Identification of imidazo[1,2-b]pyridazine TYK2 pseudokinase ligands as potent and selective allosteric inhibitors of TYK2 signalling.

As a member of the Janus (JAK) family of non-receptor tyrosine kinases, TYK2 mediates the signaling of pro-inflammatory cytokines including IL-12, IL-23 and type 1 interferon (IFN), and therefore represents an attractive potential target for treating the various immuno-inflammatory diseases in which these cytokines have been shown to play a role. Following up on our previous report that ligands to the pseudokinase domain (JH2) of TYK2 suppress cytokine-mediated receptor activation of the catalytic (JH1) domain, the imidazo[1,2-b]pyridazine (IZP) 7 was identified as a promising hit compound. Through iterative modification of each of the substituents of the IZP scaffold, the cellular potency was improved while maintaining selectivity over the JH1 domain. These studies led to the discovery of the JH2-selective TYK2 inhibitor 29, which provided encouraging systemic exposures after oral dosing in mice. Phosphodiesterase 4 (PDE4) was identified as an off-target and potential liability of the IZP ligands, and selectivity for TYK2 JH2 over this enzyme was obtained by elaborating along selectivity vectors determined from analyses of X-ray co-crystal structures of representative ligands of the IZP class bound to both proteins.

Automated mode-of-action detection by metabolic profiling.

Rapid classification and identification of the mode-of-action of bioactive compounds applied to plants can be achieved by a robust and easy-to-use metabolic-profiling method. This method uses artificial neural network analysis of one-dimensional proton NMR spectra of aqueous plant extracts to rapidly classify changes in the total metabolic profile caused by application of crop protection chemicals.

Roles of histidine 752 and glutamate 699 in the pH dependence of mouse band 3 protein-mediated anion transport.

In the accompanying paper we have shown that four different histidine residues are involved in the maintenance of mouse band 3 in a state in which it is able to execute its anion transport function. Here we focus on the functional significance of His 752 and demonstrate that this residue, together with Glu 699, plays a key role in the control of pH dependence of Cl- transport. Mouse band 3-encoding cRNA was expressed in Xenopus oocytes, and band 3-mediated Cl- transport was measured at zero membrane potential over the pH range 6.0-9.2. Transport decreased with increasing H+ concentration and was governed by a single pK of 5.8. After correction for temperature differences, this result agrees well with measurements in erythrocyte ghosts of Cl- flux by Funder and Wieth [Funder, J., & Wieth, J. O. (1976) J. Physiol. 262, 679-698] and our own determinations by 35Cl NMR spectroscopy of Cl- exchange between the substrate binding site and the medium. After mutation of either Glu 699 to Asp or of His 752 to Ser, the maximal rate of transport is reduced and the rate of anion exchange is now governed by a single pK of about 6.8-6.9. This suggests that the formation of a hydrogen bond between His 752 and Glu 699 is essential for the decrease of band 3-mediated Cl- transport at low pH. We suggest that in the wild type band 3 both the decrease of the chloride exchange between the medium and the substrate binding site and the inhibition of chloride translocation across the membrane are dominated by a common rate-limiting step and that this step involves hydrogen bond formation between Glu 699 and His 752.

Temperature dependence of anion transport in the human red blood cell.

Arrhenius plots of chloride and bromide transport yield two regions with different activation energies (Ea). Below 15 or 25 degrees C (for Cl- and Br-, respectively), Ea is about 32.5 kcal/mol; above these temperatures, about 22.5 kcal/mol (Brahm, J. (1977) J. Gen. Physiol. 70, 283-306). For the temperature dependence of SO4(2-) transport up to 37 degrees C, no such break could be observed. We were able to show that the temperature coefficient for the rate of SO4(2-) transport is higher than that for the rate of denaturation of the band 3 protein (as measured by NMR) or the destruction of the permeability barrier in the red cell membrane. It was possible, therefore, to extend the range of flux measurements up to 60 degrees C and to show that, even for the slowly permeating SO4(2-) in the Arrhenius plot, there appears a break, which is located somewhere between 30 and 37 degrees C and where Ea changes from 32.5 to 24.1 kcal/mol. At the break, the turnover number is approx. 6.9 ions/band 3 per s. Using 35Cl- -NMR (Falke, Pace and Chan (1984) J. Biol. Chem. 259, 6472-6480), we also determined the temperature dependence of Cl- -binding. We found no significant change over the entire range from 0 to 57 degrees C, regardless of whether the measurements were performed in the absence or presence of competing SO4(2-). We conclude that the enthalpy changes associated with Cl- - or SO4(2-)-binding are negligible as compared to the Ea values observed. It was possible, therefore, to calculate the thermodynamic parameters defined by transition-state theory for the transition of the anion-loaded transport protein to the activated state for Cl-, Br- and SO4(2-) below and above the temperatures at which the breaks in the Arrhenius plots are seen. We found in both regions a high positive activation entropy, resulting in a low free enthalpy of activation. Thus the internal energy required for carrying the complex between anion and transport protein over the rate-limiting energy barrier is largely compensated for by an increase of randomness in the protein and/or its aqueous environment.