Volume of Hsp90 Protein-Ligand Binding Determined by Fluorescent Pressure Shift Assay, Densitometry, and NMR

© 2016 American Chemical Society.Human heat shock protein 90 (Hsp90) is a key player in the homeostasis of the proteome and plays a role in numerous diseases, such as cancer. For the design of Hsp90 ATPase activity inhibitors, it is important to understand the relationship between an inhibitor structure and its inhibition potential. The volume of inhibitor binding is one of the most important such parameters that are rarely being studied. Here, the volumes of binding of several ligands to recombinant Hsp90 were obtained by three independent experimental techniques: fluorescent pressure shift assay, vibrating tube densitometry, and high-pressure NMR. Within the error range, all techniques provided similar volumetric parameters for the investigated protein-ligand systems. Protein-ligand binding volumes were negative, suggesting that the protein-ligand complex, together with its hydration shell, occupies less volume than the separate constituents with their hydration shells. Binding volumes of tightly binding, subnanomolar ligands were significantly more negative than those of weakly binding, millimolar ligands. The volumes of binding could be useful for designing inhibitors with desired recognition properties and further development as drugs

Characterization of megahertz X ray laser beams by multishot desorption imprints in PMMA

Proper diagnostics of intense free electron laser FEL X ray pulses is indisputably important for experimental data analysis as well as for the protection of beamline optical elements. New challenges for beam diagnostic methods are introduced by modern FEL facilities capable of delivering powerful pulses at megahertz MHz repetition rates. In this paper, we report the first characterization of a defocused MHz 13.5 nm beam generated by the free electron laser in Hamburg FLASH using the method of multi pulse desorption imprints in poly methyl methacrylate PMMA . The beam fluence profile is reconstructed in a novel and highly accurate way that takes into account the nonlinear response of material removal to total dose delivered by multiple pulses. The algorithm is applied to experimental data of single shot ablation imprints and multi shot desorption imprints at both low 10 Hz and high 1 MHz repetition rates. Reconstructed response functions show a great agreement with the theoretical desorption response function mode

Thermodynamics of Aryl-Dihydroxyphenyl-Thiadiazole Binding to Human Hsp90

The design of specific inhibitors against the Hsp90 chaperone and other enzyme relies on the detailed and correct understanding of both the thermodynamics of inhibitor binding and the structural features of the protein-inhibitor complex. Here we present a detailed thermodynamic study of binding of aryl-dihydroxyphenyl-thiadiazole inhibitor series to recombinant human Hsp90 alpha isozyme. The inhibitors are highly potent, with the intrinsic Kd approximately equal to 1 nM as determined by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). Dissection of protonation contributions yielded the intrinsic thermodynamic parameters of binding, such as enthalpy, entropy, Gibbs free energy, and the heat capacity. The differences in binding thermodynamic parameters between the series of inhibitors revealed contributions of the functional groups, thus providing insight into molecular reasons for improved or diminished binding efficiency. The inhibitor binding to Hsp90 alpha primarily depended on a large favorable enthalpic contribution combined with the smaller favorable entropic contribution, thus suggesting that their binding was both enthalpically and entropically optimized. The enthalpy-entropy compensation phenomenon was highly evident when comparing the inhibitor binding enthalpies and entropies. This study illustrates how detailed thermodynamic analysis helps to understand energetic reasons for the binding efficiency and develop more potent inhibitors that could be applied for therapeutic use as Hsp90 inhibitors

Rise and Fall of an Anti-MUC1 Specific Antibody

So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate.A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10(-10) M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells.Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology

Phase Transition Lowering in Dynamically Compressed Silicon

Silicon, being one of the most abundant elements in nature, attracts wide-ranging scientific and technological interest. Specifically, in its elemental form, crystals of remarkable purity can be produced. One may assume that this would lead to silicon being well understood, and indeed, this is the case for many ambient properties, as well as for higher-pressure behaviour under quasi-static loading. However, despite many decades of study, a detailed understanding of the response of silicon to rapid compression—such as that experienced under shock impact—remains elusive. Here, we combine a novel free-electron laser-based X-ray diffraction geometry with laser-driven compression to elucidate the importance of shear generated during shock compression on the occurrence of phase transitions. We observe lowering of the hydrostatic phase boundary in elemental silicon, an ideal model system for investigating high-strength materials, analogous to planetary constituents. Moreover, we unambiguously determine the onset of melting above 14 GPa, previously ascribed to a solid–solid phase transition, undetectable in the now conventional shocked diffraction geometry; transitions to the liquid state are expected to be ubiquitous in all systems at sufficiently high pressures and temperatures

A review of environment-dependent processes within FEL excited matter

In this review we discuss four examples of environment-dependent processes triggered by free-electron laser radiation within systems of various complexity. Those are: (i) non-thermal structural changes within irradiated solids, (ii) inverse bremsstrahlung during VUV irradiation of atomic clusters, (iii) shifts of ionic potentials due to a charged ion environment within a plasma, and (iv) fast thermalization of the electrons created after FEL irradiation. We show that FEL excitation enables access to a unique class of environment-influenced processes, depending on the radiation wavelength, pulse fluence and the structure of sample

Schlüsselreagenzien für die Proteomforschung im Hochdurchsatz

Die Entwicklung der DNA-Sequenzierungstechnologien wurde durch die Aufgabe, das gesamte menschliche Genom zu entschlüsseln, vorangetrieben. Fünf Jahre nach Ende des Humangenomprojektes ist das Verständnis der Funktion der durch die rund 23.000 Gene kodierten Proteine des menschlichen Genoms jedoch immer noch rudimentär. Einer der limitierenden Faktoren dabei ist das Fehlen einer Hochdurchsatzmethode zur Herstellung von Antikörpern für die zellbiologische und biochemische Charakterisierung der vielen unbekannten Genprodukte und ihrer Interaktionspartner. In dem innerhalb des Nationalen Genomforschungsnetzes (NGFN) geförderten Projektes „Antibody Factory“ werden deshalb Methoden entwickelt, um Antikörper in vitro in großer Zahl herstellen zu können. Die in vitro-Herstellung mittels Phagendisplay bietet die Vorteile einer einfachen Automatisierung und der preiswerten Produktion in E. coli, kommt vollständig ohne Versuchstiere aus, ist weitgehend miniaturisierbar und benötigt nur sehr geringe Mengen der meist begrenzt verfügbaren Antigene. Begleitend werden verschiedene neuartige Methoden zur Antigenherstellung und für die Validierung der Antikörper in höherem Durchsatz untersucht

Volume of Hsp90 Protein-Ligand Binding Determined by Fluorescent Pressure Shift Assay, Densitometry, and NMR

© 2016 American Chemical Society.Human heat shock protein 90 (Hsp90) is a key player in the homeostasis of the proteome and plays a role in numerous diseases, such as cancer. For the design of Hsp90 ATPase activity inhibitors, it is important to understand the relationship between an inhibitor structure and its inhibition potential. The volume of inhibitor binding is one of the most important such parameters that are rarely being studied. Here, the volumes of binding of several ligands to recombinant Hsp90 were obtained by three independent experimental techniques: fluorescent pressure shift assay, vibrating tube densitometry, and high-pressure NMR. Within the error range, all techniques provided similar volumetric parameters for the investigated protein-ligand systems. Protein-ligand binding volumes were negative, suggesting that the protein-ligand complex, together with its hydration shell, occupies less volume than the separate constituents with their hydration shells. Binding volumes of tightly binding, subnanomolar ligands were significantly more negative than those of weakly binding, millimolar ligands. The volumes of binding could be useful for designing inhibitors with desired recognition properties and further development as drugs

Volume of Hsp90 Protein-Ligand Binding Determined by Fluorescent Pressure Shift Assay, Densitometry, and NMR

© 2016 American Chemical Society.Human heat shock protein 90 (Hsp90) is a key player in the homeostasis of the proteome and plays a role in numerous diseases, such as cancer. For the design of Hsp90 ATPase activity inhibitors, it is important to understand the relationship between an inhibitor structure and its inhibition potential. The volume of inhibitor binding is one of the most important such parameters that are rarely being studied. Here, the volumes of binding of several ligands to recombinant Hsp90 were obtained by three independent experimental techniques: fluorescent pressure shift assay, vibrating tube densitometry, and high-pressure NMR. Within the error range, all techniques provided similar volumetric parameters for the investigated protein-ligand systems. Protein-ligand binding volumes were negative, suggesting that the protein-ligand complex, together with its hydration shell, occupies less volume than the separate constituents with their hydration shells. Binding volumes of tightly binding, subnanomolar ligands were significantly more negative than those of weakly binding, millimolar ligands. The volumes of binding could be useful for designing inhibitors with desired recognition properties and further development as drugs