues and in a single resistant line there can be four to eight individual mutations. Indeed, when the K76T (lysine to threonine) mutation is removed resistant parasites become sensitive to CQ. A recent study suggests that the outwards movement of CQ from the digestive vacuole (thereby reducing the binding of CQ to FP) in resistant parasites is not directly coupled to the energy supply (Bray et al., 2006). Therefore, an efflux pump and/or an active carrier-mediated transport mechanism is unlikely. The model that best suits the experimental findings is that in CQ-resistant lines a gated aqueous pore permits a passive outwards movement of the protonated form of CQ.
David Fidock (1965– ) received his PhD in microbiology from the Institute Pasteur (Paris, France) in 1994, having worked for 5 years in the group of Pierre Druilhe, an expert on Plasmodium exo-erythrocytic (EE) stages and malaria immunology. There, Fidock worked on mechanisms of protective immunity induced by irradiated sporozoites, and was awarded the prestigious Bourse Roux that led to his being recruited as a ‘Pasteurien’ scientist during his graduate years. His sabbatical to the United States led him first to work with Anthony James (University of California, Irvine, Irvine, California) on sporozoite biology and later with Thomas Wellems (National Institutes of Health (NIH), Bethesda, Maryland) on the genetic basis of CQ resistance. It was at NIH that Fidock disproved the leading gene candidate for CQ resistance (CG2) and discovered PfCRT, the gene that is now known to be the primary determinant of CQ resistance in P. falciparum. In 1999, he set up his own laboratory at the Albert Einstein College of Medicine in the Bronx (New York, New York) and in 2007 moved to Columbia University (New York, New York) where his major research focus continues to be on understanding the genetic and molecular basis of drug resistance in P. falciparum, exploring the cell biology of the P. falciparum digestive vacuole, and identifying new drug targets and lead anti-malarials.
7 Chloroquine and Hemozoin
Drug accumulation by lysosomes had first been noticed in mammalian cells by workers at the NIMR and applying this concept to CQ accumulation, Homewood, Warhurst, Peters and Baggaley (1972) were able to explain how CQ could accumulate in the acidic FVs of the Plasmodium. Warhurst and co-workers also found that quinine and WR142,490 competitively inhibited the CQ-induced morphological changes in P. berghei; later, WR142,490 would be developed as mefloquine (Warhurst and Thomas, 1975). In 1963, David Warhurst (1938– ) who received his doctor of philosophy (PhD) at Leicester University, United Kingdom (1964) took a position at National Institute for Medical Research (NIMR, London, United Kingdom) in the Division of Parasitology headed by Frank Hawking. Working under a grant from the World Health Organization (WHO, Geneva, Switzerland) he was drawn to the problem of the mode of action of chloroquine (CQ) by its morphological effects on Plasmodium bergheiinfected red cells, that is, food vacuole (FV) swelling and microscopic clumping of hemozoin due to engulfment of vacuoles in an autophagic vacuole (Warhurst and Hockley, 1967a,b). The clumping effect was seen with other species of malaria (P. cynomolgi but not with P. falciparum where digestion already occurs in a large single vacuole) and with other 4-aminoquinolines related to CQ, and this correlated with anti-plasmodial activity. In addition, a proportionality was found between anti-malarial activity for 4-aminoquinolines and quinine and the inhibition of b-hematin formation (Warhurst, 1987; Warhurst et al., 2003).
Advances in Parasitology, Volume 67 ISSN 0065-308X, DOI: 10.1016/S0065-308X(08)00407-7
#
2008 Elsevier Ltd. All rights reserved.
59
60
Irwin W. Sherman
In the early 1960s the mode of action of chloroquine was generally believed to be through binding to deoxyribonucleic acid (DNA), and the evidence, particularly from Hahn’s group at the Walter Reed Army Institute of Research (WRAIR, Washington), was thought to be conclusive (Ciak and Hahn, 1966). However, as discussed above, subsequent studies by Warhurst and colleagues demonstrated that the mechanism was incorrect. Since that time various other theories have been postulated as to the mechanism of action of CQ (Fitch, 2004; Jiang et al., 2006; Sullivan, 2002b). Chou and Fitch (1980) and Orjih et al. (1981) found that ferriprotoporphyrin (FP) and its complex with CQ were toxic for erythrocytes and malaria parasites; since CQ treatment reduced FP dimerization in vivo in CQ-sensitive P. berghei-infected red cells, Chou and Fitch (1993) claimed this explains the accumulation of toxic, undimerized FP after CQ treatment, and that the chemotherapeutic effect is due to toxic, undimerized FP that binds CQ. Sullivan (2002b) suggests that CQ binds to the FP and caps the growing crystal thereby preventing further FP incorporation; in this theory FP binding to the growing face of the hemozoin crystal is the crucial feature for the action of CQ, and this is only seen when the crystal is growing (proven by incorporation of radiolabelled drug into the hemozoin). Non-covalent binding of CQ to hemozoin would remove CQ from solution equilibrium and lead to its accumulation in the FV. Although this notion was very convincing when the polymer structure of hemozoin was believed, it is presently less so with the varied new 4-aminoquinolines that require a range of possible sites on the hemozoin (Warhurst, personal communication). The worldwide spread of CQ resistance has led to a significant resurgence of malarial morbidity and mortality concomitant with interest in the manner by which resistance develops. Investigations into the mechanisms of CQ resistance have generated several different models, including reduced influx of CQ, increased efflux of CQ, pH effects on drug accumulation and/or receptor availability, glutathione (GSH) degradation of hematin or formation of CQ-hematin complexes. It is generally accepted that CQ enters the acidic FV by passive diffusion as an uncharged species and becomes trapped in the vacuole in its di-protonated, membraneimpermeable form; the di-protonated CQ is retained in the FV as a hematin-CQ complex. If resistance involves restricted access of CQ to hematin such that there are reduced drug levels in the digestive vacuole, then possible models for how this might be achieved are: (1) efflux of CQ from the digestive vacuole via an energy-coupled mechanism, (2) leakage of CQ out of the digestive vacuole down its concentration gradient with energy driving the vacuolar proton pump such that a concentration gradient of protonated CQ is maintained (rather than energy being coupled to drug movement per se), (3) a pH-dependent reduction in CQ accumulation and (4) passive outwards movement of protonated
Chloroquine and Hemozoin
61
CQ through a gated aqueous pore (O’Neill et al., 2006). It is generally thought that differences in the digestive vacuole pH are not primarily responsible for CQ resistance, therefore, other models have received much more attention and all likely involve the P. falciparum chloroquine transporter (PfCRT; Valderramos and Fidock, 2006). The PfCRT gene was identified through the analysis of a genetic cross between a CQ-resistant and a CQ-sensitive clone (Fidock et al., 2000). The 45-kDa PfCRT protein has been localized to the digestive vacuolar membrane and it shows extraordinary amino acid sequence diversity among geographic isolates involving as many as 15 residues and in a single resistant line there can be four to eight individual mutations. Indeed, when the K76T (lysine to threonine) mutation is removed resistant parasites become sensitive to CQ. A recent study suggests that the outwards movement of CQ from the digestive vacuole (thereby reducing the binding of CQ to FP) in resistant parasites is not directly coupled to the energy supply (Bray et al., 2006). Therefore, an efflux pump and/or an active carrier-mediated transport mechanism is unlikely. The model that best suits the experimental findings is that in CQ-resistant lines a gated aqueous pore permits a passive outwards movement of the protonated form of CQ.
David Fidock (1965– ) received his PhD in microbiology from the Institute Pasteur (Paris, France) in 1994, having worked for 5 years in the group of Pierre Druilhe, an expert on Plasmodium exo-erythrocytic (EE) stages and malaria immunology. There, Fidock worked on mechanisms of protective immunity induced by irradiated sporozoites, and was awarded the prestigious Bourse Roux that led to his being recruited as a ‘Pasteurien’ scientist during his graduate years. His sabbatical to the United States led him first to work with Anthony James (University of California, Irvine, Irvine, California) on sporozoite biology and later with Thomas Wellems (National Institutes of Health (NIH), Bethesda, Maryland) on the genetic basis of CQ resistance. It was at NIH that Fidock disproved the leading gene candidate for CQ resistance (CG2) and discovered PfCRT, the gene that is now known to be the primary determinant of CQ resistance in P. falciparum. In 1999, he set up his own laboratory at the Albert Einstein College of Medicine in the Bronx (New York, New York) and in 2007 moved to Columbia University (New York, New York) where his major research focus continues to be on understanding the genetic and molecular basis of drug resistance in P. falciparum, exploring the cell biology of the P. falciparum digestive vacuole, and identifying new drug targets and lead anti-malarials.