A fluorogenic cyclic peptide for imaging and quantification of drug-induced apoptosis

Abstract

Programmed cell death or apoptosis is a central biological process that is dysregulated in many diseases, including inflammatory conditions and cancer. The detection and quantification of apoptotic cells in vivo is hampered by the need for fixatives or washing steps for non-fluorogenic reagents, and by the low levels of free calcium in diseased tissues that restrict the use of annexins. In this manuscript, we report the rational design of a highly stable fluorogenic peptide (termed Apo-15) that selectively stains apoptotic cells in vitro and in vivo in a calcium-independent manner and under wash-free conditions. Furthermore, using a combination of chemical and biophysical methods, we identify phosphatidylserine as a molecular target of Apo-15. We demonstrate that Apo-15 can be used for the quantification and imaging of drug-induced apoptosis in preclinical mouse models, thus creating opportunities for assessing the in vivo efficacy of anti-inflammatory and anti-cancer therapeutics.

Expansion of CD10neg neutrophils and CD14+HLA-DRneg/low monocytes driving proinflammatory responses in patients with acute myocardial infarction

“…neg /CD10 pos neutrophils (1x10 6 /mL) were cultured for 24 hours in RPMI 1640 medium supplemented with 5% HI-FCS and 1% PenStrep. Apoptosis rate was assessed by flow cytometry using a Vybrant™ DyeCycle™ Violet/SYTOX™ AADvanced™ Apoptosis Kit (A35135, Invitrogen) and Apotracker™ Green11 (427402; Biolegend), according to the manufacturer's protocols. Stained neutrophils were analyzed using a Gallios TM cytometer and Gallios TM software (Beckman Coulter).…”

Fluorescent Chemical Tools for Tracking Anionic Phospholipids

“…In a key recent advance another PS binding cyclic peptide was conjugated to an environmentally‐sensitive fluorophore to develop a fluorogenic PS sensor, Apo‐15, that allowed the quantification and imaging of drug induced apoptosis (Figure 11). [99] Importantly, the sensor was translated to mouse models for imaging apoptosis, indicating in vivo applicability [99] …”

“…Cy7.5 labelled CLSYYPSYC (PSP1) has been applied for imaging apoptotic cells in vivo [90b] . Interestingly, despite the small size and ease of fluorophore conjugation most peptide‐based PS sensors have only been applied to image PS externalization [90b,99] . The development of cell‐permeable peptide‐based PS sensors for detecting intracellular PS remains a goal yet to be achieved.…”

Fluorescent cyclic peptides for cell imaging

“…[ 54 ] In this case, the use of a fluorogenic label enabled to brightly stain the membranes of the fungal pathogen Aspergillus fumigatus with high signal‐to‐noise ratios and without the need for washing steps (Figure 1E). In another example, Trp‐BODIPY amino acid was employed for the preparation of a fluorescent cyclic lactadherin mimic for the detection of apoptotic bodies [ 55 ] as well as a fluorescent cyclopeptide for the quantification of drug‐induced apoptosis, [ 80 ] displaying strong binding in a Ca 2+ independent‐manner, unlike the gold standard for apoptosis detection Annexin V. Fluorophore‐bearing oligomers of cyclic dipeptides have also been implemented for the intracellular delivery of pDNA. [ 81 ] Other peptides have been described to target intracellular organelles.…”