RESUMEN
Protein kinase, membrane-associated tyrosine/threonine 1 (PKMYT1), a member of the WEE family and responsible for the regulation of CDK1 phosphorylation, has been considered a promising therapeutic target for cancer therapy. However, the highly structural conservation of the ATP-binding sites of the WEE family poses a challenge to the design of selective inhibitors for PKMYT1. Here, molecular docking, multiple microsecond-length molecular dynamics (MD) simulations and end-point free energy calculations were performed to uncover the molecular mechanism of the binding selectivity of RP-6306 toward PKMYT1 over its highly homologous kinase WEE1. The binding specificity of RP-6306 reported in previous experimental bioassays was clarified by MD simulations and binding free energy calculations. Further, the binding free energy prediction indicated that the binding selectivity of RP-6306 largely derived from the difference in the protein-ligand electrostatic interactions. The per-residue free energy decomposition suggested that the non-conserved gatekeeper residue in the hinge domain of PKMYT1/WEE1, Thr187/Asn376, is the critical factor responsible for the binding selectivity of RP-6306 toward PKMYT1. In addition, a water-mediated hydrogen bond was formed between RP-6306 and Gly191 at the hinge domain in the PKMYT1/RP-6306 complex, which was absent in the WEE1/RP-6306 complex. This study is expected to offer useful information for the design of more potent and selective PKMYT1 inhibitors.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Simulación de Dinámica Molecular , Simulación del Acoplamiento Molecular , Fosforilación , Sitios de UniónRESUMEN
Type-III copper enzymes like polyphenol oxidases (PPOs) are ubiquitous among organisms and play a significant role in the formation of pigments. PPOs comprise different enzyme groups, including tyrosinases (TYRs) and catechol oxidases (COs). TYRs catalyze the o-hydroxylation of monophenols and the oxidation of o-diphenols to the corresponding o-quinones (ECâ 1.14.18.1). In contrast, COs only catalyze the oxidation of o-diphenols to the corresponding o-quinones (ECâ 1.10.3.1). To date (August 2020), 102 PDB entries encompassing 18 different proteins from 16 organisms and several mutants have been reported, identifying key residues for tyrosinase activity. The structural similarity between TYRs and COs, especially within and around the active center, complicates the elucidation of their modes of action on a structural basis. However, mutagenesis studies illuminate residues that influence the two activities and show that crystallography on its own cannot elucidate the enzymatic activity mode. Several amino acid residues around the dicopper active center have been proposed to play an essential role in the two different activities. Herein, we critically review the role of all residues identified so far that putatively affect the two activities of PPOs.
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Catecol Oxidasa/metabolismo , Monofenol Monooxigenasa/metabolismo , Proteínas de Plantas/metabolismo , Biocatálisis , Catecol Oxidasa/química , Catecol Oxidasa/genética , Monofenol Monooxigenasa/química , Monofenol Monooxigenasa/genética , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Fenoles/química , Fenoles/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Especificidad por SustratoRESUMEN
Improvements have been made to the safety and efficacy of bumped kinase inhibitors, and they are advancing toward human and animal use for treatment of cryptosporidiosis. As the understanding of bumped kinase inhibitor pharmacodynamics for cryptosporidiosis therapy has increased, it has become clear that better compounds for efficacy do not necessarily require substantial systemic exposure. We now have a bumped kinase inhibitor with reduced systemic exposure, acceptable safety parameters, and efficacy in both the mouse and newborn calf models of cryptosporidiosis. Potential cardiotoxicity is the limiting safety parameter to monitor for this bumped kinase inhibitor. This compound is a promising pre-clinical lead for cryptosporidiosis therapy in animals and humans.
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Criptosporidiosis/tratamiento farmacológico , Cryptosporidium parvum/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Administración Oral , Animales , Animales Recién Nacidos , Bovinos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Corazón/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Interferón gamma/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Pruebas de Mutagenicidad , Embarazo , Unión Proteica , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/sangre , Inhibidores de Proteínas Quinasas/toxicidad , SeguridadRESUMEN
BACKGROUND: The determination of specific kinase substrates in vivo is challenging due to the large number of protein kinases in cells, their substrate specificity overlap, and the lack of highly specific inhibitors. In the late 90s, Shokat and coworkers developed a protein engineering-based method addressing the question of identification of substrates of protein kinases. The approach was based on the mutagenesis of the gatekeeper residue within the binding site of a protein kinase to change the co-substrate specificity from ATP to ATP analogues. One of the challenges in applying this method to other kinase systems is to identify the optimal combination of mutation in the enzyme and chemical derivative such that the ATP analogue acts as substrate for the engineered, but not the native kinase enzyme. In this study, we developed a computational protocol for estimating the effect of mutations at the gatekeeper position on the accessibility of ATP analogues within the binding site of engineered kinases. RESULTS: We tested the protocol on a dataset of tyrosine and serine/threonine protein kinases from the scientific literature where Shokat's method was applied and experimental data were available. Our protocol correctly identified gatekeeper residues as the positions to mutate within the binding site of the studied kinase enzymes. Furthermore, the approach well reproduced the experimental data available in literature. CONCLUSIONS: We have presented a computational protocol that scores how different mutations at the gatekeeper position influence the accommodation of various ATP analogues within the binding site of protein kinases. We have assessed our approach on protein kinases from the scientific literature and have verified the ability of the approach to well reproduce the available experimental data and identify suitable combinations of engineered kinases and ATP analogues.
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Adenosina Trifosfato/metabolismo , Modelos Moleculares , Mutación , Proteínas Quinasas , Adenosina Trifosfato/análisis , Sitios de Unión , Protocolos Clínicos , Humanos , Unión ProteicaRESUMEN
Many life-cycle processes in parasites are regulated by protein phosphorylation. Hence, disruption of essential protein kinase function has been explored for therapy of parasitic diseases. However, the difficulty of inhibiting parasite protein kinases to the exclusion of host orthologues poses a practical challenge. A possible path around this difficulty is the use of bumped kinase inhibitors for targeting calcium-dependent protein kinases that contain atypically small gatekeeper residues and are crucial for pathogenic apicomplexan parasites' survival and proliferation. In this article, we review efficacy against the kinase target, parasite growth in vitro, and in animal infection models, as well as the relevant pharmacokinetic and safety parameters of bumped kinase inhibitors.
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Antiprotozoarios/farmacología , Apicomplexa/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Infecciones por Protozoos/tratamiento farmacológico , Animales , Antiprotozoarios/uso terapéutico , Apicomplexa/enzimología , Bencimidazoles/química , Humanos , Imidazoles/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Infecciones por Protozoos/prevención & control , Piridinas/químicaRESUMEN
Sarcocystis neurona is the most frequent cause of equine protozoal myeloencephalitis, a debilitating neurological disease of horses that can be difficult to treat. We identified SnCDPK1, the S. neurona homologue of calcium-dependent protein kinase 1 (CDPK1), a validated drug target in Toxoplasma gondii. SnCDPK1 shares the glycine "gatekeeper" residue of the well-characterized T. gondii enzyme, which allows the latter to be targeted by bumped kinase inhibitors. This study presents detailed molecular and phenotypic evidence that SnCDPK1 can be targeted for rational drug development. Recombinant SnCDPK1 was tested against four bumped kinase inhibitors shown to potently inhibit both T. gondii (Tg) CDPK1 and T. gondii tachyzoite growth. SnCDPK1 was inhibited by low nanomolar concentrations of these BKIs and S. neurona growth was inhibited at 40-120nM concentrations. Thermal shift assays confirmed these bumped kinase inhibitors bind CDPK1 in S. neurona cell lysates. Treatment with bumped kinase inhibitors before or after invasion suggests that bumped kinase inhibitors interfere with S. neurona mammalian host cell invasion in the 0.5-2.5µM range but interfere with intracellular division at 2.5µM. In vivo proof-of-concept experiments were performed in a murine model of S. neurona infection. The experimental infected groups treated for 30days with compound BKI-1553 (n=10 mice) had no signs of disease, while the infected control group had severe signs and symptoms of infection. Elevated antibody responses were found in 100% of control infected animals, but only 20% of BKI-1553 treated infected animals. Parasites were found in brain tissues of 100% of the control infected animals, but only in 10% of the BKI-1553 treated animals. The bumped kinase inhibitors used in these assays have been chemically optimized for potency, selectivity and pharmacokinetic properties, and hence are good candidates for treatment of equine protozoal myeloencephalitis.
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Encefalomielitis/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas/efectos de los fármacos , Sarcocystis/enzimología , Sarcocistosis/tratamiento farmacológico , Animales , Línea Celular , Chlorocebus aethiops , Encefalomielitis/parasitología , Femenino , Enfermedades de los Caballos/tratamiento farmacológico , Enfermedades de los Caballos/parasitología , Caballos , Interferón gamma/genética , Masculino , Ratones , Ratones Noqueados , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Conejos , Sarcocystis/efectos de los fármacos , Temperatura , Toxoplasma/efectos de los fármacos , Toxoplasma/enzimologíaRESUMEN
Protein aggregation into ß-sheet-enriched amyloid fibrils is associated with an increasing number of human disorders. The adoption of such amyloid conformations seems to constitute a generic property of polypeptide chains. Therefore, during evolution, proteins have adopted negative design strategies to diminish their intrinsic propensity to aggregate, including enrichment of gatekeeper charged residues at the flanks of hydrophobic aggregation-prone segments. Wild type transthyretin (TTR) is responsible for senile systemic amyloidosis, and more than 100 mutations in the TTR gene are involved in familial amyloid polyneuropathy. The TTR 26-57 segment bears many of these aggressive amyloidogenic mutations as well as the binding site for heparin. We demonstrate here that Lys-35 acts as a gatekeeper residue in TTR, strongly decreasing its amyloidogenic potential. This protective effect is sequence-specific because Lys-48 does not affect TTR aggregation. Lys-35 is part of the TTR basic heparin-binding motif. This glycosaminoglycan blocks the protective effect of Lys-35, probably by neutralization of its side chain positive charge. A K35L mutation emulates this effect and results in the rapid self-assembly of the TTR 26-57 region into amyloid fibrils. This mutation does not affect the tetrameric protein stability, but it strongly increases its aggregation propensity. Overall, we illustrate how TTR is yet another amyloidogenic protein exploiting negative design to prevent its massive aggregation, and we show how blockage of conserved protective features by endogenous factors or mutations might result in increased disease susceptibility.
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Amiloide/química , Leucina/química , Lisina/química , Prealbúmina/química , Amiloide/genética , Amiloide/metabolismo , Neuropatías Amiloides Familiares/genética , Neuropatías Amiloides Familiares/metabolismo , Neuropatías Amiloides Familiares/patología , Expresión Génica , Heparina/química , Heparina/metabolismo , Humanos , Leucina/metabolismo , Lisina/metabolismo , Mutación , Prealbúmina/genética , Prealbúmina/metabolismo , Agregación Patológica de Proteínas , Multimerización de Proteína , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad EstáticaRESUMEN
The epidermal growth factor receptor (EGFR) family consists of four members that belong to the ErbB lineage of proteins (ErbB1-4). These receptors consist of an extracellular domain, a single hydrophobic transmembrane segment, and an intracellular portion with a juxtamembrane segment, a protein kinase domain, and a carboxyterminal tail. The ErbB proteins function as homo and heterodimers. Growth factor binding to EGFR induces a large conformational change in the extracellular domain. Two ligand-EGFR complexes unite to form a back-to-back dimer in which the ligands are on opposite sides of the aggregate. Following ligand binding, EGFR intracellular kinase domains form an asymmetric dimer. The carboxyterminal lobe of the activator kinase of the dimer interacts with the amino-terminal lobe of the receiver kinase thereby leading to its allosteric stimulation. Several malignancies are associated with the mutation or increased expression of members of the ErbB family including lung, breast, stomach, colorectal, head and neck, and pancreatic carcinomas. Gefitinib, erlotinib, and afatinib are orally effective protein-kinase targeted quinazoline derivatives that are used in the treatment of ERBB1-mutant lung cancer and lapatinib is an orally effective quinazoline derivative used in the treatment of ErbB2-overexpressing breast cancer. Moreover, monoclonal antibodies that target the extracellular domain of ErbB2 are used for the treatment of ErbB2-positive breast cancer and monoclonal antibodies that target ErbB1 and are used for the treatment of colorectal cancer. Cancers treated with these targeted drugs eventually become resistant to them, and a current goal of research is to develop drugs that are effective against drug-resistant tumors.
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Receptores ErbB , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biocatálisis , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos , Conformación Proteica , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Plant receptor-like kinases (RLKs) share their evolutionary origin with animal interleukin-1 receptor-associated kinase (IRAK)/Pelle family of soluble kinases and are distinguished by having tyrosine as 'gatekeeper'. This position is adjacent to the hinge region and is hidden in a hydrophobic pocket of the catalytic cleft of protein kinases and is therefore least probable to be a target for any modification. This communication illustrates the accessibility of the gatekeeper site (Y670) towards both autophosphorylation and dephosphorylation in the recombinant cytoplasmic domain of symbiosis receptor kinase from Arachis hypogaea (AhSYMRK). Autophosphorylation on gatekeeper tyrosine was detected prior to extraction but never under in vitro conditions. We hypothesize gatekeeper phosphorylation to be associated with synthesis/maturation of AhSYMRK and this phenomenon may be prevalent among RLKs.