Characterisation of Na/K-ATPase, its isoforms, and the inotropic response to ouabain in isolated failing human hearts

Objective: The aim was to determine whether failing human hearts have increased sensitivity to the inotropic and toxic effects of ouabain, and to examine alterations in Na/K-ATPase that might explain the observed higher ouabain sensitivity. Methods: For contractility studies, a total of 57 trabeculae were isolated from two non- failing (death from head injury) and 10 terminally failing, explanted human hearts. After the experiment, each trabecula was inspected under the light microscope for morphological alterations consistent with heart failure. Samples for biochemical and molecular studies were obtained from five non-failing and 13 failing hearts. Total Na/K-ATPase was measured in desoxycholate treated homogenates and expressed per unit of tissue wet or dry weight, DNA, protein, or myosin. Interference from residual bound digoxin due to previous therapy was excluded. The expression of the three α isoforms was studied at both the mRNA level using northern blots and the protein level by analysis of dissociation kinetics of the [3H]ouabain-enzyme complex. Results: Trabeculae showing morphological alterations and decreased contractility were sensitive to lower concentrations of ouabain (3-100 nM) than control trabeculae (100-1000 nM); the inotropic EC50 and the minimum toxic concentration were both reduced. [3H]Ouabain binding was significantly lower (p≪0.001) in failing than in non-failing hearts, at 293(SD 74) v 507(48) pmol·g−1 wet weight. No significant change was observed in maximum ATPase turnover rate, or in sensitivities to Na+, K+, vanadate, and dihydro-ouabain. All three α isoforms were expressed at the mRNA level in both normal and failing hearts. Conclusions: This study shows conclusively, for the first time, that failing human hearts are more sensitive to ouabain. This may be at least partly due to a mean reduction of 42% (95% confidence interval, 26 to 56%) in the concentration of Na/K-ATPase (decrease in Na,K pump reserve), but not to an alteration in its catalytic properties or in its isoform composition. Cardiovascular Research 1993;27:2229-223

Genetic resistance to JAK2 enzymatic inhibitors is overcome by HSP90 inhibition

Enzymatic inhibitors of Janus kinase 2 (JAK2) are in clinical development for the treatment of myeloproliferative neoplasms (MPNs), B cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit cytokine receptor–like factor 2 (CRLF2), and other tumors with constitutive JAK2 signaling. In this study, we identify G935R, Y931C, and E864K mutations within the JAK2 kinase domain that confer resistance across a panel of JAK inhibitors, whether present in cis with JAK2 V617F (observed in MPNs) or JAK2 R683G (observed in B-ALL). G935R, Y931C, and E864K do not reduce the sensitivity of JAK2-dependent cells to inhibitors of heat shock protein 90 (HSP90), which promote the degradation of both wild-type and mutant JAK2. HSP90 inhibitors were 100–1,000-fold more potent against CRLF2-rearranged B-ALL cells, which correlated with JAK2 degradation and more extensive blockade of JAK2/STAT5, MAP kinase, and AKT signaling. In addition, the HSP90 inhibitor AUY922 prolonged survival of mice xenografted with primary human CRLF2-rearranged B-ALL further than an enzymatic JAK2 inhibitor. Thus, HSP90 is a promising therapeutic target in JAK2-driven cancers, including those with genetic resistance to JAK enzymatic inhibitors

Leveraging the contribution of thermodynamics in drug discovery with the help of fluorescence-based thermal shift assays

The development of new drugs with better pharmacological and safety properties mandates the optimization of several parameters. Today, potency is often used as the sole biochemical parameter to identify and select new molecules. Surprisingly, thermodynamics, which is at the core of any interaction, is rarely used in drug discovery, even though it has been suggested that the selection of scaffolds according to thermodynamic criteria may be a valuable strategy. This poor integration of thermodynamics in drug discovery might be due to difficulties in implementing calorimetry experiments despite recent technological progress in this area. In this report, we show that fluorescence-based thermal shift assays could be used as pre-screening methods to identify compounds with different thermodynamic profiles. This approach allows a reduction in the number of compounds to be tested in calorimetry experiments, thus favoring greater integration of thermodynamics in drug discovery

The Central Valine Concept Provides an Entry in a New Class of Non Peptide Inhibitors of the P53-MDM2 Interaction

Disrupting the interaction between the p53 tumor suppressor and its regulator MDM2 is a promising therapeutic strategy in anticancer drug research. In our search for non peptide inhibitors of this protein-protein interaction , we have devised a ligand design concept exploiting the central position of Val 93 in the p53 binding pocket of MDM2. The design of molecules based on this concept has allowed us to rapidly identify compounds having a 3-imidazolyl indole core structure as the first representatives of a new class of potent inhibitors of the p53-MDM2 interaction

Study of the selectivity of insulin-like growth factor-1 receptor (IGF1R) inhibitors

The insulin-like growth factor-1 receptor (IGF1R) is a drug target for oncology, and many studies are ongoing to identify compounds that inhibit its tyrosine kinase activity. IGF1R is highly homologous to the insulin receptor (IR) and IGF1R inhibition might be beneficial for patients, while IR inhibition may lead to limiting toxicity. Therefore selectivity for IGF1R over IR is the aim for drug design in this context. A few compounds that selectively inhibit IGF1R over IR in cells have been identified, but none of them show the same levels of selectivity in enzymatic assays. To determine whether this discrepancy is linked to the conditions used in the enzymatic assays, we have studied the interaction between known IGF1R inhibitors (NVP-AEW541, OSI906, AG538, NVP-TAE226) and phosphorylated/unphosphorylated IGF1R/IR proteins with both biophysical (isothermal calorimetry and surface plasmon resonance) and enzymatic methods. In this report, we describe the results of this study and comment on the different degrees of selectivity IGF1R versus IR measured in biochemical and cellular assays. Finally, our study provides new information on the biochemical and mechanism of action of these small molecular weight IGF1R inhibitor

Factors influencing the inhibition of protein kinases

The protein kinase field is a very active research area in the pharmaceutical industry and many activities are ongoing to identify inhibitors of these proteins. The design of new chemical entities with improved pharmacological properties requires a deeper understanding of the factors that modulate inhibitor-kinase interactions. In this report, we study the effect of two of these factors – the magnesium ion cofactor and the protein substrate – on inhibitors of the type I insulin-like growth factor receptor. Our results show that the concentration of magnesium ion influences the potency of ATP competitive inhibitors, suggesting an explanation for the observation that such compounds retain their nanomolar potency in cells despite the presence of millimolar levels of ATP. We also show that the peptidic substrate affects the potency of these IGF1R ATP competitive inhibitors in a different manner, suggesting that the influence of this substrate on compound potency should be taken into consideration during compound optimization

Tetra-substituted imidazoles as a new class of inhibitors of the p53-MDM2 interaction

Capitalizing on crystal structure information obtained from a previous effort in the search for non peptide inhibitors of the p53-MDM2 interaction, we have discovered another new class of compounds able to disrupt this protein-protein interaction, an important target in oncology drug research. The new inhibitors, based on a tetra-substituted imidazole scaffold, have been optimized to low nanomolar potency in a biochemical assay following a structure-guided approach. An appropriate strategy has allowed us to translate the high biochemical potency achieved in significant anti-proliferative activity on a p53-dependent MDM2 amplified cell line

Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity.

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin inhibitor (mTOR) pathway is often constitutively activated in human tumor cells, providing unique opportunities for anticancer therapeutic intervention. NVP-BEZ235 is an imidazo[4,5-c]quinoline derivative that inhibits PI3K and mTOR kinase activity by binding to the ATP-binding cleft of these enzymes. In cellular settings using human tumor cell lines, this molecule is able to effectively and specifically block the dysfunctional activation of the PI3K pathway, inducing G(1) arrest. The cellular activity of NVP-BEZ235 translates well in in vivo models of human cancer. Thus, the compound was well tolerated, displayed disease stasis when administered orally, and enhanced the efficacy of other anticancer agents when used in in vivo combination studies. Ex vivo pharmacokinetic/pharmacodynamic analyses of tumor tissues showed a time-dependent correlation between compound concentration and PI3K/Akt pathway inhibition. Collectively, the preclinical data show that NVP-BEZ235 is a potent dual PI3K/mTOR modulator with favorable pharmaceutical properties. NVP-BEZ235 is currently in phase I clinical trials