RESUMEN
BACKGROUND: Persons with toxic gain-of-function variants in the gene encoding apolipoprotein L1 (APOL1) are at greater risk for the development of rapidly progressive, proteinuric nephropathy. Despite the known genetic cause, therapies targeting proteinuric kidney disease in persons with two APOL1 variants (G1 or G2) are lacking. METHODS: We used tetracycline-inducible APOL1 human embryonic kidney (HEK293) cells to assess the ability of a small-molecule compound, inaxaplin, to inhibit APOL1 channel function. An APOL1 G2-homologous transgenic mouse model of proteinuric kidney disease was used to assess inaxaplin treatment for proteinuria. We then conducted a single-group, open-label, phase 2a clinical study in which inaxaplin was administered to participants who had two APOL1 variants, biopsy-proven focal segmental glomerulosclerosis, and proteinuria (urinary protein-to-creatinine ratio of ≥0.7 to <10 [with protein and creatinine both measured in grams] and an estimated glomerular filtration rate of ≥27 ml per minute per 1.73 m2 of body-surface area). Participants received inaxaplin daily for 13 weeks (15 mg for 2 weeks and 45 mg for 11 weeks) along with standard care. The primary outcome was the percent change from the baseline urinary protein-to-creatinine ratio at week 13 in participants who had at least 80% adherence to inaxaplin therapy. Safety was also assessed. RESULTS: In preclinical studies, inaxaplin selectively inhibited APOL1 channel function in vitro and reduced proteinuria in the mouse model. Sixteen participants were enrolled in the phase 2a study. Among the 13 participants who were treated with inaxaplin and met the adherence threshold, the mean change from the baseline urinary protein-to-creatinine ratio at week 13 was -47.6% (95% confidence interval, -60.0 to -31.3). In an analysis that included all the participants regardless of adherence to inaxaplin therapy, reductions similar to those in the primary analysis were observed in all but 1 participant. Adverse events were mild or moderate in severity; none led to study discontinuation. CONCLUSIONS: Targeted inhibition of APOL1 channel function with inaxaplin reduced proteinuria in participants with two APOL1 variants and focal segmental glomerulosclerosis. (Funded by Vertex Pharmaceuticals; VX19-147-101 ClinicalTrials.gov number, NCT04340362.).
Asunto(s)
Apolipoproteína L1 , Glomeruloesclerosis Focal y Segmentaria , Proteinuria , Animales , Humanos , Ratones , Apolipoproteína L1/antagonistas & inhibidores , Apolipoproteína L1/genética , Apolipoproteínas/genética , Negro o Afroamericano , Creatinina/orina , Mutación con Ganancia de Función , Predisposición Genética a la Enfermedad , Glomeruloesclerosis Focal y Segmentaria/tratamiento farmacológico , Glomeruloesclerosis Focal y Segmentaria/genética , Células HEK293 , Proteinuria/tratamiento farmacológico , Proteinuria/genéticaRESUMEN
In our efforts to identify novel small molecule inhibitors for the treatment of adrenoleukodystrophy (ALD), we conducted a high-throughput radiometric screen for inhibitors of elongation of very long chain fatty acid 1 (ELOVL1) enzyme. We developed a series of highly potent, central nervous system (CNS)-penetrant pyrimidine ether-based compounds with favorable pharmacokinetics culminating in compound 22. Compound 22 is a selective inhibitor of ELOVL1, reducing C26:0 VLCFA synthesis in ALD patient fibroblasts and lymphocytes in vitro. Compound 22 reduced C26:0 lysophosphatidyl choline (LPC), a subtype of VLCFA, in the blood of ATP binding cassette transporter D1 (ABCD1) KO mice, a murine model of ALD to near wild-type levels. Compound 22 is a low-molecular-weight, potent ELOVL1 inhibitor that may serve as a useful tool for exploring therapeutic approaches to the treatment of ALD.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Elongasas de Ácidos Grasos/antagonistas & inhibidores , Pirimidinas/farmacología , Administración Oral , Adrenoleucodistrofia/tratamiento farmacológico , Animales , Disponibilidad Biológica , Perros , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacocinética , Éteres/química , Células HEK293 , Humanos , Macaca fascicularis , Ratones , Pirimidinas/administración & dosificación , Pirimidinas/farmacocinética , RatasRESUMEN
Accumulation of very long chain fatty acids (VLCFAs) due to defects in ATP binding cassette protein D1 (ABCD1) is thought to underlie the pathologies observed in adrenoleukodystrophy (ALD). Pursuing a substrate reduction approach based on the inhibition of elongation of very long chain fatty acid 1 enzyme (ELOVL1), we explored a series of thiazole amides that evolved into compound 27âa highly potent, central nervous system (CNS)-penetrant compound with favorable in vivo pharmacokinetics. Compound 27 selectively inhibits ELOVL1, reducing C26:0 VLCFA synthesis in ALD patient fibroblasts, lymphocytes, and microglia. In mouse models of ALD, compound 27 treatment reduced C26:0 VLCFA concentrations to near-wild-type levels in blood and up to 65% in the brain, a disease-relevant tissue. Preclinical safety findings in the skin, eye, and CNS precluded progression; the origin and relevance of these findings require further study. ELOVL1 inhibition is an effective approach for normalizing VLCFAs in models of ALD.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Elongasas de Ácidos Grasos/administración & dosificación , Pirazoles/farmacología , Adrenoleucodistrofia/tratamiento farmacológico , Adrenoleucodistrofia/patología , Amidas/química , Animales , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/uso terapéutico , Humanos , Pirazoles/química , Pirazoles/farmacocinética , Pirazoles/uso terapéutico , Relación Estructura-ActividadRESUMEN
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 (
Asunto(s)
Sondas Moleculares/metabolismo , Farmacología/métodos , Proteínas/metabolismo , Tecnología Farmacéutica/métodosRESUMEN
Biochemical screening is a major source of lead generation for novel targets. However, during the process of small molecule lead optimization, compounds with excellent biochemical activity may show poor cellular potency, making structure-activity relationships difficult to decipher. This may be due to low membrane permeability of the molecule, resulting in insufficient intracellular drug concentration. The Cell Squeeze platform increases permeability regardless of compound structure by mechanically disrupting the membrane, which can overcome permeability limitations and bridge the gap between biochemical and cellular studies. In this study, we show that poorly permeable Janus kinase (JAK) inhibitors are delivered into primary cells using Cell Squeeze, inhibiting up to 90% of the JAK pathway, while incubation of JAK inhibitors with or without electroporation had no significant effect. We believe this robust intracellular delivery approach could enable more effective lead optimization and deepen our understanding of target engagement by small molecules and functional probes.
Asunto(s)
Inhibidores de las Cinasas Janus/farmacología , Quinasas Janus/metabolismo , Dispositivos Laboratorio en un Chip , Leucocitos Mononucleares/efectos de los fármacos , Membrana Celular , Células Cultivadas , Humanos , Inhibidores de las Cinasas Janus/química , Leucocitos Mononucleares/fisiología , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The allure of phenotypic screening, combined with the industry preference for target-based approaches, has prompted the development of innovative chemical biology technologies that facilitate the identification of new therapeutic targets for accelerated drug discovery. A chemogenomic library is a collection of selective small-molecule pharmacological agents, and a hit from such a set in a phenotypic screen suggests that the annotated target or targets of that pharmacological agent may be involved in perturbing the observable phenotype. In this Review, we describe opportunities for chemogenomic screening to considerably expedite the conversion of phenotypic screening projects into target-based drug discovery approaches. Other applications are explored, including drug repositioning, predictive toxicology and the discovery of novel pharmacological modalities.
Asunto(s)
Descubrimiento de Drogas/métodos , Terapia Molecular Dirigida/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Diseño de Fármacos , Reposicionamiento de Medicamentos/métodos , Humanos , Fenotipo , Bibliotecas de Moléculas Pequeñas/toxicidadRESUMEN
Interleukin-17A (IL-17A) is a principal driver of multiple inflammatory and immune disorders. Antibodies that neutralize IL-17A or its receptor (IL-17RA) deliver efficacy in autoimmune diseases, but no small-molecule IL-17A antagonists have yet progressed into clinical trials. Investigation of a series of linear peptide ligands to IL-17A and characterization of their binding site has enabled the design of novel macrocyclic ligands that are themselves potent IL-17A antagonists.
Asunto(s)
Interleucina-17/antagonistas & inhibidores , Interleucina-17/química , Péptidos Cíclicos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Algoritmos , Sitios de Unión , Células Cultivadas , Diseño de Fármacos , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Simulación de Dinámica Molecular , Péptidos Cíclicos/química , Unión Proteica , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
The acetyl post-translational modification of chromatin at selected histone lysine residues is interpreted by an acetyl-lysine specific interaction with bromodomain reader modules. Here we report the discovery of the potent, acetyl-lysine-competitive, and cell active inhibitor PFI-3 that binds to certain family VIII bromodomains while displaying significant, broader bromodomain family selectivity. The high specificity of PFI-3 for family VIII was achieved through a novel bromodomain binding mode of a phenolic headgroup that led to the unusual displacement of water molecules that are generally retained by most other bromodomain inhibitors reported to date. The medicinal chemistry program that led to PFI-3 from an initial fragment screening hit is described in detail, and additional analogues with differing family VIII bromodomain selectivity profiles are also reported. We also describe the full pharmacological characterization of PFI-3 as a chemical probe, along with phenotypic data on adipocyte and myoblast cell differentiation assays.
Asunto(s)
Compuestos de Azabiciclo/farmacología , Sondas Moleculares/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Piridinas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Compuestos de Azabiciclo/síntesis química , Compuestos de Azabiciclo/química , Cristalografía por Rayos X , Proteínas de Unión al ADN , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Sondas Moleculares/síntesis química , Sondas Moleculares/química , Estructura Molecular , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Piridinas/síntesis química , Piridinas/química , Relación Estructura-Actividad , Especificidad por Sustrato , Factores de Transcripción/metabolismoRESUMEN
Mammalian SWI/SNF [also called Brg/Brahma-associated factors (BAFs)] are evolutionarily conserved chromatin-remodeling complexes regulating gene transcription programs during development and stem cell differentiation. BAF complexes contain an ATP (adenosine 5'-triphosphate)-driven remodeling enzyme (either BRG1 or BRM) and multiple protein interaction domains including bromodomains, an evolutionary conserved acetyl lysine-dependent protein interaction motif that recruits transcriptional regulators to acetylated chromatin. We report a potent and cell active protein interaction inhibitor, PFI-3, that selectively binds to essential BAF bromodomains. The high specificity of PFI-3 was achieved on the basis of a novel binding mode of a salicylic acid head group that led to the replacement of water molecules typically maintained in other bromodomain inhibitor complexes. We show that exposure of embryonic stem cells to PFI-3 led to deprivation of stemness and deregulated lineage specification. Furthermore, differentiation of trophoblast stem cells in the presence of PFI-3 was markedly enhanced. The data present a key function of BAF bromodomains in stem cell maintenance and differentiation, introducing a novel versatile chemical probe for studies on acetylation-dependent cellular processes controlled by BAF remodeling complexes.
Asunto(s)
Investigación Biomédica/métodos , Difusión de la Información/ética , Sondas Moleculares/química , Bibliotecas de Moléculas Pequeñas/química , Investigación Biomédica/instrumentación , Humanos , Propiedad Intelectual , Internet , Sondas Moleculares/farmacología , Peso Molecular , Sensibilidad y Especificidad , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Anisole and fluoroanisoles display distinct conformational preferences, as evident from a survey of their crystal structures. In addition to altering the free ligand conformation, various degrees of fluorination have a strong impact on physicochemical and pharmacokinetic properties. Analysis of anisole and fluoroanisole matched molecular pairs in the Pfizer corporate database reveals interesting trends: 1) PhOCF3 increases log D by ~1 log unit over PhOCH3 compounds; 2) PhOCF3 shows lower passive permeability despite its higher lipophilicity; and 3) PhOCF3 does not appreciably improve metabolic stability over PhOCH3 . Emerging from the investigation, difluoroanisole (PhOCF2 H) strikes a better balance of properties with noticeable advantages of log D and transcellular permeability over PhOCF3 . Synthetic assessment illustrates that the routes to access difluoroanisoles are often more straightforward than those for trifluoroanisoles. Whereas replacing PhOCH3 with PhOCF3 is a common tactic to optimize ADME properties, our analysis suggests PhOCF2 H may be a more attractive alternative, and greater exploitation of this motif is recommended.
Asunto(s)
Anisoles/química , Diseño de Fármacos , Flúor/química , Animales , Anisoles/metabolismo , Anisoles/farmacocinética , Línea Celular , Perros , Flúor/metabolismo , Flúor/farmacocinética , Halogenación , Humanos , Ligandos , Microsomas Hepáticos/metabolismo , PermeabilidadRESUMEN
The Structural Genomics Consortium (SGC) and its clinical, industry and disease-foundation partners are launching open-source preclinical translational medicine studies.
Asunto(s)
Línea Celular , Evaluación Preclínica de Medicamentos/métodos , Cultivo Primario de Células , Humanos , Pacientes , Asociación entre el Sector Público-Privado , Investigación Biomédica TraslacionalRESUMEN
SET domain containing (lysine methyltransferase) 7 (SETD7) is implicated in multiple signaling and disease related pathways with a broad diversity of reported substrates. Here, we report the discovery of (R)-PFI-2-a first-in-class, potent (Ki (app) = 0.33 nM), selective, and cell-active inhibitor of the methyltransferase activity of human SETD7-and its 500-fold less active enantiomer, (S)-PFI-2. (R)-PFI-2 exhibits an unusual cofactor-dependent and substrate-competitive inhibitory mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S-adenosylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition for binding to endogenous SETD7 in MCF7 cells pretreated with (R)-PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocopied the effects of Setd7 deficiency on Hippo pathway signaling, via modulation of the transcriptional coactivator Yes-associated protein (YAP) and regulation of YAP target genes. In confluent MCF7 cells, (R)-PFI-2 rapidly altered YAP localization, suggesting continuous and dynamic regulation of YAP by the methyltransferase activity of SETD7. These data establish (R)-PFI-2 and related compounds as a valuable tool-kit for the study of the diverse roles of SETD7 in cells and further validate protein methyltransferases as a druggable target class.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Epigénesis Genética/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/metabolismo , Pirrolidinas/farmacología , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Tetrahidroisoquinolinas/farmacología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Fibroblastos/efectos de los fármacos , Vía de Señalización Hippo , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Células MCF-7 , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/metabolismo , Mutación , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Pirrolidinas/química , Relación Estructura-Actividad , Sulfonamidas/química , Tetrahidroisoquinolinas/química , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
Small-molecule inhibitors that target bromodomains outside of the bromodomain and extra-terminal (BET) sub-family are lacking. Here, we describe highly potent and selective ligands for the bromodomain module of the human lysine acetyl transferase CBP/p300, developed from a series of 5-isoxazolyl-benzimidazoles. Our starting point was a fragment hit, which was optimized into a more potent and selective lead using parallel synthesis employing Suzuki couplings, benzimidazole-forming reactions, and reductive aminations. The selectivity of the lead compound against other bromodomain family members was investigated using a thermal stability assay, which revealed some inhibition of the structurally related BET family members. To address the BET selectivity issue, X-ray crystal structures of the lead compound bound to the CREB binding protein (CBP) and the first bromodomain of BRD4 (BRD4(1)) were used to guide the design of more selective compounds. The crystal structures obtained revealed two distinct binding modes. By varying the aryl substitution pattern and developing conformationally constrained analogues, selectivity for CBP over BRD4(1) was increased. The optimized compound is highly potent (Kd = 21 nM) and selective, displaying 40-fold selectivity over BRD4(1). Cellular activity was demonstrated using fluorescence recovery after photo-bleaching (FRAP) and a p53 reporter assay. The optimized compounds are cell-active and have nanomolar affinity for CBP/p300; therefore, they should be useful in studies investigating the biological roles of CBP and p300 and to validate the CBP and p300 bromodomains as therapeutic targets.
Asunto(s)
Proteína de Unión a CREB/química , Proteína p300 Asociada a E1A/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Sitios de Unión , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Técnicas de Química Sintética , Cristalografía por Rayos X , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Proteína p300 Asociada a E1A/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Genes p53 , Células HeLa/efectos de los fármacos , Humanos , Indoles/química , Isoxazoles/química , Ligandos , Microsomas Hepáticos/efectos de los fármacos , Modelos Moleculares , Estructura Molecular , Estructura Terciaria de Proteína , Bibliotecas de Moléculas Pequeñas/metabolismo , Relación Estructura-ActividadRESUMEN
Bromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) are transcriptional regulators required for efficient expression of several growth promoting and antiapoptotic genes as well as for cell-cycle progression. BET proteins are recruited on transcriptionally active chromatin via their two N-terminal bromodomains (BRD), a protein interaction module that specifically recognizes acetylated lysine residues in histones H3 and H4. Inhibition of the BET-histone interaction results in transcriptional downregulation of a number of oncogenes, providing a novel pharmacologic strategy for the treatment of cancer. Here, we present a potent and highly selective dihydroquinazoline-2-one inhibitor, PFI-1, which efficiently blocks the interaction of BET BRDs with acetylated histone tails. Cocrystal structures showed that PFI-1 acts as an acetyl-lysine (Kac) mimetic inhibitor efficiently occupying the Kac binding site in BRD4 and BRD2. PFI-1 has antiproliferative effects on leukemic cell lines and efficiently abrogates their clonogenic growth. Exposure of sensitive cell lines with PFI-1 results in G1 cell-cycle arrest, downregulation of MYC expression, as well as induction of apoptosis and induces differentiation of primary leukemic blasts. Intriguingly, cells exposed to PFI-1 showed significant downregulation of Aurora B kinase, thus attenuating phosphorylation of the Aurora substrate H3S10, providing an alternative strategy for the specific inhibition of this well-established oncology target.
Asunto(s)
Proteínas Nucleares/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Quinazolinas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Animales , Apoptosis/fisiología , Proteínas de Ciclo Celular , Procesos de Crecimiento Celular/fisiología , Niño , Regulación hacia Abajo , Humanos , Ratones , Ratones Endogámicos NOD , Modelos Moleculares , Terapia Molecular Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosforilación , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Biomarcadores Farmacológicos/química , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Sondas Moleculares/química , Biomarcadores Farmacológicos/metabolismo , Permeabilidad de la Membrana Celular , Humanos , Sondas Moleculares/metabolismo , Terapia Molecular Dirigida , Relación Estructura-Actividad , Estudios de Validación como AsuntoRESUMEN
The posttranslational modification of chromatin through acetylation at selected histone lysine residues is governed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The significance of this subset of the epigenetic code is interrogated and interpreted by an acetyllysine-specific protein-protein interaction with bromodomain reader modules. Selective inhibition of the bromo and extra C-terminal domain (BET) family of bromodomains with a small molecule is feasible, and this may represent an opportunity for disease intervention through the recently disclosed antiproliferative and anti-inflammatory properties of such inhibitors. Herein, we describe the discovery and structure-activity relationship (SAR) of a novel, small-molecule chemical probe for BET family inhibition that was identified through the application of structure-based fragment assessment and optimization techniques. This has yielded a potent, selective compound with cell-based activity (PFI-1) that may further add to the understanding of BET family function within the bromodomains.
Asunto(s)
Sondas Moleculares/farmacología , Proteínas Nucleares/metabolismo , Quinazolinonas/farmacología , Sulfonamidas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Proteínas de Ciclo Celular , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Sondas Moleculares/síntesis química , Estructura Molecular , Proteínas Nucleares/antagonistas & inhibidores , Unión Proteica , Quinazolinonas/síntesis química , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
Lead Diversification is a new technology platform developed at Pfizer for the functionalization of drug molecules using C-H activation. We describe its application to some drug programs such as P38 and gMTP and the development of some new plate based screens including a fluorination screen.