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

Abstract

Extracellular proteases including thrombin are involved in numerous biological processes and play major roles in a variety of human diseases. The spatial and temporal patterns of activation of proteases in vivo control their biological role in diseases and amenability to therapeutic targeting. Previously we developed activatable cell-penetrating peptides (ACPPs) to monitor matrix metalloproteinase (MMP) and elastase activity in tumors. Later ACPPs detect thrombin activation in atherosclerosis and brain injury. We have now modified the thrombin ACPP in two independent ways, 1) to provide a FRET-dependent emission ratiometric readout and 2) to accelerate the kinetics of cleavage by thrombin. Emission ratioing improves kinetic detection of enzyme activity, because it reflects the ratio of cleaved versus uncleaved probe but cancels out total probe concentration, illumination intensity, detection sensitivity, and tissue thickness. Because pharmacokinetic washout of the uncleaved probe is not necessary, yet the cleavage converts a diffusible substrate into an immobilized product, thrombin activity can be imaged in real time with good spatial resolution. Meanwhile, placement of norleucine-threonine (Nle-Thr) at the P4-P3 substrate positions accelerates the kinetics of thrombin cleavage by 1-2 orders of magnitude, while preserving selectivity against related proteases. The new ratiometric ACPPs detect localized thrombin activation in rapidly forming blood clots minutes after probe injection, and the signal is inhibited by thrombin specific inhibitors.Thrombin is a serine protease and a key regulator of blood coagulation. It is responsible for the proteolytic cleavage and activation of multiple coagulation factors including Factor V,

Fluorogenic protein labelling: a review of photophysical quench mechanisms and principles of fluorogen design

“…This approach has been used commonly to make fluorogenic sensors as enzyme activity probes, 51,52 where the donor fluorophore and the quencher group are linked by a substrate/inhibitor peptide such that the donor's fluorescence is quenched by the adjacent quencher through FRET. Upon cleavage of the peptide linker, the quencher is eliminated and the donor's fluorescence is restored.…”

Sidechain Engineering in Cell‐Penetrating Poly(disulfide)s

“…1 , Fig. ) [1 – 6]. The ability of CPPs to cross lipid bilayer membranes originates from the poor acidity of the guanidinium cation [2].…”

Glycosylated Cell‐Penetrating Poly(disulfide)s: Multifunctional Cellular Uptake at High Solubility

“…TEG‐CPDs 16 , however, showed much precipitation and little uptake and were not further used ( Figure ). This result was surprising considering much precedence for effective solubilization by TEGylation: At least in the context of CPDs, glycosidation was clearly more effective.…”

“…Similar, although less pronounced solubility problems were already observed with the original CPDs 2 at high concentrations, particularly in conjugation with some but not all proteins. To increase solubility of functional systems, PEGylation and the attachment of trehalose have received much attention. Glycosylation with other carbohydrates such as glucose and galactose has been successful as well, although direct applications to CPPs are remarkably rare and fully developped only recently in a breakthrough report from the Deming group .…”