Correlation of inhibitor effects on enzyme activity and thermal stability for the integral membrane protein fatty acid amide hydrolase

Abstract

The melting curves of fatty acid amide hydrolase (FAAH) in the presence of 29 reversible inhibitors were measured using a thiol-reactive fluorophore. The thermal stability (T m ) of the FAAH/inhibitor complex varied significantly depending on the chemical characteristics of the inhibitors, notably variations in the head group. Two separate distributions were observed when T m was plotted againt K i . The majority of the inhibitors showed a positive correlation between binding affinity and T m , however inhibitors with a pyridine carboxylic acid moiety in the head group fell in a distinct and uncorrelated distribution when tail groups were varied. Keywords fatty acid amide hydrolase; binding affinity; stability It has been shown that the effects of ligands on an enzyme may be observed by measuring shifts in the enzyme's thermal stability, which may in turn have implications on inhibitor potency. Indeed, a potential correlation between thermal stability and ligand affinity has been observed. 1 An additional motivation for studying the effect of ligand binding on stability is its implications towards crystallization of protein-ligand complexes since thermal stability has been linked to an increased likelihood of improving crystal diffraction. 2

Thermal Instability of ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function: Protection by Single Suppressor Mutations and Inhibiting Channel Activity

“…The ligand binds selectively to the native (functional) form and alters the structure of the target protein (or selects for a stable conformer) and thereby protects the protein from thermally-induced denaturation. Not surprisingly, these stabilizing ligands tend to be inhibitors, rather than activators (60). …”

The discovery and development of inhibitors of fatty acid amide hydrolase (FAAH)

“…In this study and in addition to demonstrating that the observed increased thermal stability qualitatively parallels an inhibitor’s potency, the 2-pyridyl-6-carboxylic acid derivatives exhibited a uniquely strong stabilizing effect among the various series examined, suggesting they constitute an especially interesting series to examine in greater detail. 34 …”

Leveraging the Contribution of Thermodynamics in Drug Discovery with the Help of Fluorescence-Based Thermal Shift Assays

“…however, several communications describe combined analyses of potency versus thermal stability. 12,14,15 Very interestingly, in their study on the fatty acid amide hydrolase, slaymaker et al 15 were able to identify two different compound populations once they plotted a graph lnK i versus t m . unfortunately, no thermodynamic measurements were realized with these compounds.…”

“…the binding of a ligand to a protein leads to the formation of a complex protein-ligand that usually exhibits a higher melting temperature, t m b , than the free protein. experiments have shown a good correlation between ligand potency measured in biochemical assays and shift in melting temperatures (Δt m = t m b -t m ) estimated in ftsa, [12][13][14][15] and ftsa is already routinely used to triage hits identified from high-throughput screening (hts) campaigns. moreover, Δt m s obtained from ftsa are directly connected to the thermodynamic parameters of the ligand-protein interaction, 7,16,17 providing a link to the thermodynamic signatures of the compounds.…”