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2.
Comput Struct Biotechnol J ; 17: 160-176, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788082

RESUMO

Targeting the "undruggable" proteome remains one of the big challenges in drug discovery. Recent innovations in the field of targeted protein degradation and manipulation of the ubiquitin-proteasome system open up new therapeutic approaches for disorders that cannot be targeted with conventional inhibitor paradigms. Proteolysis targeting chimeras (PROTACs) are bivalent ligands in which a compound that binds to the protein target of interest is connected to a second molecule that binds an E3 ligase via a linker. The E3 protein is usually either Cereblon or Von Hippel-Lindau. Several examples of selective PROTAC molecules with potent effect in cells and in vivo models have been reported. The degradation of specific proteins via these bivalent molecules is already allowing for the study of biochemical pathways and cell biology with more specificity than was possible with inhibitor compounds. In this review, we provide a comprehensive overview of recent developments in the field of small molecule mediated protein degradation, including transcription factors, kinases and nuclear receptors. We discuss the potential benefits of protein degradation over inhibition as well as the challenges that need to be overcome.

3.
Drug Discov Today ; 23(1): 12-16, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28782685

RESUMO

Since the importance of drug target residence time was first highlighted more 10 years ago, slow binding kinetics has received much attention in the drug discovery literature, and indeed within pharmaceutical research. However, the residence concept as presented in most papers is supported by rather misleading simulations and arguments, and by examples where compounds are taken out of their pharmacokinetic context. Moreover, fast association is typically more desirable than slow, and advantages of long residence time, notably a potential disconnect between pharmacodynamics (PD) and pharmacokinetics (PK), would be partially or completely offset by slow on-rate. Therefore, plain potency is likely a better predictor of drug development success than is residence time.


Assuntos
Descoberta de Drogas , Farmacocinética , Animais , Cinética , Ligantes , Modelos Biológicos , Preparações Farmacêuticas/metabolismo
4.
SLAS Discov ; 22(2): 203-209, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27789754

RESUMO

Surface plasmon resonance (SPR) is a powerful method for obtaining detailed molecular interaction parameters. Modern instrumentation with its increased throughput has enabled routine screening by SPR in hit-to-lead and lead optimization programs, and SPR has become a mainstream drug discovery technology. However, the processing and reporting of SPR data in drug discovery are typically performed manually, which is both time-consuming and tedious. Here, we present the workflow concept, design and experiences with a software module relying on a single, browser-based software platform for the processing, analysis, and reporting of SPR data. The efficiency of this concept lies in the immediate availability of end results: data are processed and analyzed upon loading the raw data file, allowing the user to immediately quality control the results. Once completed, the user can automatically report those results to data repositories for corporate access and quickly generate printed reports or documents. The software module has resulted in a very efficient and effective workflow through saved time and improved quality control. We discuss these benefits and show how this process defines a new benchmark in the drug discovery industry for the handling, interpretation, visualization, and sharing of SPR data.


Assuntos
Técnicas Biossensoriais/métodos , Análise de Dados , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/tendências , Desenho de Fármacos , Humanos , Pesquisa Farmacêutica , Software , Ressonância de Plasmônio de Superfície , Fluxo de Trabalho
5.
Drug Discov Today ; 21(3): 491-8, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26826422

RESUMO

Over the past decade biophysics has become an established discipline in HTS hit triaging, owing to its high fidelity in detecting protein-ligand interactions. Many pharma companies are using biophysical techniques to filter HTS output for false positives, as will be discussed in this review. Moreover, I will demonstrate how the earlier application of biophysics, already at the HTS assay development stage, is potentially even more impactful. Two key areas here are early mode-of-action studies and ensuring that the HTS assay and subsequent cascade are fit for purpose. Top-level results from 20 in-house projects are shown to underpin the impact of these studies.


Assuntos
Fenômenos Biofísicos , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Bioensaio , Redução de Custos , Descoberta de Drogas/economia , Ensaios de Triagem em Larga Escala/economia , Indicadores e Reagentes
6.
Bioorg Med Chem ; 19(10): 3039-53, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21515056

RESUMO

Inhibition of acetyl-CoA carboxylases has the potential for modulating long chain fatty acid biosynthesis and mitochondrial fatty acid oxidation. Hybridization of weak inhibitors of ACC2 provided a novel, moderately potent but lipophilic series. Optimization led to compounds 33 and 37, which exhibit potent inhibition of human ACC2, 10-fold selectivity over inhibition of human ACC1, good physical and in vitro ADME properties and good bioavailability. X-ray crystallography has shown this series binding in the CT-domain of ACC2 and revealed two key hydrogen bonding interactions. Both 33 and 37 lower levels of hepatic malonyl-CoA in vivo in obese Zucker rats.


Assuntos
Acetil-CoA Carboxilase/antagonistas & inibidores , Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Obesidade/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Acetil-CoA Carboxilase/metabolismo , Animais , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/enzimologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Ácidos Graxos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Malonil Coenzima A/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Obesidade/enzimologia , Ratos , Ratos Zucker , Bibliotecas de Moléculas Pequenas/farmacocinética , Bibliotecas de Moléculas Pequenas/uso terapêutico , Relação Estrutura-Atividade
8.
J Med Chem ; 51(7): 2178-86, 2008 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-18341273

RESUMO

We describe the discovery of novel inhibitors of prostaglandin D2 synthase (PGDS) through fragment-based lead generation and structure-based drug design. A library of 2500 low-molecular-weight compounds was screened using 2D nuclear magnetic resonance (NMR), leading to the identification of 24 primary hits. Structure determination of protein-ligand complexes with the hits enabled a hit optimization process, whereby we harvested increasingly more potent inhibitors out of our corporate compound collection. Two iterative cycles were carried out, comprising NMR screening, molecular modeling, X-ray crystallography, and in vitro biochemical testing. Six novel high-resolution PGDS complex structures were determined, and 300 hit analogues were tested. This rational drug design procedure culminated in the discovery of 24 compounds with an IC 50 below 1 microM in the in vitro assay. The best inhibitor (IC 50 = 21 nM) is one of the most potent inhibitors of PGDS to date. As such, it may enable new functional in vivo studies of PGDS and the prostaglandin metabolism pathway.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Lipocalinas/antagonistas & inibidores , Cristalografia por Raios X , Inibidores Enzimáticos/química , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Peso Molecular , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade
9.
Curr Top Med Chem ; 7(16): 1600-29, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17979771

RESUMO

Fragment-based lead generation (FBLG) has recently emerged as an alternative to traditional high throughput screening (HTS) to identify initial chemistry starting points for drug discovery programs. In comparison to HTS screening libraries, the screening sets for FBLG tend to contain orders of magnitude fewer compounds, and the compounds themselves are less structurally complex and have lower molecular weight. This report summarises the advent of FBLG within the industry and then describes the FBLG experience at AstraZeneca. We discuss (1) optimising the design of screening libraries, (2) hit detection methodologies, (3) evaluation of hit quality and use of ligand efficiency calculations, and (4) approaches to evolve fragment-based, low complexity hits towards drug-like leads. Furthermore, we exemplify our use of FBLG with case studies in the following drug discovery areas: antibacterial enzyme targets, GPCRs (melanocortin 4 receptor modulators), prostaglandin D2 synthase inhibitors, phosphatase inhibitors (protein tyrosine phosphotase 1B), and protease inhibitors (b-secretase).


Assuntos
Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Técnicas de Química Combinatória , Indústria Farmacêutica/métodos , Inibidores Enzimáticos , Ligantes , Ligação Proteica
10.
J Med Chem ; 50(24): 5903-11, 2007 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-17985861

RESUMO

Fragment-based lead generation was applied to find novel small-molecule inhibitors of beta-secretase (BACE-1), a key target for the treatment of Alzheimer's disease. Fragment hits coming from a 1D NMR screen were characterized by BIAcore, and the most promising compounds were soaked into protein crystals to help the rational design of more potent hit analogues. Problems arising due to our inability to grow BACE-1 crystals at the biologically relevant pH at which the screen was run were overcome by using endothiapepsin as a surrogate aspartyl protease. Among others, we identified 6-substituted isocytosines as a novel warhead against BACE-1, and the accompanying paper in this journal describes how these were optimized to a lead series of nanomolar inhibitors.1.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Citosina/análogos & derivados , Desenho de Fármacos , Inibidores Enzimáticos/química , Secretases da Proteína Precursora do Amiloide/isolamento & purificação , Ácido Aspártico Endopeptidases/isolamento & purificação , Linhagem Celular , Cristalografia por Raios X , Citosina/química , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Relação Estrutura-Atividade
11.
J Med Chem ; 50(24): 5912-25, 2007 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-17985862

RESUMO

Fragment-based lead generation has led to the discovery of a novel series of cyclic amidine-based inhibitors of beta-secretase (BACE-1). Initial fragment hits with an isocytosine core having millimolar potency were identified via NMR affinity screening. Structure-guided evolution of these fragments using X-ray crystallography together with potency determination using surface plasmon resonance and functional enzyme inhibition assays afforded micromolar inhibitors. Similarity searching around the isocytosine core led to the identification of a related series of inhibitors, the dihydroisocytosines. By leveraging the knowledge of the ligand-BACE-1 recognition features generated from the isocytosines, the dihydroisocytosines were efficiently optimized to submicromolar potency. Compound 29, with an IC50 of 80 nM, a ligand efficiency of 0.37, and cellular activity of 470 nM, emerged as the lead structure for future optimization.


Assuntos
Amidinas/síntese química , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Citosina/análogos & derivados , Modelos Moleculares , Pirimidinas/síntese química , Amidinas/química , Amidinas/farmacologia , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Linhagem Celular , Cristalografia por Raios X , Citosina/síntese química , Citosina/química , Citosina/farmacologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Pirimidinas/química , Pirimidinas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade
12.
Nature ; 447(7146): 817-22, 2007 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-17568739

RESUMO

Glutamate racemase is an enzyme essential to the bacterial cell wall biosynthesis pathway, and has therefore been considered as a target for antibacterial drug discovery. We characterized the glutamate racemases of several pathogenic bacteria using structural and biochemical approaches. Here we describe three distinct mechanisms of regulation for the family of glutamate racemases: allosteric activation by metabolic precursors, kinetic regulation through substrate inhibition, and D-glutamate recycling using a d-amino acid transaminase. In a search for selective inhibitors, we identified a series of uncompetitive inhibitors specifically targeting Helicobacter pylori glutamate racemase that bind to a cryptic allosteric site, and used these inhibitors to probe the mechanistic and dynamic features of the enzyme. These structural, kinetic and mutational studies provide insight into the physiological regulation of these essential enzymes and provide a basis for designing narrow-spectrum antimicrobial agents.


Assuntos
Isomerases de Aminoácido/química , Isomerases de Aminoácido/metabolismo , Bactérias/enzimologia , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Isomerases de Aminoácido/antagonistas & inibidores , Isomerases de Aminoácido/genética , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bactérias/patogenicidade , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Helicobacter pylori/enzimologia , Cinética , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade
13.
Biochem Biophys Res Commun ; 335(2): 361-6, 2005 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-16061201

RESUMO

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in 1H-15N correlation spectra of a 15N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the 1H-15N correlations was achieved using a suite of triple resonance NMR experiments with 15N,13C,70% 2H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbations to 1H-15N spectra revealed that the N-termini, alpha3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , DNA/química , Espectroscopia de Ressonância Magnética/métodos , Sítios de Ligação , Cristalografia por Raios X , Cinética , Modelos Moleculares , Oligonucleotídeos/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
14.
Biochemistry ; 41(48): 14176-84, 2002 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-12450381

RESUMO

The protein kinase ZAP-70 is involved in T-cell activation, and interacts with tyrosine-phosphorylated peptide sequences known as immunoreceptor tyrosine activation motifs (ITAMs), which are present in three of the subunits of the T-cell receptor. We have studied the tandem SH2 (tSH2) domains of ZAP-70, by both X-ray and NMR. Here, we present the crystal structure of the apoprotein, i.e., the tSH2 domain in the absence of ITAM. Comparison with the previously reported complex structure reveals that binding to the ITAM peptide induces surprisingly large movements between the two SH2 domains and within the actual binding sites. The conformation of the ITAM-free protein is partly governed by a hydrophobic cluster between the linker region and the C-terminal SH2 domain. Our data suggest that the two SH2 domains are able to undergo large interdomain movements. The proposed relative flexibility of the SH2 domains is further supported by the finding that no NMR signals could be detected for the two helices connecting the SH2 domains; these are likely to be broadened beyond detection due to conformational exchange. It is likely that this conformational reorientation induced by ITAM binding is the main signaling event activating the kinase domain in ZAP-70. Another NMR observation was that the N-terminal SH2 domain could bind tetrapeptides derived from the ITAM sequence, apparently without the need to interact with the C-terminal domain. In contrast, the C-terminal domain has little affinity for tetrapeptides. The opposite situation is true for binding to plain phosphotyrosine, where the C-terminal domain has a higher affinity. Distinct features in the crystal structure, showing the interdependence of both domains, explain these binding data.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas Tirosina Quinases/química , Domínios de Homologia de src , Motivos de Aminoácidos , Sequência de Aminoácidos , Apoenzimas/química , Sítios de Ligação , Simulação por Computador , Cristalização , Cristalografia por Raios X , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfotirosina/química , Fosfotirosina/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Propriedades de Superfície , Proteína-Tirosina Quinase ZAP-70
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