An engineered scorpion toxin analogue with improved Kv1.3 selectivity displays reduced conformational flexibility

The voltage-gated Kv1.3 K(+) channel plays a key role in the activation of T lymphocytes. Kv1.3 blockers selectively suppress immune responses mediated by effector memory T cells, which indicates the great potential of selective Kv1.3 inhibitors in the therapy of certain autoimmune diseases. Anuroctoxin (AnTx), a 35-amino-acid scorpion toxin is a high affinity blocker of Kv1.3, but also blocks Kv1.2 with similar potency. We designed and produced three AnTx variants: ([F32T]-AnTx, [N17A]-AnTx, [N17A/F32T]-AnTx) using solid-phase synthesis with the goal of improving the selectivity of the toxin for Kv1.3 over Kv1.2 while keeping the high affinity for Kv1.3. We used the patch-clamp technique to determine the blocking potency of the synthetic toxins on hKv1.3, mKv1.1, hKv1.2 and hKCa3.1 channels. Of the three variants [N17A/F32T]-AnTx maintained the high affinity of the natural peptide for Kv1.3 but became more than 16000-fold selective over Kv1.2. NMR data and molecular dynamics simulations suggest that the more rigid structure with restricted conformational space of the double substituted toxin compared to the flexible wild-type one is an important determinant of toxin selectivity. Our results provide the foundation for the possibility of the production and future therapeutic application of additional, even more selective toxins targeting various ion channels

Competition saturation transfer difference experiments improved with isotope editing and filtering schemes in NMR-based screening

6 páginas, 5 figuras, 1 esquema -- PAGS nros. 17148-17153Competition binding experiments are used in NMR-based screenings to match up to the binding site with that of a known ligand and to determine the strength of the interaction, thus providing a ranking of hits according to receptor affinity. These competition titration experiments must use a reference ligand for which the binding site on the receptor and the affinity of the interaction is known. These experiments rely on the observation of separate signals of the reference and hit compounds, which is very often hampered by signal overlap. Here, we present a combination of isotope editing and filtering schemes with saturation transfer difference (STD) experiments that allows the separation of the STD signals of the labeled reference ligand from that of the natural abundance hit compound even in the case of severe signal overlap. Thus, the measurement of well-defined titration curves in competition STD titration experiments is feasible and allows the quantitative determination of binding constants. Note that the method requires the availability of the reference ligand in an NMR-active, stable-isotope-labeled formWe thank Thomas Schallus for the expression vector of malectin and for sharing ideas prior to publication. K.E.K and G.B. acknowledge the support of the Hungarian Scientific Research Fund, OTKA NK-68578. C.M.-G. acknowledges the DFG Grant Mu1606/2-2. We also thank the scientific exchange program between CSIC (Spain) and Hungary for travel supportPeer reviewe