RESUMEN
Synthetic modification of cyclosporin A at P3-P4 positions led to the discovery of NIM258, a next generation cyclophilin inhibitor with excellent anti-hepatitis C virus potency, with decreased transporter inhibition, and pharmacokinetics suitable for coadministration with other drugs. Herein is disclosed the evolution of the synthetic strategy to from the original medicinal chemistry route, designed for late diversification, to a convergent and robust development synthesis. The chiral centers in the P4 fragment were constructed by an asymmetric chelated Claisen rearrangement in the presence of quinidine as the chiral ligand. Identification of advanced crystalline intermediates enabled practical supply of key intermediates. Finally, macrocyclization was carried out at 10% weight concentration by a general and unconventional "slow release" concept.
Asunto(s)
Antivirales/química , Ciclosporina/química , Hepacivirus/fisiología , Antivirales/síntesis química , Antivirales/farmacología , Ciclización , Ciclosporina/síntesis química , Ciclosporina/farmacología , Dipéptidos/síntesis química , Dipéptidos/química , Diseño de Fármacos , Quinidina/química , Estereoisomerismo , Replicación Viral/efectos de los fármacosRESUMEN
Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3ß,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2-oxysterol signalling pathway has an important role in the adaptive immune response.
Asunto(s)
Hidroxicolesteroles/farmacología , Receptores de Superficie Celular/inmunología , Animales , Formación de Anticuerpos/inmunología , Linfocitos B , Línea Celular , Movimiento Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Hidroxicolesteroles/química , Hígado/química , Ratones , Ratones Noqueados , Receptores Acoplados a Proteínas G , Ovinos , Linfocitos T/inmunologíaRESUMEN
Malaria continues to be one of the most devastating human diseases despite many efforts to limit its spread by prevention of infection or by pharmaceutical treatment of patients. We have conducted a screen for antiplasmodial compounds by using a natural product library. Here we report on cyclomarinâ A as a potent growth inhibitor of Plasmodium falciparum and the identification of its molecular target, diadenosine triphosphate hydrolase (PfAp3Aase), by chemical proteomics. Using a biochemical assay, we could show that cyclomarinâ A is a specific inhibitor of the plasmodial enzyme but not of the closest human homologue hFHIT. Co-crystallisation experiments demonstrate a unique binding mode of the inhibitor. One molecule of cyclomarinâ A binds a dimeric PfAp3Aase and prevents the formation of the enzymeâ substrate complex. These results validate PfAp3Aase as a new drug target for the treatment of malaria. We have previously elucidated the structurally unrelated regulatory subunit ClpC1 of the ClpP protease as the molecular target of cyclomarinâ A in Mycobacterium tuberculosis. Thus, cyclomarinâ A is a rare example of a natural product with two distinct and specific modes of action.
Asunto(s)
Productos Biológicos/química , Oligopéptidos/química , Ácido Anhídrido Hidrolasas/antagonistas & inhibidores , Ácido Anhídrido Hidrolasas/metabolismo , Antimaláricos/química , Antimaláricos/metabolismo , Antimaláricos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Sitios de Unión , Productos Biológicos/metabolismo , Productos Biológicos/farmacología , Endopeptidasa Clp/antagonistas & inhibidores , Endopeptidasa Clp/metabolismo , Humanos , Concentración 50 Inhibidora , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factorâ 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystinâ A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemninâ B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factorâ 1.
Asunto(s)
Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Compuestos Macrocíclicos/farmacología , Myxococcales/fisiología , Neoplasias/patología , Factor 1 de Elongación Peptídica/antagonistas & inhibidores , Antifúngicos/química , Antifúngicos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Candida albicans/efectos de los fármacos , Genómica/métodos , Humanos , Compuestos Macrocíclicos/química , Estructura Molecular , Neoplasias/tratamiento farmacológico , Factor 1 de Elongación Peptídica/genética , Factor 1 de Elongación Peptídica/metabolismo , Proteómica/métodos , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Active pharmaceutical ingredients (APIs) in the environment, primarily resulting from patient excretion, are of concern because of potential risks to wildlife. This has led to more restrictive regulatory policies. Here, we discuss the 'benign-by-design' approach, which encourages the development of environmentally friendly APIs that are also safe and efficacious for patients. We explore the challenges and opportunities associated with identifying chemical properties that influence the environmental impact of APIs. Although a straightforward application of greener properties could hinder the development of new drugs, more nuanced approaches could lead to drugs that benefit both patients and the environment. We advocate for an enhanced dialogue between research and development (R&D) and environmental scientists and development of a toolbox to incorporate environmental sustainability in drug development.
Asunto(s)
Diseño de Fármacos , Desarrollo de Medicamentos , Humanos , Desarrollo de Medicamentos/métodos , Ambiente , Animales , Preparaciones Farmacéuticas , Tecnología Química Verde/métodos , InvestigaciónRESUMEN
Redirecting E3 ligases to neo-substrates, leading to their proteasomal disassembly, known as targeted protein degradation (TPD), has emerged as a promising alternative to traditional, occupancy-driven pharmacology. Although the field has expanded tremendously over the past years, the choice of E3 ligases remains limited, with an almost exclusive focus on CRBN and VHL. Here, we report the discovery of novel ligands to the PRY-SPRY domain of TRIM58, a RING ligase that is specifically expressed in erythroid precursor cells. A DSF screen, followed by validation using additional biophysical methods, led to the identification of TRIM58 ligand TRIM-473. A basic SAR around the chemotype was established by utilizing a competitive binding assay employing a short FP peptide probe derived from an endogenous TRIM58 substrate. The X-ray co-crystal structure of TRIM58 in complex with TRIM-473 gave insights into the binding mode and potential exit vectors for bifunctional degrader design.
RESUMEN
Acute kidney injury (AKI) is a life-threatening disease with no known curative or preventive therapies. Data from multiple animal models and human studies have linked dysregulation of bone morphogenetic protein (BMP) signaling to AKI. Small molecules that potentiate endogenous BMP signaling should have a beneficial effect in AKI. We performed a high-throughput phenotypic screen and identified a series of FK506 analogs that act as potent BMP potentiators by sequestering FKBP12 from BMP type I receptors. We further showed that calcineurin inhibition was not required for this activity. We identified a calcineurin-sparing FK506 analog oxtFK through late-stage functionalization and structure-guided design. OxtFK demonstrated an improved safety profile in vivo relative to FK506. OxtFK stimulated BMP signaling in vitro and in vivo and protected the kidneys in an AKI mouse model, making it a promising candidate for future development as a first-in-class therapeutic for diseases with dysregulated BMP signaling.
Asunto(s)
Lesión Renal Aguda/tratamiento farmacológico , Proteínas Morfogenéticas Óseas/metabolismo , Tacrolimus/farmacología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Ensayos Analíticos de Alto Rendimiento , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Fenotipo , Tacrolimus/análogos & derivados , Tacrolimus/químicaRESUMEN
Biosynthesis of glycosylphosphatidylinositol (GPI) is required for anchoring proteins to the plasma membrane, and is essential for the integrity of the fungal cell wall. Here, we use a reporter gene-based screen in Saccharomyces cerevisiae for the discovery of antifungal inhibitors of GPI-anchoring of proteins, and identify the oligocyclopropyl-containing natural product jawsamycin (FR-900848) as a potent hit. The compound targets the catalytic subunit Spt14 (also referred to as Gpi3) of the fungal UDP-glycosyltransferase, the first step in GPI biosynthesis, with good selectivity over the human functional homolog PIG-A. Jawsamycin displays antifungal activity in vitro against several pathogenic fungi including Mucorales, and in vivo in a mouse model of invasive pulmonary mucormycosis due to Rhyzopus delemar infection. Our results provide a starting point for the development of Spt14 inhibitors for treatment of invasive fungal infections.
Asunto(s)
Antifúngicos/farmacología , Glicosiltransferasas/antagonistas & inhibidores , Policétidos/farmacología , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Fermentación , Genes Reporteros , Glicosilfosfatidilinositoles/biosíntesis , Células HCT116 , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Concentración 50 Inhibidora , Células K562 , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos ICR , Mucorales , Familia de Multigenes , Rhizopus , Saccharomyces cerevisiaeRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Gene knockout and knockdown strategies have been immensely successful probes of gene function, but small molecule inhibitors (SMIs) of gene products allow much greater time resolution and are particularly useful when the targets are essential for cell replication or survival. SMIs also serve as lead compounds for drug discovery. However, discovery of selective SMIs is costly and inefficient. The action of SMIs can be modeled simply by tagging gene products with an auxin-inducible degron (AID) that triggers rapid ubiquitylation and proteasomal degradation of the tagged protein upon exposure of live cells to auxin. To determine if this approach is broadly effective, we AID-tagged over 750 essential proteins in Saccharomyces cerevisiae and observed growth inhibition by low concentrations of auxin in over 66% of cases. Polytopic transmembrane proteins in the plasma membrane, Golgi complex, and endoplasmic reticulum were efficiently depleted if the AID-tag was exposed to cytoplasmic OsTIR1 ubiquitin ligase. The auxin analog 1-napthylacetic acid (NAA) was as potent as auxin on AID-tags, but surprisingly NAA was more potent than auxin at inhibiting target of rapamycin complex 1 (TORC1) function. Auxin also synergized with known SMIs when acting on the same essential protein, indicating that AID-tagged strains can be useful for SMI screening. Auxin synergy, resistance mutations, and cellular assays together suggest the essential GMP/GDP-mannose exchanger in the Golgi complex (Vrg4) as the target of a natural cyclic peptide of unknown function (SDZ 90-215). These findings indicate that AID-tagging can efficiently model the action of SMIs before they are discovered and can facilitate SMI discovery.
Asunto(s)
Ácidos Indolacéticos/farmacología , Péptidos Cíclicos/farmacología , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Saccharomyces cerevisiae/efectos de los fármacos , Factores de Transcripción/antagonistas & inhibidores , Antifúngicos/farmacología , Genética Microbiana , Proteínas de Transporte de Membrana , Ácidos Naftalenoacéticos/farmacología , Saccharomyces cerevisiae/metabolismoAsunto(s)
Antituberculosos/química , Proteínas Bacterianas/química , Proteínas de Choque Térmico/química , Mycobacterium tuberculosis/efectos de los fármacos , Oligopéptidos/química , Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Proteínas de Choque Térmico/metabolismo , Oligopéptidos/farmacología , Unión ProteicaRESUMEN
Molecular modeling based on the X-ray crystal structure of the Tang-Ghosh heptapeptide inhibitor 1 (OM99-2) of BACE led to the design and synthesis of a series of constrained P(1)' analogues. A cyclopentane ring was incorporated in 1 spanning the P(1)' Ala methyl group and the adjacent methylene carbon atom of the chain. Progressive truncation at the P(2)'-P(4)' sites led to a potent truncated analogue 5 with good selectivity over Cathepsin D. Using the same backbone replacement concept, a series of cyclopentane, cyclopentanone, tetrahydrofuran, pyrrolidine, and pyrrolidinone analogues were synthesized with considerable variation at the P and P' sites. The cyclopentanone and 2-pyrrolidinone analogues 45 and 57 showed low nM BACE inhibition. X-ray cocrystal structures of two analogues 5 and 45 revealed excellent convergence with the original inhibitor 1 structure while providing new insights into other interactions which could be exploited for future modifications.
Asunto(s)
Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ciclopentanos/síntesis química , Furanos/síntesis química , Péptidos/química , Pirrolidinas/síntesis química , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas/química , Sitios de Unión , Catepsina D/antagonistas & inhibidores , Cristalografía por Rayos X , Ciclopentanos/química , Endopeptidasas , Furanos/química , Humanos , Modelos Moleculares , Imitación Molecular , Pirrolidinas/química , Pirrolidinonas/síntesis química , Pirrolidinonas/química , Relación Estructura-ActividadRESUMEN
FR171456 is a natural product with cholesterol-lowering properties in animal models, but its molecular target is unknown, which hinders further drug development. Here we show that FR171456 specifically targets the sterol-4-alpha-carboxylate-3-dehydrogenase (Saccharomyces cerevisiae--Erg26p, Homo sapiens--NSDHL (NAD(P) dependent steroid dehydrogenase-like)), an essential enzyme in the ergosterol/cholesterol biosynthesis pathway. FR171456 significantly alters the levels of cholesterol pathway intermediates in human and yeast cells. Genome-wide yeast haploinsufficiency profiling experiments highlight the erg26/ERG26 strain, and multiple mutations in ERG26 confer resistance to FR171456 in growth and enzyme assays. Some of these ERG26 mutations likely alter Erg26 binding to FR171456, based on a model of Erg26. Finally, we show that FR171456 inhibits an artificial Hepatitis C viral replicon, and has broad antifungal activity, suggesting potential additional utility as an anti-infective. The discovery of the target and binding site of FR171456 within the target will aid further development of this compound.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Antifúngicos/química , Colesterol/análogos & derivados , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Saccharomyces cerevisiae/genética , 3-Hidroxiesteroide Deshidrogenasas/genética , Candida albicans , Colesterol/química , Farmacorresistencia Fúngica/genética , Ergosterol/biosíntesis , Mutación , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The first proteolytic step in the processing of amyloid precursor protein (APP) to amyloid-beta (Abeta) in the brain is performed by beta-site APP cleaving enzyme (BACE1). This enzyme is a membrane bound aspartic protease with high homology of the catalytic domain to renin and pepsin and of yet unknown physiologic function. It is a primary drug discovery target for Alzheimer s disease therapy. The first potent inhibitors are based on the sequence of APP around the beta-secretase cleavage site EVNL/DAEF, with the scissile Leu-Asp amide bond being replaced by a hydroxyethylene transition state analogue isostere. In addition, lipophilic sidechains have been incorporated and a crystal structure of such an octapeptidic inhibitor bound in the active site is already available. Recent progress in the field of BACE inhibition is reviewed.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores Enzimáticos/uso terapéutico , Enfermedad de Alzheimer/enzimología , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Endopeptidasas , Humanos , Conformación Proteica , Pliegue de Proteína , Relación Estructura-ActividadRESUMEN
Nonimmunosuppressive cyclophilin inhibitors have demonstrated efficacy for the treatment of hepatitis C infection (HCV). However, alisporivir, cyclosporin A, and most other cyclosporins are potent inhibitors of OATP1B1, MRP2, MDR1, and other important drug transporters. Reduction of the side chain hydrophobicity of the P4 residue preserves cyclophilin binding and antiviral potency while decreasing transporter inhibition. Representative inhibitor 33 (NIM258) is a less potent transporter inhibitor relative to previously described cyclosporins, retains anti-HCV activity in cell culture, and has an acceptable pharmacokinetic profile in rats and dogs. An X-ray structure of 33 bound to rat cyclophilin D is reported.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Ciclofilinas/antagonistas & inhibidores , Ciclosporinas/farmacología , Transportadores de Anión Orgánico/antagonistas & inhibidores , Animales , Antivirales/síntesis química , Antivirales/farmacocinética , Técnicas de Química Sintética , Cristalografía por Rayos X , Peptidil-Prolil Isomerasa F , Ciclofilinas/química , Ciclofilinas/metabolismo , Ciclosporina/química , Ciclosporina/farmacología , Ciclosporinas/química , Perros , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunosupresores/química , Inmunosupresores/farmacología , Transportador 1 de Anión Orgánico Específico del Hígado , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Ratas , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
Translation initiation is an emerging target in oncology and neurobiology indications. Naturally derived and synthetic rocaglamide scaffolds have been used to interrogate this pathway; however, there is uncertainty regarding their precise mechanism(s) of action. We exploited the genetic tractability of yeast to define the primary effect of both a natural and a synthetic rocaglamide in a cellular context and characterized the molecular target using biochemical studies and in silico modeling. Chemogenomic profiling and mutagenesis in yeast identified the eIF (eukaryotic Initiation Factor) 4A helicase homologue as the primary molecular target of rocaglamides and defined a discrete set of residues near the RNA binding motif that confer resistance to both compounds. Three of the eIF4A mutations were characterized regarding their functional consequences on activity and response to rocaglamide inhibition. These data support a model whereby rocaglamides stabilize an eIF4A-RNA interaction to either alter the level and/or impair the activity of the eIF4F complex. Furthermore, in silico modeling supports the annotation of a binding pocket delineated by the RNA substrate and the residues identified from our mutagenesis screen. As expected from the high degree of conservation of the eukaryotic translation pathway, these observations are consistent with previous observations in mammalian model systems. Importantly, we demonstrate that the chemically distinct silvestrol and synthetic rocaglamides share a common mechanism of action, which will be critical for optimization of physiologically stable derivatives. Finally, these data confirm the value of the rocaglamide scaffold for exploring the impact of translational modulation on disease.
Asunto(s)
Benzofuranos/metabolismo , Factor 4F Eucariótico de Iniciación/química , Factor 4F Eucariótico de Iniciación/metabolismo , Saccharomyces cerevisiae/metabolismo , Benzofuranos/química , Sitios de Unión , Modelos Biológicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Triterpenos/química , Triterpenos/metabolismoRESUMEN
Natural products (NPs) have been optimized in a very long natural selection process for optimal interactions with biological macromolecules. NPs are therefore an excellent source of validated substructures for the design of novel bioactive molecules. Various cheminformatics techniques can provide useful help in analyzing NPs, and the results of such studies may be used with advantage in the drug discovery process. In the present study we describe a method to calculate the natural product-likeness score--a Bayesian measure which allows for the determination of how molecules are similar to the structural space covered by natural products. This score is shown to efficiently separate NPs from synthetic molecules in a cross-validation experiment. Possible applications of the NP-likeness score are discussed and illustrated on several examples including virtual screening, prioritization of compound libraries toward NP-likeness, and design of building blocks for the synthesis of NP-like libraries.
Asunto(s)
Productos Biológicos/química , Bibliotecas de Moléculas Pequeñas , Teorema de Bayes , Técnicas Químicas Combinatorias , Diseño de Fármacos , Curva ROC , Reproducibilidad de los ResultadosRESUMEN
A hierarchical classification of chemical scaffolds (molecular framework, which is obtained by pruning all terminal side chains) has been introduced. The molecular frameworks form the leaf nodes in the hierarchy trees. By an iterative removal of rings, scaffolds forming the higher levels in the hierarchy tree are obtained. Prioritization rules ensure that less characteristic, peripheral rings are removed first. All scaffolds in the hierarchy tree are well-defined chemical entities making the classification chemically intuitive. The classification is deterministic, data-set-independent, and scales linearly with the number of compounds included in the data set. The application of the classification is demonstrated on two data sets extracted from the PubChem database, namely, pyruvate kinase binders and a collection of pesticides. The examples shown demonstrate that the classification procedure handles robustly synthetic structures and natural products.
Asunto(s)
Clasificación , Estructura Molecular , Compuestos Orgánicos/química , Bases de Datos Factuales , Ligandos , Plaguicidas , Piruvato QuinasaRESUMEN
Various heterocyclic hetero-methyl ketones of the 1-naphthyloxyacetyl-Val-Asp backbone have been prepared. A study of their structure-activity relationship (SAR) related to caspase-1, -3, -6, and -8 is reported. Their efficacy in a cellular model of cell death is also discussed. Potent broad-spectrum caspase inhibitors have been identified.
Asunto(s)
Inhibidores de Caspasas , Muerte Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Compuestos Heterocíclicos/farmacología , Cetonas/farmacología , Animales , Ácido Aspártico/química , Células Cultivadas , Compuestos Heterocíclicos/síntesis química , Cetonas/síntesis química , Ratones , Modelos Biológicos , Naftoles/química , Relación Estructura-Actividad , Valina/químicaRESUMEN
A new structural class of broad spectrum caspase inhibitors was optimized for its activity against caspases 1, 3, 6, 7, and 8. The most potent compound had low nanomolar broad spectrum activity, in particular, single digit nanomolar inhibitory activity against caspase 8.