The glucocorticoid receptor (GR) is a nuclear hormone receptor that regulates key genes controlling development, metabolism, and the immune response. GR agonists are efficacious for treatment of ...inflammatory, allergic, and immunological disorders. Steroid hormone binding to the ligand-binding domain (LBD) of GR is known to change the structural and dynamical properties of the receptor, which in turn control its interactions with DNA and various co-regulators and drive the pharmacological response. Previous biophysical studies of the GR LBD have required the use of mutant forms to overcome issues with limited protein stability and high aggregation propensity. However, these mutant variants are known to also influence the functional response of the receptor. Here we report a successful protocol for protein expression, purification, and NMR characterization of the wildtype human GR LBD. We achieved chemical shift assignments for 90% of the LBD backbone resonances, with 216 out of 240 non-proline residues assigned in the
H-
N TROSY spectrum. These advancements form the basis for future investigations of allosteric effects in GR signaling.
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 IC50 below 1 µM in the in vitro assay. The best inhibitor (IC50 = 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.
Trigger happy! High‐throughput screening was used to identify selective MMP7 inhibitors. Subsequent analysis by X‐ray crystallography revealed the underlying features for the selectivity trigger, ...unique to MMP7. In addition, further structural studies increased the understanding of the plasticity of the MMP7 active site. Finally, a single round of chemistry resulted in compounds with improved potency and retained selectivity.
The mineralocorticoid receptor (MR) is a nuclear hormone receptor involved in the regulation of body fluid and electrolyte homeostasis. In this study we explore selectivity triggers for a series of ...nonsteroidal MR antagonists to improve selectivity over other members of the oxosteroid receptor family. A biaryl sulfonamide compound was identified in a high‐throughput screening (HTS) campaign. The compound bound to MR with pKi=6.6, but displayed poor selectivity over the glucocorticoid receptor (GR) and the progesterone receptor (PR). Following X‐ray crystallography of MR in complex with the HTS hit, a compound library was designed that explored an induced‐fit hypothesis that required movement of the Met852 side chain. An improvement in MR selectivity of 11‐ to 79‐fold over PR and 23‐ to 234‐fold over GR was obtained. Given the U‐shaped binding conformation, macrocyclizations were explored, yielding a macrocycle that bound to MR with pKi=7.3. Two protein–ligand X‐ray structures were determined, confirming the hypothesized binding mode for the designed compounds.
Looking for MR right: A biarylsulfonamide was identified as a mineralocorticoid receptor (MR) antagonist in a high‐throughput screening campaign. Structure‐based drug design was used to improve selectivity over the oxosteroid receptors by exploring an induced‐fit pocket and expanding the ligands toward an area where the receptors differ from each other. X‐ray crystallography confirmed the binding mode and inspired macrocyclization to test conformationally restrained MR antagonists.
The glucocorticoid receptor (GR) is a nuclear hormone receptor that regulates key genes controlling development, metabolism, and the immune response. GR agonists are efficacious for treatment of ...inflammatory, allergic, and immunological disorders. Steroid hormone binding to the ligand-binding domain (LBD) of GR is known to change the structural and dynamical properties of the receptor, which in turn control its interactions with DNA and various co-regulators and drive the pharmacological response. Previous biophysical studies of the GR LBD have required the use of mutant forms to overcome issues with limited protein stability and high aggregation propensity. However, these mutant variants are known to also influence the functional response of the receptor. Here we report a successful protocol for protein expression, purification, and NMR characterization of the wildtype human GR LBD. We achieved chemical shift assignments for 90% of the LBD backbone resonances, with 216 out of 240 non-proline residues assigned in the
1
H–
15
N TROSY spectrum. These advancements form the basis for future investigations of allosteric effects in GR signaling.
The first known non-covalent inhibitors of rhinovirus 3C protease (3CP) have been identified through fragment based screening and hit identification activities.
Studies on the catalytic mechanism and inhibition of serine proteases are widely used as paradigms for teaching enzyme catalysis. Ground-breaking work on the structures of chymotrypsin and subtilisin ...led to the idea of a conserved catalytic triad formed by the active site Ser, His and Asp residues. An oxyanion hole, consisting of the peptide amide of the active site serine and a neighbouring glycine, was identified, and hydrogen bonding in the oxyanion hole was suggested to stabilize the two proposed tetrahedral intermediates on the catalytic pathway. Here we show electron density changes consistent with the formation of a tetrahedral intermediate during the hydrolysis of an acyl-enzyme complex formed between a natural heptapeptide and elastase. No electron density for an enzyme-product complex was observed. The structures also suggest a mechanism for the synchronization of hydrolysis and peptide release triggered by the conversion of the sp2 hybridized carbonyl carbon to an sp3 carbon in the tetrahedral intermediate. This affects the location of the peptide in the active site cleft, triggering the collapse of a hydrogen bonding network between the peptide and the β-sheet of the active site.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
38.
11th German Conference on Chemoinformatics Fechner, Uli; Evers, Andreas; Richmond, Nicola J ...
Journal of cheminformatics,
04/2016, Letnik:
8, Številka:
1
Journal Article
Time-resolved structural studies on biomolecular function are coming of age. Focus has shifted from studies on 'systems of opportunities' to a more problem-oriented approach, addressing significant ...questions in biology and chemistry. An important step in this direction has been the use of physical and chemical trapping methods to capture and then freeze reaction intermediates in crystals. Subsequent monochromatic data collection at cryogenic temperatures can produce high resolution structures of otherwise elusive intermediates. The combination of diffraction methods with spectroscopic techniques provides a means to directly correlate electronic transitions with structural transitions in the sample, eliminating much of the guesswork from experiments. Studies on cytochrome P450, isopenicillin N synthase, cytochrome cd1 nitrite reductase, copper amine oxidase and bacteriorhodopsin were selected as examples, and the results are discussed.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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