Nobilamides A-H, long-acting transient receptor potential vanilloid-1 (TRPV1) antagonists from mollusk-associated bacteria

Abstract

New compounds nobilamides A-H and related known compounds A-3302-A and A-3302-B were isolated based upon their suppression of capsaicin-induced calcium uptake in a mouse dorsal root ganglion primary cell culture assay. Two of these compounds, nobilamide B and A-3302-A, were shown to be long-acting antagonists of mouse and human TRPV1 channels, abolishing activity for >1 h after removal of drug presumably via a covalent attachment. Other derivatives also inhibited the TRPV1 channel, albeit with low potency, affording a structure-activity profile to support the proposed mechanism of action. While the activities were modest, we propose a new mechanism of action and a new site of binding for these inhibitors that may spur development of related analogues for treatment of pain.

Figure 1

Time-dependent inhibition of TRPV1 by 5. HEK-293 cells were transfected with human wild type TRPV1 and pre-treated with compound 5 (375 μM), followed by application of capsaicin (25 μM). For comparison, cells were co-treated with both agents. The change in fluorescent response to Ca²⁺ (ΔF) was measured as a function of maximum fluorescence or of fluorescence rate (n=3).

Figure 2

Stability of TRPV1 inhibition. Compound 2 (375μM) was incubated with HEK-293 cells transfected with wild-type human TRPV1. Response to capsaicin was measured over a 1 h timecourse with washes at various intervals (x-axis). The change in fluorescent response to Ca²⁺ (ΔF) was measured as a function of maximum fluorescence or of fluorescence rate.

Figure 3

Mutational analysis of human TRPV1 inhibition. A. Compounds 2 (375μM) and 5 (375 μM) were pre-incubated with HEK-293 cells transfected with wild-type and mutant TRPV1 channels. Inhibition of the capsaicin response was compared. The change in fluorescent response to Ca²⁺ (ΔF) was measured as a function of maximum fluorescence or of fluorescence rate. B. Side view of a homology model of a single subunit of human TRPV1. Highlighted residues involved in capsaicin binding (green) and nobilamide binding (yellow: Cys; red: Phe), as well as residues whose mutation does not affect nobilamide binding. C. Side view of a homology model of the TRPV1 tetramer pore loop region showing residues and intra-molecular distance between residues involved in capsaicin or nobilamide binding (colors as in B). The ion pore runs through the middle of this diagram and is presumably blocked by nobilamide binding between F660 and/or C578 and C621, either within or between subunits of the functional tetramer.