Heterobivalent agents targeting PSMA and integrin-αvβ3

Abstract

Differential expression of surface proteins on normal vs malignant cells provides the rationale for the development of receptor-, antigen-, and transporter-based, cancer-selective imaging and therapeutic agents. However, tumors are heterogeneous, and do not always express what can be considered reliable, tumor-selective markers. That suggests development of more flexible targeting platforms that incorporate multiple moieties enabling concurrent targeting to a variety of putative markers. We report the synthesis, biochemical, in vitro, and preliminary in vivo evaluation of a new heterobivalent (HtBv) imaging agent targeting both the prostate-specific membrane antigen (PSMA) and integrin-αvβ3 surface markers, each of which can be overexpressed in certain tumor epithelium and/or neovasculature. The HtBv agent was functionalized with either 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or the commercially available IRDye800CW. DOTA-conjugated HtBv probe 9 bound to PSMA or αvβ3 with affinities similar to those of monovalent (Mnv) compounds designed to bind to their targets independently. In situ energy minimization experiments support a model describing the conformations adapted by 9 that enable it to bind both targets. IRDye800-conjugated HtBv probe 10 demonstrated target-specific binding to either PSMA or integrin-αvβ3 overexpressing xenografts. HtBv agents 9 and 10 may enable dual-targeted imaging of malignant cells and tissues in an effort to address heterogeneity that confounds many cancer-targeted imaging agents.

Scheme 1. Synthesis of EUKL-DOTA (3) as a Control Mnv and DOTA-Conjugated PSMA Targeting Agent

Scheme 2. Synthesis of cRGDfK-DOTA (5) and cRGDfK-IRDye800 (6) as Control and Mnv Integrin-α_vβ₃ Targeting Agents, Respectively

Scheme 3. Synthesis of EUKL-cRGDfK-NH₂ (8)

Scheme 4. Synthesis of EUKL-cRGDfK-DOTA (9) and EUKL-cRGDfK-IRDye800 (10) as HtBv PSMA and Integrin-α_vβ₃ Targeting Agents

Figure 1

Competitive binding assays of test HtBv agents and their Mnv control agents. (A) NIR cell-based competitive binding to PSMA+ PC-3/PIP cells conducted against 10 nM of IRDye800-YC27. (B) Amplex red fluorescence assay (functional inhibition of PSMA). (C) NIR cell-based competitive binding to U87 cells performed against 50 nM of cRGDfK-IRDye800 (6). (D) Integrin-α_vβ₃ protein-based fluorescence polarization assay.

Figure 2

Proposed binding modes of compounds 3 and 9 to PSMA using an in situ ligand minimization protocol (Discovery Studio 3.1 client). The in situ experiments used 3D7H X-ray coordinates of PSMA. The protein is presented as light gray line ribbon. The binding pocket of PSMA is surrounded by a gray sphere. The bound ligand is depicted as sticks, and atoms are colored by elements in A and B; carbon (gray), nitrogen (blue), and oxygen (red). (A) Proposed binding mode of EUKL-DOTA (3). (B) Proposed binding mode of EUKL-cRGDfK-DOTA (9). (C) Superimposed compound 3 (blue) and compound 9 (cyan).

Figure 3

Proposed binding modes of compounds 5 and 9 to integrin-α_vβ₃ using an in situ ligand minimization protocol (Discovery Studio 3.1 client). The in situ experiments used 1L5G X-ray coordinates of integrin-α_vβ₃. The protein is presented as light gray line ribbon. The binding pocket of integrin-α_vβ₃ is surrounded with a gray sphere. The bound ligand is depicted as stick and atoms are colored by elements in A and B; carbon (gray), nitrogen (blue), and oxygen (red). (A) Proposed binding mode of cRGDfK-DOTA (5). (B) Proposed binding mode of EUKL-cRGDfK-DOTA (9). (C) Superimposed compound 5 (blue) and compound 9 (cyan).

Figure 4

Dose-dependent uptake of compound 10 by two different phenotypic xenografts. (A) In vivo optical imaging of three NOD/SCID mice bearing U87-MG tumors (group-1 mice). Mouse 1 received 2 nmol, mouse 2 received 1 nmol, and mouse 3 received 0.5 nmol of the HtBv agent 10. (B) In vivo optical imaging of three NOD/SCID mice bearing PC-3/PIP (forward left flank) and PSMA- PC-3/flu (forward right flank) tumors (group-2 mice). Mouse 1 received 2 nmol, mouse 2 received 1 nmol, and mouse 3 received 0.5 nmol of the HtBv agent 10. Ventral (animal supine) views were obtained at 24 h postinjection. Images were scaled to the same maximum and minimum values (arbitrary units) for each group.

Figure 5

Binding specificity demonstrated by specific blockade of 1 nmol 10 uptake by different phenotypic xenografts using specific blockers. (A) In vivo optical imaging of three NOD/SCID mice bearing U87-MG (1), PSMA+ PC3-PIP (2), and PSMA- PC3-flu (3) tumors (group-3 mice). Mouse 1 received 1 nmol of the HtBv agent 10, mouse 2 received 1 nmol of the HtBv agent 10 along with 100 nmol DCIBzL (selective PSMA ligand), and mouse 3 received a mixture of 1 nmol of the HtBv agent 10 and 300 nmol compound 5 (Mnv integrin-α_vβ₃ agent). Ventral (animal supine) views were obtained at 24 h postinjection. (B) Ex vivo Images of individually harvested organs on a Petri dish at 24 h postinjection: U87-MG (1), PC3-PIP (2), PC3-flu (3), heart (4), lung (5), liver (6), spleen (7), kidneys (8), bladder (9), GIT (10), and muscle (11). Images were scaled to the same maximum values (arbitrary units).