NMR reveals the allosteric opening and closing of Abelson tyrosine kinase by ATP-site and myristoyl pocket inhibitors

Successful treatment of chronic myelogenous leukemia is based on inhibitors binding to the ATP site of the deregulated breakpoint cluster region (Bcr)–Abelson tyrosine kinase (Abl) fusion protein. Recently, a new type of allosteric inhibitors targeting the Abl myristoyl pocket was shown in preclinical studies to overcome ATP-site inhibitor resistance arising in some patients. Using NMR and small-angle X-ray scattering, we have analyzed the solution conformations of apo Abelson tyrosine kinase (c-Abl) and c-Abl complexes with ATP-site and allosteric inhibitors. Binding of the ATP-site inhibitor imatinib leads to an unexpected open conformation of the multidomain SH3-SH2-kinase c-Abl core, whose relevance is confirmed by cellular assays on Bcr-Abl. The combination of imatinib with the allosteric inhibitor GNF-5 restores the closed, inactivated state. Our data provide detailed insights on the poorly understood combined effect of the two inhibitor types, which is able to overcome drug resistance.Protein kinases, such as Abelson tyrosine kinase (c-Abl), control numerous cellular signal pathways, and therefore require tight regulation (1). Modulation of c-Abl activity is achieved by an autoinhibitory mechanism involving the N-terminal region of the protein (1, 2). A myristoyl moiety covalently bound to Gly2 occupies a deep pocket located in the C-lobe of the kinase domain and stabilizes docking of the SH3 and SH2 domains to the kinase domain (3–5) (Fig. 1). In the oncogenic fusion protein Bcr-Abl, this crucial autoinhibition is lost as a result of chromosomal translocation, leading ultimately to chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia (6, 7).Open in a separate windowFig. 1.Domain definition and structure of the c-Abl fragment c-Abl^83–534. (A) Crystal structure of c-Abl in its fully assembled state (PDB ID code 2FO0). The inhibitor PD166326 (orange) is bound to the ATP site, whereas myristic acid (gray) occupies a deep pocket in the C-lobe of the kinase domain. Domain coloring follows that in B. Residues 2–82, which form the disordered N-terminal cap, are schematically represented by a gray dashed line. (B) Amino acid sequence and schematic representation of secondary structure elements of c-Abl^83–534. Color coding is as follows: SH3 domain (green), SH2 domain (yellow), kinase domain (blue), interdomain linkers (red), and activation loop (magenta). (C) Chemical structures of the ATP-competitive inhibitor imatinib (STI-571/Gleevec; Novartis) and the allosteric inhibitor GNF-5.The ATP-binding site inhibitors imatinib (STI-571/Gleevec), nilotinib (AMN-107/Tasigna), and dasatinib (Sprycel) constitute the front-line therapy against CML (8–11). However, spontaneous point mutations render these inhibitors ineffective and cause clinical relapse in advanced-phase patients (12, 13). Although nilotinib and dasatinib retain their efficacy against many of the imatinib-resistant mutants, the “gatekeeper” T334I mutation (T315I in Abl 1a numbering) abrogates the binding of all three inhibitors (12). Emergence of this multidrug-resistant mutant, which occurs in ∼15% of patients with resistance to imatinib, has stimulated the search for new therapeutics (14). Recently, several new ATP-competitive inhibitors that are active against the T334I mutant (15–17) have been developed, and one of these, ponatinib (Iclusig) (15), has received US Food and Drug Administration approval. An alternative approach has resulted from the discovery of allosteric inhibitors, which bind to the myristoyl-binding pocket of c-Abl (18, 19). Subsequent studies have revealed that combining allosteric inhibitors with ATP-competitive inhibitors overcomes T334I-related resistance in an in vivo model and may be a relevant therapeutic strategy (20). The importance of the myristoyl-binding pocket is further supported by the discovery of small-molecule c-Abl activators that bind to this site (21, 22).c-Abl and other tyrosine kinases are regulated by complicated allosteric interactions between their constituent domains (5). Whereas crystallographic structures have laid the foundation for our current understanding of c-Abl regulation, the vast majority of solved structures represent the isolated kinase domain in complex with small molecules. Only two reports by Kuriyan and coworkers (3, 5) provide structures of the entire minimal autoregulatory fragment of c-Abl, which comprises the SH3, SH2, and kinase (also termed SH1) domains. In both cases, the protein was complexed with an ATP-site inhibitor and a myristoyl chain attached covalently to Gly2 or added in trans. Three structural features were identified as requirements for the assembly of a “closed,” inactive state (5) (Fig. 1A): (i) docking of the SH3 domain to a polyproline helix in the SH2-kinase linker, (ii) docking of the SH2 domain to the kinase domain facilitated by the binding of the myristoyl moiety, and (iii) the clamp formed by the N-terminal cap region. The removal of these “linchpins” resulted in an “activated” c-Abl mutant with the SH3-SH2-kinase domains arranged into an elongated structure with a direct contact between the SH2 domain and the N-lobe of the kinase (5). This has inspired further studies on the role of the SH2 domain in the regulation of c-Abl and other kinases (23–25).Crystal structures represent frozen snapshots of protein states that may not reflect all physiologically relevant conformations. In particular, it is expected that the active forms of c-Abl and other kinases undergo dynamic exchange, which makes them difficult to crystallize. Indeed, for example, the apo form of c-Abl has resisted crystallization so far. In principle, solution NMR can provide much of the missing dynamic information to understand protein function (26). However, its applicability to larger systems is restricted by its inherent size limit, its low sensitivity, and the need for isotope labeling. Thus, so far, solution conformations and dynamics have been analyzed by NMR for smaller fragments of protein kinases comprising the catalytic and/or adjacent domains, for example, of protein kinase A (27, 28), MAP kinase p38 (29, 30), and Eph receptor tyrosine kinase (31), as well as c-Abl (32) (c-Abl^248-519; throughout this report the amino acid numbering follows the 1b isoform). Here, we have determined the solution conformation and domain motions of the considerably larger autoregulatory fragment c-Abl^83–534 (designated as c-Abl in the following when clear from the context), which comprises the SH3, SH2, and kinase domains (Fig. 1B), by advanced NMR experiments in combination with small-angle X-ray scattering (SAXS). The data provide the first structural information on the apo form of c-Abl in the absence of inhibitors, which is shown to adopt the “closed” conformation. Unexpectedly, the addition of the catalytic site inhibitor imatinib induces a large structural rearrangement characterized by the detachment of the SH3-SH2 domains from the kinase domain and the formation of a dynamic “open” inactive state, which is inhibited in the ATP site. As a consequence of this opening, Tyr245 within the SH2-kinase linker becomes exposed. The in vivo relevance of this unexpected opening is corroborated by an observed increase in Tyr245 phosphorylation at moderate imatinib concentrations in cellular assays on full-length Bcr-Abl. In contrast to imatinib, addition of the myristoyl pocket inhibitor GNF-5 (20) to apo c-Abl induces only local changes around the myristoyl-binding pocket and keeps the protein in the “closed” state. However, addition of GNF-5 to the “open” c-Abl•imatinib complex restores the “closed,” inactive conformation. These findings on the allosteric actions of ATP and myristoyl pocket inhibitors reveal molecular details of their recently reported synergy to overcome drug resistance and may help to devise new strategies for drug development.     

Numerical Study on the Dependency of Microstructure Morphologies of Pulsed Laser Deposited TiN Thin Films and the Strain Heterogeneities during Mechanical Testing

Numerical study of the influence of pulsed laser deposited TiN thin films’ microstructure morphologies on strain heterogeneities during loading was the goal of this research. The investigation was based on the digital material representation (DMR) concept applied to replicate an investigated thin film’s microstructure morphology. The physically based pulsed laser deposited model was implemented to recreate characteristic features of a thin film microstructure. The kinetic Monte Carlo (kMC) approach was the basis of the model in the first part of the work. The developed kMC algorithm was used to generate thin film’s three-dimensional representation with its columnar morphology. Such a digital model was then validated with the experimental data from metallographic analysis of laboratory deposited TiN(100)/Si. In the second part of the research, the kMC generated DMR model of thin film was incorporated into the finite element (FE) simulation. The 3D film’s morphology was discretized with conforming finite element mesh, and then incorporated as a microscale model into the macroscale finite element simulation of nanoindentation test. Such a multiscale model was finally used to evaluate the development of local deformation heterogeneities associated with the underlying microstructure morphology. In this part, the capabilities of the proposed approach were clearly highlighted.     

Proprioceptive contribution to oculomotor control in humans

Stretch receptors in the extraocular muscles (EOMs) inform the central nervous system about the rotation of one''s own eyes in the orbits. Whereas fine control of the skeletal muscles hinges critically on proprioceptive feedback, the role of proprioception in oculomotor control remains unclear. Human behavioural studies provide evidence for EOM proprioception in oculomotor control, however, behavioural and electrophysiological studies in the macaque do not. Unlike macaques, humans possess numerous muscle spindles in their EOMs. To find out whether the human oculomotor nuclei respond to proprioceptive feedback we used functional magnetic resonance imaging (fMRI). With their eyes closed, participants placed their right index finger on the eyelid at the outer corner of the right eye. When prompted by a sound, they pushed the eyeball gently and briefly towards the nose. Control conditions separated out motor and tactile task components. The stretch of the right lateral rectus muscle was associated with activation of the left oculomotor nucleus and subthreshold activation of the left abducens nucleus. Because these nuclei control the horizontal movements of the left eye, we hypothesized that proprioceptive stimulation of the right EOM triggered left eye movement. To test this, we followed up with an eye‐tracking experiment in complete darkness using the same behavioural task as in the fMRI study. The left eye moved actively in the direction of the passive displacement of the right eye, albeit with a smaller amplitude. Eye tracking corroborated neuroimaging findings to suggest a proprioceptive contribution to ocular alignment.     

Post-exercise oxidative stress and obesity in postmenopausal women: the role of beta3-adrenergic receptor polymorphism.

AIM: Some studies indicate that the Trp64Arg polymorphism in the gene encoding the beta3-adrenergic receptor (ADRB3) is associated with obesity, insulin resistance and earlier onset of type 2 diabetes mellitus. The aim of the present study was to evaluate the frequency of this polymorphism and its relationship with obesity and oxidative stress in postmenopausal women. MATERIAL AND METHODS: We performed the study on 200 women, aged 50-60 years. Estimation of anthropometric parameters and total body fat, android and gynoid fat deposits was carried out using dual-energy X-ray absorptiometry. Oxidative stress was estimated by measurement of thiobarbituric acid-reactive substances (TBARS) in serum. Blood for analysis was collected before, directly after and 6 h after a 30-min physical test on a cycle ergometer. ADRB3 genotyping was performed by polymerase chain reaction. RESULTS: The frequency of Trp64/Arg64 genotype in the investigated population was 12%, and of Trp64/Trp64 was 87%. The Arg64/Arg64 genotype was present in only 1% of women. Women bearing the Trp64/Arg64 genotype did not differ in any measured anthropometric parameters from women bearing the Trp64/Trp64 genotype. Moreover, genotype had no influence on oxidative stress parameters. Likewise, in both groups, mean plasma level of TBARS was increased significantly (p < 0.05) directly after the endurance test and remained elevated 6 h after the test. CONCLUSIONS: The Trp64Arg polymorphism of ADRB3 seems to not be related to obesity in postmenopausal women. Moreover, the Trp64Arg polymorphism has no influence on oxidative stress intensification after standardized physical effort in postmenopausal women.     

Management of cervical and lumbar stenosis]

Spinal stenosis is most common in elderly patients and is defined as narrowing of the spinal canal and (or) lateral nerve root canals. The underlying processes leading to spinal stenosis are degenerative changes in facet joints and intervertebral discs and buckling of the ligamentum flavum. Spinal stenosis can occur in both the cervical and the lumbar spine. Cervical stenosis mayleat to the development of radiculopathy and (or) myelopathy. The majority of patients respond to nonoperative management. Degenerative lumbar spinal stenosis presents with back and (or) leg paints of valuing severity and duration. Nonoperative treatment associated with lumbar spinal stenosis consists of restituting and avoiding those maneuvers that reproduce pain. Surgical treatment of cervical and lumbar stenosis includes decompressive lamine ctomy, often fusion and instrumentation.     

Renovascular hypertension – simultaneous aortic and renal artery reconstruction

Despite the enormous progress that has been madein the fields of vascular surgery and intensive care, reconstructive surgery of renal arteries in the presence of arteriosclerosis of the abdominal aorta remains a serious therapeutic problem and is associated with a high rate of complications. The purpose of this study was to analyze the results of treatment of patients with renovascular hypertension and so-called difficult aorta. Surgery was carried out in 68 patients with a critically stenosed renal artery and severe arteriosclerosis of the abdominal aorta. In 53 patients an aortobifemoral prosthetic graft was implanted together with endarterectomy of the renal artery in 23 patients, an aortorenal venous graft in 23 patients and a prosthetic graft in 7 patients. In the remaining 15 patients extra-anatomic anastomoses were performed between the splenic artery and the renal artery (7 patients), hepatic artery and renal artery (6) and between the superior mesenteric and renal artery (2). Postoperatively, the hypertension was cured in 55% of patients, improved in 38% and remained unchanged in 7%. After 1 year the results were respectively 47, 36, and 17%. The patients with an aortorenal prosthetic graft demonstrated a greater tendency for hypertension to recur.     

Involuntary ureteral micturition]

The paper focuses on the problem of ureteric incontinence of urine caused by dislocation of bladder ureteral orifice. A case of a 15-year old patient suffering from incontinence of urine, admitted to the Urological Clinic has been described. In order to avoid vaginoscopy, the following examinations were performed: urography, chromocystoscopy, arteriography and computer tomography. The reason for such a large number of various examinations was an unclear picture of the left kidney and the shape of the ureter, suggesting a tumour in the retroperitoneal space. The treatment ended with a removal of the kidney. The disclosed retroperitoneal space turned out to be affected by hydronephrosis.     

Effect of bilateral mediastinal lymphadenectomy on short-term pulmonary function.

OBJECTIVE: To assess if the bilateral mediastinal lymphadenectomy results in lymphatic congestion in the lungs producing clinically significant impairment of respiratory function. METHODS: In the prospective, randomized, double-blind clinical study, non-small cell lung carcinoma patients underwent preoperatively mediastinoscopy or the transcervical extended mediastinal lymphadenectomy (TEMLA). In both groups, the blood gas analysis and spirometry were measured preoperatively and on the 1st, 3rd, and 5th postoperative day, and the carbon monoxide diffusing capacity of the lung (DLCO) and lung compliance were measured preoperatively and on the 3-5 postoperative day. Any respiratory complications were also recorded. RESULTS: Forty-one patients were randomized: 21 to the TEMLA group and 20 to the mediastinoscopy group. There was no significant difference of the baseline and the 1st, 3rd, and 5th day measurements of vital capacity and forced expiratory volume (FEV1) (p>0.98), pH, pO(2), pCO(2), standard bicarbonates and base excess (p>0.31), nor significant difference of baseline and 3-5 day measurements for DLCO (p=0.91) and lung compliance (p=0.38). The incidence of respiratory insufficiency was not significantly different (p=0.51). CONCLUSIONS: (1) Complete excision of mediastinal lymph nodes stations 1, 2R, 2L, 3A, 4R, 4L, 5, 6, 7, and 8 (TEMLA) is not associated with greater incidence of respiratory insufficiency comparing with standard mediastinoscopy. (2) The TEMLA procedure does not produce greater alterations in spirometry, blood gas analysis, DLCO and lung compliance comparing with standard mediastinoscopy.     

Cyclin-dependent kinase 2 expression in human melanomas and benign melanocytic skin lesions

Cyclin-dependent kinase 2 (CDK-2) is strongly involved in regulating the progression of the cell cycle through G1/S checkpoint and S phase. Numerous studies demonstrated increased levels of CDK-2 (and also of its regulatory cyclins E and/or A) in different types of human tumours. Correlations found between the expression of those cell cycle regulators and progression and/or invasiveness of some tumours indicated the importance of CDK-2 as a potential prognostic marker. At the same time, in vitro studies of melanoma cell lines revealed melanocyte-specific regulation of CDK-2. The present study was aimed at examining levels of CDK-2 in human melanomas and benign pigmented lesions to evaluate whether it might be considered a potential molecular marker of melanoma progression. Expression of CDK-2 was determined immunohistochemically in formalin-fixed paraffin-embedded specimens comprising 76 lesions including 41 primary cutaneous melanomas, 15 lymph node melanoma metastases (in eight cases correlated with primary tumours), three melanoma recurrences (two cases correlated with both primary and metastatic melanomas) and 17 nevi. Our results demonstrate that development and progression of melanoma are associated with changes in CDK-2 expression level. Statistical significance of the observed correlations indicates that CDK-2 may be a suitable prognostic marker for melanoma and perhaps also a target for chemotherapeutic drugs.