RESUMEN
The characterization of cryptic pockets has been elusive, despite substantial efforts. Computational modeling approaches, such as molecular dynamics (MD) simulations, can provide atomic-level details of binding site motions and binding pathways. However, the time scale that MD can achieve at a reasonable cost often limits its application for cryptic pocket identification. Enhanced sampling techniques can improve the efficiency of MD simulations by focused sampling of important regions of the protein, but prior knowledge of the simulated system is required to define the appropriate coordinates. In the case of a novel, unknown cryptic pocket, such information is not available, limiting the application of enhanced sampling techniques for cryptic pocket identification. In this work, we explore the ability of SiteMap and Site Finder, widely used commercial packages for pocket identification, to detect focus points on the protein and further apply other advanced computational methods. The information gained from this analysis enables the use of computational modeling, including enhanced MD sampling techniques, to explore potential cryptic binding pockets suggested by SiteMap and Site Finder. Here, we examined SiteMap and Site Finder results on 136 known cryptic pockets from a combination of the PocketMiner dataset (a recently curated set of cryptic pockets), the Cryptosite Set (a classic set of cryptic pockets), and Natural killer group 2D (NKG2D, a protein target where a cryptic pocket is confirmed). Our findings demonstrate the application of existing, well-studied tools in efficiently mapping potential regions harboring cryptic pockets.
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Simulación de Dinámica Molecular , Sitios de Unión , Proteínas/químicaRESUMEN
The interaction between menin and histone-lysine N-methyltransferase 2A (KMT2A) is a critical dependency for KMT2A- or nucleophosmin 1 (NPM1)-altered leukemias and an emerging opportunity for therapeutic development. JNJ-75276617 is a novel, orally bioavailable, potent, and selective protein-protein interaction inhibitor of the binding between menin and KMT2A. In KMT2A-rearranged (KMT2A-r) and NPM1-mutant (NPM1c) AML cells, JNJ-75276617 inhibited the association of the menin-KMT2A complex with chromatin at target gene promoters, resulting in reduced expression of several menin-KMT2A target genes, including MEIS1 and FLT3. JNJ-75276617 displayed potent anti-proliferative activity across several AML and ALL cell lines and patient samples harboring KMT2A- or NPM1-alterations in vitro. In xenograft models of AML and ALL, JNJ-75276617 reduced leukemic burden and provided a significant dose-dependent survival benefit accompanied by expression changes of menin-KMT2A target genes. JNJ-75276617 demonstrated synergistic effects with gilteritinib in vitro in AML cells harboring KMT2A-r. JNJ-75276617 further exhibited synergistic effects with venetoclax and azacitidine in AML cells bearing KMT2A-r in vitro, and significantly increased survival in mice. Interestingly, JNJ-75276617 showed potent anti-proliferative activity in cell lines engineered with recently discovered mutations (MEN1M327I or MEN1T349M) that developed in patients refractory to the menin-KMT2A inhibitor revumenib. A co-crystal structure of menin in complex with JNJ-75276617 indicates a unique binding mode distinct from other menin-KMT2A inhibitors, including revumenib. JNJ-75276617 is being clinically investigated for acute leukemias harboring KMT2A or NPM1 alterations, as a monotherapy for relapsed/refractory (R/R) acute leukemia (NCT04811560), or in combination with AML-directed therapies (NCT05453903).
RESUMEN
Proteolysis Targeting Chimeras (PROTACs) are an emerging therapeutic modality and chemical biology tools for Targeted Protein Degradation (TPD). PROTACs contain a ligand targeting the protein of interest, a ligand recruiting an E3 ligase and a linker connecting these two ligands. There are over 600 E3 ligases known so far, but only a handful have been exploited for TPD applications. A key reason for this is the scarcity of ligands binding various E3 ligases and the paucity of structural data available, which complicates ligand design across the family. In this study, we aim to progress PROTAC discovery by proposing a shortlist of E3 ligases that can be prioritized for covalent targeting by performing systematic structural ligandability analysis on a chemoproteomic dataset of potentially reactive cysteines across hundreds of E3 ligases. One of the goals of this study is to apply AlphaFold (AF) models for ligandability evaluations, as for a vast majority of these ligases an experimental structure is not available in the protein data bank (PDB). Using a combination of pocket features, AF model quality and additional aspects, we propose a shortlist of E3 ligases and corresponding cysteines that can be prioritized to potentially discover covalent ligands and expand the PROTAC toolbox.
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Cisteína , Unión Proteica , Proteolisis , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ligandos , Cisteína/química , Cisteína/metabolismo , Humanos , Modelos Moleculares , Sitios de Unión , Bases de Datos de ProteínasRESUMEN
Membrane proteins have diverse functions within cells and are well-established drug targets. The advances in membrane protein structural biology have revealed drug and lipid binding sites on membrane proteins, while computational methods such as molecular simulations can resolve the thermodynamic basis of these interactions. Particularly, alchemical free energy calculations have shown promise in the calculation of reliable and reproducible binding free energies of protein-ligand and protein-lipid complexes in membrane-associated systems. In this review, we present an overview of representative alchemical free energy studies on G-protein-coupled receptors, ion channels, transporters as well as protein-lipid interactions, with emphasis on best practices and critical aspects of running these simulations. Additionally, we analyze challenges and successes when running alchemical free energy calculations on membrane-associated proteins. Finally, we highlight the value of alchemical free energy calculations calculations in drug discovery and their applicability in the pharmaceutical industry.
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Proteínas de la Membrana , Simulación de Dinámica Molecular , Entropía , Termodinámica , Ligandos , Lípidos , Unión ProteicaRESUMEN
The dysregulation of the PRC1/2 complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype. [1] CBX2, a key component of the canonical PRC1 complex, is an epigenetic reader, recognizing trimethylated lysine on histone 3 (H3K27me3) [2] and is overexpressed in metastatic neuroendocrine prostate cancer. [3,4] We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX2 binding to chromatin. Construct design and phosphorylation state of CBX2 were critical for successful implementation and execution of an HTS library screen. A rigorous screening funnel including counter and selectivity assays allowed us to quickly focus on true positive hit matter. Two distinct non-peptide-like chemotypes were identified and confirmed in orthogonal biochemical and biophysical assays demonstrating disruption of CBX2 binding to nucleosomes and direct binding to purified CBX2, respectively.
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Complejo Represivo Polycomb 1 , Neoplasias de la Próstata , Núcleo Celular/metabolismo , Cromatina , Histonas/metabolismo , Humanos , Masculino , Complejo Represivo Polycomb 1/genética , Neoplasias de la Próstata/metabolismoRESUMEN
The protein arginine methyltransferase 5 (PRMT5) methylates a variety of proteins involved in splicing, multiple signal transduction pathways, epigenetic control of gene expression, and mechanisms leading to protein expression required for cellular proliferation. Dysregulation of PRMT5 is associated with clinical features of several cancers, including lymphomas, lung cancer, and breast cancer. Here, we describe the characterization of JNJ-64619178, a novel, selective, and potent PRMT5 inhibitor, currently in clinical trials for patients with advanced solid tumors, non-Hodgkin's lymphoma, and lower-risk myelodysplastic syndrome. JNJ-64619178 demonstrated a prolonged inhibition of PRMT5 and potent antiproliferative activity in subsets of cancer cell lines derived from various histologies, including lung, breast, pancreatic, and hematological malignancies. In primary acute myelogenous leukemia samples, the presence of splicing factor mutations correlated with a higher ex vivo sensitivity to JNJ-64619178. Furthermore, the potent and unique mechanism of inhibition of JNJ-64619178, combined with highly optimized pharmacological properties, led to efficient tumor growth inhibition and regression in several xenograft models in vivo, with once-daily or intermittent oral-dosing schedules. An increase in splicing burden was observed upon JNJ-64619178 treatment. Overall, these observations support the continued clinical evaluation of JNJ-64619178 in patients with aberrant PRMT5 activity-driven tumors.
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Inhibidores Enzimáticos/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Proteína-Arginina N-Metiltransferasas/efectos de los fármacos , Pirimidinas/uso terapéutico , Pirroles/uso terapéutico , Animales , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Humanos , Neoplasias Pulmonares/patología , Ratones , Pirimidinas/farmacología , Pirroles/farmacologíaRESUMEN
Androgen receptor (AR) transcriptional reactivation plays a key role in the development and progression of lethal castration-resistant prostate cancer (CRPC). Recurrent alterations in the AR enable persistent AR pathway signaling and drive resistance to the treatment of second-generation antiandrogens. AR F877L, a point mutation in the ligand binding domain of the AR, was identified in patients who acquired resistance to enzalutamide or apalutamide. In parallel to our previous structure-activity relationship (SAR) studies of compound 4 (JNJ-pan-AR) and clinical stage compound 5 (JNJ-63576253), we discovered additional AR antagonists that provide opportunities for future development. Here we report a highly potent series of spirocyclic thiohydantoins as AR antagonists for the treatment of the F877L mutant and wild-type CRPC.
RESUMEN
Selective cyclooxygenase (COX)-2 inhibitors have been extensively studied for colorectal cancer (CRC) chemoprevention. Celecoxib has been reported to reduce the incidence of colorectal adenomas and CRC but is also associated with an increased risk of cardiovascular events. Here, we report a series of gut-restricted, selective COX-2 inhibitors characterized by high colonic exposure and minimized systemic exposure. By establishing acute ex vivo 18F-FDG uptake attenuation as an efficacy proxy, we identified a subset of analogues that demonstrated statistically significant in vivo dose-dependent inhibition of adenoma progression and survival extension in an APCmin/+ mouse model. However, in vitro-in vivo correlation analysis showed their chemoprotective effects were driven by residual systemic COX-2 inhibition, rationalizing their less than expected efficacies and highlighting the challenges associated with COX-2-mediated CRC disease chemoprevention.
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Antineoplásicos/farmacología , Celecoxib/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa 2/farmacología , Ciclooxigenasa 2/metabolismo , Etoricoxib/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Celecoxib/química , Celecoxib/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Inhibidores de la Ciclooxigenasa 2/química , Inhibidores de la Ciclooxigenasa 2/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Etoricoxib/química , Etoricoxib/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Numerous mechanisms of resistance arise in response to treatment with second-generation androgen receptor (AR) pathway inhibitors in metastatic castration-resistant prostate cancer (mCRPC). Among these, point mutations in the ligand binding domain can transform antagonists into agonists, driving the disease through activation of AR signaling. To address this unmet need, we report the discovery of JNJ-63576253, a next-generation AR pathway inhibitor that potently abrogates AR signaling in models of human prostate adenocarcinoma. JNJ-63576253 is advancing as a clinical candidate with potential effectiveness in the subset of patients who do not respond to or are progressing while on second-generation AR-targeted therapeutics.
Asunto(s)
Antagonistas de Receptores Androgénicos/uso terapéutico , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Dominios Proteicos/genética , Antagonistas de Receptores Androgénicos/farmacología , Animales , Línea Celular Tumoral , Humanos , Ligandos , Masculino , Ratones , Modelos Moleculares , Mutación , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Persistent androgen receptor (AR) activation drives therapeutic resistance to second-generation AR pathway inhibitors and contributes to the progression of advanced prostate cancer. One resistance mechanism is point mutations in the ligand binding domain of AR that can transform antagonists into agonists. The AR F877L mutation, identified in patients treated with enzalutamide or apalutamide, confers resistance to both enzalutamide and apalutamide. Compound 4 (JNJ-pan-AR) was identified as a pan-AR antagonist with potent activity against wild-type and clinically relevant AR mutations including F877L. Metabolite identification studies revealed a latent bioactivation pathway associated with 4. Subsequent lead optimization of 4 led to amelioration of this pathway and nomination of 5 (JNJ-63576253) as a clinical stage, next-generation AR antagonist for the treatment of castration-resistant prostate cancer (CRPC).
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Antagonistas de Receptores Androgénicos/farmacología , Nitrilos/farmacología , Picolinas/farmacología , Piperidinas/farmacología , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata/tratamiento farmacológico , Piridinas/farmacología , Compuestos de Espiro/farmacología , Antagonistas de Receptores Androgénicos/farmacocinética , Antagonistas de Receptores Androgénicos/uso terapéutico , Animales , Biotransformación , Línea Celular Tumoral , Perros , Descubrimiento de Drogas , Resistencia a Antineoplásicos/genética , Hepatocitos/metabolismo , Humanos , Masculino , Modelos Moleculares , Mutación , Nitrilos/farmacocinética , Nitrilos/uso terapéutico , Picolinas/farmacocinética , Picolinas/uso terapéutico , Piperidinas/farmacocinética , Piperidinas/uso terapéutico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata Resistentes a la Castración/genética , Piridinas/farmacocinética , Piridinas/uso terapéutico , Ratas , Compuestos de Espiro/farmacocinética , Compuestos de Espiro/uso terapéutico , Relación Estructura-ActividadRESUMEN
Protein arginine methyltransferase 5 (PRMT5) is an enzyme that can symmetrically dimethylate arginine residues in histones and nonhistone proteins by using S-adenosyl methionine (SAM) as the methyl donating cofactor. We have designed a library of SAM analogues and discovered potent, cell-active, and selective spiro diamines as inhibitors of the enzymatic function of PRMT5. Crystallographic studies confirmed a very interesting binding mode, involving protein flexibility, where both the cofactor pocket and part of substrate binding site are occupied by these inhibitors.
RESUMEN
A novel class of substituted spiro[3.4]octanes can be accessed via a [2 + 2]-cycloaddition of dichloroketene on a readily prepared exo-methylene cyclopentane building block. This reaction sequence was found to be robust on a multigram scale and afforded a central spirocyclobutanone scaffold for carbocyclic nucleosides. The reactivity of this constrained building block was evaluated and compared to the corresponding 4'-spirocyclic furanose analogues. Density functional theory calculations were performed to support the observed selectivity in the carbonyl reduction of spirocyclobutanone building blocks. Starting from novel spirocyclic intermediates, we exemplified the preparation of an undescribed class of carbocyclic nucleoside analogues and provided a proof of concept for application as inhibitors for the protein methyltransferase target PRMT5.
Asunto(s)
Ciclopentanos , Nucleósidos , Reacción de CicloadiciónRESUMEN
Despite the large variety of modified nucleosides that have been reported, the preparation of constrained 4'-spirocyclic adenosine analogues has received very little attention. We discovered that the [2+2]-cycloaddition of dichloroketene on readily available 4'-exo-methylene furanose sugars efficiently results in the diastereoselective formation of novel 4'-spirocyclobutanones. The reaction mechanism was investigated via density functional theory (DFT) and found to proceed either via a non-synchronous or stepwise reaction sequence, controlled by the stereochemistry at the 3'-position of the sugar substrate. The obtained dichlorocyclobutanones were converted into nucleoside analogues, providing access to a novel class of chiral 4'-spirocyclobutyl adenosine mimetics in eight steps from commercially available sugars. Assessment of the biological activity of designed 4'-spirocyclic adenosine analogues identified potent inhibitors for protein methyltransferase target PRMT5.
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Adenosina/química , Nucleósidos/análogos & derivados , Nucleósidos/síntesis química , Carbohidratos/química , Reacción de Cicloadición , Teoría Funcional de la Densidad , Dicloroetilenos/química , Glicosilación , Metales/química , Estructura Molecular , Oxidación-Reducción , Estereoisomerismo , TermodinámicaRESUMEN
In a continuing effort to discover novel TLR agonists, herein we report on the discovery and structure-activity relationship of novel tetrahydropyridopyrimidine TLR 7/8 agonists. Optimization of this series towards dual agonist activity and a high clearance profile resulted in the identification of compound 52a1. Evaluation in vivo revealed an interferon stimulated response (ISG) in mice with limited systemic exposure and demonstrated the potential in antiviral treatment or as a vaccine adjuvant.
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Pirimidinas/farmacología , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Administración Oral , Animales , Diseño de Fármacos , Ratones , Relación Estructura-ActividadRESUMEN
A novel series of 2,4-diaminoquinazolines was identified as potent dual Toll-like receptor (TLR) 7 and 8 agonists with reduced off-target activity. The stereochemistry of the amino alcohol was found to influence the TLR7/8 selectivity with the ( R) isomer resulting in selective TLR8 agonism. Lead optimization toward a dual agonist afforded ( S)-3-((2-amino-8-fluoroquinazolin-4-yl)amino)hexanol 31 as a potent analog, being structurally different from previously described dual agonists ( McGowan J. Med. Chem. 2016 , 59 , 7936 ). Pharmacokinetic and pharmacodynamic (PK/PD) studies revealed the desired high first pass profile aimed at limiting systemic cytokine activation. In vivo pharmacodynamic studies with lead compound 31 demonstrated production of cytokines consistent with TLR7/8 activation in mice and cynomolgus monkeys and ex vivo inhibition of hepatitis B virus (HBV).
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Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Quinazolinas/farmacología , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/metabolismo , Animales , Antivirales/química , Antivirales/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Simulación del Acoplamiento Molecular , Conformación Proteica , Quinazolinas/química , Quinazolinas/metabolismo , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Receptor Toll-Like 7/química , Receptor Toll-Like 8/químicaRESUMEN
Potent, selective and broadly characterized small molecule modulators of protein function (chemical probes) are powerful research reagents. The pharmaceutical industry has generated many high-quality chemical probes and several of these have been made available to academia. However, probe-associated data and control compounds, such as inactive structurally related molecules and their associated data, are generally not accessible. The lack of data and guidance makes it difficult for researchers to decide which chemical tools to choose. Several pharmaceutical companies (AbbVie, Bayer, Boehringer Ingelheim, Janssen, MSD, Pfizer, and Takeda) have therefore entered into a pre-competitive collaboration to make available a large number of innovative high-quality probes, including all probe-associated data, control compounds and recommendations on use (
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Sondas Moleculares/metabolismo , Farmacología/métodos , Proteínas/metabolismo , Tecnología Farmacéutica/métodosRESUMEN
The purpose of this review is to provide an overview of the complexity of the epigenetic target space. Chemical modifications of histones and nucleic acids constitute a key epigenetic mechanism. Whereas modifications such as methylation and acetylation are well-known, there are many additional, less explored modifications described here. The writers, readers and erasers of such diverse modifications, which constitute a major portion of the potential epigenetic target space, are discussed, in addition to the various other protein families that do not fall under these three categories. Finally, disease relevance and druggability of epigenetic targets are discussed with concluding remarks about the richness and diversity they will provide for future targeted therapies.
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Descubrimiento de Drogas , Epigénesis Genética/efectos de los fármacos , Acetilación/efectos de los fármacos , Animales , Metilación de ADN/efectos de los fármacos , Descubrimiento de Drogas/métodos , Histonas/genética , Humanos , Procesamiento Proteico-Postraduccional/efectos de los fármacosRESUMEN
Protein families involved in chromatin-templated events are emerging as novel target classes in oncology and other disease areas. The ability to discover selective inhibitors against chromatin factors depends on the presence of structural features that are unique to the targeted sites. To evaluate challenges and opportunities toward the development of selective inhibitors, we calculated all pair wise structural distances between 575 structures from the protein databank representing 163 unique binding pockets found in protein domains that write, read or erase post-translational modifications on histones, DNA, and RNA. We find that the structural similarity of binding sites does not always follow the sequence similarity of protein domains. Our analysis reveals increased risks of activity across target-class for compounds competing with the cofactor of protein arginine methyltransferases, lysine acetyltransferases, and sirtuins, while exploiting the conformational plasticity of a protein target is a path toward selective inhibition. The structural diversity landscape of the epigenetics pocketome can be explored via an open-access graphic user interface at thesgc.org/epigenetics_pocketome.
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Antineoplásicos/química , Epigénesis Genética , Histonas/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional , Bibliotecas de Moléculas Pequeñas/química , Programas Informáticos , Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/química , Acetiltransferasas/genética , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Sitios de Unión , Cromatina/química , Cromatina/efectos de los fármacos , ADN de Neoplasias/antagonistas & inhibidores , ADN de Neoplasias/química , ADN de Neoplasias/genética , Bases de Datos de Proteínas , Histonas/química , Histonas/genética , Humanos , Internet , Ligandos , Modelos Moleculares , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neoplasias/química , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Unión Proteica , Estructura Terciaria de Proteína , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genética , ARN Neoplásico/antagonistas & inhibidores , ARN Neoplásico/química , ARN Neoplásico/genética , Sirtuinas/antagonistas & inhibidores , Sirtuinas/química , Sirtuinas/genética , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Nuclear Factor-KkappaB (NF-kappaB) is a transcription factor whose inappropriate activation may result in the development of a number of diseases including cancer, inflammation, neurodegeneration and AIDS. Recent studies on NF-kappaB mediated pathologies, made therapeutic interventions leading to its inhibition an emerging theme in pharmaceutical research. NF-kappaB resides in the cytoplasm and is activated by several time-dependent factors, leading to proteasome-dependent degradation of its inhibitory protein (IkappaB), resulting in free NF-kappaB (p50 and p65 subunits, involved in disease states), which binds to target DNA sites, further resulting in enhanced transcription of several disease associated proteins. The complex pathway of NF-kappaB, finally leading to its DNA binding, has attracted several approaches interfering with this pathway. One such approach is that of a direct covalent modification of NF-kappaB. In this article, we present a critical review on the pharmacological agents that have been studied as inhibitors of NF-kappaB by covalently modifying redox-regulated cysteine residues in its subunits, ultimately resulting in the inhibition of kappaB DNA recognition and binding. Beginning with a general overview of NF-kappaB pathway and several possibilities of chemical interventions, the significance of redox-regulation in NF-kappaB activation and DNA binding is presented. Further, protein S-thiolation, S-nitrosylation and irreversible covalent modification are described as regular biochemical events in the cell, having provided a guideline for the development of NF-kappaB inhibitors discussed further. Although just a handful of inhibitors, with most of them being alkylating agents have been studied in the present context, this approach presents potential for the development of a new class of NF-kappaB-inhibitors.
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FN-kappa B/antagonistas & inhibidores , Alquilantes/química , Alquilantes/farmacología , Cisteína/química , FN-kappa B/metabolismo , Nitrosación , Oxidación-Reducción , Procesamiento Proteico-Postraduccional , Compuestos de Sulfhidrilo/químicaRESUMEN
TWINKLE is a DNA helicase needed for mitochondrial DNA replication. In lower eukaryotes the protein also harbors a primase activity, which is lost from TWINKLE encoded by mammalian cells. Mutations in TWINKLE underlie autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with multiple deletions in the mtDNA. Four different adPEO-causing mutations (W315L, K319T, R334Q, and P335L) are located in the N-terminal domain of TWINKLE. The mutations cause a dramatic decrease in ATPase activity, which is partially overcome in the presence of single-stranded DNA. The mutated proteins have defects in DNA helicase activity and cannot support normal levels of DNA replication. To explain the phenotypes, we use a molecular model of TWINKLE based on sequence similarities with the phage T7 gene 4 protein. The four adPEO-causing mutations are located in a region required to bind single-stranded DNA. These mutations may therefore impair an essential element of the catalytic cycle in hexameric helicases, i.e. the interplay between single-stranded DNA binding and ATP hydrolysis.