Juan Manuel Domínguez

Evidence for Irreversible Inhibition of Glycogen Synthase Kinase-3β by Tideglusib

Background: Tideglusib is a GSK-3 inhibitor currently undergoing clinical trials for Alzheimer disease and progressive supranuclear palsy. Results: Removal of unbound compound does not recover the enzyme activity, and the dissociation rate constant is close to zero. The protein shows a low turnover rate in neurons. Conclusion: Tideglusib is an irreversible inhibitor of GSK-3β. Significance: The irreversibility and the long enzyme half-life may possess interesting pharmacodynamic implications. Tideglusib is a GSK-3 inhibitor currently in phase II clinical trials for the treatment of Alzheimer disease and progressive supranuclear palsy. Sustained oral administration of the compound to a variety of animal models decreases Tau hyperphosphorylation, lowers brain amyloid plaque load, improves learning and memory, and prevents neuronal loss. We report here that tideglusib inhibits GSK-3β irreversibly, as demonstrated by the lack of recovery in enzyme function after the unbound drug has been removed from the reaction medium and the fact that its dissociation rate constant is non-significantly different from zero. Such irreversibility may explain the non-competitive inhibition pattern with respect to ATP shown by tideglusib and perhaps other structurally related compounds. The replacement of Cys-199 by an Ala residue in the enzyme seems to increase the dissociation rate, although the drug retains its inhibitory activity with decreased potency and long residence time. In addition, tideglusib failed to inhibit a series of kinases that contain a Cys homologous to Cys-199 in their active site, suggesting that its inhibition of GSK-3β obeys to a specific mechanism and is not a consequence of nonspecific reactivity. Results obtained with [35S]tideglusib do not support unequivocally the existence of a covalent bond between the drug and GSK-3β. The irreversibility of the inhibition and the very low protein turnover rate observed for the enzyme are particularly relevant from a pharmacological perspective and could have significant implications on its therapeutic potential.

Sordarin Inhibits Fungal Protein Synthesis by Blocking Translocation Differently to Fusidic Acid

Sordarin derivatives are selective inhibitors of fungal protein synthesis, which specifically impair elongation factor 2 (EF-2) function. We have studied the effect of sordarin on the ribosome-dependent GTPase activity of EF-2 fromCandida albicans in the absence of any other component of the translation system. The effect of sordarin turned out to be dependent both on the ratio of ribosomes to EF-2 and on the nature of the ribosomes. When the amount of EF-2 exceeded that of ribosomes sordarin inhibited the GTPase activity following an inverted bell-shaped dose-response curve, whereas when EF-2 and ribosomes were in equimolar concentrations sordarin yielded a typical sigmoidal dose-dependent inhibition. However, when ricin-treated ribosomes were used, sordarin stimulated the hydrolysis of GTP. These results were compared with those obtained with fusidic acid, showing that both drugs act in a different manner. All these data are consistent with sordarin blocking the elongation cycle at the initial steps of translocation, prior to GTP hydrolysis. In agreement with this conclusion, sordarin prevented the formation of peptidyl-[3H]puromycin on polysomes from Candida albicans.

Discovery of GSK837149A, an inhibitor of human fatty acid synthase targeting the β‐ketoacyl reductase reaction

GSK837149A has been identified as a selective inhibitor of human fatty acid synthase (FAS). The compound was first isolated as a minor impurity in a sample found to be active against the enzyme in a high‐throughput screening campaign. The structure of this compound was confirmed by NMR and MS studies, and evaluation of the newly synthesized molecule confirmed its activity against FAS. The compound and other analogs synthesized, all being symmetrical structures containing a bisulfonamide urea, act by inhibiting the β‐ketoacyl reductase activity of the enzyme. GSK837149A inhibits FAS in a reversible mode, with a Ki value of ∼ 30 nm, and it possibly binds to the enzyme–ketoacyl‐ACP complex. Although initial results suggest that cell penetration for these compounds is impaired, they still can be regarded as useful tools with which to probe and explore the β‐ketoacyl reductase active site in FAS, helping in the design of new inhibitors.

Thermally denatured BSA, a surrogate additive to replace BSA in buffers for high-throughput screening.

The use of thermally denatured bovine serum albumin (tdBSA) as an additive in high-throughput screening (HTS) buffers has been studied with the aim of finding a surrogate to native albumin devoid of its inconveniences, in particular its compound masking effect. The presence of aggregates in the thermally denatured material did not have any negative impact on common readout technologies used in HTS such as fluorescence intensity (FLINT), fluorescence polarization, time-resolved fluorescence resonance energy transfer (TR-FRET) and luminescence. tdBSA rendered the same beneficial effects as native albumin in several assays or even improved its performance due to the lack of specific binding properties. Although tdBSA still binds compounds nonspecifically as any other protein does, it mitigates the compound masking effect observed with native albumin and can be postulated as a convenient surrogate to BSA for HTS purposes.

Utilization of Substrate-Induced Quenching for Screening Targets Promoting NADH and NADPH Consumption

Oxidation of reduced nicotinamide adenine dinucleotides is a common event for many biochemical reactions. However, its exploitation for ultrahigh-throughput screening purposes is not an easy task and is affected by various drawbacks. It is known that such nucleotides induce quenching on the fluorescence of several dyes and that this quenching disappearswith oxidation of the nucleotide. We have made use of this property to develop an assay for high-throughput screening with NADH and NADPH-dependent reductases. Full screening campaigns have been run with excellent assay quality parameters, and interesting hits have been identified. The method is amenable to miniaturization and allows easy identification of false positives without needing extra secondary assays. Although it is based onmonitoring substrate consumption, it is demonstrated that the effect of fractional conversion on assay sensitivity is negligible.

Kinetic Considerations on the Development of Binding Assays in Single-Addition Mode Application to the Search for α2δ1 Modulators

The development of assays in single-addition mode is of great interest for screening purposes given the multiple advantages of minimizing the number of intervention steps. Binding assays seem to be more prone to this attractive format because no functional biological activity is taking place but instead a biophysical process, whose dynamics seem easier to control without introducing significant alterations, is happening. Therefore, single-addition assays based on the displacement of prebound labeled ligands can be conceived, but careful kinetic considerations must still be taken to maximize the sensitivity of the assay and to avoid jeopardizing the identification of compounds with slow-binding kinetics. This article shows the development of a single-addition, displacement-based binding assay intended to identify modulators that act by binding to the gabapentin site of the ion channel regulatory protein α2δ1. After studying the kinetics of gabapentin binding and the influence they might have on the assay sensitivity, the best conditions were identified, and the sensitivity was compared with that of the more classical two-additions competition-based assay. Although the present study focuses on α2δ1 and its interaction with gabapentin, the rationale and the methodology followed are of broad purpose and can be applied to virtually every binding assay.