The Clinically Tested Gardos Channel Inhibitor Senicapoc Exhibits Antimalarial Activity

Abstract

Senicapoc, a Gardos channel inhibitor, prevented erythrocyte dehydration in clinical trials of patients with sickle cell disease. We tested the hypothesis that senicapoc-induced blockade of the Gardos channel inhibits Plasmodium growth. Senicapoc inhibited in vitro growth of human and primate plasmodia during the clinical blood stage. Senicapoc treatment suppressed P. yoelii parasitemia in vivo in C57BL/6 mice. The reassuring safety and biochemical profile of senicapoc encourage its use in antimalarial development.

TEXT

Intracellular ion homeostasis of erythrocytes is important in the pathogenesis of sickle cell disease (SCD) and malaria. Stemming potassium loss and cellular dehydration have been explored as therapeutic strategies to prevent sickling in SCD (1). During intraerythrocyte maturation of Plasmodium, parasite swelling is coordinated with erythrocyte sodium gain and potassium loss, ultimately leading to erythrocyte rupture (2). Inhibitors of erythrocyte volume regulation have been hypothesized to function as antimalarial agents (3, 4).

The Gardos channel (KCNN4/IK-1) is a calcium-activated potassium channel abnormally active in sickle erythrocytes (5). In a phase 3 trial in patients with SCD, therapy with the Gardos channel inhibitor senicapoc improved physiological disease markers but failed to modify the frequency of vasoocclusive pain episodes (6). Senicapoc was well tolerated in human and animal studies for SCD (7, 8). Here, we report the effect of senicapoc on the growth of Plasmodium. Repurposing this well-studied safe drug may be an expeditious path to a new antimalarial agent.

The in vitro 50% inhibitory concentration (IC₅₀) values of senicapoc and other antimalarials for inhibition of multiple strains of Plasmodium falciparum and Plasmodium knowlesi are shown in Table 1. Senicapoc demonstrated antimalarial activity against blood-stage P. falciparum 3D7 with an IC₅₀ of 6.7 μM (Fig. 1A). Comparable micromolar antimalarial activity was demonstrated against P. falciparum strains with varied antimalarial sensitivities. In all human Plasmodium strains tested, the IC₉₀ was <2-fold higher than the IC₅₀. IC₅₀s of reference antimalarials determined in parallel were in the nanomolar range (Table 1).

TABLE 1.

Antimalarial activity of senicapoc against Plasmodium falciparum and Plasmodium knowlesi

Species	Strain	IC50 (mean ± 95% confidence interval)a with:
		Res	SEN (μM)	SEN (μM) IC90	CLT (μM)	MQ (nM)	DHA (nM)	CQ (nM)
P. falciparum	3D7		6.74 ± 0.25	10.17 ± 2.07	5.67 ± 1.11	10.11 ± 7.83	2.73 ± 1.55	15.55 ± 3.31
	7G8	CQ	10.88 ± 2.30	22.31 ± 9.52	2.46 ± 0.12	3.63 ± 0.58	1.65 ± 1.24	97.93 ± 20.43
	W2mef	CQ, MQ	9.24 ± 3.13	12.30 ± 1.82	8.92 ± 0.02	26.70 ± 15.53	5.16 ± 4.28	316.3 ± 135.27
P. knowlesi	H1		17.54 ± 6.74	31.36 ± 11.79
	YH-1		10.99 ± 1.38	37.43 ± 1.19

^aMean IC₅₀s of senicapoc (SEN) against a panel of P. falciparum with varied resistance (Res) patterns (3D7, 7G8, W2mef) and P. knowlesi (H1, YH-1) are shown. 95% confidence intervals determined from at least three independent experiments performed in triplicate. CQ, chloroquine; CLT, clotrimazole; MQ, mefloquine; DHA, dihydroartemisinin.

FIG 1.

Treatment with senicapoc effectively impairs parasite growth in vivo and in vitro. (A) Concentration dependence of senicapoc-mediated growth inhibition of P. falciparum 3D7 over one asexual cycle. This nonlinear regression curve is representative of seven assays, each performed in triplicate. (B) Senicapoc rapidly kills parasites in the latter stages of the asexual cycle. Arrows mark the time of drug addition. Parasitemia was determined by flow cytometry and confirmed with microscopy (not shown). Error bars are present within the data points. Data presented are representative of three replicates. (C) Senicapoc activity occurs in the later stages of the asexual parasite cycle as measured by nucleic acid replication. Mean fluorescence intensity (MFI), determined by flow cytometry, is shown as a measure of parasite viability. Error bars are present within the data points. The data point for “No drug” at 48 h was gated for schizonts only (>20% of infected cells) to eliminate signal from reinvaded ring-stage parasites. Data for other time points were not gated, as schizonts represented <5% of infected cells. (D) Structure function analysis of senicapoc congeners demonstrates that antimalarial activity is independent of Gardos activity and identifies important conserved structural elements. The scales for the axes differ because the Gardos assays were performed in serum-free medium and senicapoc is highly protein bound. (E) Chemical structures of senicapoc and congeners with similar antimalarial activity. See data in supplemental material for additional compound structures. (F) Senicapoc demonstrates prophylactic activity against parasites in an in vivo murine model. C57BL/6 mice infected with P. yoelii 17X-NL were treated by gavage with vehicle or senicapoc 400 or 800 mg/kg daily from days 2 to 10 postinfection (highlighted area) to mimic a preventive therapy. (G) Senicapoc suppressed parasite growth in an in vivo murine model following established infection. C57BL/6 mice infected with P. yoelii 17X-NL were treated with vehicle or senicapoc 400 mg/kg twice daily from day 8 to day 15 postinfection (highlighted area) to mimic treatment of an active infection. (H) Malaria infection is sustained independent of Gardos activity in an in vivo murine model. IK-1^−/− and IK-1^+/+ mice were treated with vehicle twice daily on postinoculation days 8 to 15 (highlighted area). (I) Senicapoc suppression of malaria infection is independent of Gardos activity in an in vivo murine model. IK-1^−/− and IK-1^+/+ mice were treated with senicapoc twice daily on postinoculation days 8 to 15 (highlighted area).

Senicapoc demonstrated a low micromolar IC₅₀ against the primate parasite P. knowlesi. To identify a relationship between senicapoc activity and human erythrocytes, P. knowlesi H1 cultured in rhesus erythrocytes and P. knowlesi YH-1 adapted for culture in human erythrocytes were each treated with senicapoc. There was no difference between the senicapoc IC₅₀s for H1 and YH-1 (P = 0.14) (Table 1). Senicapoc was similarly effective against parasitized human and rhesus erythrocytes.

To determine senicapoc's activity through the 48-hour asexual blood stage of the parasite, growth inhibition of P. falciparum 3D7 was characterized throughout the cycle by microscopy and flow cytometry. Parasitemia measured by flow cytometry was reduced by 48 h postinvasion (hpi) in all treated cultures compared to that in the control (P < 0.008) (Fig. 1B). Parasites within treated cultures appeared smaller and failed to form a digestive vacuole by 36 hpi, followed by rapid parasite death (see Fig. S1 in the supplemental material). Nucleic acid content measured by mean fluorescence intensity (MFI) was weaker in treated cultures at 36 h (P < 0.008), and the expected rise in MFI through schizogony was absent (Fig. 1C). Schizonts were not seen by microscopy (see Fig. S1 in the supplemental material). These data suggest that senicapoc was effective during late, metabolically active stages of parasite development.

We examined several senicapoc congeners and identified no relationship between the potency of Gardos inhibitory activity and antimalarial activity (Fig. 1D). Thus, senicapoc may not specifically target the Gardos channel for its antimalarial activity. Interestingly, halogenation of the triarylmethyl group was related to inhibition of both parasite growth and the Gardos channel. Para-position halogens were associated with more potent inhibition of parasite growth and of the channel (Fig. 1E). Unhalogenated compounds demonstrated the weakest antiparasite activity and Gardos inhibition (see Fig. S2 in the supplemental material). Halogen substitution on the phenyl group has been shown to affect compound bioavailability and in vivo stability (5). The triarylmethyl group is important for antimalarial activity in clotrimazole (9). These structural clues may be important for optimization of the antimalarial activity of senicapoc or its congeners.

To establish in vivo efficacy against blood-stage parasites, C57BL/6 mice infected with P. yoelii 17X-NL (nonlethal strain) were treated with vehicle or senicapoc. (All experiments were performed under protocols approved by the institutional animal care and use committees of the Harvard School of Public Health and of Beth Israel Deaconess Medical Center.) The expected immune-mediated clearance of 17X-NL was observed in vehicle-treated mice beyond postinoculation day 15. C57BL/6 mice exhibited partial suppression of parasite growth during treatment. When senicapoc was given as prophylaxis on postinoculation days 2 to 10, treated mice demonstrated significantly lower parasitemia by day 10 than vehicle-treated mice (4.3% versus 12%; P = 0.04). Parasitemia increased after the drug was stopped (Fig. 1F). When senicapoc was given as treatment on postinoculation days 8 to 15, treated mice demonstrated lower parasitemia beyond day 13, but the difference was not statistically significant (P = 0.058) (Fig. 1G). Considering that the shorter half-life of senicapoc in mice compared to humans may impact its effect in the murine model (8), these data demonstrate antiparasite activity of senicapoc in vivo consistent with our in vitro results.

To test whether senicapoc targets the Gardos channel in malaria-infected erythrocytes, we assessed the ability of P. yoelii to grow in the presence and absence of the channel in vivo using a Gardos knockout (IK-1^−/−) mouse model (10, 11). Unexpectedly, accelerated parasite growth was evident in IK-1^−/− mice by postinoculation day 7 (3.6% versus 2.2% in IK-1^+/+ mice; P = 0.03) (Fig. 1H). The discrepant kinetics of parasite growth in IK-1^−/− and IK-1^+/+ mice is not understood but may reflect impaired immunity in addition to absence of the ion channel from erythrocytes. For example, absent Gardos channels in some T-cell subsets might contribute to delayed parasite clearance, countering the effect of Gardos channel inhibition on parasite growth in vivo (12). We then assessed the effectiveness of senicapoc in the presence and absence of the channel in vivo. P. yoelii-infected IK-1^−/− and IK-1^+/+ mice were treated with senicapoc 400 mg/kg twice daily. Senicapoc was active against P. yoelii in IK-1^−/− mice, suggesting activity independent of the channel (Fig. 1I).

Although senicapoc's promising 12-day half-life in humans, activity across the parasite life cycle, and rapid cytocidal activity support its further exploration as an antimalarial, we identified potentially important limitations. The whole-blood concentration required for in vitro inhibition of parasite growth against P. falciparum was an order of magnitude higher than the peak concentrations achieved in human trials for SCD to date (337 nM [13]). However, in unpublished industry safety data in primates, daily doses of 1,000 mg/kg yielding a peak serum concentration of 5.3 μM were well tolerated for 9 months. Canines developed cardiotoxicity when serum concentrations exceeded 18 μM (Douglas Krafte, Pfizer, Inc., unpublished data). Thus, despite an effective antiparasite serum concentration exceeding that achieved to date in human trials of senicapoc, animal studies suggest that senicapoc is safe at high doses. Identification of a congener with an IC₅₀ in the high nanomolar range may yield an effective antimalarial with a promising safety profile.

Our in vitro data support the concept of antimalarial activity of senicapoc through a mechanism independent of the Gardos channel. As senicapoc appears to inhibit intracellular growth of the parasite, other ion channels may be involved in the mechanism of the drug and should be investigated. Building on the safety profile and antimalarial activity of senicapoc for the identification of more potent senicapoc congeners may facilitate repurposing these compounds as antimalarial agents.

Funding Statement

The Yerby Fellowship provided funding to Pedro Mejia under a Postdoctoral Research Fellowship. HHS | NIH | National Heart, Lung, and Blood Institute (NHBLI) provided funding to Venee N. Tubman under grant number T32 HL007574. The Bill and Melinda Gates Foundation provided funding to Manoj T. Duraisingh under the Grand Challenges Explorations Phase II Grant. Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID), provided funding to Manoj T. Duraisingh under grant number R01 AI091787. Harvard Catalyst (Harvard Clinical and Translational Science Center) provided funding to Venee N. Tubman under the Clinical Investigator Training Program.