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Cells
2025 May 09;1410:. doi: 10.3390/cells14100688.
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Reversible Modulation of Motile Cilia by a Benzo[e][1,2,4]triazinone: A Potential Non-Hormonal Approach to Male Contraception.
Chatzifrangkeskou M
,
Perdiou A
,
Kreouzou R
,
Zissimou GA
,
Flesariu DF
,
Koutentis PA
,
Skourides PA
.
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Motile cilia play essential roles in various physiological processes including fluid flow generation and sperm motility. In this study, we identified 1,3-diphenyl-6-(4-phenylpiperazin-1-yl)benzo[e][1,2,4]triazin-7(1H)-one as a potent and reversible modulator of ciliary function using the Xenopus laevis model. This benzotriazinone derivative inhibits ciliary-driven fluid flow by inducing cilia detachment without causing toxicity in developing embryos. Unlike traditional deciliation agents that rely on calcium signaling, this compound induces cilia loss through a shear stress-driven mechanism at the transition zone, without disrupting tissue morphology or the apical actin network. Importantly, it also induces flagellar loss and impairs sperm motility at picomolar concentrations. Our findings highlight the potential of this 6-(4-phenylpiperazin-1-yl)-substituted benzotriazinone as a non-hormonal male contraceptive and underscore a novel mechanism of cilia modulation that may have broader implications for the treatment of cilia-related disorders.
Figure 1. Benzotriazinone 10 robustly and reversibly inhibits fluid flow generation. (A) Schematic diagram illustrating the experimental workflow. Xenopus embryos were treated with chemical compounds overnight, followed by the assessment of survival scoring. Ciliary-driven fluid flow was assessed using fluorescent beads after 1 h of compound incubation. (B) Representative images of fluorescent bead tracking in embryos treated with DMSO (control) or benzotriazinone 10 (50 μΜ). Tracks indicate bead movement driven by ciliary motion (n = 4 embryos, ** p < 0.01). The chemical structure of benzotriazinone 10 is shown. Quantification of bead track speed (µm/s) demonstrated a significant reduction in fluid flow velocity in the benzotriazinone 10-treated embryos compared with the controls. (C) Dose-dependent inhibition of fluid flow by benzotriazinone 10 (n = 4 embryos, *** p < 0.001). Fluid flow decreased significantly with increasing benzotriazinone 10 concentrations.
Figure 2. Benzotriazinone 10 induces cilia shedding in Xenopus embryos. (A) Representative confocal images showing acetylated α-tubulin and phalloidin in epidermal MCCs treated with DMSO (control) or 0.5 nM benzotriazinone 10. Benzotriazinone 10 treatment led to complete cilia detachment within minutes, without disrupting the apical actin network. Scale bars: 10 µm. (B) Time-course analysis of cilia shedding in response to 0.5 nM benzotriazinone 10. Confocal images showed acetylated α-tubulin and phalloidin at various time points following benzotriazinone 10 treatment. The bottom panels represent magnified insets marked by white squares. Quantification of the bead track speed showed a significant reduction in the fluid flow velocity, correlating with cilia loss (n = 3 embryos, **** p < 0.0001, ns not significant). Scale bars: 10 µm. (C) Reversibility of benzotriazinone 10-induced cilia detachment. MCCs were treated with benzotriazinone 10 for 60 min, followed by a drug washout and recovery at 60, 80, 120, and 180 min (n = 4 embryos, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). The bottom panels represent magnified insets marked by white squares. Quantification of the bead speed demonstrated a statistically significant recovery over time. Scale bars: 10 µm.
Figure 3. Benzotriazinone 10 promotes shear stress-driven deciliation. (A) Isolated cilia on poly-L-lysine-coated cover glass following deciliation buffer or 0.5 nM benzotriazinone 10-treatment of the embryos. Acetylated α-tubulin was used to label the ciliary axonemes. Scale bar: 10 µm. (B) Stills from a time-lapse movie showing MCCs stained with a plasma membrane dye. Arrowheads show the severed cilia. Scale bar: 20 µm. (C) Immunostaining of embryos treated with either benzotriazinone 10 or NiCl2 or both. Inhibition of ciliary motility abrogated the effect of benzotriazinone 10. Scale bar: 20 µm.
Figure 4. Benzotriazinone 10 induces cilia shedding at the distal region of the transition zone. (A) Representative Z-stack images of an epidermal MCC expressing mEmerald-tagged B9D1 and RFP-Centrin to mark the basal bodies. Acetylated α-tubulin was used to label the cilia. Treatment with 0.5 nM benzotriazinone 10 showed that the transition zone marker B9D1 was preserved. Scale bar: 10 µm. (B) Representative Z-stack images of a MCC expressing mEmerald-tagged MKS1 and RFP-Centrin. Benzotriazinone 10 loss of the transition zone marker MKS1. Scale bar: 10 µm.
Figure 5. Structure–activity relationship analysis of benzotriazinone 10. (A) Summary of the chemical structures of the benzotriazinone 10 analogs and their effects on ciliary flow. The graph shows the speed of fluorescent beads in Xenopus embryos treated with the indicated compounds or DMSO (n = 3 embryos, *** p < 0.001, **** p < 0.0001, ns, not significant). (B) Quantification of fluid flow in embryos treated with benzotriazinone 10 or its analog 11 compared with the DMSO-treated controls (n = 4 embryos, * p < 0.05, ** p < 0.01). (C) Representative confocal images of Xenopus epidermal MCCs treated with DMSO or analog 11 stained for acetylated tubulin and phalloidin. Scale bar: 10 µm.
Figure 6. Effects of benzotriazinone 10 on sperm motility and morphology. (A) Representative brightfield images of Xenopus sperm from the control (DMSO) and benzotriazinone 10-treated groups. Overlaid colored tracks represent the trajectories of individual spermatozoa, illustrating their motility patterns. (B) Immunofluorescence staining of sperm from the DMSO and benzotriazinone 10-treated groups. Plasma membrane is labeled in red, -tubulin (axonemal structure) in green, and nuclei counterstained with Hoechst 33342 in blue. DMSO-treated spermatozoa exhibited intact flagella, while benzotriazinone 10-treated spermatozoa were deflagellated. Scale bar = 10um.
