In silico assessment of potential druggable pockets on the surface of α1-Antitrypsin conformers

The search for druggable pockets on the surface of a protein is often performed on a single conformer, treated as a rigid body. Transient druggable pockets may be missed in this approach. Here, we describe a methodology for systematic in silico analysis of surface clefts across multiple conformers of the metastable protein α1-antitrypsin (A1AT). Pathological mutations disturb the conformational landscape of A1AT, triggering polymerisation that leads to emphysema and hepatic cirrhosis. Computational screens for small molecule inhibitors of polymerisation have generally focused on one major druggable site visible in all crystal structures of native A1AT. In an alternative approach, we scan all surface clefts observed in crystal structures of A1AT and in 100 computationally produced conformers, mimicking the native solution ensemble. We assess the persistence, variability and druggability of these pockets. Finally, we employ molecular docking using publicly available libraries of small molecules to explore scaffold preferences for each site. Our approach identifies a number of novel target sites for drug design. In particular one transient site shows favourable characteristics for druggability due to high enclosure and hydrophobicity. Hits against this and other druggable sites achieve docking scores corresponding to a Kd in the µM–nM range, comparing favourably with a recently identified promising lead. Preliminary ThermoFluor studies support the docking predictions. In conclusion, our strategy shows considerable promise compared with the conventional single pocket/single conformer approach to in silico screening. Our best-scoring ligands warrant further experimental investigation

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

Kinetic regulation of multi-ligand binding proteins

Background: Second messengers, such as calcium, regulate the activity of multisite binding proteins in a concentration-dependent manner. For example, calcium binding has been shown to induce conformational transitions in the calcium-dependent protein calmodulin, under steady state conditions. However, intracellular concentrations of these second messengers are often subject to rapid change. The mechanisms underlying dynamic ligand-dependent regulation of multisite proteins require further elucidation. Results: In this study, a computational analysis of multisite protein kinetics in response to rapid changes in ligand concentrations is presented. Two major physiological scenarios are investigated: i) Ligand concentration is abundant and the ligand-multisite protein binding does not affect free ligand concentration, ii) Ligand concentration is of the same order of magnitude as the interacting multisite protein concentration and does not change. Therefore, buffering effects significantly influence the amounts of free ligands. For each of these scenarios the influence of the number of binding sites, the temporal effects on intermediate apo- and fully saturated conformations and the multisite regulatory effects on target proteins are investigated. Conclusions: The developed models allow for a novel and accurate interpretation of concentration and pressure jump-dependent kinetic experiments. The presented model makes predictions for the temporal distribution of multisite protein conformations in complex with variable numbers of ligands. Furthermore, it derives the characteristic time and the dynamics for the kinetic responses elicited by a ligand concentration change as a function of ligand concentration and the number of ligand binding sites. Effector proteins regulated by multisite ligand binding are shown to depend on ligand concentration in a highly nonlinear fashion

Magneto- and Electroresistance of Ultrathin Anisotropically Strained La-Sr-MnO Films

The magnetoresistance anisotropy of ultrathin La$\text{}_{0.83}$Sr$\text{}_{0.17}$Mn O$\text{}_{3}$ films deposited on NdGaO$\text{}_{3}$ substrate by metalorganic chemical vapour deposition technique was investigated. The electric-field-induced resistance change was studied up to electric fields of 10 kV/cm using ns duration electrical pulses. It was found that in ultrathin (< 10 nm) and thin (< 50 nm) films the origin of electric-field-induced resistance change is thermal. However, the films with thicknesses of about 20 nm, exhibit negative electric-field-induced resistance change, having a pure electronic nature. This effect is explained in terms of two-layer systems with imperfections located at the interface between the layers

Ultra-fast fault current limiter based on La-Ca-MnO3 thin films

A fast fault current limiter based on La0.67Ca0.33MnO3 films and designed in the shape of a co-planar 50 Ω impedance transmission line is presented. The limiter is intended to protect the input circuits of high frequency wide band electronic devices. The operation of this limiter is based on fast electroresistance phenomenon in manganites. The main behaviour of this phenomenon at temperatures close to its phase transition temperature Tm was studied. It was demonstrated that the specific resistance of the film decreases exponentially with increase of electric field. The experimental results of losses induced by the fault current limiter in a 50 Ω impedence transmission line for non-limiting and limiting regimes are presented. The phenomenological relation between input and output voltage transients' amplitudes of transmission lines protected by such limiter is obtained

Electroresistance of La-Ca-MnO Thin Films

Epitaxial, textured, and polycrystalline La$\text{}_{0.7}$Ca$\text{}_{0.3}$Mn O$\text{}_{3}$ films, having about 150 nm thickness, were prepared by pulsed laser deposition techniques onto (110) NdGaO$\text{}_{3}$, MgO and lucalox substrates and investigated using 10 ns duration, 0.5 ns rise time electrical pulses having amplitude up to 500 V. Electroresistance of the films [R(E)-R(0)]/R(0) was investigated up to 80 kV/cm electric field strengths in temperatures ranging from 300 K to 4.2 K. Strong (up to 93%) negative electroresistance was obtained in polycrystalline La$\text{}_{0.7}$Ca$\text{}_{0.3}$MnO$\text{}_{3}$ films prepared on MgO and lucalox substrates. The epitaxial films grown on NdGaO$\text{}_{3}$ substrate demonstrated only a small resistance change due to Joule heating induced by a current pulse. It was concluded that electroresistance manifests itself in strongly inhomogeneous manganites films exhibiting a large number of structural imperfections producing ferromagnetic tunnel junction nets