The 60 minute enzymatic reaction contained 5 nM Mps1 catalytic domain (aa 510–857), 60 μM (~Km-level) ATP, 5 μM phosphoacceptor peptide substrate (5FAM-RQRRSIDDTIDSTRLFE-NH2; CPC Scientific, Sunnyvale, CA), 10 mM MgCl2, 4% DMSO (with or without inhibitor), 1 mM DTT, 0.01% Tween-20, 50 mM HEPES (pH 7.5).
Abstract
Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; Ki<0.5 nM; cellular IC50 2–6 nM). Treatment of tumor cells in vitro with PF-7006 modulates expected Mps1-dependent biology as demonstrated by molecular and phenotypic measures (reduced pHH3-Ser10 levels, shorter duration of mitosis, micro-nucleation, and apoptosis). Tumor-bearing mice treated with PF-7006 exhibit tumor growth inhibition concomitant with pharmacodynamic modulation of a downstream biomarker (pHH3-Ser10). Unfortunately, efficacy only occurs at drug exposures that cause dose-limiting body weight loss, gastrointestinal toxicities, and neutropenia. Mps1 inhibitor toxicities may be mitigated by inducing G1 cell cycle arrest in Rb1-competent cells with the cyclin-dependent kinase-4/6 inhibitor palbociclib. Using an isogenic cellular model system, PF-7006 is shown to be selectively cytotoxic to Rb1-deficient cells relative to Rb1-competent cells (also a measure of kinase selectivity). Human bone marrow cells pretreated with palbociclib have decreased PF-7006-dependent apoptosis relative to cells without palbociclib pretreatment. Collectively, this study raises a concern that single agent therapies inhibiting Mps1 will not be well-tolerated clinically but may be when combined with a selective CDK4/6 drug.
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Development of a selective, sensitive and robust oxyntomodulin dual monoclonal antibody immunoassay.
Umberger, T.S., Ming, W., Cox, J.M., Konrad, R.J. and Siegel, R.W. Bioanalysis 14, no. 18 (2022): 1229-1239.
- Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, IN46285, USA
Human K2 EDTA and P800 plasma (500 μl) was spiked with proglucagon 33–61, 35–61 and 36–61 stable-isotope-labeled internal standard peptides (CPC Scientific, custom order) and diluted with I buffer (25 mmol/l Tris-HCl, 25 mmol/l HEPES, 300 mmol/l NaCl, 0.1% (v/v) octyl β-D-glucopyranoside, pH 7.5).
Line, J.E.; Seal, B.S.; Garrish, J.K. Appl. Microbiol. 2022, 2, 688–700.
Peptides were synthesized using standard solid-phase(Fmoc) chemistry with a peptide synthesizer (CPC Scientific Inc., Sunnyvale, CA 94089,USA, C12K-2β12 [..]
Kirk, N.S., Chen, Q., Wu, Y.G., Asante, A.L., Hu, H., Espinosa, J.F., Martínez-Olid, F., Margetts, M.B., Mohammed, F.A., Kiselyov, V.V. and Barrett, D.G. Nature Communications 13, no. 1 (2022): 5695.
- Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA.
Peptides were synthesized under contract by CPC Scientific, except for the N-terminally acetylated version of IM172N22 and the Glu3Arg, Glu3Ala, Glu4Arg, Glu4Ala, Glu5Ala, Glu5Arg, Trp6Ala, Gln8Ala, Ile9Ala, Glu10Ala, Glu10Arg and Tyr14Ala mutants of IM172N22
Coskun, T., Urva, S., Roell, W.C., Qu, H., Loghin, C., Moyers, J.S., O’Farrell, L.S., Briere, D.A., Sloop, K.W., Thomas, M.K. and Pirro, V. Cell Metabolism 34, no. 9 (2022): 1234-1247.
- Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
Homologous and heterologous competition experiments were performed with non-radioactive peptide analogues[127I]-Tyr1-GIP(1-42) and [127]-Tyr10-GIP(1-42) to ensure quantification of the high-affinity binding site of the GIPR. Peptide analogues were generated using synthetic [127I]-Tyr amino acid building blocks (CPC Scientific).
Cecil, D.L., Curtis, B., Gad, E., Gormley, M., Timms, A.E., Corulli, L., Bos, R., Damle, R.N., Sepulveda, M.A. and Disis, M.L. Scientific Reports 12, no. 1 (2022): 13618.
- Cancer Vaccine Institute, University of Washington, 850 Republican Street, Brotman Bld., 2nd Floor, Box 358050, Seattle, WA 98195-8050, USA.
- Janssen Research and Development LLC, Spring House, PA, USA.
- Janssen Vaccines and Prevention, Leiden, The Netherlands.
The peptides were constructed and purified by high-performance liquid chromatography (> 90% pure; CPC Scientific).
Zonari, A., Brace, L.E., Alencar-Silva, T., Porto, W.F., Foyt, D., Guiang, M., Cruz, E.A.O., Franco, O.L., Oliveira, C.R., Boroni, M. and Carvalho, J.L. Toxicology Reports 9 (2022): 1632-1638.
Peptide 14 (ETAKHWLKGI) (Sup. Fig. 1) was 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.
CPC Scientific Inc., a leading global peptide CRDMO (Contract Research, Development, and Manufacturing Organization) has invested in a new peptide API (Active Pharmaceutical Ingredient) manufacturing site, bringing many new jobs to Rocklin, California. The 41,000 sq ft facility located at 3880 Atherton Rd, Rocklin, CA 95765 will be utilized to manufacture clinical to commercial grade peptide products for increased manufacturing capacity and will diversify CPC Scientific’s supply chain.
CPC Scientific is entering an exciting period of growth and innovation for peptide and oligonucleotide therapeutic development and manufacturing, and we will continue to provide therapeutic APIs to pharmaceutical and biotech companies around the world. We are very pleased to partner with the City of Rocklin, California to bring manufacturing and Life-Science jobs to local American workers,” said Shawn Lee, PhD, CEO.
Ikeda, Z., Kakegawa, K., Kikuchi, F., Itono, S., Oki, H., Yashiro, H., Hiyoshi, H., Tsuchimori, K., Hamagami, K., Watanabe, M. and Sasaki, M. Journal of Medicinal Chemistry 65, no. 12 (2022): 8456-8477.
- Research, Takeda Pharmaceutical Company Limited, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
Subsequently, 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 2.1 μM substrate solution.
The transferred energy from a fluorescent donor is converted into molecular vibrations if the acceptor is a non-fluorescent dye (quencher). When the FRET is terminated (by separating donor and acceptor), an increase of donor fluorescence can be detected. The design and synthesis work at CPC for FRET and TR-FRET peptide substrates include modification of sequences, selection of donor/quencher pairs, improvement of FRET substrate solubility and quenching efficiency.