Paradoxically, Most Flexible Ligand Binds Most Entropy-Favored: Intriguing Impact of Ligand Flexibility and Solvation on Drug-Kinase Binding

Biophysical parameters can accelerate drug development; e.g., rigid ligands may reduce entropic penalty and improve binding affinity. We studied systematically the impact of ligand rigidification on thermodynamics using a series of fasudil derivatives inhibiting protein kinase A by crystallography, isothermal titration calorimetry, nuclear magnetic resonance, and molecular dynamics simulations. The ligands varied in their internal degrees of freedom but conserve the number of heteroatoms. Counterintuitively, the most flexible ligand displays the entropically most favored binding. As experiment shows, this cannot be explained by higher residual flexibility of ligand, protein, or formed complex nor by a deviating or increased release of water molecules upon complex formation. NMR and crystal structures show no differences in flexibility and water release, although strong ligand-induced adaptations are observed. Instead, the flexible ligand entraps more efficiently water molecules in solution <i>prior</i> to protein binding, and by release of these waters, the favored entropic binding is observed

Cysteine oxidation and disulfide formation in the ribosomal exit tunnel.

Funder: DFG graduate college: CLiC State of Hesse HMWK: BMRZUnderstanding the conformational sampling of translation-arrested ribosome nascent chain complexes is key to understand co-translational folding. Up to now, coupling of cysteine oxidation, disulfide bond formation and structure formation in nascent chains has remained elusive. Here, we investigate the eye-lens protein γB-crystallin in the ribosomal exit tunnel. Using mass spectrometry, theoretical simulations, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance and cryo-electron microscopy, we show that thiol groups of cysteine residues undergo S-glutathionylation and S-nitrosylation and form non-native disulfide bonds. Thus, covalent modification chemistry occurs already prior to nascent chain release as the ribosome exit tunnel provides sufficient space even for disulfide bond formation which can guide protein folding

Modulation of the Allosteric Communication between the Polo-Box Domain and the Catalytic Domain in Plk1 by Small Compounds

The Polo-like kinases (Plks) are an evolutionary conserved family of Ser/Thr protein kinases that possess, in addition to the classical kinase domain at the N-terminus, a C-terminal polo-box domain (PBD) that binds to phosphorylated proteins and modulates the kinase activity and its localization. Plk1, which regulates the formation of the mitotic spindle, has emerged as a validated drug target for the treatment of cancer, because it is required for numerous types of cancer cells but not for the cell division in noncancer cells. Here, we employed chemical biology methods to investigate the allosteric communication between the PBD and the catalytic domain of Plk1. We identified small compounds that bind to the catalytic domain and inhibit or enhance the interaction of Plk1 with the phosphorylated peptide PoloBoxtide in vitro. In cells, two new allosteric Plk1 inhibitors affected the proliferation of cancer cells in culture and the cell cycle but had distinct phenotypic effects on spindle formation. Both compounds inhibited Plk1 signaling, indicating that they specifically act on Plk1 in cultured cells.Fil: Raab, Monika. Goethe Universitat Frankfurt; AlemaniaFil: Sanhaji, Mourad. Goethe Universitat Frankfurt; AlemaniaFil: Pietsch, Larissa. German Cancer Research Center; Alemania. Goethe Universitat Frankfurt; AlemaniaFil: Béquignon, Isabelle. Goethe Universitat Frankfurt; AlemaniaFil: Herbrand, Amanda K.. Goethe Universitat Frankfurt; AlemaniaFil: Süß, Evelyn. Goethe Universitat Frankfurt; AlemaniaFil: Gande, Santosh L.. German Cancer Research Center; Alemania. Goethe Universitat Frankfurt; AlemaniaFil: Caspar, Birgit. Goethe Universitat Frankfurt; AlemaniaFil: Kudlinzki, Denis. Goethe Universitat Frankfurt; Alemania. German Cancer Research Center; AlemaniaFil: Saxena, Krishna. Goethe Universitat Frankfurt; AlemaniaFil: Sreeramulu, Sridhar. Goethe Universitat Frankfurt; AlemaniaFil: Schwalbe, Harald. Goethe Universitat Frankfurt; Alemania. German Cancer Research Center; AlemaniaFil: Strebhardt, Klaus. Goethe Universitat Frankfurt; Alemania. German Cancer Research Center; AlemaniaFil: Biondi, Ricardo Miguel. German Cancer Research Center; Alemania. Goethe Universitat Frankfurt; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentin

Strutural characterization of the C-terminal domain of the spliceosomal DExD/H-Box protein hPrp22

Das Spleißen von prä-mRNA ist einer der dynamischsten Prozesse in der eukaryotischen Zelle. Neben den UsnRNAs ist eine Vielzahl von Proteinen am Spleißprozeß beteiligt. Einige dieser Proteine sind mobile Faktoren, während andere Komponenten integrale Bestandteile des spleißosomalen Komplexes sind. Die meisten dieser Proteine sind nicht direkt an der Entfernung der Introns und der Ligation der Exons beteiligt, sondern vermitteln Protein-Protein- bzw. Protein-RNA-Kontakte oder katalysieren Reaktionen, die zur internen Umlagerung des Spleißosoms führen. Eine am Spleißen beteiligte Proteinklasse sind die DExD/H-Box RNA-Helikasen. Sie können interne Basenpaarungen zwischen UsnRNAs oder UsnRNA-mRNA-Basenpaarungen entwinden. Ein Protein dieser Klasse ist das DEAH-Box Protein Prp22. Es wurde gezeigt, dass das homologe Protein yPrp22 aus Saccharomyces cerevisiae ATP-abhängige RNA-Helikase Aktivität zeigt, die zur Entwindung von RNA-Interaktionen zwischen U5 snRNP und mRNA führt. Dies führt zum Ablösen der reifen mRNA vom post-spleißosomalen Komplex. Nur die vollständig prozessierte genetische Information kann denn Zellkern verlassen und wird an den Ribosomen translatiert. Im Rahmen dieser Doktorarbeit wurde die dreidimensionale Kristallstruktur der C-terminalen Domäne des spleißosomalen DExD/H-Box Proteins hPrp22 (Aminosäuren 950 - 1183) aus Homo sapiens charakterisiert. Hierzu wurden die Phasen de novo mittels anomaler Dispersion von Brom und Selen bestimmt. Die Kristallstruktur birgt ein neues Faltungsmotiv. Basierend auf der Struktur der C-terminalen Domäne von hPrp22 wurden Modelle anderer homologer C-terminaler Domänen von hPrp2, hPrp16 und hPrp43 generiert. Durch diese Modelle konnten Aussagen über die Verteilung der Oberflächenladungen getroffen werden. Es zeigte sich, dass auf einer Seite der verschiedenen Domänen die Ladungsverteilung konserviert ist, während auf der gegenüberliegenden Seite signifikante Unterschiede erkennbar sind. Weiterhin wurden zahlreiche Deletionsmutanten von hPrp22 und hPrp2, einem weiteren spleißosomalen DExD/H-Box Protein, entworfen. Einige dieser verkürzten Proteinfragmente wurden gereinigt und auf ATPase-Aktivität untersucht. Dazu wurde ein enzym-gekoppelter photometrischer ATPase-Test angepasst. Die Absorptionsabnahme durch NADH-Verbrauch wird dabei bestimmt. Erste Ergebnisse weisen darauf hin, dass die Anwesenheit der C-terminalen Domäne die ATPase-Aktivität von hPrp22 stimuliert

Impact of azidohomoalanine incorporation on protein structure and ligand binding

The impact of the incorporation of a non-natural amino acid (NNAA) on protein structure, dynamics, and ligand binding has not been studied rigorously so far. NNAAs are regularly used to modify proteins post-translationally in vivo and in vitro through click chemistry. Herein, structural characterisation of the impact of the incorporation of azidohomoalanine (AZH) into the model protein domain PDZ3 is examined by means of NMR spectroscopy and X-ray crystallography. The structure and dynamics of the apo state of AZH-modified PDZ3 remain mostly unperturbed. Furthermore, the binding of two PDZ3 binding peptides are unchanged upon incorporation of AZH. The interface of the AZH-modified PDZ3 and an azulene-linked peptide for vibrational energy transfer studies has been mapped by means of chemical shift perturbations and NOEs between the unlabelled azulene-linked peptide and the isotopically labelled protein. Co-crystallisation and soaking failed for the peptide-bound holo complex. NMR spectroscopy, however, allowed determination of the protein-ligand interface. Although the incorporation of AZH was minimally invasive for PDZ3, structural analysis of NNAA-modified proteins through the methodology presented herein should be performed to ensure structural integrity of the studied target

Identification of eph receptor signaling as a regulator of autophagy and a therapeutic target in colorectal carcinoma

Advanced colorectal carcinoma is currently incurable, and new therapies are urgently needed. We report that phosphotyrosine-dependent Eph receptor signaling sustains colorectal carcinoma cell survival, thereby uncovering a survival pathway active in colorectal carcinoma cells. We find that genetic and biochemical inhibition of Eph tyrosine kinase activity or depletion of the Eph ligand EphrinB2 reproducibly induces colorectal carcinoma cell death by autophagy. Spautin and 3-methyladenine, inhibitors of early steps in the autophagic pathway, significantly reduce autophagy-mediated cell death that follows inhibition of phosphotyrosine-dependent Eph signaling in colorectal cancer cells. A small-molecule inhibitor of the Eph kinase, NVP-BHG712 or its regioisomer NVP-Iso, reduces human colorectal cancer cell growth in vitro and tumor growth in mice. Colorectal cancers express the EphrinB ligand and its Eph receptors at significantly higher levels than numerous other cancer types, supporting Eph signaling inhibition as a potential new strategy for the broad treatment of colorectal carcinoma

Chemical Proteomics and Structural Biology Define EPHA2 Inhibition by Clinical Kinase Drugs

The receptor tyrosine kinase EPHA2 (Ephrin type-A receptor 2) plays important roles in oncogenesis, metastasis, and treatment resistance, yet therapeutic targeting, drug discovery, or investigation of EPHA2 biology is hampered by the lack of appropriate inhibitors and structural information. Here, we used chemical proteomics to survey 235 clinical kinase inhibitors for their kinase selectivity and identified 24 drugs with submicromolar affinities for EPHA2. NMR-based conformational dynamics together with nine new cocrystal structures delineated drug–EPHA2 interactions in full detail. The combination of selectivity profiling, structure determination, and kinome wide sequence alignment allowed the development of a classification system in which amino acids in the drug binding site of EPHA2 are categorized into key, scaffold, potency, and selectivity residues. This scheme should be generally applicable in kinase drug discovery, and we anticipate that the provided information will greatly facilitate the development of selective EPHA2 inhibitors in particular and the repurposing of clinical kinase inhibitors in general

Optimized Plk1 PBD Inhibitors Based on Poloxin Induce Mitotic Arrest and Apoptosis in Tumor Cells

Polo-like kinase 1 (Plk1) is a central regulator of mitosis and has been validated as a target for antitumor therapy. The polo-box domain (PBD) of Plk1 regulates its kinase activity and mediates the subcellular localization of Plk1 and its interactions with a subset of its substrates. Functional inhibition of the Plk1 PBD by low-molecular weight inhibitors has been shown to represent a viable strategy by which to inhibit the enzyme, while avoiding selectivity issues caused by the conserved nature of the ATP binding site. Here, we report structure–activity relationships and mechanistic analysis for the first reported Plk1 PBD inhibitor, Poloxin. We present the identification of the optimized analog Poloxin-2, displaying significantly improved potency and selectivity over Poloxin. Poloxin-2 induces mitotic arrest and apoptosis in cultured human tumor cells at low micromolar concentrations, highlighting it as a valuable tool compound for exploring the function of the Plk1 PBD in living cells

Molecular Mechanism of SSR128129E, an Extracellularly Acting, Small-Molecule, Allosteric Inhibitor of FGF Receptor Signaling (vol 23, pg 489, 2013)

© 2016 Elsevier Inc. (Cancer Cell 23, 489–501, April 15, 2013) In Figure 2F, the authors failed to highlight clearly that there is a split in the western blot (all rows) between the “0” and “0.1” condition. Even though all samples were run in the same experiment on the same blot, the image was split to remove samples that were simultaneously analyzed but irrelevant for this study. In the corrected Figure 2 F, the authors have now clearly separated both parts of the western blot. In Figure 5B, the image of the western blot showing total FGFR2 for the HEK293-FGFR2Y328D cell line was mistakenly replaced with the image of the western blot showing total FGFR2 for the HEK293-FGFR2WT cell line from Figure 5A. In the corrected Figure 5B, the authors have included the correct image of the western blot showing total FGFR2 for the HEK293-FGFR2Y328D cell line. The corrected Figure 2 and Figure 5 are included below. The authors apologize for any confusion these mistakes may have caused the readers.Correctionstatus: publishe