Design of Optical‐Imaging Probes by Screening of Diverse Substrate Libraries Directly in Disease‐Tissue Extracts

Abstract

Fluorescently quenched probes that are specifically activated in the cancer microenvironment have great potential application for diagnosis,e arly detection, and surgical guidance.T hese probes are often designed to target specific enzymes associated with diseases by direct optimization using single purified enzymes.However,this can result in painstaking chemistry efforts to produce ap robe with suboptimal performance when applied in vivo.W ed escribe here an alternate, unbiased activity-profiling approach in whichw hole tissue extracts are used to directly identify optimal peptide sequences for probe design. Screening of tumor extracts with ah ybrid combinatorial substrate library (HyCoSuL) identified ac ombination of natural and non-natural amino-acid residues that was used to generate highly efficient tumor-specific probes. This new strategy simplifies and enhances the process of probe optimization without any apriori knowledge of enzyme targets and has the potential to be applied to diverse disease states using clinical or animal-model tissue samples.

Peptide probes for proteases – innovations and applications for monitoring proteolytic activity

“…These include a variety of combinatorial approaches (both solid 21,22 and solution based, 23 MS substrate profiling methods, 24,25 phage libraries 26 or substrate modification of known substrates, including direct screening on complex tumour tissues. 27 Many FRET peptide probes have been developed [28][29][30] and there are reviews covering this area so here we highlight some key and novel examples 14,19 with FRET based probes developed for legumain, 31 thrombin, 30 SARS-CoV protease, 32 Granzyme B 33 and Cathepsins, 34 among others.…”

Advances and Perspectives of Peptide and Polypeptide‐Based Materials for Biomedical Imaging

“…To improve upon this, Bogyo and co‐workers reported a method to identify optimal peptide sequences for imaging platform design. [ 89 ] They designed a combinatorial library for tumor extract screening, and optimal peptide sequence was identified with high tumor specificity. The introduction of combinatorial library and screening technology did not only simplify the design and optimization of peptide imaging probes but also ensured the targeting function of specific lesions.…”

“…[111] 89 Zr-labeled peptide platform with a PEG linker was reported for tumor-specific PET/CT imaging. [112] A tumor cell-penetrating peptide TPP and a chelator for 89 Zr was conjugated with PEG to construct PET platform. Stability of this PET imaging platform was improved with the link of PEG.…”

Dual‐Mechanism Quenched Fluorogenic Probe Provides Selective and Rapid Detection of Cathepsin L Activity**

“…Thus, the 120‐fold turn‐on ratio of CTLAP is attributed to the compounding effect of two quenching mechanisms, the first being a standard off‐on mechanism caused by release of AMC, [21] whereas the second is a unique quenching event caused by the 2‐phenylthiophene residue that also endows the probe with high selectivity for CTL over competing cathepsins. This could be linked to the effect of adding a quenching dye to the probe to reduce the background of always‐on probes [26d,38] (a common strategy for CTL imaging probes) without the need to actually add such a moiety. The idea of probe components (such as a self‐immolating aromatic [32] or aliphatic [25a] spacer, or reporter and quencher location [39] ) influencing selectivity has been demonstrated, but here we show the reverse, that probe components included to increase selectivity can influence the photophysical properties of the probe.…”