All peptides were synthesized by CPC Scientific (Sunnyvale, CA) and reconstituted in dimethylformamide (DMF) unless otherwise specified. Sequences are provided in Supplementary Table S1.
Abstract
Recent years have seen the emergence of conditionally activated diagnostics and therapeutics that leverage protease-cleavable peptide linkers to enhance their specificity for cancer. However, due to a lack of methods to measure and localize protease activity directly within the tissue microenvironment, the design of protease-activated agents has been necessarily empirical, yielding suboptimal results when translated to patients. To address the need for spatially resolved protease activity profiling in cancer, we developed a new class of in situ probes that can be applied to fresh-frozen tissue sections in a manner analogous to immunofluorescence staining. These activatable zymography probes (AZP) detected dysregulated protease activity in human prostate cancer biopsy samples, enabling disease classification. AZPs were leveraged within a generalizable framework to design conditional cancer diagnostics and therapeutics and showcased in the Hi-Myc mouse model of prostate cancer, which models features of early pathogenesis. Multiplexed screening against barcoded substrates yielded a peptide, S16, that was robustly and specifically cleaved by tumor-associated metalloproteinases in the Hi-Myc model. In situ labeling with an AZP incorporating S16 revealed a potential role of metalloproteinase dysregulation in proliferative, premalignant Hi-Myc prostatic glands. Systemic administration of an in vivo imaging probe incorporating S16 perfectly classified diseased and healthy prostates, supporting the relevance of ex vivo activity assays to in vivo translation. We envision AZPs will enable new insights into the biology of protease dysregulation in cancer and accelerate the development of conditional diagnostics and therapeutics for multiple cancer types.
Table S1.
| Name | Sequence | Readout (sample type) |
|---|---|---|
| S1-Q | (5FAM)-GGPQGIWGQ-K(CPQ2)-(PEG2)-C-NH2 | Fluorescence (in vitro / ex vivo) |
| S2-Q | (5FAM)-GGLVPRGSG-K(CPQ2)-(PEG2)-C-NH2 | Fluorescence (in vitro / ex vivo) |
| S3-Q | (5FAM)-GGPVGLIG-K(CPQ2)-(PEG2)-C-NH2 | Fluorescence (in vitro / ex vivo) |
| S4-Q | (5FAM)-GGPLGVRGK-K(CPQ2)-(PEG2)-C-NH2 | Fluorescence (in vitro / ex vivo) |
| S5-Q | (5FAM)-GRQRRALEKG-K(CPQ2)-(PEG2)-GC-NH2 | Fluorescence (in vitro / ex vivo) |
| S6-Q | (5FAM)-GGGSGRSANAKG-K(CPQ2)-(PEG2)-GC-NH2 | Fluorescence (in vitro / ex vivo) |
| S7-Q | (5FAM)-GKPISLISSG-K(CPQ2)-(PEG2)-GC-NH2 | Fluorescence (in vitro / ex vivo) |
| S8-Q | (5FAM)-GILSRIVGGG-K(CPQ2)-(PEG2)-GC-NH2 | Fluorescence (in vitro / ex vivo) |
| S9-Q | (5FAM)-GRPKPVE(Nval)WRKG-K(CPQ2)-(PEG2)-GC-NH2 | Fluorescence (in vitro / ex vivo) |
| S10-Q | (5FAM)-GIQQRSLGGG-K(CPQ2)-(PEG2)-GC-NH2 | Fluorescence (in vitro / ex vivo) |
| S11-Q | (5FAM)-GGVPRGG-K(CPQ2)-(PEG2)-GC-NH2 | Fluorescence (in vitro / ex vivo) |
| S12-Q | (5FAM)-GSGSKIIGGG-K(CPQ2)-(PEG2)-GC-NH2 | Fluorescence (in vitro / ex vivo) |
| S13-Q | (5FAM)-GAANLTRG-K(CPQ2)-(PEG2)-GC-NH2 | Fluorescence (in vitro / ex vivo) |
| S14-Q | (5FAM)-GLAQAPhe(homo)RSG-K(CPQ2)-(PEG2)-GC-NH2 | Fluorescence (in vitro / ex vivo) |
| S15-Q | (5FAM)-GSPLAQAVRSSG-K(CPQ2)-(PEG2)-GC-NH2 | Fluorescence (in vitro / ex vivo) |
| S16-Q | (5FAM)-GPVPLSLVMG-K(CPQ2)-(PEG2)-GC-NH2 | Fluorescence (in vitro / ex vivo) |
| S17-Q | (5FAM)-GSQPRIVGGG-K(CPQ2)-(PEG2)-GC-NH2 | Fluorescence (in vitro / ex vivo) |
| S19-Q | (5FAM)-GGLGPKGQTG-K(CPQ2)-(PEG2)-C-NH2 | Fluorescence (in vitro / ex vivo) |
| S20-Q | (5FAM)-GGQTCKCSCK-K(CPQ2)-(PEG2)-C-NH2 | Fluorescence (in vitro / ex vivo) |
| S1-M | e(+2G)(+6V)ndneeG(+10F)(+1F)s(+1A)r-ANP-GGPQGIWGQGC | LC-MS/MS (in vitro / ex vivo) |
| S2-M | eG(+6V)ndneeGF(+1F)s(+1A)r-ANP-GGLVPRGSGGC | LC-MS/MS (in vitro / ex vivo) |
| S3-M | e(+3G)(+1V)ndneeGFFs(+4A)r-ANP-GGPVGLIGGC | LC-MS/MS (in vitro / ex vivo) |
| S4-M | e(+2G)Vndnee(+2G)FFs(+4A)r-ANP-GGPLGVRGKGC | LC-MS/MS (in vitro / ex vivo) |
| S5-M | e(+3G)(+1V)ndneeG(+10F)FsAr-ANP-GRQRRALEKGC | LC-MS/MS (in vitro / ex vivo) |
| S6-M | e(+2G)Vndnee(+2G)F(+10F)sAr-ANP-GGGSGRSANAKGC | LC-MS/MS (in vitro / ex vivo) |
| S7-M | e(+2G)(+6V)ndneeGFFsAr-ANP-GKPISLISSGC | LC-MS/MS (in vitro / ex vivo) |
| S8-M | eGVndneeGF(+10F)s(+4A)r-ANP-GILSRIVGGGC | LC-MS/MS (in vitro / ex vivo) |
| S9-M | eG(+6V)ndneeG(+10F)Fs(+4A)r-ANP-GRPKPVE(Nval)WRKGC | LC-MS/MS (in vitro / ex vivo) |
| S10-M | e(+3G)(+1V)ndnee(+2G)(+10F)Fs(+4A)r-ANP-GIQQRSLGGGC | LC-MS/MS (in vitro / ex vivo) |
| S11-M | e(+2G)Vndnee(+3G)(+10F)(+1F)s(+4A)r-ANP-GGVPRGGC | LC-MS/MS (in vitro / ex vivo) |
| S12-M | eGVndneeG(+10F)(+10F)sAr-ANP-GSGSKIIGGGC | LC-MS/MS (in vitro / ex vivo) |
| S13-M | e(+2G)(+6V)ndnee(+3G)(+10F)(+1F)s(+4A)r-ANP-GAANLTRGC | LC-MS/MS (in vitro / ex vivo) |
| S14-M | eG(+6V)ndneeG(+10F)(+10F)sAr-ANP-GLAQAPhe(homo)RSGC | LC-MS/MS (in vitro / ex vivo) |
| S15-M | e(+3G)(+1V)ndnee(+2G)(+10F)(+10F)sAr-ANPGSPLAQAVRSSGC | LC-MS/MS (in vitro / ex vivo) |
| S16-M | e(+2G)VndneeG(+10F)(+10F)s(+4A)r-ANP-GPVPLSLVMGC | LC-MS/MS (in vitro / ex vivo) |
| S17-M | eGVndnee(+2G)(+10F)(+10F)s(+4A)r-ANP-GSQPRIVGGGC | LC-MS/MS (in vitro / ex vivo) |
| S19-M | e(+2G)(+6V)ndnee(+3G)(+1F)(+1F)s(+1A)r-ANPGGLGPKGQTGGC | LC-MS/MS (in vitro / ex vivo) |
| S20-M | eG(+6V)ndnee(+3G)(+1F)Fs(+4A)r-ANP-GGQTCKCSCKGC | LC-MS/MS (in vitro / ex vivo) |
| S1-Z | U-eeeeeeee-X-GGPQGIWGQG-rrrrrrrrr-X-K(Cy3)-NH2 | Fluorescence (in situ) |
| S2-Z | U-eeeeeeee-X-GGLVPRGSGG-rrrrrrrrr-X-K(Cy5)-NH2 | Fluorescence (in situ) |
| S3-Z | U-eeeeeeee-X-GGPVGLIGG-rrrrrrrrr-X-K(5FAM)-NH2 | Fluorescence (in situ) |
| S4-Z | U-eeeeeeee-X-GPLGVRGKG-rrrrrrrrr-X-K(5FAM)-NH2 | Fluorescence (in situ) |
| S5-Z | U-eeeeeeee-X-GRQRRALEKG-rrrrrrrrr-X-K(Cy3)-NH2 | Fluorescence (in situ) |
| S6-Z | U-eeeeeeee-X-GSGRSANAG-rrrrrrrrr-X-K(Cy5)-NH2 | Fluorescence (in situ) |
| S7-Z | U-eeeeeeee-X-GKPISLISSG-rrrrrrrrr-X-K(5FAM)-NH2 | Fluorescence (in situ) |
| S8-Z | U-eeeeeeee-X-GILSRIVGGG-rrrrrrrrr-X-K(5FAM)-NH2 | Fluorescence (in situ) |
| S9-Z | U-eeeeeeee-X-GRPKPVE(Nval)WRKG-rrrrrrrrr-X-K(5FAM)-NH2 | Fluorescence (in situ) |
| S10-Z | U-eeeeeeee-X-GIQQRSLGGG-rrrrrrrrr-X-K(5FAM)-NH2 | Fluorescence (in situ) |
| S11-Z | U-eeeeeeee-X-GGGVPRGGG-rrrrrrrrr-X-K(5FAM)-NH2 | Fluorescence (in situ) |
| S12-Z | U-eeeeeeee-X-GSGSKIIGGG-rrrrrrrrr-X-K(Cy3)-NH2 | Fluorescence (in situ) |
| S13-Z | U-eeeeeeee-X-GGAANLTRGG-rrrrrrrrr-X-K(Cy3)-NH2 | Fluorescence (in situ) |
| S14-Z | U-eeeeeeee-X-GLAQAPhe(homo)RSG-rrrrrrrrr-X-K(Cy3)-NH2 | Fluorescence (in situ) |
| S15-Z | U-eeeeeeee-X-GSPLAQAVRSSG-rrrrrrrrr-X-K(Cy3)-NH2 | Fluorescence (in situ) |
| S16-Z | U-eeeeeeee-X-GPVPLSLVMG-rrrrrrrrr-X-K(5FAM)-NH2 | Fluorescence (in situ) |
| S17-Z | U-eeeeeeee-X-GSQPRIVGGG-rrrrrrrrr-X-K(Cy5)-NH2 | Fluorescence (in situ) |
| S18-Z | U-eeeeeeee-X-GGGHARLVHVG-rrrrrrrrr-X-K(Cy3)-NH2 | Fluorescence (in situ) |
| S19-Z | U-eeeeeeee-X-GGLGPKGQTGG-rrrrrrrrr-X-K(Cy3)-NH2 | Fluorescence (in situ) |
| S20-Z | U-eeeeeeee-X-GGQTCKCSCKG-rrrrrrrrr-X-K(Cy5)-NH2 | Fluorescence (in situ) |
| dS6-Z | U-eeeeeeee-X-GsGrsanaG-rrrrrrrrr-X-K(Cy5)-NH2 | Fluorescence (in situ) |
| S6-QZ | (QSY21)-eeeeeeeee-c-o-GSGRSANAG-rrrrrrrrr-K(Cy5)-NH2 | Fluorescence (in situ) |
| dS6-QZ | (QSY21)-eeeeeeeee-c-o-GsGrsanaG-rrrrrrrrr-K(Cy5)-NH2 | Fluorescence (in situ) |
| dS16-Z | U-eeeeeeee-X-GpvplslvmG-rrrrrrrrr-X-K(5FAM)-NH2 | Fluorescence (in situ) |
| S16-QZ | (QSY21)-eeeeeeeee-c-o-GPVPLSLVMG-rrrrrrrrr-K(Cy5)-NH2 | Fluorescence (in situ / in vivo) |
| dS16-QZ | (QSY21)-eeeeeeeee-c-o-GpvplslvmG-rrrrrrrrr-K(Cy5)-NH2 | Fluorescence (in situ / in vivo) |
| S16 | GPVPLSLVMG | Cleavage motif |
| polyR | rrrrrrrrr-X-K(Cy7)-NH2 | Fluorescence (in situ) |
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Shurtleff, V.W., Layton, M.E., Parish, C.A., Perkins, J.J., Schreier, J.D., Wang, Y., Adam, G.C., Alvarez, N., Bahmanjah, S., Bahnck-Teets, C.M. and Boyce, C.W. Journal of Medicinal Chemistry 67, no. 5 (2024): 3935-3958.
- Center for Discovery and Innovation, Hackensack Meridian Health. 111 Ideation Way. Nutley, New Jersey 07110, United States.
- Merck & Co., Inc., Rahway, New Jersey 07065, United States.
The enzymatic activity of recombinantly expressed 3CLPro enzymes from different coronaviruses was measured using the following synthetic quenched FRET peptide: CP488-ESATLQSGLRKAK- (CPQ2)-NH2 (CPC Scientific, San Jose, CA.
Goh, Joleen Pei Zhen. Nanyang Technological University (2023)
9 generic fluorogenic substrates (CPC Scientific) (Figure S4) were added to the final concentration of 20 μM. Fluorescence was measured at Excitation/Emission=330/390 nm on BioTek Synergy H1 microplate reader and proteolytic activity was calculated as a change in relative fluorescence units per sec using the slope of the linear range for this signal.
Design-rules for stapled peptides with in vivo activity and their application to Mdm2/X antagonists.
Chandramohan, A., Josien, H., Yuen, T.Y., Duggal, R., Spiegelberg, D., Yan, L., Juang, Y.C.A., Ge, L., Aronica, P.G., Kaan, H.Y.K. and Lim, Y.H. Nature Communications 15, no. 1 (2024): 489.
- Merck & Co., Inc., Kenilworth, NJ 07033, USA.
- Merck & Co., Inc., Boston, MA 02115, USA
- Merck & Co., Inc., West Point, PA 19486, USA
- Genentech, South San Francisco, CA 94080, USA
We thank Evans (Chen) Ge, Mike (Dixin) Xue, and Simon (Junhua) Li at Chinese Peptide Company (CPC) for peptide synthesis support.
Hao, Liangliang, Renee T. Zhao, Nicole L. Welch, Edward Kah Wei Tan, Qian Zhong, Nour Saida Harzallah, Chayanon Ngambenjawong et al. Nature Nanotechnology (2023): 1-10.
All peptides and oligonucleotides were synthesized and HPLC purified by CPC Scientific and Integrated DNA Technologies (IDT), respectively. Peptide–oligonucleotide conjugates were generated by copper-free click chemistry.
Kikuchi, F., Ikeda, Z., Kakegawa, K., Nishikawa, Y., Sasaki, S., Fukuda, K., Takami, K., Banno, Y., Nishikawa, H., Taya, N. and Nakahata, T. Bioorganic & Medicinal Chemistry 93 (2023): 117462.
- Research, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
- Pharmaceutical Sciences, Takeda Pharmaceutical Company Ltd., 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
5FAM-Abu-Gly-Asp-Asp-Asp-Lys-Ile-Val-Gly-Gly-Lys(CPQ2)-Lys-Lys-NH2 (purity: 97.2%, CPC Scientific, Inc.) was diluted with an assay buffer to prepare a 5.4 μM substrate solution.
Senotherapeutic peptide treatment reduces biological age and senescence burden in human skin models.
Zonari, A.; Brace, L. E.; Al-Katib, K.; Porto, W. F.; Foyt, D.; Guiang, M.; Cruz, E. A. O.; Marshall, B.; Gentz, M.; Guimaraes, G. R.; Franco, O. L.; Oliveira, C. R.; Boroni, M.; Carvalho, J. L., NPJ Aging 2023, 9 (1), 10.
The top hit peptides selected (Pep 14, 144, 156, 195, and 393) from the screening and the fluorescence labeled peptide (5FAM-PEG2-Pep 14) were purchased from CPC Scientific Inc. (USA), which synthesized the peptide by solid phase (Fmoc) on a Rink amide resin, with >95% purity, in the form of acetate salt.
Synthetic oligonucleotides constitute an important class of therapeutics developed to treat a variety of indications. Two main synthetic approaches exist for the conjugation of a peptide to an oligonucleotide: parallel and linear. The primary benefit of the linear approach is the one-pot solid-phase assembly and compatibility with machine automation. However, in cases where poor compatibility of peptide and oligo chemistries exist or long peptide and oligo fragments are required, preparing both components separately and linking both compounds together may offer the simplest solution.
Solid-phase peptide synthesis (SPPS) approaches require that the side chains of certain amino acids be protected from undesired reactivity during synthesis. The installation and removal of these protection groups results in a lower atom economy in the production process. Removal of the protection groups often requires large volumes of trifluoroacetic acid (TFA) or other strong acids which can result in lower yields and pose a significant risk to the environment.
Schiemer, J., Maxwell, A., Horst, R., Liu, S., Uccello, D.P., Borzilleri, K., Rajamohan, N., Brown, M.F. and Calabrese, M.F. Nature Communications 14, no. 1 (2023): 1189.
- Discovery Sciences, Pfizer Worldwide Research and Development, Groton, CT, USA
After 3 h the resin was washed with binding buffer until no protein was detected, followed by elution with 0.2 mg ml−1 FLAG peptide DYKDDDDK (CPC Scientific Peptide company)
