RESUMEN
Many reagents have emerged to study the function of specific enzymes in vitro. On the other hand, target specific reagents are scarce or need improvement, allowing investigations of the function of individual enzymes in their native cellular context. Here we report the development of a target-selective fluorescent small-molecule activity-based DUB probe that is active in live cells and an in vivo animal model. The probe labels active ubiquitin carboxy-terminal hydrolase L1 (UCHL1), also known as neuron-specific protein PGP9.5 (PGP9.5) and Parkinson disease 5 (PARK5), a DUB active in neurons that constitutes 1 to 2% of the total brain protein. UCHL1 variants have been linked with neurodegenerative disorders Parkinson's and Alzheimer's diseases. In addition, high levels of UCHL1 also correlate often with cancer and especially metastasis. The function of UCHL1 activity or its role in cancer and neurodegenerative disease is poorly understood and few UCHL1-specific activity tools exist. We show that the reagents reported here are specific to UCHL1 over all other DUBs detectable by competitive activity-based protein profiling and by mass spectrometry. Our cell-penetrable probe, which contains a cyanimide reactive moiety, binds to the active-site cysteine residue of UCHL1 in an activity-dependent manner. Its use is demonstrated by the fluorescent labeling of active UCHL1 both in vitro and in live cells. We furthermore show that this probe can selectively and spatiotemporally report UCHL1 activity during the development of zebrafish embryos. Our results indicate that our probe has potential applications as a diagnostic tool for diseases with perturbed UCHL1 activity.
Asunto(s)
Colorantes Fluorescentes/química , Bibliotecas de Moléculas Pequeñas/química , Ubiquitina Tiolesterasa/análisis , Ubiquitina Tiolesterasa/metabolismo , Proteínas de Pez Cebra/análisis , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Supervivencia Celular , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/farmacología , Células HEK293 , Humanos , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Proteínas de Pez Cebra/antagonistas & inhibidoresRESUMEN
Bioorthogonal chemistry allows the selective modification of biomolecules in complex biological samples. One application of this methodology is in two-step activity-based protein profiling (ABPP), a methodology that is particularly attractive where direct ABPP using fluorescent or biotinylated probes is ineffective. Herein we describe a set of norbornene-modified, mechanism-based proteasome inhibitors aimed to be selective for each of the six catalytic sites of human constitutive proteasomes and immunoproteasomes. The probes developed for ß1i, ß2i, ß5c, and ß5i proved to be useful two-step ABPs that effectively label their developed proteasome subunits in both Raji cell extracts and living Raji cells through inverse-electron-demand Diels-Alder (iEDDA) ligation. The compound developed for ß1c proved incapable of penetrating the cell membrane, but effectively labels ß1c in vitro. The compound developed for ß2c proved not selective, but its azide-containing analogue LU-002c proved effective in labeling of ß2c via azide-alkyne click ligation chemistry both in vitro and inâ situ. In total, our results contribute to the growing list of proteasome activity tools to include five subunit-selective activity-based proteasome probes, four of which report on proteasome activities in living cells.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Alquinos/química , Alquinos/farmacología , Azidas/química , Azidas/farmacología , Dominio Catalítico/efectos de los fármacos , Línea Celular Tumoral , Humanos , Estructura Molecular , Inhibidores de Proteasoma/síntesis química , Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/farmacologíaRESUMEN
The linear synthesis of the N-terminal domain of mISG15 has been developed which enables the synthesis of full-length mISG15 and the activity-based probe Rho-mISG15-PA via native chemical ligation. Pilot experiments showed that the synthetic proteins were properly folded and recognized by endogenous enzymes. Our synthesis strategy allows the generation of other mISG15-based probes and reagents that can accelerate the research in this field.
Asunto(s)
Citocinas/síntesis química , Citocinas/metabolismo , Técnicas de Síntesis en Fase Sólida/métodos , Secuencia de Aminoácidos , Colorantes Fluorescentes/química , Interleucinas/metabolismo , Ligandos , Peso Molecular , Unión Proteica , Dominios Proteicos , Rodaminas/química , Relación Estructura-Actividad , Ubiquitinas/síntesis química , Ubiquitinas/metabolismoRESUMEN
Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality that might be present. Recent developments in the field include orthogonal bioorthogonal reactions to modify multiple biomolecules simultaneously. During our research, we observed that the reaction rates for the bioorthogonal inverse-electron-demand Diels-Alder (iEDDA) reactions between nonstrained vinylboronic acids (VBAs) and dipyridyl-s-tetrazines were exceptionally higher than those between VBAs and tetrazines bearing a methyl or phenyl substituent. As VBAs are mild Lewis acids, we hypothesised that coordination of the pyridyl nitrogen atom to the boronic acid promoted tetrazine ligation. Herein, we explore the molecular basis and scope of VBA-tetrazine ligation in more detail and benefit from its unique reactivity in the simultaneous orthogonal tetrazine labelling of two proteins modified with VBA and norbornene, a widely used strained alkene. We further show that the two orthogonal iEDDA reactions can be performed in living cells by labelling the proteasome by using a nonselective probe equipped with a VBA and a subunit-selective VBA bearing a norbornene moiety.
Asunto(s)
Alquenos/química , Ácidos Borónicos/química , Reacción de Cicloadición/métodos , Norbornanos/química , Proteínas/química , Compuestos de Vinilo/química , Alquenos/síntesis química , Ácidos Borónicos/síntesis química , Proteínas Fluorescentes Verdes/síntesis química , Proteínas Fluorescentes Verdes/química , Compuestos Heterocíclicos con 1 Anillo/síntesis química , Compuestos Heterocíclicos con 1 Anillo/química , Humanos , Norbornanos/síntesis química , Proteínas/síntesis química , Albúmina Sérica Humana/síntesis química , Albúmina Sérica Humana/química , Compuestos de Vinilo/síntesis químicaRESUMEN
The development of γ-thionorleucine (ThioNle) as a handle for native chemical ligation-desulfurization is reported here. ThioNle is a new addition to the expanding thiolated amino acid toolbox and serves as a methionine substitute in NCL with the advantage that it lacks the undesirable oxidation-prone thioether moiety. Its usefulness for N-terminal ubiquitination is demonstrated by efficient preparation of fully synthetic linear diubiquitin with preserved protein folding compared to the expressed material. Interestingly, gel-based deubiquitinating assays revealed that the methionine to norleucine substitution did affect diubiquitin cleavage, which may indicate a more profound role for methionine in the interaction between ubiquitin and the deubiquitinating enzymes than has been known so far.
Asunto(s)
Metionina/química , Norleucina/química , Ubiquitinas/química , Ubiquitinas/metabolismoRESUMEN
Most mammalian tissues contain a single proteasome species: constitutive proteasomes. Tissues able to express, next to the constitutive proteasome catalytic activities (ß1c, ß2c, ß5c), the three homologous activities, ß1i, ß2i and ß5i, may contain numerous distinct proteasome particles: immunoproteasomes (composed of ß1i, ß2i and ß5i) and mixed proteasomes containing a mix of these activities. This work describes the development of new subunit-selective activity-based probes and their use in an activity-based protein profiling assay that allows the detection of various proteasome particles. Tissue extracts are treated with subunit-specific probes bearing distinct fluorophores and subunit-specific inhibitors. The samples are resolved by native polyacrylamide gel electrophoresis, after which fluorescence-resonance energy transfer (FRET) reports on the nature of proteasomes present.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Electroforesis en Gel de Poliacrilamida Nativa , Complejo de la Endopetidasa Proteasomal/química , Sitios de Unión , Catálisis , Citoplasma/metabolismo , Colorantes Fluorescentes/química , Células HeLa , Humanos , Inhibidores de Proteasoma/farmacología , Estructura Secundaria de ProteínaRESUMEN
Idiopathic inflammatory myopathies (IIMs) are diseases with muscle weakness, morphologically characterized by inflammatory infiltration and increased expression of MHC class I molecule on myofibers. Immunoproteasome, as a proteolytic complex that shapes the repertoire of antigenic peptides, has been previously demonstrated to be over-expressed in IIMs at mRNA level. In this study, we investigated the expression and the function of the immunoproteasome in IIMs in more detail. As shown by immunofluorescence staining, expression of relevant players of the immunoproteasome was detectable in the inflamed skeletal muscle tissue from IIM patients. In fact, two subunits of the immunoproteasome, ß1i or ß5i were upregulated in sporadic inclusion body myositis, immune-mediated necrotizing myopathies and dermatomyositis muscle biopsies and co-localized with the MHC class I expressing myofibers. Double immunofluorescence revealed that both myofibers and muscle infiltrating cells, including CD8+ T-cells and CD68 + macrophages in IIMs expressed ß1i or ß5i. In addition, we have also investigated the role of the immunoproteasome in myoblasts during in vitro inflammatory conditions. Using human primary myoblasts cultures we found that pro-inflammatory cytokines, TNF-α or IFN-γ upregulate ß1i or ß5i. Selective inhibition or depletion of ß5i amplified the TNF-α or IFN-γ mediated expression of cytokines/chemokines (myokines) in myoblasts. Furthermore, we demonstrated that specific inhibitors of ß1i or ß5i reduced the cell surface expression of MHC class I in myoblasts induced by IFN-γ. Taken together, our data suggest that the immunoproteasome is involved in pathologic MHC class I expression and maintenance of myokine production in IIMs. Thus, induction of the immunoproteasome was identified as a pathomechanism underlying inflammation in IIMs.
Asunto(s)
Citocinas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Músculo Esquelético/inmunología , Miositis/inmunología , Complejo de la Endopetidasa Proteasomal/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Células Cultivadas , Preescolar , Citocinas/genética , Citocinas/metabolismo , Dermatomiositis/genética , Dermatomiositis/inmunología , Dermatomiositis/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Interferón gamma/farmacología , Masculino , Microscopía Fluorescente , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mioblastos/efectos de los fármacos , Mioblastos/inmunología , Mioblastos/metabolismo , Miositis/genética , Miositis/metabolismo , Pancreatitis Aguda Necrotizante/genética , Pancreatitis Aguda Necrotizante/inmunología , Pancreatitis Aguda Necrotizante/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/inmunología , Subunidades de Proteína/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/farmacología , Adulto JovenRESUMEN
Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) is a promising therapeutic target for the treatment of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL). Several research groups have reported on the development of Malt1 inhibitors and activity-based probes for in vitro and in situ monitoring and modulating Malt1 activity. In this paper, we report on two activity-based Malt1 probes (6 and 7) and a focused library of 19 new Malt1 inhibitors. Our peptide-based probe 6 labels Malt1 in an activity-based manner. In contrast, probe 7, derived from the known covalent inhibitor MI-2, labels both wild type and catalytically inactive Cys to Ala mutant Malt1, suggesting that MI-2 inhibits Malt1 by reacting with a nucleophilic residue other than the active site cysteine. Furthermore, two of our inhibitors (9, apparent IC50 3.0µM, and 13, apparent IC50 2.1µM) show good inhibitory activity against Malt1 and outperform MI-2 (apparent IC50 7.8µM) in our competitive activity-based protein profiling assay.
Asunto(s)
Inhibidores de Caspasas/síntesis química , Inhibidores de Caspasas/farmacología , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Carbamatos/síntesis química , Carbamatos/química , Carbamatos/farmacología , Inhibidores de Caspasas/química , Caspasas/metabolismo , Línea Celular Tumoral , Descubrimiento de Drogas/métodos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Células Jurkat , Linfoma de Células B Grandes Difuso/enzimología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/metabolismo , Triazoles/síntesis química , Triazoles/química , Triazoles/farmacologíaRESUMEN
Proteasomes are therapeutic targets for various cancers and autoimmune diseases. Constitutively expressed proteasomes have three active sites, ß1c, ß2c, and ß5c. Lymphoid tissues also express the immunoproteasome subunits ß1i, ß2i, and ß5i. Rapid and simultaneous measurement of the activity of these catalytic subunits would assist in the discovery of new inhibitors, improve analysis of proteasome inhibitors in clinical trials, and simplify analysis of subunit expression. In this work, we present a cocktail of activity-based probes that enables simultaneous gel-based detection of all six catalytic human proteasome subunits. We used this cocktail to develop specific inhibitors for ß1c, ß2c, ß5c, and ß2i, to compare the active-site specificity of clinical proteasome inhibitors, and to demonstrate that many hematologic malignancies predominantly express immunoproteasomes. Furthermore, we show that selective and complete inhibition of ß5i and ß1i is cytotoxic to primary cells from acute lymphocytic leukemia (ALL) patients.
Asunto(s)
Sondas Moleculares/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Dominio Catalítico , HumanosRESUMEN
Peptide epoxyketones are potent and selective proteasome inhibitors. Selectivity is governed by the epoxyketone dual electrophilic warhead, which reacts with the N-terminal threonine 1,2-amino alcohol uniquely present in proteasome active sites. We studied a series of C-terminally modified oligopeptides featuring adjacent electrophiles based on the epoxyketone warhead. We found that the carbonyl moiety in the natural warhead is essential, but that the adjacent epoxide can be replaced by a carbonyl, though with considerable loss of activity.
Asunto(s)
Electrones , Compuestos Epoxi/química , Péptidos/química , Inhibidores de Proteasoma/química , Carbonilación Proteica , Células HEK293 , Humanos , Oligopéptidos/químicaRESUMEN
Proteasome inhibitors (PIs) are a backbone of multiple myeloma (MM) therapy. The proteasome harbors six proteolytically active subunits (ß1, ß2, ß5), while ß5 was identified as rate-limiting and is a primary target of clinically available PIs. The most effective pattern of subunit inhibition provided by these PIs for cytotoxic activity in MM is unknown. A head-to-head comparison of clinically available PIs shows that in the clinically relevant setting only the co-inhibition of ß1 or ß2 with ß5 activity achieves meaningful functional proteasome inhibition and cytotoxicity, while the selective ß2/ß5 inhibition of both constitutive and immunoproteasome is the most cytotoxic. In the long-term setting, selective inhibition of ß5 subunit is sufficient to induce cytotoxicity in PI-sensitive, but not in PI-resistant MM, and the ß5/ß2 co-inhibition is the most cytotoxic in PI-resistant MM. These results give a rational basis for selecting individual PIs for the treatment of MM.
Asunto(s)
Antineoplásicos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/metabolismo , Anciano , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Bortezomib/metabolismo , Bortezomib/farmacología , Bortezomib/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Complejo de la Endopetidasa Proteasomal/química , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , Distribución Tisular , Células Tumorales CultivadasRESUMEN
Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal relevance of individual proteasome active sites. Whereas the inhibitors for the ß1c, ß1i, ß5c, and ß5i subunits exploit the differences in the substrate-binding channels identified by X-ray crystallography, compounds selectively targeting ß2c or ß2i could not yet be rationally designed because of the high structural similarity of these two subunits. Here, we report the development, chemical synthesis, and biological screening of a compound library that led to the identification of the ß2c- and ß2i-selective compounds LU-002c (4; IC50 ß2c: 8 nM, IC50 ß2i/ß2c: 40-fold) and LU-002i (5; IC50 ß2i: 220 nM, IC50 ß2c/ß2i: 45-fold), respectively. Co-crystal structures with ß2 humanized yeast proteasomes visualize protein-ligand interactions crucial for subunit specificity. Altogether, organic syntheses, activity-based protein profiling, yeast mutagenesis, and structural biology allowed us to decipher significant differences of ß2 substrate-binding channels and to complete the set of subunit-selective proteasome inhibitors.
Asunto(s)
Oligopéptidos/farmacología , Inhibidores de Proteasoma/farmacología , Subunidades de Proteína/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Dominio Catalítico , Línea Celular Tumoral , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Ratones , Mutación , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Biblioteca de Péptidos , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/síntesis química , Inhibidores de Proteasoma/metabolismo , Unión Proteica , Ingeniería de Proteínas , Subunidades de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/metabolismo , EstereoisomerismoRESUMEN
This work reports the development of highly potent and selective inhibitors of the ß5c catalytic activity of human constitutive proteasomes. The work describes the design principles, large hydrophobic P3 residue and small hydrophobic P1 residue, that led to the synthesis of a panel of peptide epoxyketones; their evaluation and the selection of the most promising compounds for further analyses. Structure-activity relationships detail how in a logical order the ß1c/i, ß2c/i, and ß5i activities became resistant to inhibition as compounds were diversified stepwise. The most effective compounds were obtained as a mixture of cis- and trans-biscyclohexyl isomers, and enantioselective synthesis resolved this issue. Studies on yeast proteasome structures complexed with some of the compounds provide a rationale for the potency and specificity. Substitution of the N-terminus in the most potent compound for a more soluble equivalent led to a cell-permeable molecule that selectively and efficiently blocks ß5c in cells expressing both constitutive proteasomes and immunoproteasomes.
Asunto(s)
Diseño de Fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasoma/síntesis química , Inhibidores de Proteasoma/química , Relación Estructura-ActividadRESUMEN
A series of 2-(1H-imidazol-2-yl) pyridine derivatives (CLW01-CLW31) have been designed and synthesized, and they were screened for BRAF kinase inhibitory activity. Besides, their biological activities were evaluated in vitro and in vivo. All the compounds were reported for the first time, and compounds CLW14 and CLW27 displayed the most potent antiproliferative activity against cell line A375 in vitro, with IC50 values of 4.26 and 2.93 µM, respectively, which were comparable with the positive control Sorafenib. Those two compounds were further evaluated for the in vivo efficacy using an A375 xenograft nude mice model. The results showed that the growth of A375 cancer cells xenografts was suppressed by factors of 35.68% and 42.50% (percent tumor growth inhibition values) after intragastric (ig) administration of compound CLW14 and CLW27 at concentration of 50 mg/kg. Thus they may be promising lead compounds to be developed as an alternative for current Sorafenib therapy.
Asunto(s)
Antineoplásicos/farmacología , Diseño de Fármacos , Imidazoles/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Piridinas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células Hep G2 , Humanos , Imidazoles/síntesis química , Imidazoles/química , Células MCF-7 , Ratones , Ratones Desnudos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Neoplasias Experimentales/patología , Niacinamida/síntesis química , Niacinamida/química , Niacinamida/farmacología , Compuestos de Fenilurea/síntesis química , Compuestos de Fenilurea/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas B-raf/metabolismo , Piridinas/síntesis química , Piridinas/química , Sorafenib , Relación Estructura-ActividadRESUMEN
Mammalian genomes encode seven catalytic proteasome subunits, namely, ß1c, ß2c, ß5c (assembled into constitutive 20S proteasome core particles), ß1i, ß2i, ß5i (incorporated into immunoproteasomes), and the thymoproteasome-specific subunit ß5t. Extensive research in the past decades has yielded numerous potent proteasome inhibitors including compounds currently used in the clinic to treat multiple myeloma and mantle cell lymphoma. Proteasome inhibitors that selectively target combinations of ß1c/ß1i, ß2c/ß2i, or ß5c/ß5i are available, yet ligands truly selective for a single proteasome activity are scarce. In this work we report the development of cell-permeable ß1i and ß5i selective inhibitors that outperform existing leads in terms of selectivity and/or potency. These compounds are the result of a rational design strategy using known inhibitors as starting points and introducing structural features according to the X-ray structures of the murine constitutive and immunoproteasome 20S core particles.
Asunto(s)
Diseño de Fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Línea Celular , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Complejo de la Endopetidasa Proteasomal/química , Inhibidores de Proteasoma/síntesis química , Inhibidores de Proteasoma/química , Relación Estructura-ActividadRESUMEN
A three-dimensional (3D) quantitative pharmacophore model was developed from a training set of structurally diverse substituted ureas against Raf-1 kinase, which was well validated to be highly predictive by two methods, namely, test set prediction and Cat-Scramble method. Then a virtual database searching was performed with the pharmacophore model as a 3D query, and the bioactivities of the retrieved hits were predicted by the pharmacophore. Subsequently, molecular docking was carried out on the selected hits whose estimated IC(50) was less than 1 microM. Finally, 29 hits were identified as potential leads against Raf-1 kinase, which exhibited good estimated activities, high docking scores, similar binding mode to experimentally proven compounds and favorable drug-like properties. The study may facilitate the discovery and rational design of novel leads with potent inhibitory activity targeting Raf-1 kinase.