Erythrocyte G Protein as a Novel Target for Malarial Chemotherapy

BACKGROUND: Malaria remains a serious health problem because resistance develops to all currently used drugs when their parasite targets mutate. Novel antimalarial drug targets are urgently needed to reduce global morbidity and mortality. Our prior results suggested that inhibiting erythrocyte G(s) signaling blocked invasion by the human malaria parasite Plasmodium falciparum. METHODS AND FINDINGS: We investigated the erythrocyte guanine nucleotide regulatory protein G(s) as a novel antimalarial target. Erythrocyte “ghosts” loaded with a G(s) peptide designed to block G(s) interaction with its receptors, were blocked in β-adrenergic agonist-induced signaling. This finding directly demonstrates that erythrocyte G(s) is functional and that propranolol, an antagonist of G protein–coupled β-adrenergic receptors, dampens G(s) activity in erythrocytes. We subsequently used the ghost system to directly link inhibition of host G(s) to parasite entry. In addition, we discovered that ghosts loaded with the peptide were inhibited in intracellular parasite maturation. Propranolol also inhibited blood-stage parasite growth, as did other β(2)-antagonists. β-blocker growth inhibition appeared to be due to delay in the terminal schizont stage. When used in combination with existing antimalarials in cell culture, propranolol reduced the 50% and 90% inhibitory concentrations for existing drugs against P. falciparum by 5- to 10-fold and was also effective in reducing drug dose in animal models of infection. CONCLUSIONS: Together these data establish that, in addition to invasion, erythrocyte G protein signaling is needed for intracellular parasite proliferation and thus may present a novel antimalarial target. The results provide proof of the concept that erythrocyte G(s) antagonism offers a novel strategy to fight infection and that it has potential to be used to develop combination therapies with existing antimalarials

Evidence for continued two-brood replication of Plasmodium falciparum in vivo during quinine treatment.

To investigate the relationship between fever and parasite clearance in falciparum malaria, we studied 54 adults with Plasmodium falciparum infections who were all treated with quinine. The median oral temperature profile showed peaks at 24 h intervals during the first 3 days. Although there was no equivalent pattern evident in the median parasite clearance curve, we hypothesize that small numbers of two distinct parasite broods continued to develop in antiphase through schizogony despite quinine therapy. These data are consistent with previous reports of two dominant broods in untreated humans and monkeys infected with P. falciparum, and highlight the need for an adequate duration of quinine treatment

Quinine in severe falciparum malaria: evidence of declining efficacy in Thailand.

Between 1981 and 1992, 196 Thai adults with severe falciparum malaria were treated with a quinine loading dose regimen. Nineteen patients died (10%) and 6 developed late hypoglycaemia. There was no serious cardiovascular or nervous system toxicity. Although there was no evidence of high grade resistance, and no change in the mortality rate, in recent years an increasing proportion of patients had a delayed clinical and parasitological response to treatment. Since 1988, 78% (29/37) of patients with cerebral malaria were unconscious for > 72 h compared with 41% (11/27) between 1981 and 1987 (P = 0.002). In the past 2 years parasite clearance times have exceeded 96 h in 33% (26/78) of patients compared with 14% (15/102) previously (P = 0.006). Quinine remains an effective treatment for severe multi-drug resistant falciparum malaria in this area, but there is now evidence of a decline in the immediate therapeutic response, and its efficacy will need close monitoring as resistance increases further

Abnormal circulatory control in falciparum malaria: the effects of antimalarial drugs.

We have studied blood pressure and heart rate responses to standing in 29 previously ambulant adult Thai patients with acute uncomplicated falciparum malaria before and after treatment with quinine or mefloquine. There was significant, symptomatic, and usually profound orthostatic hypotension in 12 patients (41%) before antimalarial treatment. The median maximum fall in systolic pressure was 24 mm Hg, significantly greater than the maximum fall in diastolic pressure 16 mm Hg. Blood pressure fell in two phases: an initial transient and usually asymptomatic fall immediately on standing, and a progressive, usually symptomatic fall, worsening over several minutes without a rise in pulse rate. Orthostatic hypotension was associated with core temperature (r = 0.37, P = 0.05). Antimalarial treatment accentuated the delayed orthostatic hypotension during malaria, despite (in the case of quinine) a significant reduction in fever. Both antimalarial drugs attenuated the cardioacceleratory response to symptomatic postural hypotension; the mean reduction in heart rate at the time of lowest blood pressure was 22 beats.min-1. The electrocardiograph ratio of RR intervals at the 30th and 15th beats was reduced significantly in acute malaria, but was not affected further by the drugs. When restudied in convalescence all the patients had normal postural cardiovascular responses. Acute falciparum malaria is associated with impaired circulatory control and the tendency to postural hypotension is worsened significantly by antimalarial treatment with the quinoline antimalarials quinine and mefloquine

The effect of plasma free fatty acids and long-chain triglycerides on glucose metabolism in uncomplicated falciparum malaria.

To investigate the therapeutic potential of increased plasma free fatty acid (FFA) and triglyceride concentrations in hypoglycaemic patients receiving quinine, 32 untreated Thai adults with uncomplicated falciparum malaria were allocated at random to one of 4 regimens: 2 mg/kg/min dextrose infused over 60 min either alone (group A) or with a prior injection of 5000 units of heparin and simultaneous Intralipid infusion (group C), or 4 min/kg/min dextrose alone (group B) or with heparin and Intralipid (group D). Quinine (10 mg/kg) was also infused over 60 min in all cases. In patients of groups A and C, mean changes in plasma glucose concentrations from the beginning to the end of the infusion were 0.1 (SD 0.8) and 1.0 (SD 0.7) mmol/L respectively (P = 0.015). In groups B and D, plasma glucose increased by 1.8 (SD 1.2) and 2.2 (SD 0.4) mmol/L respectively (P < 0.5). Plasma FFA levels fell by approximately 50% during the infusion in groups A and B but increased by a similar percentage in groups C and D. Despite significant mean increases in plasma insulin during the infusion (from 12.2 milliunits (mu)/L in group A to 38.8 mu/L in group D), no rebound hypoglycaemia was observed in any patient during the ensuing 7 h. These data suggest that the glycaemic response to dextrose given at high rates, which match average glucose utilization in a severely ill patient with malaria, is not augmented by increased plasma FFA and long-chain triglycerides. However, this strategy increases the glycaemic efficacy of lower dextrose infusion rates and the combination could, therefore, reduce the volumes of hypertonic dextrose required to prevent hypoglycaemia in severely ill patients in whom optimal fluid balance is crucial

Assessment of the neurotoxicity of oral dihydroartemisinin in mice.

High doses of the oil-soluble antimalarial artemisinin derivatives artemether and arteether, given by intramuscular injection to experimental mammals, produce an unusual pattern of selective damage to brainstem centres predominantly involved in auditory processing and vestibular reflexes. We have shown recently, in adult Swiss albino mice, that constant exposure either from depot intramuscular injection of oil-based artemisinin derivatives, or constant oral intake carries relatively greater neurotoxic potential than other methods of drug administration. Using the same model, oral dihydroartemisinin suspended in water was administered once or twice daily at different doses ranging from 50 to 300 mg/kg/day for 28 days. The neurotoxic potential of the oral dihydroartemisinin was assessed and compared to that of oral artemether and artesunate. Oral artemether, artesunate, and dihydroartemisinin had similar neurotoxic effects with no significant clinical or neuropathological evidence of toxicity at doses below 200 mg/kg/day. These data indicate that once and twice daily oral administration of artemether, artesunate and dihydroartemisinin is relatively safe when compared to intramuscular administration of the oil-based compounds