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Design of Optical-Imaging Probes by Screening of Diverse Substrate Libraries Directly in Disease-Tissue Extracts
Martina Tholen1, Joshua J Yim1,2, Katarzyna Groborz3
1Department of Pathology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA, 94305, USA.
Abstract:
Fluorescently quenched probes that are specifically activated in the cancer microenvironment have great potential application for diagnosis, early detection, and surgical guidance. These 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 a probe with suboptimal performance when applied in vivo. We describe here an alternate, unbiased activity-profiling approach in which whole tissue extracts are used to directly identify optimal peptide sequences for probe design. Screening of tumor extracts with a hybrid combinatorial substrate library (HyCoSuL) identified a combination 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 a priori knowledge of enzyme targets and has the potential to be applied to diverse disease states using clinical or animal-model tissue samples.
Insights
Researchers developed a new method using whole tissue extracts to find optimal peptide sequences for cancer-detecting fluorescent probes. This unbiased approach enhances probe design for improved in vivo performance in cancer diagnosis and surgical guidance.
Area of Science:
- Biochemistry
- Chemical Biology
- Molecular Imaging
Background:
- Fluorescent probes activated in the tumor microenvironment show promise for cancer diagnosis, early detection, and surgical guidance.
- Current probe design often relies on optimizing against single purified enzymes, which can lead to suboptimal in vivo performance.
- This enzyme-centric approach requires extensive chemical synthesis and may not fully represent the complex in vivo environment.
Purpose of the Study:
- To develop an unbiased activity-profiling approach for identifying optimal peptide sequences for fluorescent probe design.
- To bypass the limitations of traditional enzyme-specific optimization methods.
- To create highly efficient and tumor-specific fluorescent probes using a novel screening strategy.
Main Methods:
- Utilized whole tissue extracts for direct activity profiling to identify optimal peptide sequences.
- Employed a hybrid combinatorial substrate library (HyCoSuL) for screening tumor extracts.
- Generated and tested novel tumor-specific fluorescent probes based on identified peptide sequences.
Main Results:
- Identified a unique combination of natural and non-natural amino acid residues for probe design.
- Developed highly efficient tumor-specific fluorescent probes with improved performance.
- Demonstrated a simplified and enhanced process for probe optimization without prior enzyme target knowledge.
Conclusions:
- The described activity-profiling approach using whole tissue extracts offers a more efficient and effective strategy for designing fluorescent probes.
- This method enhances the development of tumor-specific probes, improving their potential for in vivo applications.
- The strategy is versatile and can be applied to various disease states using different tissue samples.
