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1.
J Biol Chem ; 298(12): 102659, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36328246

RESUMO

Self-association of WT ß2-microglobulin (WT-ß2m) into amyloid fibrils is associated with the disorder dialysis related amyloidosis. In the familial variant D76N-ß2m, the single amino acid substitution enhances the aggregation propensity of the protein dramatically and gives rise to a disorder that is independent of renal dysfunction. Numerous biophysical and structural studies on WT- and D76N-ß2m have been performed in order to better understand the structure and dynamics of the native proteins and their different potentials to aggregate into amyloid. However, the structural properties of transient D76N-ß2m oligomers and their role(s) in assembly remained uncharted. Here, we have utilized NMR methods, combined with photo-induced crosslinking, to detect, trap, and structurally characterize transient dimers of D76N-ß2m. We show that the crosslinked D76N-ß2m dimers have different structures from those previously characterized for the on-pathway dimers of ΔN6-ß2m and are unable to assemble into amyloid. Instead, the crosslinked D76N-ß2m dimers are potent inhibitors of amyloid formation, preventing primary nucleation and elongation/secondary nucleation when added in substoichiometric amounts with D76N-ß2m monomers. The results highlight the specificity of early protein-protein interactions in amyloid formation and show how mapping these interfaces can inform new strategies to inhibit amyloid assembly.


Assuntos
Amiloidose , Microglobulina beta-2 , Humanos , Microglobulina beta-2/química , Amiloide/química , Proteínas Amiloidogênicas/genética , Substituição de Aminoácidos , Amiloidose/genética , Fenômenos Biofísicos , Polímeros
2.
J Biol Chem ; 295(35): 12474-12484, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661194

RESUMO

The D76N variant of human ß2-microglobulin (ß2m) is the causative agent of a hereditary amyloid disease. Interestingly, D76N-associated amyloidosis has a distinctive pathology compared with aggregation of WT-ß2m, which occurs in dialysis-related amyloidosis. A folding intermediate of WT-ß2m, known as the IT-state, which contains a nonnative trans Pro-32, has been shown to be a key precursor of WT-ß2m aggregation in vitro However, how a single amino acid substitution enhances the rate of aggregation of D76N-ß2m and gives rise to a different amyloid disease remained unclear. Using real-time refolding experiments monitored by CD and NMR, we show that the folding mechanisms of WT- and D76N-ß2m are conserved in that both proteins fold slowly via an IT-state that has similar structural properties. Surprisingly, however, direct measurement of the equilibrium population of IT using NMR showed no evidence for an increased population of the IT-state for D76N-ß2m, ruling out previous models suggesting that this could explain its enhanced aggregation propensity. Producing a kinetically trapped analog of IT by deleting the N-terminal six amino acids increases the aggregation rate of WT-ß2m but slows aggregation of D76N-ß2m, supporting the view that although the folding mechanisms of the two proteins are conserved, their aggregation mechanisms differ. The results exclude the IT-state as the origin of the rapid aggregation of D76N-ß2m, suggesting that other nonnative states must cause its high aggregation rate. The results highlight how a single substitution at a solvent-exposed site can affect the mechanism of aggregation and the resulting disease.


Assuntos
Amiloide/química , Simulação de Dinâmica Molecular , Agregados Proteicos , Microglobulina beta-2/química , Substituição de Aminoácidos , Amiloide/genética , Cristalografia por Raios X , Humanos , Mutação de Sentido Incorreto , Microglobulina beta-2/genética
3.
Nucleic Acids Res ; 47(3): 1493-1504, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30476241

RESUMO

Trans-splicing of trypanosomatid polycistronic transcripts produces polyadenylated monocistronic mRNAs modified to form the 5' cap4 structure (m7Gpppm36,6,2'Apm2'Apm2'Cpm23,2'U). NMR and X-ray crystallography reveal that Leishmania has a unique type of N-terminally-extended cap-binding protein (eIF4E4) that binds via a PAM2 motif to PABP1. This relies on the interactions of a combination of polar and charged amino acid side-chains together with multiple hydrophobic interactions, and underpins a novel architecture in the Leishmania cap4-binding translation factor complex. Measurements using microscale thermophoresis, fluorescence anisotropy and surface plasmon resonance characterize the key interactions driving assembly of the Leishmania translation initiation complex. We demonstrate that this complex can accommodate Leishmania eIF4G3 which, unlike the standard eukaryotic initiation complex paradigm, binds tightly to eIF4E4, but not to PABP1. Thus, in Leishmania, the chain of interactions 5'cap4-eIF4E4-PABP1-poly(A) bridges the mRNA 5' and 3' ends. Exceptionally, therefore, by binding tightly to two protein ligands and to the mRNA 5' cap4 structure, the trypanosomatid N-terminally extended form of eIF4E acts as the core molecular scaffold for the mRNA-cap-binding complex. Finally, the eIF4E4 N-terminal extension is an intrinsically disordered region that transitions to a partly folded form upon binding to PABP1, whereby this interaction is not modulated by poly(A) binding to PABP1.


Assuntos
Fator de Iniciação 4E em Eucariotos/química , Leishmania/genética , Proteína I de Ligação a Poli(A)/química , Trans-Splicing/genética , Cristalografia por Raios X , Fator de Iniciação 4E em Eucariotos/genética , Ligantes , Espectroscopia de Ressonância Magnética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteína I de Ligação a Poli(A)/genética , Proteínas de Ligação ao Cap de RNA/química , Proteínas de Ligação ao Cap de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética
4.
J Biomol NMR ; 71(1): 1-9, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29752607

RESUMO

We present Farseer-NMR ( https://git.io/vAueU ), a software package to treat, evaluate and combine NMR spectroscopic data from sets of protein-derived peaklists covering a range of experimental conditions. The combined advances in NMR and molecular biology enable the study of complex biomolecular systems such as flexible proteins or large multibody complexes, which display a strong and functionally relevant response to their environmental conditions, e.g. the presence of ligands, site-directed mutations, post translational modifications, molecular crowders or the chemical composition of the solution. These advances have created a growing need to analyse those systems' responses to multiple variables. The combined analysis of NMR peaklists from large and multivariable datasets has become a new bottleneck in the NMR analysis pipeline, whereby information-rich NMR-derived parameters have to be manually generated, which can be tedious, repetitive and prone to human error, or even unfeasible for very large datasets. There is a persistent gap in the development and distribution of software focused on peaklist treatment, analysis and representation, and specifically able to handle large multivariable datasets, which are becoming more commonplace. In this regard, Farseer-NMR aims to close this longstanding gap in the automated NMR user pipeline and, altogether, reduce the time burden of analysis of large sets of peaklists from days/weeks to seconds/minutes. We have implemented some of the most common, as well as new, routines for calculation of NMR parameters and several publication-quality plotting templates to improve NMR data representation. Farseer-NMR has been written entirely in Python and its modular code base enables facile extension.


Assuntos
Bases de Dados de Proteínas , Espectroscopia de Ressonância Magnética/métodos , Software , Conjuntos de Dados como Assunto , Proteínas/química
5.
Biochem Soc Trans ; 46(6): 1753-1770, 2018 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-30545934

RESUMO

The receptor tyrosine kinase family of fibroblast growth factor receptors (FGFRs) play crucial roles in embryonic development, metabolism, tissue homeostasis and wound repair via stimulation of intracellular signalling cascades. As a consequence of FGFRs' influence on cell growth, proliferation and differentiation, FGFR signalling is frequently dysregulated in a host of human cancers, variously by means of overexpression, somatic point mutations and gene fusion events. Dysregulation of FGFRs is also the underlying cause of many developmental dysplasias such as hypochondroplasia and achondroplasia. Accordingly, FGFRs are attractive pharmaceutical targets, and multiple clinical trials are in progress for the treatment of various FGFR aberrations. To effectively target dysregulated receptors, a structural and mechanistic understanding of FGFR activation and regulation is required. Here, we review some of the key research findings from the last couple of decades and summarise the strategies being explored for therapeutic intervention.


Assuntos
Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/química , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Humanos , Transdução de Sinais/fisiologia
6.
J Biol Chem ; 291(4): 1703-1718, 2016 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-26565026

RESUMO

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions.


Assuntos
Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína Son Of Sevenless de Drosófila/química , Proteína Son Of Sevenless de Drosófila/metabolismo , Sítio Alostérico , Domínio Catalítico , Fluorescência , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Son Of Sevenless de Drosófila/genética
7.
J Biomol NMR ; 69(1): 31-44, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28879611

RESUMO

Recently, 15N-detected multidimensional NMR experiments have been introduced for the investigation of proteins. Utilization of the slow transverse relaxation of nitrogen nuclei in a 15N-TROSY experiment allowed recording of high quality spectra for high molecular weight proteins, even in the absence of deuteration. Here, we demonstrate the applicability of three 15N-detected H-N correlation experiments (TROSY, BEST-TROSY and HSQC) to RNA. With the newly established 15N-detected BEST-TROSY experiment, which proves to be the most sensitive 15N-detected H-N correlation experiment, spectra for five RNA molecules ranging in size from 5 to 100 kDa were recorded. These spectra yielded high resolution in the 15N-dimension even for larger RNAs since the increase in line width with molecular weight is more pronounced in the 1H- than in the 15N-dimension. Further, we could experimentally validate the difference in relaxation behavior of imino groups in AU and GC base pairs. Additionally, we showed that 15N-detected experiments theoretically should benefit from sensitivity and resolution advantages at higher static fields but that the latter is obscured by exchange dynamics within the RNAs.


Assuntos
Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular/métodos , RNA/química
8.
Org Biomol Chem ; 14(15): 3782-6, 2016 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-27005701

RESUMO

α-Helix proteomimetics represent an emerging class of ligands that can be used to inhibit an array of helix mediated protein-protein interactions. Within this class of inhibitor, aromatic oligobenzamide foldamers have been widely and successfully used. This manuscript describes alternative syntheses of these compounds that can be used to access mimetics that are challenging to synthesize using previously described methodologies, permitting access to compounds functionalized with multiple sensitive side chains and accelerated library assembly through late stage derivatisation.


Assuntos
Benzamidas/síntese química , Benzamidas/química , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Biomimética/métodos , Modelos Moleculares , Dobramento de Proteína , Estrutura Secundária de Proteína
9.
Angew Chem Int Ed Engl ; 54(16): 4764-7, 2015 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-25693499

RESUMO

An NMR-based approach marries the two traditional screening technologies (phenotypic and target-based screening) to find compounds inhibiting a specific enzymatic reaction in bacterial cells. Building on a previous study in which it was demonstrated that hydrolytic decomposition of meropenem in living Escherichia coli cells carrying New Delhi metallo-ß-lactamase subclass 1 (NDM-1) can be monitored in real time by NMR spectroscopy, we designed a cell-based NMR screening platform. A strong NDM-1 inhibitor was identified with cellular IC50 of 0.51 µM, which is over 300-fold more potent than captopril, a known NDM-1 inhibitor. This new screening approach has great potential to be applied to targets in other cell types, such as mammalian cells, and to targets that are only stable or functionally competent in the cellular environment.


Assuntos
Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Espectroscopia de Prótons por Ressonância Magnética , beta-Lactamases/química , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Meropeném , Ligação Proteica , Tienamicinas/química , Tienamicinas/metabolismo , beta-Lactamases/metabolismo
10.
Chembiochem ; 15(8): 1083-7, 2014 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-24782431

RESUMO

The therapeutically relevant hypoxia inducible factor HIF-1α-p300 protein-protein interaction can be orthosterically inhibited with α-helix mimetics based on an oligoamide scaffold that recapitulates essential features of the C-terminal helix of the HIF-1α C-TAD (C-terminal transactivation domain). Preliminary SAR studies demonstrated the important role of side-chain size and hydrophobicity/hydrophilicity in determining potency. These small molecules represent the first biophysically characterised HIF-1α-p300 PPI inhibitors and the first examples of small-molecule aromatic oligoamide helix mimetics to be shown to have a selective binding profile. Although the compounds were less potent than HIF-1α, the result is still remarkable in that the mimetic reproduces only three residues from the 42-residue HIF-1α C-TAD from which it is derived.


Assuntos
Amidas/farmacologia , Materiais Biomiméticos/farmacologia , Proteína p300 Associada a E1A/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Amidas/síntese química , Amidas/química , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Biomimética , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Proteína p300 Associada a E1A/química , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
11.
Angew Chem Int Ed Engl ; 53(8): 2130-3, 2014 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-24458501

RESUMO

Disconnections between in vitro responses and those observed in whole cells confound many attempts to design drugs in areas of serious medical need. A method based on 1D (1)H NMR spectroscopy is reported that affords the ability to monitor the hydrolytic decomposition of the carbapenem antibiotic meropenem inside Escherichia coli cells expressing New Delhi metallo-ß-lactamase subclass 1 (NDM-1), an emerging antibiotic-resistance threat. Cell-based NMR studies demonstrated that two known NDM-1 inhibitors, L-captopril and ethylenediaminetetraacetic acid (EDTA), inhibit the hydrolysis of meropenem in vivo. NDM-1 activity in cells was also shown to be inhibited by spermine, a porin inhibitor, although in an in vitro assay, the influence of spermine on the activity of isolated NDM-1 protein is minimal. This new approach may have generic utility for monitoring reactions involving diffusible metabolites in other complex biological matrices and whole-cell settings, including mammalian cells.


Assuntos
Escherichia coli/enzimologia , beta-Lactamases/análise , Antibacterianos/química , Antibacterianos/metabolismo , Captopril/química , Captopril/metabolismo , Farmacorresistência Bacteriana , Ácido Edético/química , Ácido Edético/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Hidrólise , Espectroscopia de Ressonância Magnética , Meropeném , Espermina/química , Espermina/metabolismo , Tienamicinas/química , Tienamicinas/metabolismo , beta-Lactamases/metabolismo
12.
Nat Commun ; 13(1): 1040, 2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-35210421

RESUMO

Human islet amyloid polypeptide (hIAPP) self-assembles into amyloid fibrils which deposit in pancreatic islets of type 2 diabetes (T2D) patients. Here, we applied chemical kinetics to study the mechanism of amyloid assembly of wild-type hIAPP and its more amyloidogenic natural variant S20G. We show that the aggregation of both peptides involves primary nucleation, secondary nucleation and elongation. We also report the discovery of two structurally distinct small-molecule modulators of hIAPP assembly, one delaying the aggregation of wt hIAPP, but not S20G; while the other enhances the rate of aggregation of both variants at substoichiometric concentrations. Investigation into the inhibition mechanism(s) using chemical kinetics, native mass spectrometry, fluorescence titration, SPR and NMR revealed that the inhibitor retards primary nucleation, secondary nucleation and elongation, by binding peptide monomers. By contrast, the accelerator predominantly interacts with species formed in the lag phase. These compounds represent useful chemical tools to study hIAPP aggregation and may serve as promising starting-points for the development of therapeutics for T2D.


Assuntos
Diabetes Mellitus Tipo 2 , Ilhotas Pancreáticas , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/metabolismo
13.
Nat Commun ; 12(1): 4045, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193876

RESUMO

RAS mutations are the most common oncogenic drivers across human cancers, but there remains a paucity of clinically-validated pharmacological inhibitors of RAS, as druggable pockets have proven difficult to identify. Here, we identify two RAS-binding Affimer proteins, K3 and K6, that inhibit nucleotide exchange and downstream signaling pathways with distinct isoform and mutant profiles. Affimer K6 binds in the SI/SII pocket, whilst Affimer K3 is a non-covalent inhibitor of the SII region that reveals a conformer of wild-type RAS with a large, druggable SII/α3 pocket. Competitive NanoBRET between the RAS-binding Affimers and known RAS binding small-molecules demonstrates the potential to use Affimers as tools to identify pharmacophores. This work highlights the potential of using biologics with small interface surfaces to select unseen, druggable conformations in conjunction with pharmacophore identification for hard-to-drug proteins.


Assuntos
Produtos Biológicos/farmacologia , Técnicas de Visualização da Superfície Celular/métodos , Descoberta de Drogas/métodos , Neoplasias/tratamento farmacológico , Proteínas ras/antagonistas & inibidores , Sítio Alostérico , Produtos Biológicos/química , Humanos , Neoplasias/química , Neoplasias/enzimologia , Transdução de Sinais , Proteínas ras/metabolismo
14.
Biomol NMR Assign ; 12(2): 231-235, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29582384

RESUMO

Fibroblast growth factors receptors (FGFR) are transmembrane protein tyrosine kinases involved in many cellular process, including growth, differentiation and angiogenesis. Dysregulation of FGFR enzymatic activity is associated with developmental disorders and cancers; therefore FGFRs have become attractive targets for drug discovery, with a number of agents in late-stage clinical trials. Here, we present the backbone resonance assignments of FGFR3 tyrosine kinase domain in the ligand-free form and in complex with the canonical FGFR kinase inhibitor PD173074. Analysis of chemical shift changes upon inhibitor binding highlights a characteristic pattern of allosteric network perturbations that is of relevance for future drug discovery activities aimed at development of conformationally-selective FGFR inhibitors.


Assuntos
Apoproteínas/química , Apoproteínas/metabolismo , Ressonância Magnética Nuclear Biomolecular , Pirimidinas/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Apoproteínas/antagonistas & inibidores , Humanos , Ligação Proteica , Domínios Proteicos , Pirimidinas/farmacologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores
15.
Structure ; 26(3): 446-458.e8, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29478821

RESUMO

Receptor tyrosine kinase FGFR3 is involved in many signaling networks and is frequently mutated in developmental disorders and cancer. The Hsp90/Cdc37 chaperone system is essential for function of normal and neoplastic cells. Here we uncover the mechanistic inter-relationships between these proteins by combining approaches including NMR, HDX-MS, and SAXS. We show that several disease-linked mutations convert FGFR3 to a stronger client, where the determinant underpinning client strength involves an allosteric network through the N-lobe and at the lobe interface. We determine the architecture of the client kinase/Cdc37 complex and demonstrate, together with site-specific information, that binding of Cdc37 to unrelated kinases induces a common, extensive conformational remodeling of the kinase N-lobe, beyond localized changes and interactions within the binary complex. As further shown for FGFR3, this processing by Cdc37 deactivates the kinase and presents it, in a specific orientation established in the complex, for direct recognition by Hsp90.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Mutação , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Sítio Alostérico , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Espalhamento a Baixo Ângulo , Difração de Raios X
16.
Chem Commun (Camb) ; 52(31): 5421-4, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-27009828

RESUMO

Using the HIF-1α transcription factor as a model, this manuscript illustrates how an extended sequence of α-amino acids in a polypeptide can be replaced with a non-natural topographical mimic of an α-helix comprised from an aromatic oligoamide. The resultant hybrid is capable of reproducing the molecular recognition profile of the p300 binding sequence of HIF-1α from which it is derived.


Assuntos
Amidas/química , Materiais Biomiméticos/química , Proteína p300 Associada a E1A/química , Hidrocarbonetos Aromáticos/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Peptídeos/química , Amidas/metabolismo , Sítios de Ligação , Materiais Biomiméticos/metabolismo , Biônica , Proteína p300 Associada a E1A/metabolismo , Humanos , Hidrocarbonetos Aromáticos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Modelos Moleculares , Peptídeos/metabolismo , Conformação Proteica em alfa-Hélice
17.
Oncotarget ; 7(17): 24252-68, 2016 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-26992226

RESUMO

Frequent genetic alterations discovered in FGFRs and evidence implicating some as drivers in diverse tumors has been accompanied by rapid progress in targeting FGFRs for anticancer treatments. Wider assessment of the impact of genetic changes on the activation state and drug responses is needed to better link the genomic data and treatment options. We here apply a direct comparative and comprehensive analysis of FGFR3 kinase domain variants representing the diversity of point-mutations reported in this domain. We reinforce the importance of N540K and K650E and establish that not all highly activating mutations (for example R669G) occur at high-frequency and conversely, that some "hotspots" may not be linked to activation. Further structural characterization consolidates a mechanistic view of FGFR kinase activation and extends insights into drug binding. Importantly, using several inhibitors of particular clinical interest (AZD4547, BGJ-398, TKI258, JNJ42756493 and AP24534), we find that some activating mutations (including different replacements of the same residue) result in distinct changes in their efficacy. Considering that there is no approved inhibitor for anticancer treatments based on FGFR-targeting, this information will be immediately translatable to ongoing clinical trials.


Assuntos
Benzamidas/farmacologia , Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/patologia , Mutação , Neoplasias/genética , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Humanos , Camundongos , Células NIH 3T3 , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
18.
Nat Commun ; 6: 7877, 2015 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-26203596

RESUMO

Protein tyrosine kinases differ widely in their propensity to undergo rearrangements of the N-terminal Asp-Phe-Gly (DFG) motif of the activation loop, with some, including FGFR1 kinase, appearing refractory to this so-called 'DFG flip'. Recent inhibitor-bound structures have unexpectedly revealed FGFR1 for the first time in a 'DFG-out' state. Here we use conformationally selective inhibitors as chemical probes for interrogation of the structural and dynamic features that appear to govern the DFG flip in FGFR1. Our detailed structural and biophysical insights identify contributions from altered dynamics in distal elements, including the αH helix, towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib. We conclude that the αC-ß4 loop and 'molecular brake' regions together impose a high energy barrier for this conformational rearrangement, and that this may have significance for maintaining autoinhibition in the non-phosphorylated basal state of FGFR1.


Assuntos
Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Escherichia coli , Humanos , Imidazóis , Cinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Piridazinas , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores
19.
Mol Biosyst ; 11(10): 2738-49, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26135796

RESUMO

The HIF-1α/p300 protein-protein interaction plays a key role in tumor metabolism and thus represents a high value target for anticancer drug-development. Although several studies have identified inhibitor candidates using rationale design, more detailed understanding of the interaction and binding interface is necessary to inform development of superior inhibitors. In this work, we report a detailed biophysical analysis of the native interaction with both peptide and Adhiron phage display experiments to identify novel binding motifs and binding regions of the surface of p300 to inform future inhibitor design.


Assuntos
Proteína p300 Associada a E1A/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Proteína p300 Associada a E1A/química , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Modelos Moleculares , Biblioteca de Peptídeos , Ligação Proteica/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Estrutura Secundária de Proteína
20.
J Med Chem ; 58(5): 2265-74, 2015 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-25695162

RESUMO

Constitutively active mutant KRas displays a reduced rate of GTP hydrolysis via both intrinsic and GTPase-activating protein-catalyzed mechanisms, resulting in the perpetual activation of Ras pathways. We describe a fragment screening campaign using X-ray crystallography that led to the discovery of three fragment binding sites on the Ras:SOS complex. The identification of tool compounds binding at each of these sites allowed exploration of two new approaches to Ras pathway inhibition by stabilizing or covalently modifying the Ras:SOS complex to prevent the reloading of Ras with GTP. Initially, we identified ligands that bound reversibly to the Ras:SOS complex in two distinct sites, but these compounds were not sufficiently potent inhibitors to validate our stabilization hypothesis. We conclude by demonstrating that covalent modification of Cys118 on Ras leads to a novel mechanism of inhibition of the SOS-mediated interaction between Ras and Raf and is effective at inhibiting the exchange of labeled GDP in both mutant (G12C and G12V) and wild type Ras.


Assuntos
Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína SOS1/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Estrutura Molecular , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína SOS1/química , Bibliotecas de Moléculas Pequenas/química
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