[17] The kinetics of reversible tight-binding inhibition

Proprotein convertases (PCs) are involved in the pathogenesis of various diseases, making them promising drug targets. Most assays for PCs have been performed with few standard substrates, regardless of differences in cleavage efficiencies. Derived from studies on substrate-analogue inhibitors, 11 novel substrates were synthesized and characterized with five PCs. H-Arg-Arg-Tle-Lys-Arg-AMC is the most efficiently cleaved furin substrate based on its k_cat/K_M value. Due to its higher k_cat value, acetyl-Arg-Arg-Tle-Arg-Arg-AMC was selected for further measurements to demonstrate the benefit of this improved substrate. Compared to our standard conditions, its use allowed a 10-fold reduction of the furin concentration, which enabled K_i value determinations of previously described tight-binding inhibitors under classical conditions. Under these circumstances, a slow-binding behavior was observed for the first time with inhibitor MI-1148. In addition to furin, four additional PCs were used to characterize these substrates. The most efficiently cleaved PC1/3 substrate was acetyl-Arg-Arg-Arg-Tle-Lys-Arg-AMC. The highest k_cat/K_M values for PC2 and PC7 were found for the N-terminally unprotected analogue of this substrate, although other substrates possess higher k_cat values. The highest efficiency for PC5/6A was observed for the substrate acetyl-Arg-Arg-Tle-Lys-Arg-AMC. In summary, we have identified new substrates for furin, PC1/3, PC2, and PC7 suitable for improved enzyme-kinetic measurements.

Effective drug discovery relies on the considered deployment of screening assays from the outset of a project. Selected carefully, the data from screening assays can guide series optimization and give insight into the likely performance of compounds in a patient population. Established assays can provide information on a wide variety of compound properties, ranging from target affinity and functional assessment through to pharmacokinetic and toxicological properties, and with capacity on a scale from high-throughput plate formats down to isolated bespoke experiments. Selecting the assays in which to screen a compound at a given stage in a discovery project is a balance between resource cost and information value, and can have a profound impact on the pace and outcomes of the project. This article presents commonly evaluated properties, their physiological relevance, caveats to their interpretation, and the integration of in silico, in vitro and in vivo data to characterize the holistic profiles of discovery compounds and series.

The accurate quantitative determination of affinity and binding kinetics (BK) for tight binding inhibition is extraordinary important from both the continuous optimization of compounds, particularly in developing structure–activity relationships (SAR), and the prediction of in vivo target occupancy (TO). Due to the unique properties for tight binding inhibition that the inhibitors are characterized by the ultrahigh-affinity, relatively fast association to the target enzyme combined with extremely slow dissociation of the inhibitor-enzyme binary complex, the classical steady state equilibrium methods are no longer valid. Here, we made several recommendations of how to design the optimal experiments and apply special mathematical calculation approaches to quantitatively evaluate the accurate affinity and BK as the examples of two tight binding inhibitors against the xanthine oxidase (XO), as well as compared the differences in the results calculated from the different data analytical methods and analyzed the influence of these differences on the XO engagement in human. Analysis of the results displayed that the accurate apparent dissociation constant (K_i^{*, app}) was 0.2 ± 0.06 nM for topiroxotstat and was 0.45 ± 0.2 nM for febuxostat; that on-rate (k_on) was (4.3 ± 1.1) × 10⁶ M⁻¹s⁻¹ for topiroxotstat and was(133.3 ± 3.5) × 10⁶ M⁻¹s⁻¹ for febuxostat, and off-rate (k_off) was (1.0$\pm$0.2) × 10⁻⁵ s⁻¹ for topiroxotstat and was ≤ 0.16 × 10⁻⁵ s⁻¹for febuxostat. Moreover, there were significant differences in the K_i^{*, app} and k_off values estimated using the appropriate specialized methods for tight binding inhibition versus classical steady state equilibrium methods, with the substantial differences of 14-fold and 32-fold reduction for topiroxostat, respectively, and of 9.6-fold and ≥ 213-fold reduction for febuxostat, while the k_on values remain the moderate differences for the two inhibitors. The obvious greater AUC of XO engagement time courses and longer durations of above 70% engagement by the appropriate specialized methods for tight binding inhibition were observed that the results display the differences of 70.1% and 88%, respectively for topiroxostat and of 38.1% and 35.0%, respectively for febuxostat in human liver cell than by classical steady state equilibrium methods. Again, our studies provide several valuable recommendations of the optimal experiment protocols and appropriate analytical approaches for accurately quantitatively assessing the affinity and BK parameters as well as demonstrate the ability of our recommended methods to generate reliable data for tight binding inhibitors against XO.

Enzymes are essential, physiological catalysts involved in all processes of life, including metabolism, cellular signaling and motility, as well as cell growth and division. They are attractive drug targets because of the presence of defined substrate-binding pockets, which can be exploited as binding sites for pharmaceutical enzyme inhibitors. Understanding the reaction mechanisms of enzymes and the molecular mode of action of enzyme inhibitors is indispensable for the discovery and development of potent, efficacious, and safe novel drugs. The combination of classical concepts of enzymology with new experimental and data analysis methods opens new routes for drug discovery.

Despite possessing only 32 residues, the tsetse thrombin inhibitor (TTI) is among the most potent anticoagulants described, with sub-picomolar inhibitory activity against thrombin. Unexpectedly, TTI isolated from the fly is 2000-fold more active and 180 Da heavier than synthetic and recombinant variants. We predicted the presence of a tyrosine O-sulfate post-translational modification of TTI, prompting us to investigate the effect of the modification on anticoagulant activity. A combination of chemical synthesis and functional assays was used to reveal that sulfation significantly improved the inhibitory activity of TTI against thrombin. Using X-ray crystallography, we show that the N-terminal sulfated segment of TTI binds the basic exosite II of thrombin, establishing interactions similar to those of physiologic substrates, while the C-terminal segment abolishes the catalytic activity of thrombin. This non-canonical mode of inhibition, coupled with its potency and small size, makes TTI an attractive scaffold for the design of novel antithrombotics.