Plates were incubated for 210 min at 25 °C followed by quenching with 25 uL water with 2% formic acid containing 600 nM of the internal standard with the sequence KTEEISEVN[L-U7]-OH (CPC Scientific).
Abstract
As a label-free technology, mass spectrometry (MS) enables assays to be generated that monitor the conversion of substrates with native sequences to products without the requirement for substrate modifications or indirect detection methods. Although traditional liquid chromatography (LC)–MS methods are relatively slow for a high-throughput screening (HTS) paradigm, with cycle times typically ≥60 s per sample, the Agilent RapidFire High-Throughput Mass Spectrometry (HTMS) System, with a cycle time of 5–7 s per sample, enables rapid analysis of compound numbers compatible with HTS. By monitoring changes in mass directly, HTMS assays can be used as a triaging tool by eliminating large numbers of false positives resulting from fluorescent compound interference or from compounds interacting with hydrophobic fluorescent dyes appended to substrates. Herein, HTMS assays were developed for multiple protease programs, including cysteine, serine, and aspartyl proteases, and applied as a confirmatory assay. The confirmation rate for each protease assay averaged <30%, independent of the primary assay technology used (i.e., luminescent, fluorescent, and time-resolved fluorescent technologies). Importantly, >99% of compounds designed to inhibit the enzymes were confirmed by the corresponding HTMS assay. Hence, HTMS is an effective tool for removing detection-based false positives from ultrahigh-throughput screening, resulting in hit lists enriched in true actives for downstream dose response titrations and hit-to-lead efforts.
SOCIAL MEDIA
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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)
