Sildenafil 100 mg: Efficacy and Onset of Action

31Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, 40127 Bologna, Italy 32Friedrich-Baur-Institute, Department of Neurology, LMU University Hospital, Ludwig-Maximilians-Universität München, 80336 Munich, Germany 33Munich Cluster for Systems Neurology (SyNergy), 81377 Munich, Germany 34German Center for Neurodegenerative Diseases (DZNE), 81377 Munich, Germany 35Genome Engineering and Model Development lab (GEMD), IUF-Leibniz Research Institute for Environmental Medicine, 40223 Düsseldorf, Germany 36NeuroCure Cluster of Excellence, 10117 Berlin, Germany 37German Center for Cardiovascular Research (DZHK), partner site Lower Saxony and Berlin, 10785 Berlin, Germany 38National Center for Tumor Diseases (NCT), German Cancer Consortium (DKTK), 13125 Berlin, Germany 39German Center for Neurodegenerative Diseases (DZNE), 10117 Berlin, Germany 40Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, 37077 Göttingen, Germany 41Department of Neurology, University Medical Center Göttingen, 37075 Göttingen, Germany 42Human and Animal Physiology, Wageningen University, 6708 WD Wageningen, the Netherlands 43Neuropsychiatry and Laboratory of Molecular Psychiatry, Department of Psychiatry and Neurosciences, Charité - Universitätsmedizin Berlin, 10117 Berlin, Germany 44Department of Molecular Biology, Institute of Genetics and Animal Biotechnology, Polish Academy of Sciences, 05-552 Jastrzebiec n/Warsaw, Poland 45HiLife, University of Helsinki, 0004 Helsinki, Finland 46HUS Diagnostics, Helsinki University Hospital, 00290 Helsinki, Finland 47Department of Clinical Sciences and Community Health, Dipartimento di Eccellenza 2023-2027, University of Milan, 20122 Milan, Italy 48IKERBASQUE, Basque Foundation for Science, 48009 Bilbao, Spain 49CIC bioGUNE-BRTA (Basque Research and Technology Alliance), Bizkaia Technology Park, 48160 Derio, Spain 50German Center for Child and Adolescent Health (DZKJ), Section CNS Development and Neurological Disease, partner site Berlin, 13353 Berlin, Germany Received April 14, 2025; Revised August 19, 2025; Accepted February 6, 2026; Published online March 11, 2026 DOI: 10.1016/j.cell.2026.02.008 External LinkAlso available on ScienceDirect External Link Repurposable drug screen in Leigh syndrome neural cells identifies PDE5 inhibitors PDE5 inhibitor sildenafil rescues the neurodevelopmental disease signature in vitro PDE5 inhibitor sildenafil extends the lifespan of Leigh syndrome animal models in vivo Sildenafil treatment on an individual basis clinically improves symptoms in six LS patients Mitochondrial disease encompasses inherited disorders affecting mitochondrial function. A severe and untreatable form of mitochondrial disease is Leigh syndrome (LS), causing psychomotor regression and metabolic crises. To accelerate drug discovery for LS, we screen a library of 5,632 repurposable compounds in neural cells from LS-patient-derived induced pluripotent stem cells (iPSCs). We identify phosphodiesterase type 5 (PDE5) inhibitors as leads and prioritize sildenafil for its clinical safety. Sildenafil corrects mitochondrial membrane potential defects, restores neurodevelopmental pathways, and normalizes calcium responses in LS brain organoids.

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31Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, 40127 Bologna, Italy 32Friedrich-Baur-Institute, Department of Neurology, LMU University Hospital, Ludwig-Maximilians-Universität München, 80336 Munich, Germany 33Munich Cluster for Systems Neurology (SyNergy), 81377 Munich, Germany 34German Center for Neurodegenerative Diseases (DZNE), 81377 Munich, Germany 35Genome Engineering and Model Development lab (GEMD), IUF-Leibniz Research Institute for Environmental Medicine, 40223 Düsseldorf, Germany 36NeuroCure Cluster of Excellence, 10117 Berlin, Germany 37German Center for Cardiovascular Research (DZHK), partner site Lower Saxony and Berlin, 10785 Berlin, Germany 38National Center for Tumor Diseases (NCT), German Cancer Consortium (DKTK), 13125 Berlin, Germany 39German Center for Neurodegenerative Diseases (DZNE), 10117 Berlin, Germany 40Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, 37077 Göttingen, Germany 41Department of Neurology, University Medical Center Göttingen, 37075 Göttingen, Germany 42Human and Animal Physiology, Wageningen University, 6708 WD Wageningen, the Netherlands 43Neuropsychiatry and Laboratory of Molecular Psychiatry, Department of Psychiatry and Neurosciences, Charité - Universitätsmedizin Berlin, 10117 Berlin, Germany 44Department of Molecular Biology, Institute of Genetics and Animal Biotechnology, Polish Academy of Sciences, 05-552 Jastrzebiec n/Warsaw, Poland 45HiLife, University of Helsinki, 0004 Helsinki, Finland 46HUS Diagnostics, Helsinki University Hospital, 00290 Helsinki, Finland 47Department of Clinical Sciences and Community Health, Dipartimento di Eccellenza 2023-2027, University of Milan, 20122 Milan, Italy 48IKERBASQUE, Basque Foundation for Science, 48009 Bilbao, Spain 49CIC bioGUNE-BRTA (Basque Research and Technology Alliance), Bizkaia Technology Park, 48160 Derio, Spain 50German Center for Child and Adolescent Health (DZKJ), Section CNS Development and Neurological Disease, partner site Berlin, 13353 Berlin, Germany Received April 14, 2025; Revised August 19, 2025; Accepted February 6, 2026; Published online March 11, 2026 DOI: 10.1016/j.cell.2026.02.008 External LinkAlso available on ScienceDirect External Link Repurposable drug screen in Leigh syndrome neural cells identifies PDE5 inhibitors PDE5 inhibitor sildenafil rescues the neurodevelopmental disease signature in vitro PDE5 inhibitor sildenafil extends the lifespan of Leigh syndrome animal models in vivo Sildenafil treatment on an individual basis clinically improves symptoms in six LS patients Mitochondrial disease encompasses inherited disorders affecting mitochondrial function. A severe and untreatable form of mitochondrial disease is Leigh syndrome (LS), causing psychomotor regression and metabolic crises. To accelerate drug discovery for LS, we screen a library of 5,632 repurposable compounds in neural cells from LS-patient-derived induced pluripotent stem cells (iPSCs). We identify phosphodiesterase type 5 (PDE5) inhibitors as leads and prioritize sildenafil for its clinical safety. Sildenafil corrects mitochondrial membrane potential defects, restores neurodevelopmental pathways, and normalizes calcium responses in LS brain organoids.

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In small and large mammalian models of LS, sildenafil extends lifespan and ameliorates disease phenotypes. Off-label treatment on an individual basis with sildenafil in six LS patients improves their motor function and resistance to metabolic crises. Collectively, the findings highlight the potential of iPSC-driven drug discovery and position sildenafil as a promising drug candidate for mitochondrial disease. Mitochondrial disease encompasses rare genetic conditions caused by dysfunction in the cell’s energy-producing organelles.1 A severe form of mitochondrial disease is Leigh syndrome (LS, OMIM #256000), which is characterized by neurodevelopmental regression and muscle weakness, with early death following metabolic crises.2,3 LS can result from pathogenic variants in more than 100 genes in nuclear DNA or mitochondrial DNA (mtDNA) involved in oxidative phosphorylation (OXPHOS),4 including complex V (CV) gene MT-ATP6 (mitochondrially encoded ATP synthase subunit 6),5 complex IV (CIV) assembly factor gene SURF1 (SURFEIT1),6 and complex I gene NDUFS4 (nicotinamide adenine dinucleotide [NAD] + hydrogen [NADH] dehydrogenase [ubiquinone] iron-sulfur protein 4).7 Currently, there are no treatments for LS.8 One obstacle to therapy discovery is the paucity of model systems.9 The difficulties in mtDNA editing have hindered the establishment of models for mtDNA defects.10 A pig model of LS due to SURF1 knockout (KO) showed neurodevelopmental defects and early lethality,11 but Surf1 KO mice failed to replicate LS phenotypes.12,13 The most widely used model of LS is the homozygous Ndufs4 KO mouse, characterized by progressive encephalopathy, growth retardation, and premature death.14,15 This model has been used to propose therapeutic interventions, including antioxidants,16,17,18 rapamycin,19 interferon-gamma-targeting therapies,20 cannabidiol,21 or hypoxia.22,23 Only a few of these strategies have been tested in LS individuals, and no successful results have been obtained. New approach methodologies (NAMs) with LS patient-derived induced pluripotent stem cells (iPSCs)24 have been instrumental in uncovering pathological mechanisms.

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These include neuronal outgrowth defects,25 altered calcium homeostasis,26,27 and glutamate toxicity in neuronal cultures,28 as well as impaired cortical development and neuromorphogenesis in brain organoids.25,29,30 Despite these advances, large-scale drug screens in iPSC models of mitochondrial disease have yet to be performed. We previously demonstrated that iPSC-derived neural precursor cells (NPCs) are an effective drug discovery platform for mtDNA diseases.27 LS NPCs carrying a MT-ATP6 variant exhibited abnormal mitochondrial membrane potential (MMP), a feature that can be exploited for high-throughput screens.31 Here, we leveraged this MMP phenotype to screen a library of 5,632 repurposable drug candidates in LS NPCs. We identified phosphodiesterase type 5 (PDE5) inhibitors as lead compounds and prioritized sildenafil for its safety profile.32 Sildenafil rescued the neurodevelopmental disease signature, promoted neuronal outgrowth, and normalized calcium homeostasis in human LS models. In vivo, sildenafil extended the lifespan of Ndufs4 KO mice and SURF1 KO pigs. Mechanistically, sildenafil’s action appeared to be mediated through cyclic GMP (cGMP)-dependent protein kinase 1 (PRKG1). In small and large mammalian models of LS, sildenafil extends lifespan and ameliorates disease phenotypes. Off-label treatment on an individual basis with sildenafil in six LS patients improves their motor function and resistance to metabolic crises. Collectively, the findings highlight the potential of iPSC-driven drug discovery and position sildenafil as a promising drug candidate for mitochondrial disease. Mitochondrial disease encompasses rare genetic conditions caused by dysfunction in the cell’s energy-producing organelles.1 A severe form of mitochondrial disease is Leigh syndrome (LS, OMIM #256000), which is characterized by neurodevelopmental regression and muscle weakness, with early death following metabolic crises.2,3 LS can result from pathogenic variants in more than 100 genes in nuclear DNA or mitochondrial DNA (mtDNA) involved in oxidative phosphorylation (OXPHOS),4 including complex V (CV) gene MT-ATP6 (mitochondrially encoded ATP synthase subunit 6),5 complex IV (CIV) assembly factor gene SURF1 (SURFEIT1),6 and complex I gene NDUFS4 (nicotinamide adenine dinucleotide [NAD] + hydrogen [NADH] dehydrogenase [ubiquinone] iron-sulfur protein 4).7 Currently, there are no treatments for LS.8 One obstacle to therapy discovery is the paucity of model systems.9 The difficulties in mtDNA editing have hindered the establishment of models for mtDNA defects.10 A pig model of LS due to SURF1 knockout (KO) showed neurodevelopmental defects and early lethality,11 but Surf1 KO mice failed to replicate LS phenotypes.12,13 The most widely used model of LS is the homozygous Ndufs4 KO mouse, characterized by progressive encephalopathy, growth retardation, and premature death.14,15 This model has been used to propose therapeutic interventions, including antioxidants,16,17,18 rapamycin,19 interferon-gamma-targeting therapies,20 cannabidiol,21 or hypoxia.22,23 Only a few of these strategies have been tested in LS individuals, and no successful results have been obtained. New approach methodologies (NAMs) with LS patient-derived induced pluripotent stem cells (iPSCs)24 have been instrumental in uncovering pathological mechanisms. These include neuronal outgrowth defects,25 altered calcium homeostasis,26,27 and glutamate toxicity in neuronal cultures,28 as well as impaired cortical development and neuromorphogenesis in brain organoids.25,29,30 Despite these advances, large-scale drug screens in iPSC models of mitochondrial disease have yet to be performed.

We previously demonstrated that iPSC-derived neural precursor cells (NPCs) are an effective drug discovery platform for mtDNA diseases.27 LS NPCs carrying a MT-ATP6 variant exhibited abnormal mitochondrial membrane potential (MMP), a feature that can be exploited for high-throughput screens.31 Here, we leveraged this MMP phenotype to screen a library of 5,632 repurposable drug candidates in LS NPCs. We identified phosphodiesterase type 5 (PDE5) inhibitors as lead compounds and prioritized sildenafil for its safety profile.32 Sildenafil rescued the neurodevelopmental disease signature, promoted neuronal outgrowth, and normalized calcium homeostasis in human LS models. In vivo, sildenafil extended the lifespan of Ndufs4 KO mice and SURF1 KO pigs.

Mechanistically, sildenafil’s action appeared to be mediated through cyclic GMP (cGMP)-dependent protein kinase 1 (PRKG1). Off-label treatment on an individual basis with sildenafil in six patients with LS carrying MT-ATP6 variants sildenafil 20 mg tablets improved muscle strength and resistance to metabolic crises. The data implicate sildenafil as a repurposable drug for mitochondrial disorders. To determine whether MMP hyperpolarization observed in LS NPCs carrying the m.9185T>C variant in the MT-ATP6 gene27 represented a robust phenotype, we generated NPCs from 7 LS patients carrying different MT-ATP6 variants (3 m.9185T>C, 1 m.8993T>C, 2 m.8993T>G, and one m.9176T>G) (Figure S1A).27,33,34,35,36 For controls, we used NPCs from 8 healthy individuals, including 2 patients’ mothers carrying MT-ATP6 variants at low heteroplasmy (<2%) (Figure S10H).

LS NPCs expressed NPC markers similarly to control NPCs (Figures S1E and S1F) and retained mtDNA heteroplasmy of parental iPSCs (Figure S10H).

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RV revealed early hypertrophic molecular changes similar to LV during LV pressure-overload, and sildenafil inhibited these in both ventricles

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LS NPCs showed defective bioenergetics (Figure S1C), disrupted MT-ATP6 protein expression (Figure S1B) and CV assembly structure (Figures S4A and S4B), and MMP hyperpolarization (Figure 1A). We applied a well-curated library of 5,632 repurposable molecules37 to modulate the MMP of LS NPCs. We used live-cell high-content analysis (HCA) based on tetramethylrhodamine methyl ester (TMRM) intensity31 (Figures 1B, S2A, and S2B). Compounds were applied with 0.05% maximal concentration of DMSO, which was not toxic in LS NPCs (Figure S2C). We included MMP values from one control NPC line for baseline MMP after having confirmed that various control NPC lines displayed comparable MMP (Figure S2D).

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Off-label treatment on an individual basis with sildenafil in six patients with LS carrying MT-ATP6 variants sildenafil 20 mg tablets improved muscle strength and resistance to metabolic crises. The data implicate sildenafil as a repurposable drug for mitochondrial disorders. To determine whether MMP hyperpolarization observed in LS NPCs carrying the m.9185T>C variant in the MT-ATP6 gene27 represented a robust phenotype, we generated NPCs from 7 LS patients carrying different MT-ATP6 variants (3 m.9185T>C, 1 m.8993T>C, 2 m.8993T>G, and one m.9176T>G) (Figure S1A).27,33,34,35,36 For controls, we used NPCs from 8 healthy individuals, including 2 patients’ mothers carrying MT-ATP6 variants at low heteroplasmy (<2%) (Figure S10H). LS NPCs expressed NPC markers similarly to control NPCs (Figures S1E and S1F) and retained mtDNA heteroplasmy of parental iPSCs (Figure S10H). LS NPCs showed defective bioenergetics (Figure S1C), disrupted MT-ATP6 protein expression (Figure S1B) and CV assembly structure (Figures S4A and S4B), and MMP hyperpolarization (Figure 1A).

Key takeaway

We applied a well-curated library of 5,632 repurposable molecules37 to modulate the MMP of LS NPCs. We used live-cell high-content analysis (HCA) based on tetramethylrhodamine methyl ester (TMRM) intensity31 (Figures 1B, S2A, and S2B). Compounds were applied with 0.05% maximal concentration of DMSO, which was not toxic in LS NPCs (Figure S2C). We included MMP values from one control NPC line for baseline MMP after having confirmed that various control NPC lines displayed comparable MMP (Figure S2D). We established the Z′ factor38 to be ∼0.68–0.85 across plates (Figures S2E–S2G) and identified 5 μM as the optimal screening concentration achieving an ∼5% hit rate with low toxicity (Figure S2H) and high reproducibility (Figure S2I).

S1 Fig. Western blot of p-ERK1/2 in the RV myocardium.

We classified compounds based on their depolarizing effect in LS NPCs (MMP inhibition) relative to the standard deviation (SD) of DMSO-treated LS NPCs (SD = 6.51%). We identified 767 mild uncouplers (13.0%–19.5% inhibition, >2 SD), 520 intermediate uncouplers (19.5%–32.5% inhibition, >3 SD), and 187 strong uncouplers (>5 SD) (Figure 1C). We excluded toxic compounds based on cell sildenafil 50g count (cut-off: 29.86%). We focused on mild uncouplers that depolarized the MMP of LS NPCs to values similar to control NPCs (Figure 1C). Among those, we identified two PDE5 inhibitors (PDE5i), T-0156 and vardenafil hydrochloride, which showed dose-dependent MMP depolarization in LS NPCs without loss of viability (Figures S3A–S3D). We established the Z′ factor38 to be ∼0.68–0.85 across plates (Figures S2E–S2G) and identified 5 μM as the optimal screening concentration achieving an ∼5% hit rate with low toxicity (Figure S2H) and high reproducibility (Figure S2I). We classified compounds based on their depolarizing effect in LS NPCs (MMP inhibition) relative to the standard deviation (SD) of DMSO-treated LS NPCs (SD = 6.51%).

• Sildenafil 100g refers to a high dosage of the medication used to treat erectile dysfunction.
• This dosage is significantly higher than the common 50mg or 25mg tablets.
• Always consult a healthcare provider before using sildenafil in such high amounts.
• Overuse can cause severe side effects like dangerous drops in blood pressure.
• Sildenafil 100g is typically used only under strict medical supervision for specific cases.

We identified 767 mild uncouplers (13.0%–19.5% inhibition, >2 SD), 520 intermediate uncouplers (19.5%–32.5% inhibition, >3 SD), and 187 strong uncouplers (>5 SD) (Figure 1C). We excluded toxic compounds based on cell sildenafil 50g count (cut-off: 29.86%).

• Storage of sildenafil should be in a cool, dry place away from direct sunlight.
• Common side effects include dizziness, upset stomach, or vision changes.
• The dosage of sildenafil should be tailored to individual health needs.
• Sildenafil can interact with other medications, affecting its safety and efficacy.
• Always seek medical guidance before using sildenafil, especially at high doses.

We focused on mild uncouplers that depolarized the MMP of LS NPCs to values similar to control NPCs (Figure 1C).

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As our previous proof-of-concept screen in LS NPCs highlighted the PDE5i avanafil,27 we prioritized PDE5i employed in pediatric settings. Sildenafil emerged as a strong candidate, given its safety profile in children treated for pulmonary arterial hypertension (PAH) or lymphatic malformations.39,40,41,42 Sildenafil led to dose-dependent MMP depolarization in LS NPCs (Figure 1D) without toxicity (Figures S3E–S3G) and with a half-maximal inhibitory concentration (IC50) of ∼3 μM at 16 h (Figure S3H). MMP normalization occurred similarly in LS NPCs carrying different MT-ATP6 variants with overall MMP depolarization of ∼16% (Figure 1E). Sildenafil did not modify the MMP in control NPCs (Figures S3I and S3J). Bioenergetically, sildenafil did not lead to a measurable improvement in CV activity (Figure 1F) or in CV assembly structure43,44 (Figure S4C).

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The protein levels of known mitochondrial modulators were also not significantly impacted (Figures S4D and S4E).

Statistical analysis

Among those, we identified two PDE5 inhibitors (PDE5i), T-0156 and vardenafil hydrochloride, which showed dose-dependent MMP depolarization in LS NPCs without loss of viability (Figures S3A–S3D). As our previous proof-of-concept screen in LS NPCs highlighted the PDE5i avanafil,27 we prioritized PDE5i employed in pediatric settings.

Sildenafil emerged as a strong candidate, given its safety profile in children treated for pulmonary arterial hypertension (PAH) or lymphatic malformations.39,40,41,42 Sildenafil led to dose-dependent MMP depolarization in LS NPCs (Figure 1D) without toxicity (Figures S3E–S3G) and with a half-maximal inhibitory concentration (IC50) of ∼3 μM at 16 h (Figure S3H). MMP normalization occurred similarly in LS NPCs carrying different MT-ATP6 variants with overall MMP depolarization of ∼16% (Figure 1E). Sildenafil did not modify the MMP in control NPCs (Figures S3I and S3J). Bioenergetically, sildenafil did not lead to a measurable improvement in CV activity (Figure 1F) or in CV assembly structure43,44 (Figure S4C). The protein levels of known mitochondrial modulators were also not significantly impacted (Figures S4D and S4E).