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1.
ACS Omega ; 2(11): 7881-7891, 2017 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30023565

RESUMO

Overexpression of the eukaryotic initiation factor 4E (eIF4E) is linked to a variety of cancers. Both mitogen-activated protein kinases-interacting kinases 1 and 2 (Mnk1/2) activate the oncogene eIF4E through posttranslational modification (phosphorylating it at the conserved Ser209). Inhibition of Mnk prevents eIF4E phosphorylation, making the Mnk-eIF4E axis a potential therapeutic target for oncology. Recently, the design and synthesis of a series of novel potent compounds inhibiting the Mnk1/2 kinases were carried out in-house. Here, we describe computational models of the interactions between Mnk1/2 kinases and these inhibitors. Molecular modeling combined with free energy calculations show that these compounds bind to the inactive forms of the kinases. All compounds adopt similar conformations in the catalytic sites of both kinases, stabilized by hydrogen bonds with the hinge regions and with the catalytic Lys78 (Mnk1) and Lys113 (Mnk2). These hydrogen bond interactions clearly play a critical role in determining the conformational stability and potency of the compounds. We also find that van der Waals interactions with an allosteric pocket are key to their binding and potency. Two distinct hydration sites that appear to further stabilize the ligand binding/interactions were observed. Critically, the inclusion of explicit water molecules in the calculations results in improving the agreement between calculated and experimental binding free energies.

2.
Oncotarget ; 8(68): 112825-112840, 2017 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-29348869

RESUMO

Murine double minute 4 protein (MDMX) is crucial for the regulation of the tumor suppressor protein p53. Phosphorylation of the N-terminal domain of MDMX is thought to affect its binding with the transactivation domain of p53, thus playing a role in p53 regulation. In this study, the effects of MDMX phosphorylation on the binding of p53 were investigated using molecular dynamics simulations. It is shown that in addition to the previously proposed mechanism in which phosphorylated Y99 of MDMX inhibits p53 binding through steric clash with P27 of p53, the N-terminal lid of MDMX also appears to play an important role in regulating the phosphorylation-dependent interactions between MDMX and p53. In the proposed mechanism, phosphorylated Y99 aids in pulling the lid into the p53-binding pocket, thus inhibiting the binding between MDMX and p53. Rebinding of p53 appears to be facilitated by the subsequent phosphorylation of Y55, which draws the lid away from the binding pocket by electrostatic attraction of the lid's positively charged N-terminus. The ability to target these mechanisms for the proper regulation of p53 could have important implications for understanding cancer biology and for drug development.

3.
Structure ; 25(1): 188-194, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-27916520

RESUMO

Ligand binding pockets in proteins contain water molecules, which play important roles in modulating protein-ligand interactions. Available crystallographic data for the 5' mRNA cap-binding pocket of the translation initiation factor protein eIF4E shows several structurally conserved waters, which also persist in molecular dynamics simulations. These waters engage an intricate hydrogen-bond network between the cap and protein. Two crystallographic waters in the cleft of the pocket show a high degree of conservation and bridge two residues, which are part of an evolutionarily conserved scaffold. This appears to be a preformed recognition module for the cap with the two structural waters facilitating an efficient interaction. This is also recapitulated in a new crystal structure of the apo protein. These findings open new windows for the design and screening of compounds targeting eIF4E.


Assuntos
Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/metabolismo , Capuzes de RNA/metabolismo , Água/metabolismo , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica
4.
J Phys Chem Lett ; 7(17): 3452-7, 2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27532490

RESUMO

Protein flexibility poses a major challenge in binding site identification. Several computational pocket detection methods that utilize small-molecule probes in molecular dynamics (MD) simulations have been developed to address this issue. Although they have proven hugely successful at reproducing experimental structural data, their ability to predict new binding sites that are yet to be identified and characterized has not been demonstrated. Here, we report the use of benzenes as probe molecules in ligand-mapping MD (LMMD) simulations to predict the existence of two novel binding sites on the surface of the oncoprotein MDM2. One of them was serendipitously confirmed by biophysical assays and X-ray crystallography to be important for the binding of a new family of hydrocarbon stapled peptides that were specifically designed to target the other putative site. These results highlight the predictive power of LMMD and suggest that predictions derived from LMMD simulations can serve as a reliable basis for the identification of novel ligand binding sites in structure-based drug design.


Assuntos
Benzeno/química , Sítios de Ligação , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica
5.
Genes Dev ; 30(3): 281-92, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26798135

RESUMO

The extant jawless vertebrates, represented by lampreys and hagfish, are the oldest group of vertebrates and provide an interesting genomic evolutionary pivot point between invertebrates and jawed vertebrates. Through genome analysis of one of these jawless vertebrates, the Japanese lamprey (Lethenteron japonicum), we identified all three members of the important p53 transcription factor family--Tp53, Tp63, and Tp73--as well as the Mdm2 and Mdm4 genes. These genes and their products are significant cellular regulators in human cancer, and further examination of their roles in this most distant vertebrate relative sheds light on their origin and coevolution. Their important role in response to DNA damage has been highlighted by the discovery of multiple copies of the Tp53 gene in elephants. Expression of lamprey p53, Mdm2, and Mdm4 proteins in mammalian cells reveals that the p53-Mdm2 interaction and the Mdm2/Mdm4 E3 ligase activity existed in the common ancestor of vertebrates and have been conserved for >500 million years of vertebrate evolution. Lamprey Mdm2 degrades human p53 with great efficiency, but this interaction is not blocked by currently available small molecule inhibitors of the human HDM2 protein, suggesting utility of lamprey Mdm2 in the study of the human p53 signaling pathway.


Assuntos
Lampreias/genética , Lampreias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Sequência Conservada , Genoma , Humanos , Lampreias/classificação , Camundongos , Modelos Moleculares , Filogenia , Ligação Proteica , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
6.
PLoS One ; 9(8): e104914, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25115702

RESUMO

As key negative regulator of the p53 tumour suppressor, Mdm2 is an attractive therapeutic target. Small molecules such as Nutlin have been developed to antagonise Mdm2, resulting in p53-dependent death of tumour cells. We have recently described a mutation in Mdm2 (M62A), which precludes binding of Nutlin, but not p53. This Nutlin-resistant variant is not, however, refractory to binding and inhibition by stapled peptide antagonists targeting the same region of Mdm2. A detailed understanding of how stapled peptides are recalcitrant to Mdm2 mutations conferring Nutlin-resistance will aid in the further development of potent Mdm2 antagonists. Here, we report the 2.00 Å crystal structure of a stapled peptide antagonist bound to Nutlin resistant Mdm2. The stapled peptide relies on an extended network of interactions along the hydrophobic binding cleft of Mdm2 for high affinity binding. Additionally, as seen in other stapled peptide structures, the hydrocarbon staple itself contributes to binding through favourable interactions with Mdm2. The structure highlights the intrinsic plasticity present in both Mdm2 and the hydrocarbon staple moiety, and can be used to guide future iterations of both small molecules and stapled peptides for improved antagonists of Mdm2.


Assuntos
Imidazóis/química , Piperazinas/química , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/química , Sequência de Aminoácidos , Ligação Competitiva , Linhagem Celular , Cristalografia por Raios X , Polarização de Fluorescência , Humanos , Imidazóis/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Piperazinas/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo
7.
J Am Chem Soc ; 136(17): 6159-62, 2014 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-24494589

RESUMO

We demonstrate the use of fluorescent molecular rotors as probes for detecting biomolecular interactions, specifically peptide-protein interactions. Molecular rotors undergo twisted intramolecular charge transfer upon irradiation, relax via the nonradiative torsional relaxation pathway, and have been typically used as viscosity probes. Their utility as a tool for detecting specific biomolecular interactions has not been explored. Using the well characterized p53-Mdm2 interaction as a model system, we designed a 9-(2-carboxy-2-cyanovinyl) julolidine-based p53 peptide reporter, JP1-R, which fluoresces conditionally only upon Mdm2 binding. The reporter was used in a rapid, homogeneous assay to screen a fragment library for antagonists of the p53-Mdm2 interaction, and several inhibitors were identified. Subsequent validation of these hits using established secondary assays suggests increased sensitivity afforded by JP1-R. The fluorescence of molecular rotors contingent upon target binding makes them a versatile tool for detecting specific biomolecular interactions.


Assuntos
Corantes Fluorescentes/metabolismo , Nitrilas/metabolismo , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Quinolizinas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Avaliação Pré-Clínica de Medicamentos/métodos , Corantes Fluorescentes/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Nitrilas/química , Peptídeos/química , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Quinolizinas/química , Espectrometria de Fluorescência/métodos , Proteína Supressora de Tumor p53/antagonistas & inibidores , Viscosidade
8.
Cell Cycle ; 12(3): 394-404, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23324352

RESUMO

The interaction of p53 with its regulators MDM2 and MDMX plays a major role in regulating the cell cycle. Inhibition of this interaction has become an important therapeutic strategy in oncology. Although MDM2 and MDMX share a very high degree of sequence/structural similarity, the small-molecule inhibitor nutlin appears to be an efficient inhibitor only of the p53-MDM2 interaction. Here, we investigate the mechanism of interaction of nutlin with these two proteins and contrast it with that of p53 using Brownian dynamics simulations. In contrast to earlier attempts to examine the bound states of the partners, here we locate initial reaction events in these interactions by identifying the regions of space around MDM2/MDMX, where p53/nutlin experience associative encounters with prolonged residence times relative to that in bulk solution. We find that the initial interaction of p53 with MDM2 is long-lived relative to nutlin, but, unlike nutlin, it takes place at the N- and C termini of the MDM2 protein, away from the binding site, suggestive of an allosteric mechanism of action. In contrast, nutlin initially interacts with MDM2 directly at the clefts of the binding site. The interaction of nutlin with MDMX, however, is very short-lived compared with MDM2 and does not show such direct initial interactions with the binding site. Comparison of the topology of the electrostatic potentials of MDM2 and MDMX and the locations of the initial encounters with p53/nutlin in tandem with structure-based sequence alignment revealed that the origin of the diminished activity of nutlin toward MDMX relative to MDM2 may stem partly from the differing topologies of the electrostatic potentials of the two proteins. Glu25 and Lys51 residues underpin these topological differences and appear to collectively play a key role in channelling nutlin directly toward the binding site on the MDM2 surface and are absent in MDMX. The results, therefore, provide new insight into the mechanism of p53/nutlin interactions with MDM2 and MDMX and could potentially have a broader impact on anticancer drug optimization strategies.


Assuntos
Imidazóis/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Piperazinas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Ciclo Celular , Proteínas de Ciclo Celular , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Alinhamento de Sequência , Eletricidade Estática
9.
ACS Chem Biol ; 8(3): 506-12, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23214419

RESUMO

By using a phage display derived peptide as an initial template, compounds have been developed that are highly specific against Mdm2/Mdm4. These compounds exhibit greater potency in p53 activation and protein-protein interaction assays than a compound derived from the p53 wild-type sequence. Unlike Nutlin, a small molecule inhibitor of Mdm2/Mdm4, the phage derived compounds can arrest cells resistant to p53 induced apoptosis over a wide concentration range without cellular toxicity, suggesting they are highly suitable for cyclotherapy.


Assuntos
Peptídeos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ciclo Celular , Humanos , Modelos Moleculares , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Peptídeos/química , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/química
10.
Antimicrob Agents Chemother ; 57(3): 1513-7, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23263008

RESUMO

Emerging resistance to current antibiotics raises the need for new microbial drug targets. We show that targeting branched-chain amino acid (BCAA) biosynthesis using sulfonylurea herbicides, which inhibit the BCAA biosynthetic enzyme acetohydroxyacid synthase (AHAS), can exert bacteriostatic effects on several pathogenic bacteria, including Burkholderia pseudomallei, Pseudomonas aeruginosa, and Acinetobacter baumannii. Our results suggest that targeting biosynthetic enzymes like AHAS, which are lacking in humans, could represent a promising antimicrobial drug strategy.


Assuntos
Acetolactato Sintase/antagonistas & inibidores , Acinetobacter baumannii/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Burkholderia pseudomallei/efeitos dos fármacos , Herbicidas/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Compostos de Sulfonilureia/farmacologia , Acetolactato Sintase/metabolismo , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/crescimento & desenvolvimento , Aminoácidos de Cadeia Ramificada/antagonistas & inibidores , Aminoácidos de Cadeia Ramificada/biossíntese , Animais , Proteínas de Bactérias/metabolismo , Burkholderia pseudomallei/enzimologia , Burkholderia pseudomallei/crescimento & desenvolvimento , Feminino , Melioidose/tratamento farmacológico , Melioidose/microbiologia , Melioidose/mortalidade , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/mortalidade , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Análise de Sobrevida
11.
PLoS One ; 7(8): e43985, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22952838

RESUMO

Atomistic simulations of a set of stapled alpha helical peptides derived from the BH3 helix of MCL-1 (Stewart et al. (2010) Nat Chem Biol 6: 595-601) complexed to a fragment (residues 172-320) of MCL-1 revealed that the highest affinity is achieved when the staples engage the surface of MCL-1 as has also been demonstrated for p53-MDM2 (Joseph et al. (2010) Cell Cycle 9: 4560-4568; Baek et al. (2012) J Am Chem Soc 134: 103-106). Affinity is also modulated by the ability of the staples to pre-organize the peptides as helices. Molecular dynamics simulations of these stapled BH3 peptides were carried out followed by determination of the energies of interactions using MM/GBSA methods. These show that the location of the staple is a key determinant of a good binding stapled peptide from a bad binder. The good binder derives binding affinity from interactions between the hydrophobic staple and a hydrophobic patch on MCL-1. The position of the staple was varied, guiding the design of new stapled peptides with higher affinities.


Assuntos
Desenho de Fármacos , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas/farmacologia , Sequência de Aminoácidos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Soluções
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