Drug-target kinetics and thermodynamics in drug discovery: Using of p-NPP to characterize the ligands of Na+/K+-ATPase in a cheap way

Drug-target kinetics and thermodynamics in drug discovery: Using of p-NPP to characterize the ligands of Na+/K+-ATPase in a cheap way

• Author
• Pedro Azalim
• Co-authors
• François Noël , Simone Silva , José Villar , Leandro Barbosa , George O’Doherty , Luis Quintas
• Abstract

• Introduction: Drug discovery approach are usually performed using affinity or potency parameters. Copeland et al. (Nat. Rev. Drug Discov. 2006, 9:730-739) proposed that the time that a ligand spends in its binding site (residence time, 1/koff) could have a major influence on its clinical efficacy. There is an interest in searching for new Na⁺/K⁺-ATPase (NKA, EC 3.6.3.9) modulators targeting clinical conditions such as cancer (Slingerland et al., New Drugs 2013, 31:1087–1094). We performed a Structure-Kinetic Relationship to study the influence of kinetics of NKA inhibition. 

  Methods: 17 CS natural and synthesized (Wang et al., ACS Med. Chem. Lett. 2011, 2:264-269) was tested to inhibit the K⁺-dependent?phosphatase activity performed at different temperature and ionic condition. The association rate (k_on) and dissociation rate (k_off) constants were obtained by globally fitting the inhibition vs. time of each compound using the following equation: y?=?Imax ([I]/([I]+Ki))1?e(?1(kon[I]+koff)t).

  Results: Changing conditions (from ATPase to p-NPPase activity) resulted in an increase of K_i of the cardenolides due to a reduction of k_on, in contrast to the K_i of the structurally related bufadienolide. When evaluating the kinetics of 17 natural and semi-synthetic CTS, we observed that both k_on and k_off correlated with K_i (Spearman test), showing that 4 CTS has the same potence but different kinetics. A rhamnose in C3 of the steroidal nucleus enhanced the inhibitory potency by a reduction of k_off rather than an increase of k_on. Rising the temperature did not alter the koff of a glycosylated CS, generating a ?H^‡ (k_off) of -10.4 ± 4.3 kJ/mol, suggesting a complex dissociation mechanism. 

   

  Conclusion: Based on a simple and inexpensive methodology, we determined the values of k_on, k_off, and K_i of the CTS and provided original kinetics and thermodynamics differences between CTS that could help the design of new compounds.

• Keywords
• Kinetics; Thermodynamics; Na, K-ATPase; Cardiotonic Steroids; Drug Discovery
• Subject Area
• Na Pump

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