Imatinib disassembles the regulatory core of Abelson kinase by binding to its ATP site and not by binding to its myristoyl pocket

It was recently reported (Xie et al., 2022) that the Abelson tyrosine kinase (Abl) ATP-site inhibitor imatinib also binds to Abl's myristoyl binding pocket, which is the target of allosteric Abl inhibitors. This was based on a crystal structure of a truncated Abl kinase domain construct in complex with imatinib bound to the allosteric site as well as further isothermal titration calorimetry (ITC), NMR, and kinase activity data. Although imatinib's affinity for the allosteric site is significantly weaker (10 µ M) than for the ATP site (10 nM), imatinib binding to the allosteric site may disassemble the regulatory core of Abl, thereby stimulating kinase activity, in particular for Abl mutants with reduced imatinib ATP-site affinity. It was argued that the previously observed imatinib-induced opening of the Abl regulatory core (Skora et al., 2013; Sonti et al., 2018) may be caused by the binding of imatinib to the allosteric site and not to the ATP site. We show here that this is not the case but that indeed imatinib binding to the ATP site induces the opening of the regulatory core at nanomolar concentrations. This agrees with findings that other type-II ATP-site inhibitors (nilotinib, ponatinib) disassemble the regulatory core despite demonstrated negligible binding to the allosteric site

In Vivo Metabolism of [1,6-C-13(2)]Glucose Reveals Distinct Neuroenergetic Functionality between Mouse Hippocampus and Hypothalamus

Glucose is a major energy fuel for the brain, however, less is known about specificities of its metabolism in distinct cerebral areas. Here we examined the regional differences in glucose utilization between the hypothalamus and hippocampus using in vivo indirect C-13 magnetic resonance spectroscopy (H-1-[C-13]-MRS) upon infusion of [1,6-C-13(2)]glucose. Using a metabolic flux analysis with a 1-compartment mathematical model of brain metabolism, we report that compared to hippocampus, hypothalamus shows higher levels of aerobic glycolysis associated with a marked gamma-aminobutyric acid-ergic (GABAergic) and astrocytic metabolic dependence. In addition, our analysis suggests a higher rate of ATP production in hypothalamus that is accompanied by an excess of cytosolic nicotinamide adenine dinucleotide (NADH) production that does not fuel mitochondria via the malate-aspartate shuttle (MAS). In conclusion, our results reveal significant metabolic differences, which might be attributable to respective cell populations or functional features of both structures

Solid-state NMR at natural isotopic abundance for the determination of conformational polymorphism - the case of designed beta-turn peptides containing di-prolines

The proton double quantum-carbon single quantum correlation experiment has been applied to designed peptides in the solid state in natural isotopic abundance. Analogous to nOe studies in solution, through-space double-quantum connectivities have been exploited to obtain the cis-trans conformational polymorphism of diproline residues occurring at beta-turns in the peptides

<em>In Vitro</em> Drug Metabolism Studies Using Human Liver Microsomes

Metabolism of most pharmaceutical drugs occurs in the liver. In drug metabolism, enzymes convert drugs to highly water-soluble metabolites to facilitate excretion from the body. Thus, in vitro models for studying drug metabolism usually target hepatocytes or subcellular liver fractions like microsomes, cytosols, or S9 fractions with high concentrations of specific enzymes. The most popular subcellular fraction used during drug discovery tends to be the microsomes, as these are easy to prepare and store, are amenable to high throughput screening, and are a relatively low-cost option. Understanding the metabolic stability and kinetics of glucuronidation of an investigational drug is crucial for predicting the pharmacokinetic parameters that support dosing and dose frequency. This chapter provides detailed information about metabolite profiling, metabolic stability, glucuronidation kinetics, reactive metabolites identification, CYP enzyme inhibition, and general protocols using human liver microsomes

Designed three-stranded beta-sheet in an alpha/beta hybrid peptide

The incorporation of beta-amino acid residues into the antiparallel beta-strand segments of a multi-stranded beta-sheet peptide is demonstrated for a 19-residue peptide, Boc-LV(beta)FV(D)PGL(beta)FVVL(D)PGLVL(beta)FVV-OMe (BBH19). Two centrally positioned (D)Pro-Gly segments facilitate formation of a stable three-stranded beta-sheet, in which beta-phenylalanine ((beta)Phe) residues occur at facing positions 3, 8 and 17. Structure determination in methanol solution is accomplished by using NMR-derived restraints obtained from NOEs, temperature dependence of amide NH chemical shifts, rates of H/D exchange of amide protons and vicinal coupling constants. The data are consistent with a conformationally well-defined three-stranded beta-sheet structure in solution. Cross-strand interactions between (beta)Phe3/(beta)Phe17 and (beta)Phe3/Val15 residues define orientations of these side-chains. The observation of close contact distances between the side-chains on the N- and C-terminal strands of the three-stranded beta-sheet provides strong support for the designed structure. Evidence is presented for multiple side-chain conformations from an analysis of NOE data. An unusual observation of the disappearance of the Gly NH resonances upon prolonged storage in methanol is rationalised on the basis of a slow aggregation step, resulting in stacking of three-stranded beta-sheet structures, which in turn influences the conformational interconversion between type I' and type II' beta-turns at the two (D)Pro-Gly segments. Experimental evidence for these processes is presented. The decapeptide fragment Boc-LV(beta)FV(D)PGL(beta)FVV-OMe (BBH10), which has been previously characterized as a type I' beta-turn nucleated hairpin, is shown to favour a type II' beta-turn conformation in solution, supporting the occurrence of conformational interconversion at the turn segments in these hairpin and sheet structures

NMR Analysis of Cross Strand Aromatic Interactions in an 8 Residue Hairpin and a 14 Residue Three Stranded β‑Sheet Peptide

Cross strand aromatic interactions between a facing pair of phenylalanine residues in antiparallel β-sheet structures have been probed using two structurally defined model peptides. The octapeptide Boc-LFV^DP^LPLFV-OMe (peptide <b>1</b>) favors the β-hairpin conformation nucleated by the type II′ β-turn formed by the ^DPro-^LPro segment, placing Phe2 and Phe7 side chains in proximity. Two centrally positioned ^DPro-^LPro segments facilitate the three stranded β-sheet formation in the 14 residue peptide Boc-LFV^DP^LPLFVA^DP^LPLFV-OMe (peptide <b>2</b>) in which the Phe2/Phe7 orientations are similar to that in the octapeptide. The anticipated folded conformations of peptides <b>1</b> and <b>2</b> are established by the delineation of intramolecularly hydrogen bonded NH groups and by the observation of specific cross strand NOEs. The observation of ring current shifted aromatic protons is a diagnostic of close approach of the Phe2 and Phe7 side chains. Specific assignment of aromatic proton resonances using HSQC and HSQC-TOCSY methods allow an analysis of interproton NOEs between the spatially proximate aromatic rings. This approach facilitates specific assignments in systems containing multiple aromatic rings in spectra at natural abundance. Evidence is presented for a dynamic process which invokes a correlated conformational change about the C^α-C^β(χ^<b>1</b>) bond for the pair of interacting Phe residues. NMR results suggest that aromatic ring orientations observed in crystals are maintained in solution. Anomalous temperature dependence of ring current induced proton chemical shifts suggests that solvophobic effects may facilitate aromatic ring clustering in apolar solvents

The molecular basis of Abelson kinase regulation by its αI-helix

Abelson tyrosine kinase (Abl) is regulated by the arrangement of its regulatory core, consisting sequentially of the SH3, SH2, and kinase (KD) domains, where an assembled or disassembled core corresponds to low or high kinase activity, respectively. It was recently established that binding of type II ATP site inhibitors, such as imatinib, generates a force from the KD N-lobe onto the SH3 domain and in consequence disassembles the core. Here, we demonstrate that the C-terminal alpha I-helix exerts an additional force toward the SH2 domain, which correlates both with kinase activity and type II inhibitor-induced disassembly. The alpha I-helix mutation E528K, which is responsible for the ABL1 malformation syndrome, strongly activates Abl by breaking a salt bridge with the KD C-lobe and thereby increasing the force onto the SH2 domain. In contrast, the allosteric inhibitor asciminib strongly reduces Abl's activity by fixating the alpha I-helix and reducing the force onto the SH2 domain. These observations are explained by a simple mechanical model of Abl activation involving forces from the KD N-lobe and the alpha I-helix onto the KD/SH2SH3 interface.ISSN:2050-084

C-12 Helices in Long Hybrid (alpha gamma)(n) Peptides Composed Entirely of Unconstrained Residues with Proteinogenic Side Chains

Unconstrained gamma(4) amino acid residues derived by homologation of proteinogenic amino acids facilitate helical folding in hybrid (alpha gamma)(n) sequences. The C-12 helical conformation for the decapeptide, Boc-Leu-gamma(4)(R)Val](5)-OMe, is established in crystals by X-ray diffraction. A regular C-12 helix is demonstrated by NMR studies of the 18 residue peptide, Boc-Leu-gamma(4)(AR)Val](9)-OMe, and a designed 16 residue (alpha gamma)(n) peptide, incorporating variable side chains. Unconstrained (alpha gamma)(n) peptides show an unexpectedly high propensity for helical folding in long polypeptide sequences

Directing peptide conformation with centrally positioned pre-organized dipeptide segments: studies of a 12-residue helix and beta-hairpin

Secondary structure formation in oligopeptides can be induced by short nucleating segments with a high propensity to form hydrogen bonded turn conformations. Type I/III turns facilitate helical folding while type II'/I' turns favour hairpin formation. This principle is experimentally verified by studies of two designed dodecapeptides, Boc-Val-Phe-Leu-Phe-Val-Aib-Aib-Val-Phe-Leu-Phe-Val-OMe 1 and Boc-Val-Phe-Leu-Phe-Val- (D) Pro- (L) Pro-Val-Phe-Leu-Phe-Val-OMe 2. The N- and C-terminal flanking pentapeptide sequences in both cases are identical. Peptide 1 adopts a largely alpha-helical conformation in crystals, with a small 3(10) helical segment at the N-terminus. The overall helical fold is maintained in methanol solution as evidenced by NMR studies. Peptide 2 adopts an antiparallel beta-hairpin conformation stabilized by 6 interstrand hydrogen bonds. Key nuclear Overhauser effects (NOEs) provide evidence for the antiparallel beta-hairpin structure. Aromatic proton chemical shifts provide a clear distinction between the conformation of peptides 1 (helical) and 2 (beta-hairpin). The proximity of facing aromatic residues positioned at non-hydrogen bonding positions in the hairpin results in extensively ring current shifted proton resonances in peptide 2

Rotating Structure Modeling and Damping Measurements

The structural damping is of importance to suppress the vibration amplitude of compressor blades rotating at high angular velocity under a high cycle impact. To avoid the appearance of the high cycle fatigue (HCF), damping materials may be applied to the compressor blades. To quantify the effect while using damping materials, a numerical tool needs to be developed for the damping prediction of a dynamic rotating blade. This thesis is divided into two parts: Paper A develops a dynamic model of a rotating blade and Paper B a damping structure model including measurements. In Paper A, a dynamic rotating blade model is developed by using a plate model at an arbitrary stagger angle. Hamilton’s principle is applied to derive a system of equations of motion and the corresponding boundary conditions. Numerical simulation is implemented to perform eigenfrequency analysis by the Extended Galerkin method. In addition, parametric analysis is performed with respect to rotation speed and stagger angle, respectively. Results show a good agreement with those of the finite element method. Finally, forced response analysis is determined for two cases; a point force and a distribution force, using a proportional damping model. In Paper B, unconstrained and constrained damping techniques are applied to increase the structural damping of the blades, including measurement and modeling results. Two specimens, titanium and stainless steel, are treated by aluminum oxide and epoxy coating material. Measurement results show that both treatments give damping increase, where aluminum oxide is more effective for damping improvement than the corresponding epoxy treatment. The unconstrained damping layer model is used to predict the total material damping of the combined structure as well as the material damping of coating layer. Furthermore, the constrained-layer model is used to optimize the damping configuration. Two compressor blades in titanium and stainless steel are tested in air and vacuum. One reason is being that the radiation loss factor increases the total damping comparing with that under vacuum condition. The calculation of radiation loss factor is performed to match the measurement data. Finally, increased material damping decreases peak stress and therefore increases the life time of the compressor blades.QC 20110311</p