Atovaquone-resistant Parasites Research Articles

Clinically relevant atovaquone-resistant human malaria parasites fail to transmit by mosquito

Long-acting injectable medications, such as atovaquone, offer the prospect of a “chemical vaccine” for malaria, combining drug efficacy with vaccine durability. However, selection and transmission of drug-resistant parasites is of concern. Laboratory studies have indicated that atovaquone resistance disadvantages parasites in mosquitoes, but lack of data on clinically relevant Plasmodium falciparum has hampered integration of these variable findings into drug development decisions. Here we generate atovaquone-resistant parasites that differ from wild type parent by only a Y268S mutation in cytochrome b, a modification associated with atovaquone treatment failure in humans. Relative to wild type, Y268S parasites evidence multiple defects, most marked in their development in mosquitoes, whether from Southeast Asia (Anopheles stephensi) or Africa (An. gambiae). Growth of asexual Y268S P. falciparum in human red cells is impaired, but parasite loss in the mosquito is progressive, from reduced gametocyte exflagellation, to smaller number and size of oocysts, and finally to absence of sporozoites. The Y268S mutant fails to transmit from mosquitoes to mice engrafted with human liver cells and erythrocytes. The severe-to-lethal fitness cost of clinically relevant atovaquone resistance to P. falciparum in the mosquito substantially lessens the likelihood of its transmission in the field.

Abbreviated atovaquone-proguanil prophylaxis regimens in travellers after leaving malaria-endemic areas: A systematic review.

We evaluated existing data on the prophylactic efficacy of atovaquone-proguanil (AP) in order to determine whether prophylaxis in travellers can be discontinued on the day of return from a malaria-endemic area instead of seven days after return as per currently recommended post-travel schedule. PubMed and Embase databases were searched to identify relevant studies. This PROSPERO-registered systematic review followed PRISMA guidelines. The search strategy included terms or synonyms relevant to AP combined with terms to identify articles relating to prophylactic use of AP and inhibitory and half-life properties of AP. Studies considered for inclusion were: randomized controlled trials, cohort studies, quasi-experimental studies, open-label trials, patient-control studies, cross-sectional studies; as well as case-series and non-clinical studies. Data on study design, characteristics of participants, interventions, and outcomes were extracted. Primary outcomes considered relevant were prophylactic efficacy and prolonged inhibitory activity and half-life properties of AP. The initial search identified 1,482 publications, of which 40 were selected based on screening. Following full text review, 32 studies were included and categorized into two groups, namely studies in support of the current post-travel regimen (with a total of 2,866 subjects) and studies in support of an alternative regimen (with a total of 533 subjects). There is limited direct and indirect evidence to suggest that an abbreviated post-travel regimen for AP may be effective. Proguanil, however, has a short half-life and is essential for the synergistic effect of the combination. Stopping AP early may result in mono-prophylaxis with atovaquone and possibly select for atovaquone-resistant parasites. Furthermore, the quality of the studies in support of the current post-travel regimen outweighs the quality of the studies in support of an alternative short, post-travel regimen, and the total sample size of the studies to support stopping AP early comprises a small percentage of the total sample size of the studies performed to establish the efficacy of the current AP regimen. Additional research is required - especially from studies evaluating impact on malaria parasitaemia and clinical illness and conducted among travellers in high malaria risk settings - before an abbreviated regimen can be recommended in current practice. CRD42017055244.

Transmission blocked by drug resistance

Malaria Drugs Resistance to the antimalarial drug atovaquone might prove to be this parasite's weak spot. Resistance develops rapidly via mutations in the drug's target: the parasite's mitochondrial cytochrome b complex. Goodman et al. have discovered that although resistant Plasmodium berghei parasites persist in mice, in blood-sucking malarial mosquitoes, the mutations disable female parasites too much for them to reproduce. The human-specific Plasmodium falciparum can only be investigated experimentally in mosquitoes, but a similar effect was seen. Thus, atovaquone-resistant parasites cannot be transmitted to another mammal or person. Science , this issue p. [349][1] [1]: /lookup/doi/10.1126/science.aad9279

Molecular basis of extreme resistance in Plasmodium falciparum to atovaquone and other mitochondrial inhibitors

Background Atovaquone is a safe, effective antimalarial drug that inhibits the mitochondrial electron transport chain (mtETC) of P. falciparum. Clinical resistance is conferred by SNPs in cytochrome b. Although multiple non-synonymous SNPs can be selected in vitro ,t hus far clinical resistance is limite dt o amino acid substitutions at position 268 (e.g., Y268S). We are investigating additional mechanisms by which some isolates of P. falciparum became extremely resistant to atovaquone and other mtETC inhibitors by selecting for in vitro resistance to the dihydroorotate dehydrogenase (DHODH) inhibitor DSM-1. Methods We characterized isolates from patients that failed treatment in atovaquone phase II studies by using a series of chemotypes that target mitochondrial function. We defined their structure-activity relationships and observed broad resistance (5-10,000 fold in atovaquone), suggesting that cytochrome b mutations alone are not sufficient. We tested this by selecting for DSM-1 resistance in atovaquone-susceptible and resistant parasites (paired patient admission and recrudescent isolates). Following selection, the parasites were sequenced for mutations in cytochrome b, DHODH and other candidate mtETC genes. Results The extreme atovaquone resistance phenotype accompanies a high-grade resistance to all mtETC inhibitors tested including DSM-1. Selection studies of atovaquone susceptible parasites rapidly generated resistance in vitro to both atovaquone and DSM-1 alone (10x IC50 concentration) yet atovaquone resistant parasites could only generate low resistance to DSM-1 (2x IC50), and one population reverted the aa268 mutation to wild-type to cope with DSM-1 pressure. Furthermore, we successfully recapitulated the clinical Y268S mutation in vitro in TM90-C2A, the pre-treatment parent for TM90-C2B. Conclusions Cytochrome b mutations are not sufficient to explain extreme mtETC resistance, and selective pressure on the DHODH enzyme revealed that the Y268S cytochrome b mutation incurs a fitness cost on the parasite that cannot be overcome under DSM-1 drug pressure, yet resistance was easily obtained in atovaquone-susceptible parasites. This suggests that resistance to atovaquone (Y268S) and DSM-1 are mutually incompatible. Selection of the Y268S mutation in TM90-C2A indicates that the parasite genetic background is critical in the selection of clinical atovaquone resistance, since selection attempts in multiple other strains results in mutations in positions other than aa268. Further studies are required to determine the impact of this phenotype on the development of new mitochondria-targeted drugs.

Identification and Validation of Tetracyclic Benzothiazepines as Plasmodium falciparum Cytochrome bc1 Inhibitors

Here we report the discovery of tetracyclic benzothiazepines (BTZs) as highly potent and selective antimalarials along with the identification of the Plasmodium falciparum cytochrome bc(1) complex as the primary functional target of this novel compound class. Investigation of the structure activity relationship within this previously unexplored chemical scaffold has yielded inhibitors with low nanomolar activity. A combined approach employing genetically modified parasites, biochemical profiling, and resistance selection validated inhibition of cytochrome bc(1) activity, an essential component of the parasite respiratory chain and target of the widely used antimalarial drug atovaquone, as the mode of action of this novel compound class. Resistance to atovaquone is eroding the efficacy of this widely used antimalarial drug. Intriguingly, BTZ-based inhibitors retain activity against atovaquone resistant parasites, suggesting this chemical class may provide an alternative to atovaquone in combination therapy.

A Chemical Genomic Analysis of Decoquinate, a Plasmodium falciparum Cytochrome b Inhibitor

Decoquinate has single-digit nanomolar activity against in vitro blood stage Plasmodium falciparum parasites, the causative agent of human malaria. In vitro evolution of decoquinate-resistant parasites and subsequent comparative genomic analysis to the drug-sensitive parental strain revealed resistance was conferred by two nonsynonymous single nucleotide polymorphisms in the gene encoding cytochrome b. The resultant amino acid mutations, A122T and Y126C, reside within helix C in the ubiquinol-binding pocket of cytochrome b, an essential subunit of the cytochrome bc1 complex. As with other cytochrome bc1 inhibitors, such as atovaquone, decoquinate has low nanomolar activity against in vitro liver stage P. yoelii and provides partial prophylaxis protection when administered to infected mice at 50 mg kg–1. In addition, transgenic parasites expressing yeast dihydroorotate dehydrogenase are >200-fold less sensitive to decoquinate, which provides additional evidence that this drug inhibits the parasite’s mitochondrial electron transport chain. Importantly, decoquinate exhibits limited cross-resistance to a panel of atovaquone-resistant parasites evolved to harbor various mutations in cytochrome b. The basis for this difference was revealed by molecular docking studies, in which both of these inhibitors were shown to have distinctly different modes of binding within the ubiquinol-binding site of cytochrome b.

Plasmodium chabaudi chabaudi malaria parasites can develop stable resistance to atovaquone with a mutation in the cytochrome b gene

BackgroundPlasmodium falciparum, has developed resistance to many of the drugs in use. The recommended treatment policy is now to use drug combinations. The atovaquone-proguanil (AP) drug combination, is one of the treatment and prophylaxis options. Atovaquone (ATQ) exerts its action by inhibiting plasmodial mitochondria electron transport at the level of the cytochrome bc1 complex. Plasmodium falciparum in vitro resistance to ATQ has been associated with specific point mutations in the region spanning codons 271-284 of the cytochrome b gene. ATQ -resistant Plasmodium yoelii and Plasmodium berghei lines have been obtained and resistant lines have amino acid mutations in their CYT b protein sequences. Plasmodium chabaudi model for studying drug-responses and drug-resistance selection is a very useful rodent malaria model but no ATQ resistant parasites have been reported so far. The aim of this study was to determine the ATQ sensitivity of the P. chabaudi clones, to select a resistant parasite line and to perform genotypic characterization of the cytb gene of these clones.MethodsTo select for ATQ resistance, Plasmodium. chabaudi chabaudi clones were exposed to gradually increasing concentrations of ATQ during several consecutive passages in mice. Plasmodium chabaudi cytb gene was amplified and sequenced.ResultsATQ resistance was selected from the clone AS-3CQ. In order to confirm whether an heritable genetic mutation underlies the response of AS-ATQ to ATQ, the stability of the drug resistance phenotype in this clone was evaluated by measuring drug responses after (i) multiple blood passages in the absence of the drug, (ii) freeze/thawing of parasites in liquid nitrogen and (iii) transmission through a mosquito host, Anopheles stephensi. ATQ resistance phenotype of the drug-selected parasite clone kept unaltered. Therefore, ATQ resistance in clone AS-ATQ is genetically encoded. The Minimum Curative Dose of AS-ATQ showed a six-fold increase in MCD to ATQ relative to AS-3CQ.ConclusionsA mutation was found on the P. chabaudi cytb gene from the AS-ATQ sample a substitution at the residue Tyr268 for an Asn, this mutation is homologous to the one found in P. falciparum isolates resistant to ATQ.

Antimalarial quinolones: Synthesis, potency, and mechanistic studies

In the present article we examine the antiplasmodial activities of novel quinolone derivatives bearing extended alkyl or alkoxy side chains terminated by a trifluoromethyl group. In the series under investigation, the IC 50 values ranged from 1.2 to ≈30 nM against chloroquine-sensitive and multidrug-resistant Plasmodium falciparum strains. Modest to significant cross-resistance was noted in evaluation of these haloalkyl- and haloalkoxyquinolones for activity against the atovaquone-resistant clinical isolate Tm90-C2B, indicating that a primary target for some of these compounds is the parasite cytochrome bc 1 complex. Additional evidence to support this biochemical mechanism includes the use of oxygen biosensor plate technology to show that the quinolone derivatives block oxygen consumption by parasitized red blood cells in a fashion similar to atovaquone in side-by-side experiments. Atovaquone is extremely potent and is the only drug in clinical use that targets the Plasmodium bc 1 complex, but rapid emergence of resistance to it in both mono- and combination therapy is evident and therefore additional drugs are needed to target the cytochrome bc 1 complex which are active against atovaquone-resistant parasites. Our study of a number of halogenated alkyl and alkoxy 4(1 H)-quinolones highlights the potential for development of “endochin-like quinolones” (ELQ), bearing an extended trifluoroalkyl moiety at the 3-position, that exhibit selective antiplasmodial effects in the low nanomolar range and inhibitory activity against chloroquine and atovaquone-resistant parasites. Further studies of halogenated alkyl- and alkoxy-quinolones may lead to the development of safe and effective therapeutics for use in treatment or prevention of malaria and other parasitic diseases.

Lengthy Antimalarial Activity of Atovaquone in Human Plasma following Atovaquone-Proguanil Administration

Recently, it was reported that sera from healthy volunteers given atovaquone-proguanil (Malarone) inhibited parasite transmission and asexual blood stage development for up to 6 weeks after treatment (1). The lengthy persistence of drug activity was quite unexpected because earlier studies had shown that proguanil and atovaquone had elimination half-lives of about 14 to 20 h (2-4, 10, 11) and 2 to 3 days, (5, 9-11), respectively. This drug combination acts synergistically against malaria parasites and avoids the rapid selection of atovaquone-resistant parasites whenever parasites are exposed to the action of atovaquone alone (7). The present study was designed to quantitate the persistence of atovaquone in three Caucasian volunteers who had participated in a study to assess the effects of repeated subclinical infections on the development of immunity to Plasmodium falciparum (8). The three volunteers (mean weight, 84.8 ± 21.9 kg) had been inoculated intravenously with about 30 parasitized erythrocytes of P. falciparum (3D7 strain) on three occasions at 35-day intervals, and on each occasion, treatment with atovaquone-proguanil (1,000 mg atovaquone-400 mg proguanil daily for 3 days) was started 8 days later. Blood samples were collected at 6, 20, and 35 days after the onset of treatment (Fig. ​(Fig.1).1). None of the volunteers developed symptoms of malaria. FIG. 1. Mean plasma atovaquone (ATQ) concentrations versus time after repeated atovaquone-proguanil treatment for uncomplicated Plasmodium falciparum malaria in three volunteers infected on three occasions. Plasma ATQ concentrations were measured by HPLC (•) ... Mean plasma atovaquone concentrations-versus-time curves measured by high-pressure liquid chromatography (HPLC) (9) were similar after each treatment with atovaquone-proguanil (Fig. ​(Fig.1).1). Comparable values were obtained by bioassay of samples using the 3D7 atovaquone-sensitive strain (6). The average elimination half-life of atovaquone in the three volunteers was 5.9 days by HPLC and 4.9 days by bioassay. The prolonged persistence of atovaquone was further illustrated by the complete suppression of schizont formation when plasma collected up to 35 days after treatment was incubated with parasites of the 3D7 strain (50% inhibitory concentration [IC50], ∼2 ng/ml). In contrast, plasma collected 6 days after treatment failed to inhibit the maturation of parasites of three atovaquone-resistant strains (TM90-C2b, TM91-C32b, TM93-C1088), with mean IC50 values exceeding 4,000 ng/ml (7). These findings suggest that residual atovaquone may have contributed to the suppression of malaria in the volunteers challenged with the 3D7 strain 35 days after treatment (8). The lengthy 5- to 6-day elimination half-life of atovaquone in these three Caucasian volunteers is twice as long as observed previously in African and Asian patients treated with atovaquone (5, 10, 11). Although proguanil concentrations were not measured in this study, numerous previous studies have shown that proguanil and its cycloguanil metabolite are eliminated much more quickly than atovaquone (2-4, 10, 11), and none would be present within a week after atovaquone-proguanil treatment to potentiate the antimalarial activity of atovaquone. The prolonged presence of atovaquone is of little concern as long as this expensive drug continues to be used mainly by travelers to malarious areas. However, if atovaquone-proguanil were used more widely by expatriate residents living in areas of endemicity, long-lasting low atovaquone concentrations in the absence of any residual proguanil may facilitate the rapid selection of atovaquone-resistant parasites.

Short communication: Prevalence of mutations associated with resistance to atovaquone and to the antifolate effect of proguanil in Plasmodium falciparum isolates from northern Ghana.

Atovaquone-proguanil has recently been introduced for the treatment and prophylaxis of malaria. However, resistance of Plasmodium falciparum is increasingly reported. We assessed P. falciparum polymorphisms associated with resistance to atovaquone (cytochrome b, cytb) and to cycloguanil, the active compound of proguanil (dihydrofolate reductase, dhfr) in 100 isolates from northern Ghana. None of these exhibited cytb codon 268 mutations. Moreover, no dhfr V16A, S108T or I164L mutations linked with cycloguanil resistance were detected. However, dhfr triple mutants (S108N-I51L-C59R) conferring resistance to proguanil and sulphadoxine-pyrimethamine were seen in 51% of the isolates. In northern Ghana, P. falciparum cytb codon 268 mutations associated with atovaquone resistance are absent. Although proguanil appears to act synergistically with atovaquone in a way different from its antifolate property, the abundance of dhfr polymorphisms will likely compromise the prevention of dissemination of atovaquone-resistant parasites once emerged.

Characterisation of atovaquone resistance in Leishmania infantum promastigotes

Atovaquone, an antiparasitic agent, could possibly represent an alternative therapy after relapse following classical treatment for visceral leishmaniasis. Atovaquone-resistant strains were selected in vitro by stepwise drug pressure to study the mechanism of resistance in Leishmania. Characteristics of a promastigote strain resistant to 250μg/ml of atovaquone were compared with those of the wild type (WT) strain. Resistant strains were shown to have a high level of resistance (45 times). They were stable in drug-free medium for 6 months, and showed no cross-resistance with other antileishmanial drugs. Rhodamine uptake and efflux were studied. They were not modified in the resistant strain, indicating the absence of P-glycoprotein overexpession. The effect of atovaquone on membrane lipidic composition was determined in both WT and atovaquone-resistant promastigotes. Analysis of lipid composition of the atovaquone-resistant strain showed that sterol biosynthesis was decreased in atovaquone-resistant parasites. Cholesterol was found to be the major membrane sterol as opposed to the WT strain. Cholesterol, due to its ordering effect, could decrease membrane fluidity and subsequently block the passage of atovaquone through the membrane. Increased membrane cholesterol content and altered drug membrane fluidity resulted from possible decrease of ergosterol biosynthesis by atovaquone, incorporation of cholesterol by promastigotes in the culture medium, solubilisation of atovaquone by cholesterol and co-passage of the two compounds or influence of dimethylsulfoxide. These results indicate that different cellular alterations may participate in the resistant phenotype, by altering drug membrane permeability.

Plasmodium falciparum: The Effects of Atovaquone Resistance on Respiration

Suswam, E., Kyle, D., and Lang-Unnasch, N. 2001. Plasmodium falciparum: The effects of atovaquone resistance on respiration. Experimental Parasitology98, 180–187. Atovaquone is an antimalarial agent that specifically inhibits the cytochrome bc1 complex of the cytochrome pathway. High-level atovaquone resistance is associated with a point mutation in the cytochrome b gene. A pair of isogenic clinical isolates of Plasmodium falciparum derived from before and after the acquisition of atovaquone resistance was used to determine whether the change in the cytochrome b gene resulted in changes in respiration in response to atovaquone. Since P. falciparum appears to utilize a branched respiratory system comprising both the cytochrome and an alternative respiratory pathway, the proportion of each pathway utilized by the sensitive and resistant parasites was investigated. Atovaquone inhibited total parasite oxygen consumption by up to 66% in the sensitive isolate but only up to 28% in the resistant isolate. Both the atovaquone-sensitive and the atovaquone-resistant parasites were comparably sensitive to the alternative pathway inhibitor, salicylhydroxamic acid. Atovaquone appeared to partially inhibit the rate of oxygen consumed through the alternative pathway in only the atovaquone-sensitive isolate. Cross resistance was noted between atovaquone and a new antimalarial agent WR243251. However, the level of WR243251 resistance was very modest compared to the level of atovaquone resistance. WR243251 was shown to rapidly reduce the rate of parasite oxygen consumption by almost 80% in the atovaquone-sensitive isolate and by 57% in the atovaquone-resistant isolate. Drug interaction studies suggest that atovaquone and WR243251 may inhibit growth additively or with mild synergy. Together, these results suggest that while WR243251 may inhibit respiration, its target of action probably differs from that of atovaquone.

Characterization of cytochrome b from Toxoplasma gondii and Q o domain mutations as a mechanism of atovaquone-resistance

Atovaquone is active in vitro against the tachyzoites of Toxoplasma gondii at nanomolar concentrations and is used clinically to treat acute cases of human toxoplasmosis. In pursuit of the mechanism of action of atovaquone against T. gondii and to understand how resistance might arise, drug-resistant mutants were generated and examined. The previously uncloned cytochrome b gene of T. gondii was cloned and sequenced from wild type and resistant strains as this was a likely candidate for the target of the drug and thus a source of resistance. Mutations are present within the cytochrome b gene of atovaquone-resistant parasites (M129L and I254L) and represent alterations in two different regions of the ubiquinol-binding pocket (Q o domain) of cytochrome b, suggesting that atovaquone interferes with electron transport at the cytochrome bc 1 complex in T. gondii. A structural model for how this hydroxynaphthoquinone is binding within the Q o domain is presented. Further analysis of the cytochrome b gene suggested that the protein may differ from other homologues by terminating within the mitochondrial membrane. Cytochrome b becomes the first complete mitochondrial gene and cognate protein to be described for T. gondii.