GHRELIN O-ACYLTRANSFERASE (GOAT) IMAGING AGENTS

Abstract

Imaging agents that can bind to ghrelin O-acyltransferase (GOAT) without binding to the ghrelin receptor (GHS-R1a). The imaging agents comprise a base structure for selective binding to GOAT that is coupled via an amino acid linker to a chemical group to enable imaging such as a fluorescent label, radioactive tracer, or metal chelator. For example, the imaging agent may comprise a ghrelin substrate mimetic inhibitor incorporating an unmodified 2,3-diaminopropanoic acid (Dap) group at the site analogous to serine 3. These agents enable specific detection and imaging of GOAT versus the GHS-R1a receptor in a variety of biological contexts.

Claims

1. An imaging agent, comprising a compound having having the formula ##STR00001## coupled to a fluorescent label at a C-terminus of the compound via an amino acid linker.

2. The imaging agent of claim 1, wherein the imaging agent comprises molecule of fluorescein.

3. The imaging agent of claim 2, wherein the amino acid linker comprises SEQ ID NO: 2 (LSPEHQ).

4. The imaging agent of claim 3, wherein the compound further comprises an unmodified 2,3-diaminopropanoic acid (Dap) group.

5. An imaging agent, comprising a compound having the formula: ##STR00002## where R1 is selected from the group consisting of H and CH3, R2 is selected from the group of consisting of H and OH, R3 is selected from the group consisting of H, CH3, a linear alkane having two to nine carbons, a branched saturated hydrocarbon having two to nine carbons, an unsaturated hydrocarbon having two to nine carbons, a monounsaturated linear hydrocarbon having a terminal aromatic group, and a polyunsaturated linear hydrocarbon having a terminal aromatic group, R4 is selected from group consisting of phenyl, indole, or an aromatic group, R5 is selected from the group consisting of leucine, isoleucine, methionine, and phenylalanine, R6 is selected from the group consisting of H and OH, and Y is an imaging agent.

6. The imaging agent of claim 5, wherein the imaging agent comprises a fluorescent label.

7. The imaging agent of claim 5, wherein the imaging agent comprises a radioactive label.

8. The imaging agent of claim 5, wherein the imaging agent comprises a chelator.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0007] The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which:

[0008] FIG. 1 is a schematic of an imaging agent for ghrelin O-acyl-transferase (GOAT) according to the present invention.

[0009] FIG. 2 is a schematic of the N- and C-terminal length dependence of Dap-containing ghrelin mimetic peptide GOAT inhibitors;

[0010] FIG. 3 is a schematic of a generic platform for the design of imaging agents for ghrelin O-acyl-transferase (GOAT) according to the present invention;

[0011] FIG. 4 is a chart of certain compounds according to the present invention and the IC.sub.50 values for those compounds in GHS-R1A and GOAT assays;

[0012] FIG. 5 is a graph of hGOAT activity and inhibition in the presence of SulfoCy5-3, a fluorescently labeled version of the proposed GOAT imaging agent according to FIGS. 3 and 4;

[0013] FIG. 6A is a series of images of cell membrane binding in PC3 (GOAT positive) cells; and

[0014] FIG. 6B is a series of images of peptide internalization in PC3 (GOAT positive) and HEK293 (GOAT negative) cells using a fluorescently labeled GOAT imaging agent per FIG. 4.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Referring to the figures, wherein like numeral refer to like parts throughout, there is seen in FIG. 1 a schematic of a GOAT imaging agent according to the present invention that is a ghrelin substrate mimetic inhibitor based on the first six amino acids of ghrelin, GSSFLS (SEQ ID NO: 1), that has a sarcosine substitution at the G1 position and an unmodified 2,3-diaminopropanoic acid (Dap) group at the site analogous to serine 3. As seen in FIG. 1, the imaging agent is coupled via an amino acid linker LSPEHQ (SEQ ID NO: 2) to a fluorescent label for imaging, thereby resulting in the complex SarSDapFLSPEHQ-Fluorescein.

[0016] The present invention stems from comparison between the structure-activity relationships governing ghrelin binding to GOAT and to the GHS-R1a receptor. Proceeding from the N-terminus of ghrelin, binding to both GOAT and GHS-R1a is severely diminished by acetylation of the N-terminal amino group of ghrelin. A sarcosine substitution at the G1 position leads to a >25-fold loss in binding affinity for the ghrelin receptor as reflected by IC.sub.50 values in a competition binding assay, while the same substitution strengthens binding to GOAT by 60 percent, as seen below in Table 1:

TABLE-US-00001 TABLE 1 Impact of nitrogen methylation on Dap peptide inhibitor potency against hGOAT. Methylation site(s) Peptide sequence IC.sub.50 (M) none GSDapFL 0.14 0.02 G1 SarSDapFL 0.088 0.001 S2 G.sub.N-MeSDapFL >100 F4 GSDap.sub.N-MeFL 0.097 0.013 L5 GSDapF.sub.N-MeL 0.062 0.009 G1, F4 SarSDap.sub.N-MeF 1.5 0.1 G1, F4, L5 SarSDap.sub.N-MeF.sub.N-MeL 6 1

[0017] The marked differences in ligand binding requirements between GOAT and GHS-R1a, particularly at the G1 and S3 positions of ghrelin-derived peptides, support the potential for designing molecules that specifically target either of these ghrelin-interacting proteins for use in studying and modulating the ghrelin signaling pathway.

[0018] There is seen in FIG. 3 a schematic of alternative approaches for designing a GOAT imaging agent. In FIG. 3, all stereocenters shown as L-amino acids; D-amino acids are also possible at all positions. R1 may comprise H, CH3. R2 may comprise H (alanine), OH (serine). R3 may comprise H, CH3, or a linear alkane with 2-9 carbons, mono- or polyunsaturated linear hydocarbons with 2-9 carbons, branched saturated or unsaturated hydrocarbons with length 2-9 carbons, or mono- or poly-unsaturated linear hydocarbons with terminal aromatic groups. R4 may comprise phenyl (phenylalanine), indole (tryptophan), or other aromatic group. R5 may comprise leucine, isoleucine, methionine, or phenylalanine. R6 may comprise H (alanine), OH (serine). X comprises a peptide sequence from 0-4 amino acids (for example PEHQ, PTHQ, PEFQ). Y comprises an imaging modality (e.g. fluorescent group (fluorescein, TAMRA, coumarin, etc), a radioactive group (group incorporating 18F, 14C, 3H), or a chelator for imaging metal (lanthanide, Tc, etc).

[0019] Referring to FIG. 4, the IC.sub.50 values were determined for certain compounds of the present invention using three different ligands using GHS-R1A and GOAT assays. Referring to FIG. 5, SulfoCy5-3 inhibition studies demonstrated that the compound could be used to determine hGOAT binding. SulfoCy5-3 labeling of GOAT in PC3 prostate cancer cells and HEK293 cells was also used to determine cell membrane binding by incubating with the SulfoCy5-3 ligand and then imaging by fluorescence microscopy at 20 magnification (scale bar is 10 mM). In FIG. 6A, the incubation of PC3 cells at 4 C. to minimize membrane recycling led to plasma membrane binding. In FIG. 6B, the incubation of PC3 cells and HEK293 cells at 37 C. showed no labeling with the HEK293 cells and peptide label internalization with PC3 cells.