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1.
Cell ; 187(7): 1666-1684.e26, 2024 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-38490194

RESUMO

Diminished hepatocyte regeneration is a key feature of acute and chronic liver diseases and after extended liver resections, resulting in the inability to maintain or restore a sufficient functional liver mass. Therapies to restore hepatocyte regeneration are lacking, making liver transplantation the only curative option for end-stage liver disease. Here, we report on the structure-based development and characterization (nuclear magnetic resonance [NMR] spectroscopy) of first-in-class small molecule inhibitors of the dual-specificity kinase MKK4 (MKK4i). MKK4i increased liver regeneration upon hepatectomy in murine and porcine models, allowed for survival of pigs in a lethal 85% hepatectomy model, and showed antisteatotic and antifibrotic effects in liver disease mouse models. A first-in-human phase I trial (European Union Drug Regulating Authorities Clinical Trials [EudraCT] 2021-000193-28) with the clinical candidate HRX215 was conducted and revealed excellent safety and pharmacokinetics. Clinical trials to probe HRX215 for prevention/treatment of liver failure after extensive oncological liver resections or after transplantation of small grafts are warranted.


Assuntos
Inibidores Enzimáticos , Falência Hepática , MAP Quinase Quinase 4 , Animais , Humanos , Camundongos , Hepatectomia/métodos , Hepatócitos , Fígado , Hepatopatias/tratamento farmacológico , Falência Hepática/tratamento farmacológico , Falência Hepática/prevenção & controle , Regeneração Hepática , Suínos , MAP Quinase Quinase 4/antagonistas & inibidores , Inibidores Enzimáticos/uso terapêutico
2.
Biochem J ; 477(22): 4383-4395, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-33111951

RESUMO

A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule that increased the activity of the fungal glycoside hydrolase TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified improved compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate-binding site, generating a productive conformation for the enzyme-substrate complex.


Assuntos
Ativadores de Enzimas/química , Proteínas Fúngicas/química , Hypocreales/química , beta-Glucosidase/química , Ressonância Magnética Nuclear Biomolecular
3.
J Biomol NMR ; 74(10-11): 521-529, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32901320

RESUMO

No matter the source of compounds, drug discovery campaigns focused directly on the target are entirely dependent on a consistent stream of reliable data that reports on how a putative ligand interacts with the protein of interest. The data will derive from many sources including enzyme assays and many types of biophysical binding assays such as TR-FRET, SPR, thermophoresis and many others. Each method has its strengths and weaknesses, but none is as information rich and broadly applicable as NMR. Here we provide a number of examples of the utility of NMR for enabling and providing ongoing support for the early pre-clinical phase of small molecule drug discovery efforts. The examples have been selected for their usefulness in a commercial setting, with full understanding of the need for speed, cost-effectiveness and ease of implementation.


Assuntos
Descoberta de Drogas/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Cristalografia/métodos , Ensaios de Triagem em Larga Escala , Ligantes , Proteínas/isolamento & purificação , Relação Quantitativa Estrutura-Atividade , Bibliotecas de Moléculas Pequenas/química , Soluções/química
4.
Biomol NMR Assign ; 14(2): 269, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32654087

RESUMO

In the original publication of the article, the name of one of the authors is incorrect. The author's name is Eiso AB, but was modified to A. B. Eiso. The correct name is given in this Correction.

5.
Angew Chem Int Ed Engl ; 59(46): 20508-20514, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-32533782

RESUMO

The single-domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, which is one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, which is attributed to an enzyme-induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction.


Assuntos
Glicosídeo Hidrolases/metabolismo , Hidrólise , Cinética , Ligantes , Modelos Moleculares , Ligação Proteica
6.
Int J Mol Sci ; 20(15)2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31357624

RESUMO

The divisome is a large protein complex that regulates bacterial cell division and therefore represents an attractive target for novel antibacterial drugs. In this study, we report on the ligandability of FtsQ, which is considered a key component of the divisome. For this, the soluble periplasmic domain of Escherichia coli FtsQ was immobilized and used to screen a library of 1501 low molecular weight (< 300 Da), synthetic compounds for those that interact with the protein. A primary screen was performed using target immobilized NMR screening (TINS) and yielded 72 hits. Subsequently, these hits were validated in an orthogonal assay. At first, we aimed to do this using surface plasmon resonance (SPR), but the lack of positive control hampered optimization of the experiment. Alternatively, a two-dimensional heteronuclear single quantum coherence (HSQC) NMR spectrum of FtsQ was obtained and used to validate these hits by chemical shift perturbation (CSP) experiments. This resulted in the identification of three fragments with weak affinity for the periplasmic domain of FtsQ, arguing that the ligandability of FtsQ is low. While this indicates that developing high affinity ligands for FtsQ is far from straightforward, the identified hit fragments can help to further interrogate FtsQ interactions.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular , Divisão Celular , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Relação Estrutura-Atividade
7.
Essays Biochem ; 61(5): 485-493, 2017 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-29118095

RESUMO

NMR spectroscopy is a powerful technique that can provide valuable structural information for drug discovery endeavors. Here, we discuss the strengths (and limitations) of NMR applications to structure-based drug discovery, highlighting the different levels of resolution and throughput obtainable. Additionally, the emerging field of paramagnetic NMR in drug discovery and recent developments in approaches to speed up and automate protein-observed NMR data collection and analysis are discussed.


Assuntos
Desenho de Fármacos , Drogas em Investigação/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Bibliotecas de Moléculas Pequenas/química , Sítios de Ligação , Descoberta de Drogas/métodos , Drogas em Investigação/síntese química , Humanos , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Proteínas/agonistas , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Relação Estrutura-Atividade
8.
J Am Chem Soc ; 139(28): 9523-9533, 2017 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-28691806

RESUMO

Methyl groups are powerful probes for the analysis of structure, dynamics and function of supramolecular assemblies, using both solution- and solid-state NMR. Widespread application of the methodology has been limited due to the challenges associated with assigning spectral resonances to specific locations within a biomolecule. Here, we present Methyl Assignment by Graph Matching (MAGMA), for the automatic assignment of methyl resonances. A graph matching protocol examines all possibilities for each resonance in order to determine an exact assignment that includes a complete description of any ambiguity. MAGMA gives 100% accuracy in confident assignments when tested against both synthetic data, and 9 cross-validated examples using both solution- and solid-state NMR data. We show that this remarkable accuracy enables a user to distinguish between alternative protein structures. In a drug discovery application on HSP90, we show the method can rapidly and efficiently distinguish between possible ligand binding modes. By providing an exact and robust solution to methyl resonance assignment, MAGMA can facilitate significantly accelerated studies of supramolecular machines using methyl-based NMR spectroscopy.


Assuntos
Automação , Proteínas de Choque Térmico HSP90/química , Ressonância Magnética Nuclear Biomolecular , Algoritmos , Proteínas de Choque Térmico HSP90/genética , Humanos , Substâncias Macromoleculares/química , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida
9.
J Biol Chem ; 287(10): 7146-58, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22130672

RESUMO

Retinoblastoma-binding protein-6 (RBBP6) plays a facilitating role, through its RING finger-like domain, in the ubiquitination of p53 by Hdm2 that is suggestive of E4-like activity. Although the presence of eight conserved cysteine residues makes it highly probable that the RING finger-like domain coordinates two zinc ions, analysis of the primary sequence suggests an alternative classification as a member of the U-box family, the members of which do not bind zinc ions. We show here that despite binding two zinc ions, the domain adopts a homodimeric structure highly similar to those of a number of U-boxes. Zinc ions could be replaced by cadmium ions without significantly disrupting the structure or the stability of the domain, although the rate of substitution was an order of magnitude slower than any previous measurement, suggesting that the structure is particularly stable, a conclusion supported by the high thermal stability of the domain. A hallmark of U-box-containing proteins is their association with chaperones, with which they cooperate in eliminating irretrievably unfolded proteins by tagging them for degradation by the proteasome. Using a yeast two-hybrid screen, we show that RBBP6 interacts with chaperones Hsp70 and Hsp40 through its N-terminal ubiquitin-like domain. Taken together with the structural similarities to U-box-containing proteins, our data suggest that RBBP6 plays a role in chaperone-mediated ubiquitination and possibly in protein quality control.


Assuntos
Proteínas de Transporte/química , Proteínas de Ligação a DNA/química , Cádmio/química , Cádmio/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Ligação Proteica/fisiologia , Domínios RING Finger , Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases , Ubiquitinação/fisiologia , Zinco/química , Zinco/metabolismo
10.
J Biomol Screen ; 15(8): 978-89, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20817886

RESUMO

Fragment-based drug discovery (FBDD) has become a widely accepted tool that is complementary to high-throughput screening (HTS) in developing small-molecule inhibitors of pharmaceutical targets. Because a fragment campaign can only be as successful as the hit matter found, it is critical that the first stage of the process be optimized. Here the authors compare the 3 most commonly used methods for hit discovery in FBDD: high concentration screening (HCS), solution ligand-observed nuclear magnetic resonance (NMR), and surface plasmon resonance (SPR). They selected the commonly used saturation transfer difference (STD) NMR spectroscopy and the proprietary target immobilized NMR screening (TINS) as representative of the array of possible NMR methods. Using a target typical of FBDD campaigns, the authors find that HCS and TINS are the most sensitive to weak interactions. They also find a good correlation between TINS and STD for tighter binding ligands, but the ability of STD to detect ligands with affinity weaker than 1 mM K(D) is limited. Similarly, they find that SPR detection is most suited to ligands that bind with K(D) better than 1 mM. However, the good correlation between SPR and potency in a bioassay makes this a good method for hit validation and characterization studies.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Proteínas Imobilizadas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/isolamento & purificação , Ressonância de Plasmônio de Superfície/métodos , Técnicas Biossensoriais/métodos , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas Imobilizadas/metabolismo , Proteínas Imobilizadas/farmacologia , Ligantes , Modelos Biológicos , Terapia de Alvo Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Biblioteca de Peptídeos , Ligação Proteica , Bibliotecas de Moléculas Pequenas/análise
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