Discovery and Optimization of the First ATP Competitive Type-III c-MET Inhibitor.

Recent clinical reports have highlighted the need for wild-type (WT) and mutant dual inhibitors of c-MET kinase for the treatment of cancer. We report herein a novel chemical series of ATP competitive type-III inhibitors of WT and D1228V mutant c-MET. Using a combination of structure-based drug design and computational analyses, ligand 2 was optimized to a highly selective chemical series with nanomolar activities in biochemical and cellular settings. Representatives of the series demonstrate excellent pharmacokinetic profiles in rat in vivo studies with promising free-brain exposures, paving the way for the design of brain permeable drugs for the treatment of c-MET driven cancers.

Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance.

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.

What is the Role of Hyperosmolar Therapy in Hemispheric Stroke Patients?

The role of hyperosmolar therapy (HT) in large hemispheric ischemic or hemorrhagic strokes remains a controversial issue. Past and current stroke guidelines state that it represents a reasonable therapeutic measure for patients with either neurological deterioration or intracranial pressure (ICP) elevations documented by ICP monitoring. However, the lack of evidence for a clear effect of this therapy on radiological tissue shifts and clinical outcomes produces uncertainty with respect to the appropriateness of its implementation and duration in the context of radiological mass effect without clinical correlates of neurological decline or documented elevated ICP. In addition, limited data suggest a theoretical potential for harm from the prophylactic and protracted use of HT in the setting of large hemispheric lesions. HT exerts effects on parenchymal volume, cerebral blood volume and cerebral perfusion pressure which may ameliorate global ICP elevation and cerebral blood flow; nevertheless, it also holds theoretical potential for aggravating tissue shifts promoted by significant interhemispheric ICP gradients that may arise in the setting of a large unilateral supratentorial mass lesion. The purpose of this article is to review the literature in order to shed light on the effects of HT on brain tissue shifts and clinical outcome in the context of large hemispheric strokes, as well as elucidate when HT should be initiated and when it should be avoided.

Promiscuous G-Protein-Coupled Receptor Inhibition of Transient Receptor Potential Melastatin 3 Ion Channels by Gßγ Subunits.

Transient receptor potential melastatin 3 (TRPM3) is a nonselective cation channel that is inhibited by Gßγ subunits liberated following activation of Gαi/o protein-coupled receptors. Here, we demonstrate that TRPM3 channels are also inhibited by Gßγ released from Gαs and Gαq Activation of the Gs-coupled adenosine 2B receptor and the Gq-coupled muscarinic acetylcholine M1 receptor inhibited the activity of TRPM3 heterologously expressed in HEK293 cells. This inhibition was prevented when the Gßγ sink ßARK1-ct (C terminus of ß-adrenergic receptor kinase-1) was coexpressed with TRPM3. In neurons isolated from mouse dorsal root ganglion (DRG), native TRPM3 channels were inhibited by activating Gs-coupled prostaglandin-EP2 and Gq-coupled bradykinin B2 (BK2) receptors. The Gi/o inhibitor pertussis toxin and inhibitors of PKA and PKC had no effect on EP2- and BK2-mediated inhibition of TRPM3, demonstrating that the receptors did not act through Gαi/o or through the major protein kinases activated downstream of G-protein-coupled receptor (GPCR) activation. When DRG neurons were dialyzed with GRK2i, which sequesters free Gßγ protein, TRPM3 inhibition by EP2 and BK2 was significantly reduced. Intraplantar injections of EP2 or BK2 agonists inhibited both the nocifensive response evoked by TRPM3 agonists, and the heat hypersensitivity produced by Freund's Complete Adjuvant (FCA). Furthermore, FCA-induced heat hypersensitivity was completely reversed by the selective TRPM3 antagonist ononetin in WT mice and did not develop in Trpm3-/- mice. Our results demonstrate that TRPM3 is subject to promiscuous inhibition by Gßγ protein in heterologous expression systems, primary neurons and in vivo, and suggest a critical role for this ion channel in inflammatory heat hypersensitivity.SIGNIFICANCE STATEMENT The ion channel TRPM3 is widely expressed in the nervous system. Recent studies showed that Gαi/o-coupled GPCRs inhibit TRPM3 through a direct interaction between Gßγ subunits and TRPM3. Since Gßγ proteins can be liberated from other Gα subunits than Gαi/o, we examined whether activation of Gs- and Gq-coupled receptors also influence TRPM3 via Gßγ. Our results demonstrate that activation of Gs- and Gq-coupled GPCRs in recombinant cells and sensory neurons inhibits TRPM3 via Gßγ liberation. We also demonstrated that Gs- and Gq-coupled receptors inhibit TRPM3 in vivo, thereby reducing pain produced by activation of TRPM3, and inflammatory heat hypersensitivity. Our results identify Gßγ inhibition of TRPM3 as an effector mechanism shared by the major Gα subunits.

An Ethical (Descriptive) Framework for Judgment of Actions and Decisions in the Construction Industry and Engineering-Part I.

The construction industry is usually characterized as a fragmented system of multiple-organizational entities in which members from different technical backgrounds and moral values join together to develop a particular business or project. The greatest challenge in the construction process for the achievement of a successful practice is the development of an outstanding reputation, which is built on identifying and applying an ethical framework. This framework should reflect a common ethical ground for myriad people involved in this process to survive and compete ethically in today's turbulent construction market. This study establishes a framework for ethical judgment of behavior and actions conducted in the construction process. The framework was primarily developed based on the essential attributes of business management identified in the literature review and subsequently incorporates additional attributes identified to prevent breaches in the construction industry and common ethical values related to professional engineering. The proposed judgment framework is based primarily on the ethical dimension of professional responsibility. The Ethical Judgment Framework consists of descriptive approaches involving technical, professional, administrative, and miscellaneous terms. The framework provides the basis for judging actions as either ethical or unethical. Furthermore, the framework can be implemented as a form of preventive ethics, which would help avoid ethical dilemmas and moral allegations. The framework can be considered a decision-making model to guide actions and improve the ethical reasoning process that would help individuals think through possible implications and consequences of ethical dilemmas in the construction industry.

G protein ßγ subunits inhibit TRPM3 ion channels in sensory neurons.

Transient receptor potential (TRP) ion channels in peripheral sensory neurons are functionally regulated by hydrolysis of the phosphoinositide PI(4,5)P2 and changes in the level of protein kinase mediated phosphorylation following activation of various G protein coupled receptors. We now show that the activity of TRPM3 expressed in mouse dorsal root ganglion (DRG) neurons is inhibited by agonists of the Gi-coupled µ opioid, GABA-B and NPY receptors. These agonist effects are mediated by direct inhibition of TRPM3 by Gßγ subunits, rather than by a canonical cAMP mediated mechanism. The activity of TRPM3 in DRG neurons is also negatively modulated by tonic, constitutive GPCR activity as TRPM3 responses can be potentiated by GPCR inverse agonists. GPCR regulation of TRPM3 is also seen in vivo where Gi/o GPCRs agonists inhibited and inverse agonists potentiated TRPM3 mediated nociceptive behavioural responses.

A Moral (Normative) Framework for the Judgment of Actions and Decisions in the Construction Industry and Engineering: Part II.

The construction industry is typically characterized as a fragmented, multi-organizational setting in which members from different technical backgrounds and moral values join together to develop a particular business or project. The most challenging obstacle in the construction process is to achieve a successful practice and to identify and apply an ethical framework to manage the behavior of involved specialists and contractors and to ensure the quality of all completed construction activities. The framework should reflect a common moral ground for myriad people involved in this process to survive and compete ethically in today's turbulent construction market. This study establishes a framework for moral judgment of behavior and actions conducted in the construction process. The moral framework provides the basis of judging actions as "moral" or "immoral" based on three levels of moral accountability: personal, professional, and social. The social aspect of the proposed framework is developed primarily from the essential attributes of normative business decision-making models identified in the literature review and subsequently incorporates additional attributes related to professional and personal moral values. The normative decision-making models reviewed are based primarily on social attributes as related to moral theories (e.g., utilitarianism, duty, rights, virtue, etc.). The professional and moral attributes are established by identifying a set of common moral values recognized by professionals in the construction industry and required to prevent common construction breaches. The moral framework presented here is the complementary part of the ethical framework developed in Part I of this article and is based primarily on the personal behavior or the moral aspect of professional responsibility. The framework can be implemented as a form of preventive personal ethics, which would help avoid ethical dilemmas and moral implications in the first place. Furthermore, the moral framework can be considered as a decision-making model to guide actions and improve the moral reasoning process, which would help individuals think through possible implications and the consequences of ethical and moral issues in the construction industry.

Changes in intracuff pressure of a cuffed endotracheal tube during surgery for congenital heart disease using cardiopulmonary bypass.

BACKGROUND: With the development of newer polyurethane cuffed endotracheal tubes (cETTs), there has been a shift in clinical practice among pediatric anesthesiologists. Despite improvements in design, excessive inflation of the cuff can still compromise tracheal mucosal perfusion. Several perioperative factors can affect the intracuff pressure (CP), and there is no consensus on safe CP in pediatric patients undergoing repair of congenital cardiac disease (CHD) utilizing cardiopulmonary bypass (CPB). In the current study, the CP was continuously monitored in pediatric patients undergoing surgery for CHD. METHODS: After IRB approval, this observational study was conducted on pediatric patients who underwent repair of CHD using CPB with a cETT in place. After anesthetic induction and endotracheal intubation, the cuff was inflated using the air leak technique while maintaining a continuous positive airway pressure of 20 cmH2 O. After inflation, the CP was continuously monitored throughout the procedure. In addition, temperature and mean arterial pressure (MAP) were also recorded. RESULTS: The study included 33 patients who ranged in age from 1 month to 15.3 years. Their weight ranged from 4.0 to 83.6 kg. Six patients were excluded from the analysis due to the need to add or remove air from the cuff, leaving 27 patients for data analysis for cuff pressure over time. The baseline CP at the time of inflation was 16.1 ± 7.6 cmH2 O. With the use of CPB and initiation of hypothermia, when compared to the baseline, the CP decreased by -0.7 ± 5.8 cmH2 O at 35-37°C, -9.1 ± 8.4 cmH2 O at 31-33°C, -7.8 ± 6.2 cmH2 O at 27-29°C, and -11.1 ± 6.0 cmH2 O at <27°C. With rewarming, the CP increased back to the baseline level (-3.5 ± 7.0 cmH2 O). CONCLUSION: There was a significant decrease in the CP during CPB and associated hypothermia. This may offer some protection for mucosal perfusion during CPB which is usually associated with lower than normal MAP. However, the decrease in the CP may compromise the tracheal seal which may not offer the intended protection for the airway from aspiration.

Targeted silencing of anthrax toxin receptors protects against anthrax toxins.

Anthrax spores can be aerosolized and dispersed as a bioweapon. Current postexposure treatments are inadequate at later stages of infection, when high levels of anthrax toxins are present. Anthrax toxins enter cells via two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). We hypothesized that host cells would be protected from anthrax toxins if anthrax toxin receptor expression was effectively silenced using RNA interference (RNAi) technology. Thus, anthrax toxin receptors in mouse and human macrophages were silenced using targeted siRNAs or blocked with specific antibody prior to challenge with anthrax lethal toxin. Viability assays were used to assess protection in macrophages treated with specific siRNA or antibody as compared with untreated cells. Silencing CMG2 using targeted siRNAs provided almost complete protection against anthrax lethal toxin-induced cytotoxicity and death in murine and human macrophages. The same results were obtained by prebinding cells with specific antibody prior to treatment with anthrax lethal toxin. In addition, TEM8-targeted siRNAs also offered significant protection against lethal toxin in human macrophage-like cells. Furthermore, silencing CMG2, TEM8, or both receptors in combination was also protective against MEK2 cleavage by lethal toxin or adenylyl cyclase activity by edema toxin in human kidney cells. Thus, anthrax toxin receptor-targeted RNAi has the potential to be developed as a life-saving, postexposure therapy against anthrax.