Thermodynamics of ligand binding to histone deacetylase like amidohydrolase from Bordetella/Alcaligenes

Thermodynamic studies on ligand–protein binding have become increasingly important in the process of drug design. In combination with structural data and molecular dynamics simulations, thermodynamic studies provide relevant information about the mode of interaction between compounds and their target proteins and therefore build a sound basis for further drug optimization. Using the example of histone deacetylases (HDACs), particularly the histone deacetylase like amidohydrolase (HDAH) from Bordetella/Alcaligenes, a novel sensitive competitive fluorescence resonance energy transfer-based binding assay was developed and the thermodynamics of interaction of both fluorescent ligands and inhibitors to histone deacetylase like amidohydrolase were investigated. The assay consumes only small amounts of valuable target proteins and is suitable for fast kinetic and mechanistic studies as well as high throughput screening applications. Binding affinity increased with increasing length of aliphatic spacers (n?=?4–7) between the hydroxamate moiety and the dansyl head group of ligand probes. Van't Hoff plots revealed an optimum in enthalpy contribution to the free energy of binding for the dansyl-ligand with hexyl spacer. The selectivity in the series of dansyl-ligands against human class I HDAC1 but not class II HDACs 4 and 6 increased with the ratio of deltaH0/deltaG0. The data clearly emphasize the importance of thermodynamic signatures as useful general guidance for the optimization of ligands or rational drug design

The Many Faces of FKBP51

The FK506-binding protein 51 (FKBP51) has emerged as a key regulator of endocrine stress responses in mammals and as a potential therapeutic target for stress-related disorders (depression, post-traumatic stress disorder), metabolic disorders (obesity and diabetes) and chronic pain. Recently, FKBP51 has been implicated in several cellular pathways and numerous interacting protein partners have been reported. However, no consensus on the underlying molecular mechanisms has yet emerged. Here, we review the protein interaction partners reported for FKBP51, the proposed pathways involved, their relevance to FKBP51’s physiological function(s), the interplay with other FKBPs, and implications for the development of FKBP51-directed drugs

Enantioselective Synthesis of a Tricyclic, sp³‐Rich Diazatetradecanedione: an Amino Acid‐Based Natural Product‐Like Scaffold

6‐, 7‐, and 8‐membered rings are assembled from a linear precursor by successive cyclisation reactions to construct a tricyclic diazatricyclo[6.5.1.0⁴,⁹]‐tetradecanedione scaffold. Advanced building blocks based on d‐aspartic acid and l‐pyroglutamic acid were combined by a sp³−sp² Negishi coupling. A carbamate‐guided syn‐diastereoselective epoxidation followed by an intramolecular epoxide opening allowed the construction of the piperidine ring. An efficient one‐pot hydroxyl‐group protection twofold deprotection reaction prepared the ground for the cyclisation to the bicycle. A final deprotection of the orthogonal protecting groups and lactamisation led to the novel, sp³‐rich tricycle. The final compound is a substrate mimic of peptidyl‐prolyl cis‐trans isomerases featuring a locked trans‐amide bond. Cheminformatic analysis of 179 virtual derivatives indicates favourable physicochemical properties and drug‐like characteristics. As proof of concept we, show a low micromolar activity in a fluorescence polarisation assay towards the FK506‐binding protein 12

Mechanism‐Based Design of the First GlnA4‐Specific Inhibitors

γ‐Glutamylamine synthetases are an important class of enzymes that play a key role in glutamate‐based metabolism. Methionine sulfoximine (MSO) is a well‐established inhibitor for the archetypal glutamine synthetase (GS) but inhibitors for most GS‐like enzymes are unknown. Assuming a conserved catalytic mechanism for GS and GS‐like enzymes, we explored if subtype‐selective inhibitors can be obtained by merging MSO with the cognate substrates of the respective GS‐like enzymes. Using GlnA4Sc from Streptomyces coelicolor, an enzyme recently shown to produce γ‐glutamylethanolamine, we demonstrate that MSO can be reengineered in a straightforward fashion into potent and selective GlnA4Sc inhibitors. Linkage chemistry as well as linker length between the MSO moiety and the terminal hydroxyl group derived from ethanolamine were in agreement with the postulated phosphorylated catalytic intermediate. The best GlnA4 inhibitor 7 b potently blocked S. coelicolor growth in the presence of ethanolamine as the sole nitrogen source. Our results provide the first GlnA4Sc‐specific inhibitors and suggest a general strategy to develop mechanism‐based inhibitors for GS‐like enzymes

Binding pocket stabilization by high-throughput screening of yeast display libraries

Protein dynamics have a great influence on the binding pockets of some therapeutic targets. Flexible protein binding sites can result in transient binding pocket formation which might have a negative impact on drug screening efforts. Here, we describe a protein engineering strategy with FK506-binding protein 51 (FKBP51) as a model protein, which is a promising target for stress-related disorders. High-throughput screening of yeast display libraries of FKBP51 resulted in the identification of variants exhibiting higher affinity binding of conformation-specific FKBP51 selective inhibitors. The gene libraries of a random mutagenesis and site saturation mutagenesis of the FK1 domain of FKBP51 encoding sequence were used to create a yeast surface display library. Fluorescence-activated cell sorting for FKBP51 variants that bind conformation-specific fluorescently labeled ligands with high affinity allowed for the identification of 15 different protein variants with improved binding to either, or both FKBP51-specific ligands used in the screening, with improved affinities up to 34-fold compared to the wild type. These variants will pave the way to a better understanding of the conformational flexibility of the FKBP51 binding pocket and may enable the isolation of new selective ligands that preferably and selectively bind the active site of the protein in its open conformation state

Discovery of a Potent Proteolysis Targeting Chimera Enables Targeting the Scaffolding Functions of FK506‐Binding Protein 51 (FKBP51)

The FK506‐binding protein 51 (FKBP51) is a promising target in a variety of disorders including depression, chronic pain, and obesity. Previous FKBP51‐targeting strategies were restricted to occupation of the FK506‐binding site, which does not affect core functions of FKBP51. Here, we report the discovery of the first FKBP51 proteolysis targeting chimera (PROTAC) that enables degradation of FKBP51 abolishing its scaffolding function. Initial synthesis of 220 FKBP‐focused PROTACs yielded a plethora of active PROTACs for FKBP12, six for FKBP51, and none for FKBP52. Structural analysis of a binary FKBP12:PROTAC complex revealed the molecular basis for negative cooperativity. Linker‐based optimization of first generation FKBP51 PROTACs led to the PROTAC SelDeg51 with improved cellular activity, selectivity, and high cooperativity. The structure of the ternary FKBP51:SelDeg51:VCB complex revealed how SelDeg51 establishes cooperativity by dimerizing FKBP51 and the von Hippel‐Lindau protein (VHL) in a glue‐like fashion. SelDeg51 efficiently depletes FKBP51 and reactivates glucocorticoid receptor (GR)‐signalling, highlighting the enhanced efficacy of full protein degradation compared to classical FKBP51 binding

Summary and Conclusions of the First DESY Test Beam User Workshop

On October 5/6, 2017, DESY hosted the first DESY Test Beam User Workshop [1] which took place in Hamburg. Fifty participants from different user communities, ranging from LHC (ALICE, ATLAS, CMS, LHCb) to FAIR (CBM, PANDA), DUNE, Belle-II, future linear colliders (ILC, CLIC) and generic detector R&D presented their experiences with the DESY II Test Beam Facility, their concrete plans for the upcoming years and a first estimate of their needs for beam time in the long-term future beyond 2025. A special focus was also on additional improvements to the facility beyond its current capabilities

[4.3.1]Bicyclic FKBP Ligands Inhibit Legionella Pneumophila Infection by LpMip‐Dependent and LpMip‐Independent Mechanisms

Legionella pneumophila is the causative agent of Legionnaires’ disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506‐binding proteins (FKBPs), plays a major role in the proliferation of the gram‐negative bacterium in host organisms. In this work, we test our library of >1000 FKBP‐focused ligands for inhibition of LpMip. The [4.3.1]‐bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non‐toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection‐assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]‐bicyclic sulfonamides as anti‐legionellal agents, although their anti‐infective properties cannot be explained by inhibition of LpMip alone

Research and Design of a Routing Protocol in Large-Scale Wireless Sensor Networks

无线传感器网络，作为全球未来十大技术之一，集成了传感器技术、嵌入式计算技术、分布式信息处理和自组织网技术，可实时感知、采集、处理、传输网络分布区域内的各种信息数据，在军事国防、生物医疗、环境监测、抢险救灾、防恐反恐、危险区域远程控制等领域具有十分广阔的应用前景。 本文研究分析了无线传感器网络的已有路由协议，并针对大规模的无线传感器网络设计了一种树状路由协议，它根据节点地址信息来形成路由，从而简化了复杂繁冗的路由表查找和维护，节省了不必要的开销，提高了路由效率，实现了快速有效的数据传输。 为支持此路由协议本文提出了一种自适应动态地址分配算——ADAR（AdaptiveDynamicAddre...As one of the ten high technologies in the future, wireless sensor network, which is the integration of micro-sensors, embedded computing, modern network and Ad Hoc technologies, can apperceive, collect, process and transmit various information data within the region. It can be used in military defense, biomedical, environmental monitoring, disaster relief, counter-terrorism, remote control of haz...学位：工学硕士院系专业：信息科学与技术学院通信工程系_通信与信息系统学号：2332007115216

Selectivity in histone deacetylase inhibition: A biophysical approach

The development of selective and save compounds is an important task in drug discovery and during the past 25 years biophysical methods for the characterization of protein-ligand interactions have been developed to a valuable tool. On the one hand these methods allow to validate screening hits and on the other hand they complement traditional approaches in drug discovery by providing deep insights into the thermodynamics, kinetics and the mechanism of molecular interactions. Based on these information, approaches were developed which allow to identify promising lead structures for further development. Histone deacetylases (HDACs) are a protein family which are investigated as therapeutic targets for the treatment of different diseases like cancer neurodegenerative disorders and parasitic infections. By catalyzing the deacetylation of the ε-amino function of lysines of histones and other proteins they fulfill an important function for the epigenetic regulation. With SAHA, FK228, PXD101 and LBH589 four HDAC inhibitors were approved for the treatment of different cancers. They cause several unwanted side effects. SAHA, PXD101 and LBH589 are unselective inhibitors which inhibit most HDAC isotypes. In order to minimize unwanted side effects and to provide a more specific therapy isotype selective inhibitors are developed. The present work deals with the question how information from an analysis of the thermodynamics, kinetics and binding mechanisms can be exploited to characterize the selectivity of inhibitors of the HDAC family. The generated results of this doctoral thesis are provided in the cumulative part which consists of five articles published in peer reviewed journals and one article submitted for peer review. One the one hand they extend the methodology on the identification and biophysical characterization of HDAC inhibitors and on the other hand they contribute to the deep comprehension of the molecular basis of the inhibition of HDACs and of protein-ligand interactions. The first part of this work covers methods developed for the identification and biophysical characterization of HDAC inhibitors. In a cooperation with the research group of Prof. Wessig at the University of Potsdam ligands were generated whose fluorescence lifetime change upon binding to histone deacetylases. By a displacement of the ligands from the active site the binding of HDAC inhibitors can be monitored. A developed competitive binding assay is outstandingly suited for high throughput screening applications and the determination of binding constants. Furthermore, the developed assay is also applicable for class IIa HDACs resulting in the first binding assay for this HDAC class. For the determination of kinetic parameters and the binding mechanism of the binding inhibitors to histone deacetylases an established fluorescence spectroscopic binding assay was modified, so that a time-resolved monitoring of the binding of nonfluorescent ligands is allowed. The determined kinetic traces were subjected to a global fit analysis. With this method the reaction mechanisms of the binding of four ligands to the human histone deacetylases 1, 6 and 8 as well as to a bacterial histone deacetylase-like amidohydrolase were evaluated. In the second part of this work it was evaluated on the basis of a histone deacetylase-like amidohydrolase from Pseudomonas aeruginosa, HDAHpa, which structural elements of HDAC inhibitors affect the selectivity and how mechanistic and thermodynamic parameters can be applied to assess the selectivity. Therefore, the reaction mechanisms and the thermodynamic signature of structurally related inhibitors of the known HDAC inhibitor N-hydroxy-N′-phenyloctanediamide (SAHA) were determined and evaluated for their selectivity against the human histone deacetylases 1–8. Generally, SAHA and other compounds with a hydroxamic acid as zinc binding moiety exhibit a good selectivity against class IIa HDACs. In contrast, the compound SATFMK, where the hydroxamic acid moiety is exchanged by a trifluoromethyl ketone moiety, exhibits a good to very good selectivity against the HDACs 1–3 and 6 but a lower selectivity against class IIa HDACs and HDAC8. With an at least 1000-fold selectivity over human HDACs the highest selectivity was determined for the compound PFSAHA. This compound exhibits, compared to SAHA and SATFMK, a sterically more demanding perflourinated spacer. Furthermore, it was determined in another study that upon an exchange of the cap group the high selectivity is preserved in most cases and can be improved by methylation and chlorination. Surprisingly, an analysis of the determined binding mechanisms suggests that PFSAHA and SATFMK bind each to other protein conformations as the other ligands. These results support the assumption that selective ligands can be identified on the basis of their binding mechanism. In order to deepen the knowledge about the molecular recognition of inhibitors by HDAHpa the binding reactions were studied by isothermal titration calorimetry. The determination of the thermodynamic parameters revealed that the binding of PFSAHA to HDAHpa occurs with unfavorable entropic contributions and is solely enthallpically driven, while for the binding of SATFMK the entropic and enthalpic contributions are balanced. This indicates indeed that for selective binders the enthalpic contributions are more pronounced. However, the enthalpic contributions to binding are of limited suitability for the prediction of the selectivity of HDAC inhibitors. Only a newly defined enthalpy weighted binding constant exhibits a good correlation to the determined selectivity of the HDAC inhibitors. Certainly, the applicability and transferability to other protein targets have yet to be evaluated. In a further study, the binding of PFSAHA and SATFMK to HDAHpa and a to HDAHpa homologous histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes were investigated in more detail by protein crystallography and isothermal titration calorimetry. It was shown, that the thermodynamic signature and the mechanism of the binding reaction is largely influenced by flexibility and accessibility of the active site. With a better accessibility and a higher flexibility the binding occurs in an apparent one-step reaction and with more favorable enthalpic contributions to binding. But these are balanced by enthalpy-entropy compensation resulting in an unchanged binding affinity. These findings seem to be exceptionally important for the development of HDAC inhibitors, since HDACs interact with several proteins and it is likely that these interactions alter the flexibility of the HDACs and with it the recognition of ligands. Taken together these studies indicate that generally a solely consideration of thermodynamic signatures is insufficient for the identification of selective compounds, but especially in combination with mechanistic investigations useful information about the potential selectivity of compounds are provided, which supplement existing parameters for the early identification of leads with a high probability of success