Tumor imaging by means of proteolytic activation of cell-penetrating peptides

Abstract

We have devised and tested a new strategy for selectively delivering molecules to tumor cells. Cellular association of polyargininebased, cell-penetrating peptides (CPPs) is effectively blocked when they are fused to an inhibitory domain made up of negatively charged residues. We call these fusions activatable CPPs (ACPPs) because cleavage of the linker between the polycationic and polyanionic domains, typically by a protease, releases the CPP portion and its attached cargo to bind to and enter cells. Association with cultured cells typically increases 10-fold or more upon linker cleavage. In mice xenografted with human tumor cells secreting matrix metalloproteinases 2 and 9, ACPPs bearing a far-red-fluorescent cargo show in vivo contrast ratios of 2-3 and a 3.1-fold increase in standard uptake value for tumors relative to contralateral normal tissue or control peptides with scrambled linkers. Ex vivo slices of freshly resected human squamous cell carcinomas give similar or better contrast ratios. Because CPPs are known to import a wide variety of nonoptical contrast and therapeutic agents, ACPPs offer a general strategy toward imaging and treating disease processes associated with linker-cleaving activities such as extracellular proteases.cancer ͉ molecular imaging ͉ polycation ͉ transduction

Multistep Synthesis on SU-8: Combining Microfabrication and Solid-Phase Chemistry on a Single Material

“…A nonapeptide (HIV-Protease 1 substrate), was chosen as the target (13, Val-SerGln-Asn-Tyr-Pro-Ile-Val-Gln). [29][30][31] Nonapeptide 13 was synthesized on SU-8 prepared on Al (11a) since this had given the best results for the synthesis of Leu-Enkephalin (12) (Scheme 3). As peptide 13 was not commercially available, a synthetic standard of 14 was prepared by standard SPPS using standard PS resin with a Wang linker, 15.…”

How molecular imaging is speeding up antiangiogenic drug development

“…Later, similar approaches were used for imaging MMP expression in the heart after myocardial infarction where an near-IR fluorescent probe was activated upon proteolytic cleavage by MMP-2 and MMP-9 (75). In another report, cellular association of polyargininebased cell-penetrating peptides is effectively blocked when they are fused to an inhibitory domain composed of negatively charged residues, which was called ''activatable cell-penetrating peptides'' (76). Cleavage of the MMPsensitive linker between the polycationic and polyanionic domains releases the cell-penetrating peptide portion and its attached cargo to bind to and enter cells.…”

Ratiometric Activatable Cell‐Penetrating Peptides Provide Rapid In Vivo Readout of Thrombin Activation

“…This method provides detection of spatially localized enzymatic activity in living tissues through the accumulation of cleaved probe. ACPPs have been previously reported that target MMPs [16,17] and elastases [20] in tumors. A thrombin-activated ACPP with cleavage sequence DPRSFL, from the PAR1 receptor was recently reported for monitoring thrombin activation in atherosclerotic plaques.…”

“…[15] Activatable cell-penetrating peptides (ACPPs) target various cargos, including fluorescent imaging agents, to sites of protease activity in vivo. [16][17][18][19] ACPPs consist of a polycationic cell-penetrating peptide attached to a cargo and a polyanionic inhibitory domain with a protease-cleavable linker. Probe activation and cargo uptake depend on localized proteolysis of the linker sequence that connects the polyanionic and polycationic domains, which converts the probe to an adherent form.…”