Denis Kudlinzki

Molecular Mechanism of SSR128129E, an Extracellularly Acting, Small-Molecule, Allosteric Inhibitor of FGF Receptor Signaling

The fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling network plays an important role in cell growth, survival, differentiation, and angiogenesis. Deregulation of FGFR signaling can lead to cancer development. Here, we report an FGFR inhibitor, SSR128129E (SSR), that binds to the extracellular part of the receptor. SSR does not compete with FGF for binding to FGFR but inhibits FGF-induced signaling linked to FGFR internalization in an allosteric manner, as shown by crystallography studies, nuclear magnetic resonance, Fourier transform infrared spectroscopy, molecular dynamics simulations, free energy calculations, structure-activity relationship analysis, and FGFR mutagenesis. Overall, SSR is a small molecule allosteric inhibitor of FGF/FGFR signaling, acting via binding to the extracellular part of the FGFR.

Influence of Heparin Mimetics on Assembly of the FGF-FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM6), octasaccharide (HM8), and decasaccharide (HM10), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM8 and HM10 are significantly more potent than HM6 in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM8 relative to HM6 in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM8 to bind and dimerize FGF2. Notably FGF2·HM8 exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.

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.

Structure and Biophysical Characterization of the S-adenosylmethionine Dependent O-methyltransferase PaMTH1, a Putative Enzyme Accumulating during Senescence of Podospora anserina

Background: PaMTH1, a putative O-methyltransferase protects Podospora anserina from oxidative stress during senescence and acts as a longevity assurance factor. Results: Crystal structures of PaMTH1/PaMTH1-SAM/SAH co-complexes and NMR-based characterization of enzymatic methylation of its substrate were obtained. Conclusion: PaMTH1 catalyzes methyl group transfer from the co-factor to the substrate in a cation-dependent manner. Significance: This work facilitates the identification of endogenous polyphenolic compounds directly involved in metal-induced oxidative stress. Low levels of reactive oxygen species (ROS) act as important signaling molecules, but in excess they can damage biomolecules. ROS regulation is therefore of key importance. Several polyphenols in general and flavonoids in particular have the potential to generate hydroxyl radicals, the most hazardous among all ROS. However, the generation of a hydroxyl radical and subsequent ROS formation can be prevented by methylation of the hydroxyl group of the flavonoids. O-Methylation is performed by O-methyltransferases, members of the S-adenosyl-l-methionine (SAM)-dependent O-methyltransferase superfamily involved in the secondary metabolism of many species across all kingdoms. In the filamentous fungus Podospora anserina, a well established aging model, the O-methyltransferase (PaMTH1) was reported to accumulate in total and mitochondrial protein extracts during aging. In vitro functional studies revealed flavonoids and in particular myricetin as its potential substrate. The molecular architecture of PaMTH1 and the mechanism of the methyl transfer reaction remain unknown. Here, we report the crystal structures of PaMTH1 apoenzyme, PaMTH1-SAM (co-factor), and PaMTH1-S-adenosyl homocysteine (by-product) co-complexes refined to 2.0, 1.9, and 1.9 Å, respectively. PaMTH1 forms a tight dimer through swapping of the N termini. Each monomer adopts the Rossmann fold typical for many SAM-binding methyltransferases. Structural comparisons between different O-methyltransferases reveal a strikingly similar co-factor binding pocket but differences in the substrate binding pocket, indicating specific molecular determinants required for substrate selection. Furthermore, using NMR, mass spectrometry, and site-directed active site mutagenesis, we show that PaMTH1 catalyzes the transfer of the methyl group from SAM to one hydroxyl group of the myricetin in a cation-dependent manner.

Recombinant expression and purification of human TATA binding protein using a chimeric fusion.

The TATA binding protein (TBP) is the central core protein of the transcription factor II D that binds directly to the TATA box and therefore plays an integral part in eukaryotic transcription. This pivotal position of TBP is underlined by the vast number of interaction partners involved. Expression and purification of human TATA binding protein (hTBP) has remained a challenge due to protein instability and the protein loss during expression and purification involved. Here, we present a novel approach for high yield expression and purification of human TBP core (hTBPc) protein. Protein fold and activity are verified by nuclear magnetic resonance (NMR) spectroscopy and microscale thermophoresis (MST).

Chemoproteomics-Aided Medicinal Chemistry for the Discovery of EPHA2 Inhibitors.

The receptor tyrosine kinase EPHA2 has gained attention as a therapeutic drug target for cancer and infectious diseases. However, EPHA2 research and EPHA2‐based therapies have been hampered by the lack of selective small‐molecule inhibitors. Herein we report the synthesis and evaluation of dedicated EPHA2 inhibitors based on the clinical BCR‐ABL/SRC inhibitor dasatinib as a lead structure. We designed hybrid structures of dasatinib and the previously known EPHA2 binders CHEMBL249097, PD‐173955, and a known EPHB4 inhibitor in order to exploit both the ATP pocket entrance as well as the ribose pocket as binding epitopes in the kinase EPHA2. Medicinal chemistry and inhibitor design were guided by a chemical proteomics approach, allowing early selectivity profiling of the newly synthesized inhibitor candidates. Concomitant protein crystallography of 17 inhibitor co‐crystals delivered detailed insight into the atomic interactions that underlie the structure–affinity relationship. Finally, the anti‐proliferative effect of the inhibitor candidates was confirmed in the glioblastoma cell line SF‐268. In this work, we thus discovered a novel EPHA2 inhibitor candidate that features an improved selectivity profile while maintaining potency against EPHA2 and anticancer activity in SF‐268 cells.

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 prior to protein binding, and by release of these waters, the favored entropic binding is observed.

Expression and Purification of EPHA2 Tyrosine Kinase Domain for Crystallographic and NMR Studies.

The receptor tyrosine kinase EPHA2 is overexpressed in several cancers (breast, head and neck, non‐small‐cell lung cancer). Small‐molecule‐based inhibition of the EPHA2 kinase domain (KD) is seen as an important strategy for therapeutic intervention. However, obtaining structural information by crystallography or NMR spectroscopy for drug discovery is severely hampered by the lack of pure, homogeneous protein. Here, different fragments of the EPHA2 KD were expressed and purified from both bacterial (Escherichia coli, BL21(DE3) cells) and insect cells (Spodoptera frugiperda, Sf9 cells).1H,15N HSQC was used to determine the proper folding and homogeneity of all the constructs. Protein from E. coli was well‐folded but unstable, and it did not crystallize. However, a construct (D596–G900) produced in Sf9 cells yielded homogenous, well‐folded protein that crystallized readily, thereby resulting in eleven new EPHA2–ligand crystal structures. We have also established a strategy for selective and uniform 15N‐amino acid labeling of EPHA2 KD in Sf9 cells for investigating dynamics and EPHA2–drug interactions by NMR.

High-resolution crystal structure of cAMP-dependent protein kinase from Cricetulus griseus.

Protein kinases (PKs) are dynamic regulators of numerous cellular processes. Their phosphorylation activity is determined by the conserved kinase core structure, which is maintained by the interaction and dynamics with associated domains or interacting proteins. The prototype enzyme for investigations to understand the activity and regulation of PKs is the catalytic subunit of cAMP-dependent protein kinase (PKAc). Major effects of functional regulation and ligand binding are driven by only minor structural modulations in protein-protein interactions. In order to resolve such minor structural differences, very high resolution structures are required. Here, the high-resolution X-ray structure of PKAc from Cricetulus griseus is reported.

MOTOR: Model assisted software for NMR structure determination

Eukaryotic proteins with important biological function can be partially unstructured, conformational flexible, or heterogenic. Crystallization trials often fail for such proteins. In NMR spectroscopy, parts of the polypeptide chain undergoing dynamics in unfavorable time regimes cannot be observed. De novo NMR structure determination is seriously hampered when missing signals lead to an incomplete chemical shift assignment resulting in an information content of the NOE data insufficient to determine the structure ab initio. We developed a new protein structure determination strategy for such cases based on a novel NOE assignment strategy utilizing a number of model structures but no explicit reference structure as it is used for bootstrapping like algorithms. The software distinguishes in detail between consistent and mutually exclusive pairs of possible NOE assignments on the basis of different precision levels of measured chemical shifts searching for a set of maximum number of consistent NOE assignments in agreement with 3D space. Validation of the method using the structure of the low molecular‐weight‐protein tyrosine phosphatase A (MptpA) showed robust results utilizing protein structures with 30–45% sequence identity and 70% of the chemical shift assignments. About 60% of the resonance assignments are sufficient to identify those structural models with highest conformational similarity to the real structure. The software was benchmarked by de novo solution structures of fibroblast growth factor 21 (FGF21) and the extracellular fibroblast growth factor receptor domain FGFR4 D2, which both failed in crystallization trials and in classical NMR structure determination. Proteins 2013; 81:2007–2022.