RESUMO
Transcription factors are generally considered challenging, if not "undruggable", targets but they promise new therapeutic options due to their fundamental involvement in many diseases. In this study, we aim to assess the ligandability of the C-terminal Rel-homology domain of nuclear factor of activated T cells 1 (NFAT1), a TF implicated in T-cell regulation. Using a combination of experimental and computational approaches, we demonstrate that small molecule fragments can indeed bind to this protein domain. The newly identified binder is the first small molecule binder to NFAT1 validated with biophysical methods and an elucidated binding mode by X-ray crystallography. The reported eutomer/distomer pair provides a strong basis for potential exploration of higher potency binders on the path toward degrader or glue modalities.
Assuntos
Fatores de Transcrição NFATC , Sítios de Ligação , Cristalografia por Raios X , Ligantes , Fatores de Transcrição NFATC/metabolismo , Fatores de Transcrição NFATC/química , Ligação Proteica , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
Since the late 1980s, mutations in the RAS genes have been recognized as major oncogenes with a high occurrence rate in human cancers. Such mutations reduce the ability of the small GTPase RAS to hydrolyze GTP, keeping this molecular switch in a constitutively active GTP-bound form that drives, unchecked, oncogenic downstream signaling. One strategy to reduce the levels of active RAS is to target guanine nucleotide exchange factors, which allow RAS to cycle from the inactive GDP-bound state to the active GTP-bound form. Here, we describe the identification of potent and cell-active small-molecule inhibitors which efficiently disrupt the interaction between KRAS and its exchange factor SOS1, a mode of action confirmed by a series of biophysical techniques. The binding sites, mode of action, and selectivity were elucidated using crystal structures of KRASG12C-SOS1, SOS1, and SOS2. By preventing formation of the KRAS-SOS1 complex, these inhibitors block reloading of KRAS with GTP, leading to antiproliferative activity. The final compound 23 (BAY-293) selectively inhibits the KRAS-SOS1 interaction with an IC50 of 21 nM and is a valuable chemical probe for future investigations.
Assuntos
Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteína SOS1/antagonistas & inibidores , Linhagem Celular , Cristalografia por Raios X , Descoberta de Drogas , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína SOS1/química , Proteína SOS1/metabolismo , Transdução de SinaisRESUMO
Inhibitors of the ATPase function of bacterial DNA gyrase, located in the GyrB subunit and its related ParE subunit in topoisomerase IV, have demonstrated antibacterial activity. In this study we describe an NMR fragment-based screening effort targeting Staphylococcus aureus GyrB that identified several attractive and novel starting points with good ligand efficiency. Fragment hits were further characterized using NMR binding studies against full-length S. aureus GyrB and Escherichia coli ParE. X-ray co-crystal structures of select fragment hits confirmed binding and suggested a path for medicinal chemistry optimization. The identification, characterization, and elaboration of one of these fragment series to a 0.265 µM inhibitor is described herein.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , DNA Girase/química , Inibidores da Topoisomerase II/química , Adenosina Trifosfatases/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , DNA Girase/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/metabolismo , Desenho de Fármacos , Escherichia coli/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Staphylococcus aureus/enzimologia , Inibidores da Topoisomerase II/metabolismoRESUMO
The backbone 1H, 13C and 15N resonance assignment of Ubiquitin Specific Protease 7 catalytic domain (residues 208-554) was performed in its complex with a small molecule ligand and in its apo form as a reference. The amide 1H-15N signal intensities were boosted by an amide hydrogen exchange protocol, where expressed 2H, 13C, 15N-labeled protein was unfolded and re-folded to ensure exchange of amide deuterons to protons. The resonance assignments were used to determine chemical shift perturbations on ligand binding, which are consistent with the binding site observed by crystallography.
Assuntos
Domínio Catalítico , Ressonância Magnética Nuclear Biomolecular , Humanos , Ligantes , Isótopos de NitrogênioRESUMO
Protein kinase A (PKA) exists as several tissue-specific isoforms that through phosphorylation of serine and threonine residues of substrate proteins act as key regulators of a number of cellular processes. We here demonstrate that the human sperm-specific isoform of PKA named Cα2 is important for sperm motility and thus male fertility. Furthermore, we report on the first three-dimensional crystal structure of human apo Cα2 to 2.1 Å. Apo Cα2 displays an open conformation similar to the well-characterized apo structure of murine Cα1. The asymmetric unit contains two molecules and the core of the small lobe is rotated by almost 13° in the A molecule relative to the B molecule. In addition, a salt bridge between Lys72 and Glu91 was observed for Cα2 in the apo-form, a conformation previously found only in dimeric or ternary complexes of Cα1. Human Cα2 and Cα1 share primary structure with the exception of the amino acids at the N-terminus coded for by an alternative exon 1. The N-terminal glycine of Cα1 is myristoylated and this aliphatic chain anchors the N-terminus to an intramolecular hydrophobic pocket. Cα2 cannot be myristoylated and the crystal structure revealed that the equivalent hydrophobic pocket is unoccupied and exposed. Nuclear magnetic resonance (NMR) spectroscopy further demonstrated that detergents with hydrophobic moieties of different lengths can bind deep into this uncovered pocket. Our findings indicate that Cα2 through the hydrophobic pocket has the ability to bind intracellular targets in the sperm cell, which may modulate protein stability, activity and/or cellular localization.
Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Espermatozoides/metabolismo , Cristalografia por Raios X , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Humanos , Espectroscopia de Ressonância Magnética , MasculinoRESUMO
Inhibitors of MAPKAP kinase 2 (MK2) are expected to attenuate the p38alpha signal transduction pathway in macrophages in a similar way to p38alpha inhibitors and to have a lower propensity for toxic side effects that have slowed the clinical development of the latter. Therefore, novel MK2 inhibitors may find therapeutic application in acute and chronic, TNFalpha-mediated inflammatory conditions like rheumatoid arthritis and others. Herein we have applied fragment screening, using physiologically relevant bioassays and fragment binding mode mapping by protein-observed NMR spectroscopy to the discovery of novel efficient chemical starting points for MK2.
Assuntos
Bioensaio/métodos , Inibidores Enzimáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Fragmentos de Peptídeos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
TSG-6 is an inflammation-associated hyaluronan (HA)-binding protein that has anti-inflammatory and protective functions in arthritis and asthma as well as a critical role in mammalian ovulation. The interaction between TSG-6 and HA is pH-dependent, with a marked reduction in affinity on increasing the pH from 6.0 to 8.0. Here we have investigated the mechanism underlying this pH dependence using a combined approach of site-directed mutagenesis, NMR, isothermal titration calorimetry and microtiter plate assays. Analysis of single-site mutants of the TSG-6 Link module indicated that the loss in affinity above pH 6.0 is mediated by the change in ionization state of a histidine residue (His(4)) that is not within the HA-binding site. To understand this in molecular terms, the pH-dependent folding profile and the pK(a) values of charged residues within the Link module were determined using NMR. These data indicated that His(4) makes a salt bridge to one side-chain oxygen atom of a buried aspartate residue (Asp(89)), whereas the other oxygen is simultaneously hydrogen-bonded to a key HA-binding residue (Tyr(12)). This molecular network transmits the change in ionization state of His(4) to the HA-binding site, which explains the loss of affinity at high pH. In contrast, simulations of the pH affinity curves indicate that another histidine residue, His(45), is largely responsible for the gain in affinity for HA between pH 3.5 and 6.0. The pH-dependent interaction of TSG-6 with HA (and other ligands) provides a means of differentially regulating the functional activity of this protein in different tissue microenvironments.
Assuntos
Moléculas de Adesão Celular/química , Ácido Hialurônico/química , Dobramento de Proteína , Aminoácidos/química , Aminoácidos/metabolismo , Moléculas de Adesão Celular/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Concentração de Íons de Hidrogênio , Ligantes , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
The TipAL protein, a bacterial transcriptional regulator of the MerR family, is activated by numerous cyclic thiopeptide antibiotics. Its C-terminal drug-binding domain, TipAS, defines a subfamily of broadly distributed bacterial proteins including Mta, a central regulator of multidrug resistance in Bacillus subtilis. The structure of apo TipAS, solved by solution NMR [Brookhaven Protein Data Bank entry 1NY9], is composed of a globin-like alpha-helical fold with a deep surface cleft and an unfolded N-terminal region. Antibiotics bind within the cleft at a position that is close to the corresponding heme pocket in myo- and hemoglobin, and induce folding of the N-terminus. Thus the classical globin fold is well adapted not only for accommodating its canonical cofactors, heme and other tetrapyrroles, but also for the recognition of a variety of antibiotics where ligand binding leads to transcriptional activation and drug resistance.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Transativadores/química , Transativadores/metabolismo , Sequência de Aminoácidos , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Relação Estrutura-Atividade , Transativadores/genéticaRESUMO
The solution structure of the Link module from human TSG-6, a hyaladherin with important roles in inflammation and ovulation, has been determined in both its free and hyaluronan-bound conformations. This reveals a well defined hyaluronan-binding groove on one face of the Link module that is closed in the absence of ligand. The groove is lined with amino acids that have been implicated in mediating the interaction with hyaluronan, including two tyrosine residues that appear to form essential intermolecular hydrogen bonds and two basic residues capable of supporting ionic interactions. This is the first structure of a non-enzymic hyaladherin in its active state, and identifies a ligand-induced conformational change that is likely to be conserved across the Link module superfamily. NMR and isothermal titration calorimetry experiments with defined oligosaccharides have allowed us to infer the minimum length of hyaluronan that can be accommodated within the binding site and its polarity in the groove; these data have been used to generate a model of the complex formed between the Link module and a hyaluronan octasaccharide.
Assuntos
Moléculas de Adesão Celular/metabolismo , Ácido Hialurônico/metabolismo , Inflamação/fisiopatologia , Ovulação/fisiologia , Sequência de Aminoácidos , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/fisiologia , Escherichia coli/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
The N-terminal domain of the vaccinia virus protein E3L (Z alpha(E3L)) is essential for full viral pathogenicity in mice. It has sequence similarity to the high-affinity human Z-DNA-binding domains Z alpha(ADAR1) and Z alpha(DLM1). Here, we report the solution structure of Z alpha(E3L) and the chemical shift map of its interaction surface with Z-DNA. The global structure and the Z-DNA interaction surface of Z alpha(E3L) are very similar to the high-affinity Z-DNA-binding domains Z alpha(ADAR1) and Z alpha(DLM1). However, the key Z-DNA contacting residue Y48 of Z alpha(E3L) adopts a different side chain conformation in unbound Z alpha(E3L), which requires rearrangement for binding to Z-DNA. This difference suggests a molecular basis for the significantly lower in vitro affinity of Z alpha(E3L) to Z-DNA compared with its homologues.
Assuntos
DNA Forma Z/química , Proteínas de Ligação a RNA/química , Tirosina , Proteínas Virais/química , Sequência de Aminoácidos , Sítios de Ligação , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , SoluçõesRESUMO
Adhesive interactions involving CD44, the cell surface receptor for hyaluronan, underlie fundamental processes such as inflammatory leukocyte homing and tumor metastasis. Regulation of such events is critical and appears to be effected by changes in CD44 N-glycosylation that switch the receptor "on" or "off" under appropriate circumstances. How altered glycosylation influences binding of hyaluronan to the lectin-like Link module in CD44 is unclear, although evidence suggests additional flanking sequences peculiar to CD44 may be involved. Here we show using X-ray crystallography and NMR spectroscopy that these sequences form a lobular extension to the Link module, creating an enlarged HA binding domain and a formerly unidentified protein fold. Moreover, the disposition of key N-glycosylation sites reveals how specific sugar chains could alter both the affinity and avidity of CD44 HA binding. Our results provide the necessary structural framework for understanding the diverse functions of CD44 and developing novel therapeutic strategies.