Synthesis and in vitro Evaluation of ADAM10 and ADAM17 Highly Selective Bioimaging Probes

Abstract

A disintegrin and metalloproteinase (ADAMs) are membrane-bound metalloproteases responsible for the ectodomain shedding of various transmembrane proteins and play important roles in multiple relevant biological processes. Their altered expression is involved in several pathological conditions, and in particular ADAM10 or ADAM17 overexpression is found in various forms of cancer. To better understand how they are regulated in the cellular context, it is useful to visualize the specific ADAMs pathway by means of molecular imaging techniques. For this purpose, we synthesized bioactive fluorescent probes suitable for cell imaging and that are able to specifically target ADAM10 or ADAM17. Two previously developed ADAM17- and ADAM10-selective inhibitors were chosen for conjugation, respectively, to a Cy5.5 dye and to Cy5.5 and FITC dyes. Herein we also report the synthesis of a gold-labeled compound as an additional bioimaging probe for ADAM10. The newly synthesized ligands were found to be active in vitro on human recombinant ADAM10 and/or ADAM17, showing IC values in the nanomolar range and a good selectivity over matrix metalloproteinases (MMPs). Finally, these newly developed probes were successfully used for ADAMs staining on different lymphoma cell lines and lymph node mesenchymal stromal cells.

Specific ADAM10 inhibitors localize in exosome-like vesicles released by Hodgkin lymphoma and stromal cells and prevent sheddase activity carried to bystander cells

“…To further evaluate the localization of ADAM10 inhibitors, we performed transmission electron microscopy (TEM) on MSC16412 exposed for 24 h to the gold-conjugated CAM29 (CAM49) compound. 30 As shown in Figure 3A , CAM49 was detectable in microvesicles, one appearing as a blebbing structure ( Figure 3A , quadrant in a enlarged in b), indicative of membrane internalization by endocytosis. 31 Thus, we set up a series of experiments using ExoV purified from conditioned medium of MSC or HL cell lines.…”

“…LT4 was synthesized as described, 21 and CAM29, showing a IC 50 selective for ADAM10 (Supplemental Data and Suppl.Table 1), or CAM29 conjugated with cyanine 5.5 (Cy5.5, CAM36) or with fluorescein-isothiocyanate (FITC, CAM50), were newly synthesized. 30 As reported in our previous publication, 21 LT4 is a sulfonamido-based hydroxamate that inhibits ADAM10 by interacting with its catalytic domain by chelating the zinc ion, critical for both substrate binding and cleavage. CAM29 is its PEGylated analogue used to prepare the fluorescent probes and the CAM49 gold nanoprobe prepared by conjugating multivalent gold nanoparticles with CAM29.…”

“… 29 To determine the subcellular distribution of the ADAM10 inhibitors that we reported to be active on HL cells, 20,21 we performed a series of experiments using the previously described LT4, the newly synthesized CAM29, both showing a IC 50 selective for ADAM10 (Supplemental Data and Suppl.Table 1), or CAM29 conjugated with cyanine 5.5 (Cy5.5, CAM36) or with fluorescein isothiocyanate (FITC, CAM50). 30 First, upon exposure to LT4 ( Figure 2A ), and CAM29 (not shown), ADAM10 time-dependent compartmentalization in endolysosomes increased in MSC16412, possibly interfering with ADAM10 stability due to retention in the degradative pathway while decreasing membrane localization ( Figure 2A , right blot). Second, exposure of MSC16412 ( Figure 2B ) or MSC773 (not shown) to CAM29 led to a reduction of surface reactivity of the anti-ADAM10 mAb and this interference was dose-dependent ( Figure 2B ), thus supporting that the inhibitor induces a conformational change and/or a subcellular redistribution of ADAM10 that becomes unavailable for antibody binding.…”

“…CAM29 is its PEGylated analogue used to prepare the fluorescent probes and the CAM49 gold nanoprobe prepared by conjugating multivalent gold nanoparticles with CAM29. 30 Compound ST178, a Cy5.5- N -isopropyl amide unable to bind ADAM10, was used as a negative control for the fluorescent probes.…”

“…ExoV were observed under a transmission electronic microscope Zeiss Leo EM 900 at 80 kV. For TEM analysis of MSC16412 exposed to gold-conjugated CAM29 (CAM49) overnight at 2.5 µM concentration, cells were processed as described 30 (see Supplemental Methods).…”