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The serine/threonine kinase SGK1 is an activator of the β-catenin pathway and a powerful stimulator of cartilage degradation that is found to be upregulated under genomic control in diseased osteoarthritic cartilage. Today, no oral disease-modifying treatments are available and chronic treatment in this indication sets high requirements for the drug selectivity, pharmacokinetic, and safety profile. We describe the identification of a highly selective druglike 1

We here report on non-equilibrium targeted Molecular Dynamics simulations as tool for the estimation of protein-ligand unbinding kinetics. Correlating simulations with experimental data from SPR kinetics measurements and X-ray crystallography on two small molecule compound libraries bound to the N-terminal domain of the chaperone Hsp90, we show that the mean non-equilibrium work computed in an ensemble of trajectories of enforced ligand unbinding is a promising predictor for ligand unbinding rates. We furthermore investigate the molecular basis determining unbinding rates within the compound libraries. We propose ligand conformational changes and protein-ligand nonbonded interactions to impact on unbinding rates. Ligands may remain longer at the protein if they exhibit strong electrostatic and/or van der Waals interactions with the target. In the case of ligands with rigid chemical scaffold that exhibit longer residence times however, transient electrostatic interactions with the protein appear to facilitate unbinding. Our results imply that understanding the unbinding pathway and the protein-ligand interactions along this path is crucial for the prediction of small molecule ligands with defined unbinding, This unedited version of the article may be downloaded for personal use only. Any other use requires prior permission of the author and the American Chemical Society. This article appeared in J. Chem. Inf. Model. (2019), 10.1021/acs.jcim.9b00592 and may be found at https://pubs.acs.org/doi/10.1021/acs.jcim.9b00592

Drug-target residence time (τ), one of the main determinants of drug efficacy, remains highly challenging to predict computationally and, therefore, is usually not considered in the early stages of drug design. Here, we present an efficient computational method, τ-random acceleration molecular dynamics (τRAMD), for the ranking of drug candidates by their residence time and obtaining insights into ligand-target dissociation mechanisms. We assessed τRAMD on a data set of 70 diverse drug-like ligands of the N-terminal domain of HSP90α, a pharmaceutically important target with a highly flexible binding site, obtaining computed relative residence times with an accuracy of about 2.3τ for 78% of the compounds and less than 2.0τ within congeneric series. Analysis of dissociation trajectories reveals features that affect ligand unbinding rates, including transient polar interactions and steric hindrance. These results suggest that τRAMD will be widely applicable as a computationally efficient aid to improving drug residence times during lead optimization.

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 fromCricetulus griseusis reported.

A key component to success in structure-based drug design is reliable information on protein-ligand interactions. Recent development in NMR techniques has accelerated this process by overcoming some of the limitations of X-ray crystallography and computational protein-ligand docking. In this work we present a new scoring protocol based on NMR-derived interligand INPHARMA NOEs to guide the selection of computationally generated docking modes. We demonstrate the performance in a range of scenarios, encompassing traditionally difficult cases such as docking to homology models and ligand dependent domain rearrangements. Ambiguities associated with sparse experimental information are lifted by searching a consensus solution based on simultaneously fitting multiple ligand pairs. This study provides a previously unexplored integration between molecular modeling and experimental data, in which interligand NOEs represent the key element in the rescoring algorithm. The presented protocol should be widely applicable for protein-ligand docking also in a different context from drug design and highlights the important role of NMR-based approaches to describe intermolecular ligand-receptor interactions.

From an HTS hit, a series of potent and selective inhibitors of GSK3β have been designed based on a Cdk2-homology model and with the help of several crystal structures of the compounds within Cdk2.

The crystal structure of the hypothetical protein MJ1247 from Methanococccus jannaschii at 2 A resolution, a detailed sequence analysis, and biochemical assays infer its molecular function to be 3-hexulose-6-phosphate isomerase (PHI). In the dissimilatory ribulose monophosphate (RuMP) cycle, ribulose-5-phosphate is coupled to formaldehyde by the 3-hexulose-6-phosphate synthase (HPS), yielding hexulose-6-phosphate, which is then isomerized to fructose-6-phosphate by the enzyme 3-hexulose-6-phosphate isomerase. MJ1247 is an alpha/beta structure consisting of a five-stranded parallel beta sheet flanked on both sides by alpha helices, forming a three-layered alpha-beta-alpha sandwich. The fold represents the nucleotide binding motif of a flavodoxin type. MJ1247 is a tetramer in the crystal and in solution and each monomer has a folding similar to the isomerase domain of glucosamine-6-phosphate synthase (GlmS).

Interleukin 4 (IL-4) is a pleiotropic cytokine which induces T-cell differentiation and class switching of B cells. It therefore plays a central role in the development of allergies and asthma. An IL-4 variant in which Glu9 was mutated to alanine shows an 800-fold drop in binding affinity towards its high-affinity receptor chain. As shown by surface plasmon resonance measurements, this mostly arises from a decreased association rate. Here, the crystal structure of this mutant is reported. It reveals that the protein has a virtually identical structure to the wild type, showing that the unusual behaviour of the mutated protein is not a consequence of misfolding. The possibility that polar interactions in the encounter complex have a steering effect is discussed.

Publisher Summary The transforming growth factor β (TGF β) superfamily is a large group of soluble, dimeric proteins that initiate and control proliferation and differentiation of many cell types in animals, thus playing important roles in embryonal development and adult tissue homeostasis. The complex formation in TGF β signaling is initiated by high-affinity interactions between the ligand and type II receptor TGFβRII. This primary complex subsequently recruits the low-affinity type I receptor (TGFβRI), which is not able to bind to the ligands in the absence of TGFβRII. Affinity for type II receptors is slightly increased in the presence of type I receptors, but, even in the absence of the latter, BMP2 is able to interact with type II chains and the cooperative effect observed with TGFβ is less pronounced. A population of bone morphogenetic protein (BMP) receptors exists in preformed complexes composed of BMPRII and BMPRIA or BMPRIB prior to ligand binding. Binding of BMP2 to either such preformed receptor complexes or in a sequential way first to the high-affinity BMPRI receptors and then to BMPRII, yielding ligand-induced receptor complexes, results in induction of different signaling pathways. The ActRII-ActA complexes reveal the inherent flexibility of the activin ligand. In one of the analyzed crystals the activin adopts a rather folded-up conformation in which the bound receptor chains are in direct contact which each other, while in the second crystal the activin protomer chains are spread apart in a similar fashion as in free ActA or BMP7 and BMP2 and the receptor chains are not in mutual contact.

Die gezielte Mutation seiner Bindungsstelle bewirkt, dass das Proteinhormon IL-4 zwar noch an seinen Rezeptor andocken kann, aber keine Signalubertragung mehr auslost. Da IL-4 spezifisch fur den allergischen Zweig des Immunsystems verantwortlich ist, konnte seine Hemmung fur die Behandlung allergischer Erkrankungen vollig neue Therapiewege eroffnen. Derzeit werden IL-4-Antagonisten fur die Behandlung von allergischem Asthma klinisch getestet.

APRV (Automatic Processing, Refinement and Visualization) is a new program that enables high-throughput batch processing of crystallographic data. The program combines processing of raw diffraction images, initial structure refinement and visual inspection of resulting electron density into a seamless one-step procedure, during which all relevant parameters are refined automatically. It is controlled by a user-friendly graphical interface, facilitating operation by non-experts.

The transforming growth factor β (TGFβ) superfamily is a large group of soluble, dimeric factors that initiate and control proliferation and differentiation of many cell types in animals, thus playing important roles in embryonal development and adult tissue homeostasis. Malfunctioning of this tightly controlled signaling system leads to developmental disorders, and severe defects in organ function and is the cause of several diseases, including various types of cancer. Signaling by these factors requires binding of the ligand to two homologous but functionally distinct types of transmembrane serine/threonine receptor kinases, designated type I and type II. Upon ligand-complex formation, the constitutively active type II receptor kinases activate the type I receptor kinases through phosphorylation in a conserved juxtamembrane region (GS-box). Activated type I receptors are then able to phosphorylate targets further downstream of the signaling pathways. Despite the large number of ligands in the TGFp superfamily, the number of receptors is comparatively small. Also, in contrast to other highly specific receptor-ligand systems, the members of the TGPP superfamily display some promiscuity, in that several ligands are able to recognize different receptors and most of the receptors can bind different Hgands. With a focus on BMP-2 and TGFp, this chapter summarizes current knowledge about the initiating events in signal transduction within the TGpp superfamily based on crystal and solution structures of ligands, receptor ectodomains, and receptor-ligand complexes.

BMPs and other members of the TGF-13 superfamily are powerful secreted signalling proteins that determine development and homoeostasis of many organs and tissues [1, 2]. These comprise bone, cartilage and teeth as well as heart, kidney, muscle, skin, hair, reproductive tract, and several others. Despite the diversity of the biological functions, all ligands and receptors in this superfamily show on a molecular level many similarities in structure and function [3, 4].

BACKGROUND Bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs) belong to the large transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. Signaling of the BMPs requires the binding of the BMP to the BMP cell surface receptors BMPR-IA, BMPR-IB, and BMPR-II. Similar to other cytokines, members of the TGF-beta superfamily exhibit stringent specificity in their ligand-receptor interactions, which may be a reason for the qualitative and quantitative differences in cellular responses. To understand how BMPs and GDFs activate their receptors, it is important to determine structure and binding mechanisms of ligand-receptor complexes. We have used BMP-2 as a key representative of the BMPs to identify the epitopes for type I and type II receptor binding by mutational interaction analyses and have solved the crystal structure of a BMP2:BMPR-IA receptor ectodomain complex. METHODS To identify amino acid side chains involved in receptor binding, a collection of in vitro mutagenized human BMP-2 variants was prepared and subjected to interaction analyses with use of the receptor ectodomains of BMPR-IA, BMPR-II, and ActR-II immobilized on a biosensor system. The biological activity of the BMP-2 variants was measured by BMP-2 dependent expression of alkaline phosphatase (ALP) in C2C12 cells. For crystallization, a complex of BMP-2 and the ectodomain of BMPR-IA was formed in solution, purified, and crystallized as described(12). RESULTS The ligand-receptor interaction analysis of the BMP-2 variants identified distinct epitopes for type I and type II receptor binding. Because the structure of TGF-beta-like proteins has been compared with that of an open hand, the binding epitope for the type I receptor was-on the basis of its location-termed "wrist" epitope. The crystal structure of the BMP-2:BMPR-IA ectodomain complex revealed a key feature of the ligand-receptor interaction: a large hydrophobic residue (Phe85) within a hydrophobic patch of BMPR-IA fit into a hydrophobic pocket composed of residues of both BMP-2 monomers. A second epitope identified by alanine mutagenesis scanning was termed the "knuckle" epitope on the basis of its location on the outer side of the "finger" segments of BMP-2. Mutations in either the wrist epitope or the knuckle epitope produced variants with altered biological activities. Variants with antagonistic properties were exclusively generated by mutations in the knuckle epitope of BMP-2. CONCLUSIONS AND CLINICAL RELEVANCE The identification and characterization of the two receptor binding epitopes in BMP-2 provide new insight into the primary steps of BMP-receptor activation. Because of the structural similarities between members of the TGF-beta superfamily, it can be assumed that the data presented in this work are transferable to other TGF-beta receptor systems. Because of the association with various diseases, the generation of antagonists of other TGF-beta superfamily members might generate potent tools for basic research and therapeutic approaches.