The antagonism of folate receptor by dolutegravir: developmental toxicity reduction by supplemental folic acid

Abstract

Objective: Maternal folate (vitamin B9) status is the largest known modifier of neural tube defect risk, so we evaluated folate-related mechanisms of action for dolutegravir (DTG) developmental toxicity.

Design: Folate receptor 1 (FOLR1) was examined as a target for DTG developmental toxicity using protein and cellular interaction studies and an animal model.

Methods: FOLR1 competitive binding studies were used to test DTG for FOLR1 antagonism. Human placenta cell line studies were used to test interactions with DTG, folate, and cations. Zebrafish were selected as an animal model to examine DTG-induced developmental toxicity and rescue strategies.

Results: FOLR1 binding studies indicate DTG is a noncompetitive FOLR1 antagonist at therapeutic concentrations. In-vitro testing indicates calcium (2 mmol/l) increases FOLR1-folate interactions and alters DTG-FOLR1-folate interactions and cytotoxicity. DTG does not inhibit downstream folate metabolism by dihydrofolate reductase. Early embryonic exposure to DTG is developmentally toxic in zebrafish, and supplemental folic acid can mitigate DTG developmental toxicity.

Conclusion: Folates and FOLR1 are established modifiers of risk for neural tube defects, and binding data indicates DTG is a partial antagonist of FOLR1. Supplemental folate can ameliorate increased developmental toxicity due to DTG in zebrafish. The results from these studies are expected to inform and guide future animal models and clinical studies of DTG-based antiretroviral therapy in women of childbearing age.

Figure 1.. Binding Curves for DTG Antagonism of FOLR1

A) The binding curves produced by DTG and FOLR1-folate show concentration-dependent decreased signal intensities. Maximum FA-HRP is decreased and curves are marginally shifted left in a concentration-dependent manner (P=0.06). A left shift is expected with competitive antagonists. These data are most consistent with partial or non-competitive antagonism. B) Normalized analysis of FA (competitive) binding to FOLR1. C) Normalized analysis of DTG indicates partial antagonist activity (56% signal at 16μM) by DTG, showing allosteric noncompetitive antagonism of FOLR1 by DTG.

Figure 2.. Calcium Modifies Folate Binding to FOLR1 and DTG Interactions

Calcium (2mM) modified folate binding to FOLR1 in the microtiter protein-ligand binding assay. In the presence of Ca⁺2 (2mM) and absence of DTG, folate receptor bound 42.3% more folate (FA-HRP). The response to DTG in the presence of 2mM Ca⁺2 appeared bi-phasic. The DTG-Ca-FOLR1 interaction below 4μM increased folate binding, but at concentration above 4μM, the binding fell to 77%, or decreased −23% compared to untreated wells.

Figure 3.. Calcium Alters DTG-FA-680 Interactions in HTR-8/SVneo

Images of HTR-8/SVneo cells exposed to 20μM DTG, 50nM FA-680 (red), and Hoechst 3342 (blue, live-nuclei) in buffer (1X DPBS, 1% FBS) (A) without additional Ca⁺2 or (B) with 2mM Ca⁺2 for 1 hour. Bar = 200μm. After 24hrs in RPMI (1% FBS, 50nM FA-680) and variable concentrations of DTG, nuclei showed DTG dependent (C) nuclear contraction (* P = 0.0285, **** P < 0.0001) and (D) decreased cell counts (r = −0.9318, R² = 0.8683, P < 0.0001, dotted lines show 95% confidence interval).

Figure 4.. Folic Acid Rescue of DTG Developmental Toxicity

Folate exposure rescues DTG toxicity in zebrafish embryos. (A-C) Representative images of zebrafish embryos exposed to folate and/or dolutegravir (DTG) starting at 3 hours post fertilization (hpf). Embryos were imaged at 1-day post fertilization. B-B’ represents DTG-induced toxicity in embryos that is rescued upon co-treatment with folate (C). (D-E) Quantification of normal (D) and dead (E) embryos following folate, DTG, or DTG+folate treatment starting at 3 hpf. Each dot represents the mean percent from a clutch of embryos. Bars represent the average of 4 clutches. DTG exposure induces 100% toxicity in embryos while co-exposure with folate rescues toxicity. (F-G) Representative images of zebrafish embryos exposed to vehicle (DMSO) or DTG starting at 5 hpf. Embryos were imaged at 1-day post fertilization. (H-I) Quantification of normal (H) and dead (I) embryos following vehicle or DTG treatment starting at 3 or 5 hpf. Each dot represents the mean percent from a single clutch of embryos. Bars represent the average of 3–4 clutches. The 3 hpf clutches are the same groups from panels B, B’, D, E. DTG exposure is significantly reduced when starting exposure at 5 hpf compared to 3 hpf.

Figure 5.. Critical Period of DTG Developmental Toxicity in Zebrafish

The critical window of exposure for DTG was determined over time by testing the start of DTG exposure at different times (1, 3, 5, and 6–8 hpf). For analysis, the 6–8 hpf range was plot at 7hpf. The normal phenotype (% normal, A) and mortality-morbidity (% dead or deformed, B) observed at 24 hpf was plot as a function of DTG treatment (100μM) start time. Linear regression was fit to the resultant data (R² = 0.86, P < 0.05, for DTG A & B). The mortality-morbidity for DTG exposure was highest with early pre-gastrulation exposures (1–3hpf), differed significantly over time (1–3 hpf vs 5–8hpf, T-test P = 0.001), and was lowest at 6–8hpf. Co-exposure of folic acid (FA, 60ng/mL) with DTG (100μM) results in rescue from developmental toxicity.