Repurposing the Kinase Inhibitor Mavelertinib for Giardiasis Therapy

Abstract

A phenotypic screen of the ReFRAME compound library was performed to identify cell-active inhibitors that could be developed as therapeutics for giardiasis. A primary screen against Giardia lamblia GS clone H7 identified 85 cell-active compounds at a hit rate of 0.72%. A cytotoxicity counterscreen against HEK293T cells was carried out to assess hit compound selectivity for further prioritization. Mavelertinib (PF-06747775), a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), was identified as a potential new therapeutic based on indication, activity, and availability after reconfirmation. Mavelertinib has in vitro efficacy against metronidazole-resistant 713-M3 strains. Other EGFR-TKIs screened in follow-up assays exhibited insignificant inhibition of G. lamblia at 5 μM, suggesting that the primary molecular target of mavelertinib may have a different mechanistic binding mode from human EGFR-tyrosine kinase. Mavelertinib, dosed as low as 5 mg/kg of body weight or as high as 50 mg/kg, was efficacious in the acute murine Giardia infection model. These results suggest that mavelertinib merits consideration for repurposing and advancement to giardiasis clinical trials while its analogues are further developed.

Keywords: EGFR tyrosine kinase inhibitors; Giardia lamblia strains; ReFRAME library.

FIG 1

Flowchart of screening workflow and selection of high-value hits for resupply. The primary screening against G. lamblia GS clone H7 (assemblage B) was at a final concentration of 5 μM. The majority of the compounds that reconfirmed in dose response either were cytotoxic (29), had a chemical scaffold similar to the standard of care (18), or were unavailable for resupply (4), leaving only 2 selective compounds with a novel chemotype available for resupply. Based on reconfirmation in the G. lamblia CBG99 luciferase strain, mavelertinib and pelletierine were selected for resupply for testing in the mouse model of giardiasis.

FIG 2

Chemical structure of selective hits with potential novel mechanism of action/chemotypes. The 6 primary hits, PR-104, CHF-6001, mavelertinib, propyl red, caroverine, and pelletierine, reconfirmed in follow-up assay have anti-Giardia EC₅₀s of 1.98 μM, 2.03 μM, 2.34 μM, 5.78 μM, 13.70 μM, and 20.30 μM, respectively. Selectivity indices (defined as CC₅₀/EC₅₀ against G. lamblia GS clone H7 trophozoites versus toxicity in mammalian cell cultures) were >13, >12, >11, >4, >2, and >1 for PR-104, CHF-6001, mavelertinib, propyl red, caroverine, and pelletierine, respectively. The chemical structure of mavelertinib showing the reactive covalently binding acrylamide chemical functional group is shown.

FIG 3

(A) Noninvasive imaging of G. lamblia CBG99 strain trophozoite growth in mice. Treatment started 6 days postinfection after confirmation of infection. Mice were treated with mavelertinib at 50 mg/kg × 1 (1 day), 20 mg/kg QD × 2 (2 days), 5 mg/kg QD × 2, and 2.5 mg/kg QD × 2. Metronidazole, dosed at 20 mg/kg QD × 3 (3 days), was used as a control. Positive controls included compound 1717 at 50 mg/kg QD × 2, while a group of mice dosed with the blank vehicle served as the untreated control. (B) Radiance plot and mouse images show absence of luminescence signal after a single 50 mg/kg treatment, 20 mg/kg QD × 2, and 5 mg/kg QD × 2 for mavelertinib as well as 5 mg/kg QD × 2 of compound 1717 relative to the untreated or metronidazole-treated controls. (C) Plot of the average measured photons (radiance) of bioluminescence of G. lamblia CBG99 trophozoites in mice treated with 1 mg/kg of mavelertinib and in untreated mice. In this experiment, the in vivo efficacy of mavelertinib (red plot) dosed at 1 mg/kg QD on days 6 to 9 postinfection in G. lamblia CBG99-infected B6 IFN-γ KO mice was measured. Treatment control for the study included 20 mg/kg of metronidazole (blue plot), vehicle (untreated) infected mice, and uninfected background controls (BKGD). Mavelertinib dosed at 1 mg/kg did not clear the infection after 4 doses.

FIG 4

Pharmacokinetic analysis of blood at treatment concentrations. The time at which maximum plasma concentration (C_max) was observed (T_max) was 0.5 h in all cases. The 50 mg/kg dose had an average C_max of 9.3 μM at 0.5 h postdose. The 20 mg/kg and 5 mg/kg C_maxs were 3.5 μM and 0.63 μM, respectively. For the group treated at 1 mg/kg QD, mavelertinib was barely detectable in the plasma samples at all time points sampled. All dosage groups had plasma concentrations below the detection limit at 24 h postdose.