Senicapoc, a Gardos Channel Inhibitor Developed to Treat Sickle Cell Disease, Exhibits Antimalarial Activity

BACKGROUND: The inhibitor of the erythrocyte Gardos channel (KCNN-4, IK-1), senicapoc, was developed for treatment of sickle cell disease. Activation of the Gardos channel constitutes a major potassium leak pathway contributing to cellular dehydration. Administration of senicapoc to patients with sickle cell disease was well tolerated and reduced hemolysis through attenuation of sickle red cell dehydration, but failed to reduce the frequency of vaso-occlusive pain crises (Ataga KI et al. Br J Haematol 2011;153:92-104). Red cell volume regulation is critical both to the pathology of sickle cell disease and to the growth of Plasmodium, the parasites that cause malaria, since Plasmodium depends upon ion flow across the host membrane for growth (Glushakova S et al. Curr Biol 2010; 20:1117-1121). We tested the hypothesis that senicapoc-induced blockade of the Gardos channel inhibits intraerythrocytic growth and development of malarial parasites.

METHODS: Blood type O+ erythrocytes were infected with P. falciparum strains 3D7, W2mef, and 7G8, and P. knowlesi strains H-1 and YH-1. Parasites were cultured in the presence of senicapoc and in parallel, with drugs with known antimalarial properties (e.g. mefloquine, chloroquine, clotrimazole, and dihydroartemisinin). Parasites were also cultured in the presence of each of eight senicapoc analogs with variable inhibitory potency for the Gardos channel. To define interactions between these drugs, parasites were cultured in fixed-ratio combinations of senicapoc and senicapoc analogs using the fixed-ratio isobologram method. Parasite growth and maturation were determined by microscopy and flow cytometry. The effect of senicapoc in vivo was determined using wild-type C57Bl/6 mice along with IK-1^+/+ and IK-1^-/- pups on a C57Bl/6 background. Mice were infected with the murine parasite, P. yoelii and treated with senicapoc or vehicle control (methylcellulose 0.5%).

RESULTS: We observed that senicapoc blocked the intraerythrocyte growth of P. falciparum 3D7 with an IC₅₀ value of 7 uM and P. knowlesi H1 with an IC₅₀ value of 18 uM. Senicapoc inhibited growth leading to death of intracellular parasites at and beyond the late trophozoite stage, demonstrating minimal effect on ring stage parasites, and an intermediate effect on mature schizonts. Analogs with IC₅₀ values for inhibition of parasite growth similar to senicapoc were potent inhibitors of the Gardos channel. These analogs also shared common elements of the molecular structure. Combined treatment with senicapoc and analogs with potent inhibition of the Gardos channel demonstrated an additive effect on inhibition of parasite growth. Both additive and synergistic interactions were demonstrated when parasites were cultured with senicapoc and analogs without potent inhibition of the Gardos channel. In the murine model, C57Bl/6 mice treated with senicapoc exhibited suppression in parasite growth, confirming the efficacy of senicapoc against Plasmodia in vivo. Parasite growth was suppressed in IK^+/+ mice treated with senicapoc, though the effect was not sustained through the period of treatment. Surprisingly, parasite growth was also suppressed in IK^-/-mice treated with senicapoc, though the response was delayed. The kinetics of parasite growth differed between mice with and without the Gardos channel.

CONCLUSIONS: Senicapoc demonstrates antimalarial activity against P. falciparum and P. knowlesiin vitro and in vivo against P. yoelii in mice. Our in vivo studies suggest a mechanism for inhibition of parasite growth in mice that includes both Gardos-dependent and independent components. The excellent safety profile of senicapoc and its long half-life in humans demonstrated in clinical trials suggest its possible utility in antimalarial development either as a lead compound or in combination with other antimalarials.