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. A method of imaging ghrelin O-acyltransferase (GOAT) activity, comprising the step of providing an imaging agent comprised of a peptide sequence including a ghrelin O-acyltransferase binding ligand and an imaging modality coupled to the peptide sequence, wherein the peptide sequence has a reduced binding affinity to a ghrelin receptor compared to ghrelin.

2. The method of claim 1, wherein the imaging agent has the formula ##STR00001## where R1 is selected from the group consisting of H and CH.sub.3, R2 is selected from the group of consisting of H and OH, R3 is selected from the group consisting of H, CH.sub.3, 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 a phenyl, an indole, and 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, X is a peptide sequence, and Y is an imaging modality.

3. The method of claim 1, wherein the peptide sequence has the formula ##STR00002##

4. The method of claim 3, wherein the imaging modality is a fluorescent label.

5. The imaging agent of claim 3, wherein the imaging modality is a radioactive label.

6. The imaging agent of claim 3, wherein the imaging modality is a chelator.

7. A ghrelin O-acyltransferase (GOAT) imaging agent, comprising a peptide sequence including a ghrelin O-acyltransferase binding ligand and an imaging modality coupled to the peptide sequence, wherein the peptide sequence has a reduced binding affinity to a ghrelin receptor compared to ghrelin.

8. The imaging agent of claim 7, wherein the imaging agent has the formula ##STR00003## where R1 is selected from the group consisting of H and CH.sub.3, R2 is selected from the group of consisting of H and OH, R3 is selected from the group consisting of H, CH.sub.3, 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 a phenyl, an indole, and 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, X is a peptide sequence, and Y is an imaging modality.

9. The imaging agent of claim 8, wherein the peptide sequence has the formula ##STR00004##

10. The imaging agent of claim 9, wherein the imaging modality is a fluorescent label.

11. The imaging agent of claim 9, wherein the imaging modality is a radioactive label.

12. The imaging agent of claim 9, wherein the imaging modality is 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 (μM) none GSDapFL 0.14 ± 0.02 G1 SarSDapFL 0.088 ± 0.001 S2 G.sub.N.Math.MeSDapFL >100 F4 GSDap.sub.N.Math.MeFL 0.097 ± 0.013 L5 GSDapF.sub.N.Math.MeL 0.062 ± 0.009 G1, F4 SarSDap.sub.N.Math.MeF 1.5 ± 0.1 G1, F4, L5 SarSDap.sub.N.Math.MeF.sub.N.Math.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.