RESUMEN
The protein kinase DYRK1A is involved in Alzheimer's disease, Down syndrome, diabetes, viral infections, and leukemia. Leucettines, a family of 2-aminoimidazolin-4-ones derived from the marine sponge alkaloid Leucettamine B, have been developed as pharmacological inhibitors of DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases). We report here on the synthesis and structure-activity relationship (SAR) of 68 Leucettines. Leucettines were tested on 11 purified kinases and in 5 cellular assays: (1) CLK1 pre-mRNA splicing, (2) Threonine-212-Tau phosphorylation, (3) glutamate-induced cell death, (4) autophagy and (5) antagonism of ligand-activated cannabinoid receptor CB1. The Leucettine SAR observed for DYRK1A is essentially identical for CLK1, CLK4, DYRK1B, and DYRK2. DYRK3 and CLK3 are less sensitive to Leucettines. In contrast, the cellular SAR highlights correlations between inhibition of specific kinase targets and some but not all cellular effects. Leucettines deserve further development as potential therapeutics against various diseases on the basis of their molecular targets and cellular effects.
Asunto(s)
Imidazoles/química , Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Empalme del ARN , Receptor Cannabinoide CB1/antagonistas & inhibidores , Animales , Autofagia , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Ratones , Neuronas/efectos de los fármacos , Neuronas/enzimología , Fosforilación , Relación Estructura-ActividadRESUMEN
Isobutene is a high value gaseous alkene used as fuel additive and a chemical building block. As an alternative to fossil fuel derived isobutene, we here develop a modified mevalonate pathway for the production of isobutene from glucose in vivo. The final step in the pathway consists of the decarboxylation of 3-methylcrotonic acid, catalysed by an evolved ferulic acid decarboxylase (Fdc) enzyme. Fdc belongs to the prFMN-dependent UbiD enzyme family that catalyses reversible decarboxylation of (hetero)aromatic acids or acrylic acids with extended conjugation. Following a screen of an Fdc library for inherent 3-methylcrotonic acid decarboxylase activity, directed evolution yields variants with up to an 80-fold increase in activity. Crystal structures of the evolved variants reveal that changes in the substrate binding pocket are responsible for increased selectivity. Solution and computational studies suggest that isobutene cycloelimination is rate limiting and strictly dependent on presence of the 3-methyl group.
Asunto(s)
Alquenos/metabolismo , Carboxiliasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Mononucleótido de Flavina/química , Glucosa/metabolismo , Alquenos/química , Biocatálisis , Carboxiliasas/genética , Crotonatos/metabolismo , Evolución Molecular Dirigida/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fermentación , Mononucleótido de Flavina/metabolismo , Glucosa/química , Hypocreales/enzimología , Hypocreales/genética , Ácido Mevalónico/metabolismo , PrenilaciónRESUMEN
Growing evidence supports the implication of DYRK1A in the development of cognitive deficits seen in Down syndrome (DS) and Alzheimer's disease (AD). We here demonstrate that pharmacological inhibition of brain DYRK1A is able to correct recognition memory deficits in three DS mouse models with increasing genetic complexity [Tg(Dyrk1a), Ts65Dn, Dp1Yey], all expressing an extra copy of Dyrk1a Overexpressed DYRK1A accumulates in the cytoplasm and at the synapse. Treatment of the three DS models with the pharmacological DYRK1A inhibitor leucettine L41 leads to normalization of DYRK1A activity and corrects the novel object cognitive impairment observed in these models. Brain functional magnetic resonance imaging reveals that this cognitive improvement is paralleled by functional connectivity remodelling of core brain areas involved in learning/memory processes. The impact of Dyrk1a trisomy and L41 treatment on brain phosphoproteins was investigated by a quantitative phosphoproteomics method, revealing the implication of synaptic (synapsin 1) and cytoskeletal components involved in synaptic response and axonal organization. These results encourage the development of DYRK1A inhibitors as drug candidates to treat cognitive deficits associated with DS and AD.
Asunto(s)
Disfunción Cognitiva/complicaciones , Disfunción Cognitiva/tratamiento farmacológico , Dioxoles/farmacología , Dioxoles/uso terapéutico , Síndrome de Down/complicaciones , Imidazoles/farmacología , Imidazoles/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Biocatálisis , Encéfalo/efectos de los fármacos , Encéfalo/patología , Encéfalo/fisiopatología , Disfunción Cognitiva/patología , Citoplasma/metabolismo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Dioxoles/química , Modelos Animales de Enfermedad , Síndrome de Down/patología , Imidazoles/química , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Red Nerviosa/efectos de los fármacos , Red Nerviosa/fisiopatología , Fosforilación , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteómica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Sinapsinas/química , Sinapsinas/metabolismo , Quinasas DyrKRESUMEN
Following the discovery of (R)-roscovitine's beneficial effects in three polycystic kidney disease (PKD) mouse models, cyclin-dependent kinases (CDKs) inhibitors have been investigated as potential treatments. We have used various affinity chromatography approaches to identify the molecular targets of roscovitine and its more potent analog (S)-CR8 in human and murine polycystic kidneys. These methods revealed casein kinases 1 (CK1) as additional targets of the two drugs. CK1ε expression at the mRNA and protein levels is enhanced in polycystic kidneys of 11 different PKD mouse models as well as in human polycystic kidneys. A shift in the pattern of CK1α isoforms is observed in all PKD mouse models. Furthermore, the catalytic activities of both CK1ε and CK1α are increased in mouse polycystic kidneys. Inhibition of CK1ε and CK1α may thus contribute to the long-lasting attenuating effects of roscovitine and (S)-CR8 on cyst development. CDKs and CK1s may constitute a dual therapeutic target to develop kinase inhibitory PKD drug candidates.
Asunto(s)
Caseína Cinasa 1 épsilon/antagonistas & inhibidores , Caseína Quinasa Ialfa/antagonistas & inhibidores , Riñón/efectos de los fármacos , Enfermedades Renales Poliquísticas/prevención & control , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Piridinas/farmacología , Roscovitina/farmacología , Animales , Caseína Cinasa 1 épsilon/genética , Caseína Cinasa 1 épsilon/metabolismo , Caseína Quinasa Ialfa/genética , Caseína Quinasa Ialfa/metabolismo , Catálisis , Cromatografía de Afinidad/métodos , Modelos Animales de Enfermedad , Humanos , Riñón/enzimología , Riñón/patología , Ratones Transgénicos , Enfermedades Renales Poliquísticas/enzimología , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/patología , Unión Proteica , Inhibidores de Proteínas Quinasas/metabolismo , Purinas/metabolismo , Piridinas/metabolismo , Roscovitina/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
A large diversity of 2-aminoimidazolone alkaloids is produced by various marine invertebrates, especially by the marine Calcareous sponges Leucetta and Clathrina. The phylogeny of these sponges and the wide scope of 2-aminoimidazolone alkaloids they produce are reviewed in this article. The origin (invertebrate cells, associated microorganisms, or filtered plankton), physiological functions, and natural molecular targets of these alkaloids are largely unknown. Following the identification of leucettamine B as an inhibitor of selected protein kinases, we synthesized a family of analogues, collectively named leucettines, as potent inhibitors of DYRKs (dual-specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) and potential pharmacological leads for the treatment of several diseases, including Alzheimer's disease and Down syndrome. We assembled a small library of marine sponge- and ascidian-derived 2-aminoimidazolone alkaloids, along with several synthetic analogues, and tested them on a panel of mammalian and protozoan kinases. Polyandrocarpamines A and B were found to be potent and selective inhibitors of DYRKs and CLKs. They inhibited cyclin D1 phosphorylation on a DYRK1A phosphosite in cultured cells. 2-Aminoimidazolones thus represent a promising chemical scaffold for the design of potential therapeutic drug candidates acting as specific inhibitors of disease-relevant kinases, and possibly other disease-relevant targets.
Asunto(s)
Alcaloides/farmacología , Imidazoles/farmacología , Poríferos/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Urocordados/química , Alcaloides/síntesis química , Alcaloides/uso terapéutico , Enfermedad de Alzheimer/tratamiento farmacológico , Aminas/síntesis química , Aminas/farmacología , Aminas/uso terapéutico , Animales , Antiprotozoarios/síntesis química , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Síndrome de Down/tratamiento farmacológico , Humanos , Imidazoles/síntesis química , Imidazoles/química , Imidazoles/uso terapéutico , Fosforilación , Filogenia , Poríferos/genética , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/uso terapéutico , Infecciones por Protozoos/tratamiento farmacológico , Proteínas Protozoarias/antagonistas & inhibidores , Relación Estructura-Actividad , Quinasas DyrKRESUMEN
3,6-Disubstituted imidazo[1,2-b]pyridazine derivatives were synthesized to identify new inhibitors of various eukaryotic kinases, including mammalian and protozoan kinases. Among the imidazo[1,2-b]pyridazines tested as kinase inhibitors, several derivatives were selective for DYRKs and CLKs, with IC50 < 100 nM. The characterization of the kinome of several parasites, such as Plasmodium and Leishmania, has pointed out profound divergences between protein kinases of the parasites and those of the host. This led us to investigate the activities of the prepared compounds against 11 parasitic kinases. 3,6-Disubstituted imidazo[1,2-b]pyridazines showed potent inhibition of Plasmodium falciparum CLK1 (PfCLK1). Compound 20a was found to be the most selective product against CLK1 (IC50 = 82 nM), CLK4 (IC50 = 44 nM), DYRK1A (IC50 = 50 nM), and PfCLK1 (IC50 = 32 nM). The compounds were also tested against Leishmania amazonensis. Several compounds showed anti-leishmanial activity at rather high (10 µM) concentration, but were not toxic at 1 µM or 10 µM, as judged by viability assays carried out using a neuroblastoma cell line.
Asunto(s)
Piridazinas/farmacología , Antiparasitarios/síntesis química , Antiparasitarios/química , Antiparasitarios/farmacología , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Eucariontes/efectos de los fármacos , Concentración 50 Inhibidora , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas/síntesis química , Piridazinas/químicaRESUMEN
USER friendly DNA recombination (USERec) is based on near homology-independent recombination of DNA fragments (~40-400 bp) that are efficiently reassembled into full-length genes, proven for up to ten fragments. USERec requires a minimal crossover sequence (a 5'-AN(4-8)T-3' motif) of the fragments that can be implemented wherever structural or functional comparisons suggest a fragment boundary. The greatly reduced sequence constraints of this method facilitate directional assembly of gene fragments for applications such as exon or domain shuffling, loop grafting, reassembly of natural modular biosynthetic assembly lines, and rearrangement of structurally (but not sequence) homologous proteins.
Asunto(s)
Biblioteca de Genes , Clonación Molecular , Barajamiento de ADN , Recombinación GenéticaRESUMEN
Modular natural products are biosynthesized by series of enzymes that activate, assemble, and process a nascent chain of building blocks. Adenylation domains are gatekeepers in nonribosomal peptide biosynthesis, providing the entry point for assembly of typical peptide-based natural products. We report the directed evolution of an adenylation domain based on a strategy of using a weak, promiscuous activity as a springboard for reprogramming the biosynthetic assembly line. Randomization of residues invoked in a "specificity-conferring code" and selection for a non-native substrate lead to mutant G2.1, favoring smaller amino acids with a specificity change of 10(5): a 170-fold improvement for L-alanine corresponds to a 10(3)-fold decrease for its original substrate (L-phenylalanine). These results establish directed evolution as a method to change gatekeeper domain specificity and suggest that adaptation of modules in combinatorial biosynthesis is achievable with few mutations during evolution.
Asunto(s)
Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Péptido Sintasas/química , Péptido Sintasas/metabolismo , Alanina/metabolismo , Aminoácidos/metabolismo , Sitios de Unión , Evolución Molecular Dirigida , Interacciones Hidrofóbicas e Hidrofílicas , Mutagénesis Sitio-Dirigida , Mutación , Péptido Sintasas/genética , Fenilalanina/metabolismo , Estructura Terciaria de Proteína , Distribución Aleatoria , Solventes/química , Especificidad por SustratoRESUMEN
The catalytic potential of tyrocidine synthetase 1 (TycA) was probed by the kinetic characterization of its adenylation activity. We observed reactions with 30 substrates, thus suggesting some substrate promiscuity. However, although the TycA adenylation (A) domain was able to accommodate alternative substrates, their k(cat)/K(M) values ranged over six orders of magnitude. A comparison of the activities allowed the systematic mapping of the substrate specificity determinants of the TycA A-domain. Hydrophobicity plays a major role in the recognition of substrate analogues but can be combined with shape complementarity, conferring higher activity, and/or steric exclusion, leading to substantial discrimination against larger substrates. A comparison of the k(cat)/K(M) values of the TycA A-domain and phenylalanyl-tRNA synthetase showed that the level of discrimination was comparable in the two enzymes for the adenylation reaction and suggested that TycA was also subjected to high selective pressure. The specificity patterns observed and the quantification of alternative activities provide a basis for exploring possible paths for the future directed evolution of A-domain specificity.
Asunto(s)
Péptido Sintasas/química , Péptido Sintasas/metabolismo , Adenosina Trifosfato/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Difosfatos/metabolismo , Cinética , Modelos Moleculares , Estructura Terciaria de Proteína , Estereoisomerismo , Especificidad por SustratoRESUMEN
We report a new format for measuring ATP/[(32)P]pyrophosphate exchange in a higher throughput assay of adenylation domains (A-domains) of non-ribosomal peptide synthetases. These enzymes are key specificity determinants in the assembly line biosynthesis of non-ribosomal peptides, an important class of natural products with an activity spectrum ranging from antibiotic to antitumor activities. Our assay in 96-well format allows the rapid measurement of approximately 1000 data points per week as a basis for precise assessment of the kinetics of A-domains. The assay also allows quantitative high-throughput screening of the substrate specificity of A-domains identifying alternative, promiscuous substrates. We show that our assay is able to give high quality data for the T278A mutant of the A-domain of the tyrocidine synthetase module TycA with a 330-fold lower k(cat)/K(M). The large dynamic range of this assay will be useful for the screening of libraries of mutant A-domains. Finally we describe and evaluate a procedure for the high-throughput purification of A-domains in 96-well format for the latter purpose. Our approach will be of utility for mechanistic analysis, substrate profiling and directed evolution of the A-domains, to ultimately enable the combinatorial biosynthesis of non-natural analogues of non-ribosomal peptides that may have potential as alternative drug candidates.