Macrocyclic Gq Protein Inhibitors FR900359 and/or YM-254890-Fit for Translation?

Abstract

Guanine nucleotide-binding proteins (G proteins) transduce extracellular signals received by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. While GPCRs represent the largest class of drug targets, G protein inhibition has only recently been recognized as a novel strategy for treating complex diseases such as asthma, inflammation, and cancer. The structurally similar macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent selective inhibitors of the Gq subfamily of G proteins. FR and YM differ in two positions, FR being more lipophilic than YM. Both compounds are utilized as pharmacological tools to block Gq proteins in vitro and in vivo. However, no detailed characterization of FR and YM has been performed, which is a prerequisite for the compounds' translation into clinical application. Here, we performed a thorough study of both compounds' physicochemical, pharmacokinetic, and pharmacological properties. Chemical stability was high across a large range of pH values, with FR being somewhat more stable than YM. Oral bioavailability and brain penetration of both depsipeptides were low. FR showed lower plasma protein binding and was metabolized significantly faster than YM by human and mouse liver microsomes. FR accumulated in lung after chronic intratracheal or intraperitoneal application, while YM was more distributed to other organs. Most strikingly, the previously observed longer residence time of FR resulted in a significantly prolonged pharmacologic effect as compared to YM in a methacholine-induced bronchoconstriction mouse model. These results prove that changes within a molecule which seem marginal compared to its structural complexity can lead to crucial pharmacological differences.

Figure 1

Chemical structures of the macrocyclic depsipeptidic Gq protein inhibitors FR (1) and YM (2).

Figure 2

Chemical stability of FR and YM A. in simulated gastric fluid, pH 1, B. at pH 9, and C. at pH 11 (100 μM starting concentration). Solutions were prepared by adding 50 μL of a 1 mM stock solution in DMSO of FR or YM to 450 μL of an aqueous solution A, B, or C. Values represent means ± SEM from three independent experiments. For details, see Methods.

Figure 3

Proposed isomerization reactions (FR: R₁ = CH₂CH₃, R₂ = CH(CH₃)₂; YM: R₁ = CH₃, R₂ = CH₃). The secondary alcohol is displayed in green. Esters which possibly take part in the isomerization reaction are highlighted in blue and labeled A or B. Ester bonds newly formed after the reaction are highlighted in dark orange.

Figure 4

Stability of G protein inhibitors under various conditions: A. human liver microsomes (FR, YM); B. mouse liver microsomes (FR, YM); C. stability in mouse plasma (FR); and D. mouse lung tissue (FR). Data represent means ± SEM (n = 3).

Figure 5

A. Concentration of FR ± SEM and B. concentration of YM ± SEM in mouse tissues after intratracheal application of 5 μg of drug on 7 consecutive days. FR and YM levels in organs from three mice were determined.

Figure 6

A. Concentration of FR ± SEM in various mouse tissues after intratracheal application of 2.5 μg of FR twice a day for 3 weeks. B. Concentration of FR ± SEM in various mouse tissues after intraperitoneal application of 10 μg of FR for 3 weeks (administration from Monday to Friday). FR levels in organs from three mice were determined.

Figure 7

Repeated enhanced pause (Penh) measurements after intratracheal application of FR and YM. Bronchoconstriction was induced by application of methacholine (MCh). FR/YM (2.5 μg) or DMSO was administered intratracheally on day 0. Whole-body plethysmography was carried out after 4 h, 24 h, 48 h, 72 h, and 96 h in the presence of increasing concentrations of MCh which was applied as an aerosol by a nebulizer in the plethysmograph. Data represent means ± SEM (FR, DMSO n = 5; YM n = 4). Statistical significance was assessed by two-way repeated measures ANOVA with Bonferroni’s post hoc test. ****, ***, and ** represent a p value < 0.0001, 0.001, and 0.01, respectively.