RESUMEN
PKCtheta (protein kinase Ctheta) is a central signalling molecule in the T-cell receptor activation pathway and is a target for treatment of a number of diseases. Several PKC inhibitors are in the drug-discovery pharmaceutical programmes today for the treatment of cancer, diabetes and arthritis. CD4(+) T-lymphocytes also play a critical role in the initiation and progression of allergic airway inflammation. Our goal is the development of PKCtheta antagonists as a means to control asthma and autoimmune diseases, using the strategy based on developing small-molecule agents that would block the enzyme's catalytic activity. Here, we discuss our work on the discovery of lead chemical series and review our X-ray structural and modelling approaches, including a structure-surrogate strategy that helped guide us in the lead compound optimizations.
Asunto(s)
Isoenzimas/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Isoenzimas/metabolismo , Modelos Moleculares , Estructura Molecular , Proteína Quinasa C/metabolismo , Proteína Quinasa C-theta , Inhibidores de Proteínas Quinasas/metabolismo , Especificidad por SustratoRESUMEN
A ring of aligned glutamate residues named the intermediate ring of charge surrounds the intracellular end of the acetylcholine receptor channel and dominates cation conduction (Imoto et al. 1988). Four of the five subunits in mouse-muscle acetylcholine receptor contribute a glutamate to the ring. These glutamates were mutated to glutamine or lysine, and combinations of mutant and native subunits, yielding net ring charges of -1 to -4, were expressed in Xenopus laevis oocytes. In all complexes, the alpha subunit contained a Cys substituted for alphaThr244, three residues away from the ring glutamate alphaGlu241. The rate constants for the reactions of alphaThr244Cys with the neutral 2-hydroxyethyl-methanethiosulfonate, the positively charged 2-ammonioethyl-methanethiosulfonate, and the doubly positively charged 2-ammonioethyl-2'-ammonioethanethiosulfonate were determined from the rates of irreversible inhibition of the responses to acetylcholine. The reagents were added in the presence and absence of acetylcholine and at various transmembrane potentials, and the rate constants were extrapolated to zero transmembrane potential. The intrinsic electrostatic potential in the channel in the vicinity of the ring of charge was estimated from the ratios of the rate constants of differently charged reagents. In the acetylcholine-induced open state, this potential was -230 mV with four glutamates in the ring and increased linearly towards 0 mV by +57 mV for each negative charge removed from the ring. Thus, the intrinsic electrostatic potential in the narrow, intracellular end of the open channel is almost entirely due to the intermediate ring of charge and is strongly correlated with alkali-metal-ion conductance through the channel. The intrinsic electrostatic potential in the closed state of the channel was more positive than in the open state at all values of the ring charge. These electrostatic properties were simulated by theoretical calculations based on a simplified model of the channel.