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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Diao, J., Komura, R., Sano, T., Pantua, H., Storek, K.M., Inaba, H., Ogawa, H., Noland, C.L., Peng, Y., Gloor, S.L. and Yan, D. bioRxiv (2020): 2020-10.
- Departments of Infectious Diseases, Structural Biology, Biochemical and Cellular Pharmacology, Translational Immunology, Pathology, and Early Discovery Biochemistry, Genentech, South San Francisco, CA 94080 USA
The standard assay consists of 6 μL reaction mixture with 3 nM Lgt-DDM, 50 μM phosphatidylglycerol (1,2-dipalmitoyl-sn-glycero-3-phospho-(1’-rac-glycerol), Avanti), 12.5 μM Pal-IAAC peptide substrate derived from the Pal lipoprotein (MQLNKVLKGLMIALPVMAIAACSSNKN, synthesized by CPC Scientific)
Pantua, H., Skippington, E., Braun, M.G., Noland, C.L., Diao, J., Peng, Y., Gloor, S.L., Yan, D., Kang, J., Katakam, A.K. and Reeder, J. MBio 11, no. 5 (2020): 10-1128.
- Departments of Infectious Diseases, OMNI Bioinformatics, Chemistry, Structural Biology, Biochemical and Cellular Pharmacology, Translational Immunology, Pathology, Molecular Biology, Genentech, South San Francisco, California, USA
MQLNKV-L(U13C6,15N)-KGL(U13C6,15N)MIALPVMAIAA-dipalmitoyl2C-SSNKNGG-K-biotin, which upon cleavage by LspA, yields the product peptide dipalmitoyl2C-SSNKNGG-K-biotin. A product standard [dipalmitoyl2C-SSNKNAAK-(NHCH2CH2NH)-biotin; CPC Scientific] was in the reaction mixture as an internal standard for normalization of product quantitation.
Protein-protein and protein-peptide interactions play critical roles in all types of cellular processing. Peptides are natural partners to proteins and, as ligands, bind to proteins with high affinity due to their capacity to adapt to the often flexible protein surface. Despite this, peptides have drawbacks as drug candidates that include low plasma bioavailability, instability from proteolytic enzymes, and poor passive membrane permeability. Some success has been achieved with linear peptides, particularly peptides that maintain α-helical secondary structures. These motifs can be introduced to stabilized α-helical motifs by common “peptide-stapling” approaches, but stapled peptides can suffer from low bioactivity and poor solubility. Another strategy to maintain peptide secondary structure is modification by macrocyclization.
SAN JOSE, CA., Sept 3, 2020 /CPCNewswire/ — CPC Scientific Inc. is pleased to announce that the European Commission (EC) has granted the Conditional Marketing Authorization (CMA) for MYR Pharmaceuticals lead compound HEPCLUDEX®. CPC Scientific serves as a supplier and partner to MYR Pharmaceuticals for the development and manufacturing of bulevirtide (Hepcludex). This drug is […]
Curreli, Francesca, Sofia MB Victor, Shahad Ahmed, Aleksandra Drelich, Xiaohe Tong, Chien-Te K. Tseng, Christopher D. Hillyer, and Asim K. Debnath. Mbio 11, no. 6 (2020): e02451-20.
We have synthesized (CPC Scientific, Inc.) four stapled peptides, as depicted in Figure 2. We also synthesized the linear peptide, NYBSP-C, as a control. Besides, we purchased a linear peptide, SBP1, to use as a control, which was reported recently to bind to SARS-CoV-2 RBD with high affinity (KD = 47nM).
Abdulganiyyu, I. A.; Kaczmarek, K.; Zabrocki, J.; Nachman, R. J.; Marchal, E.; Schellens, S.; Verlinden, H.; Broeck, J. V.; Marco, H.; Jackson, G. E. Insect Biochemistry and Molecular Biology 2020, 103362.
A head-to-tail cyclic, octapeptide analog of Locmi-AKH-I, cycloAKH (cyclo[LNFTPNWG]) was synthesized to severely restrict the conformational freedom of the AKH structure.
Clairfeuille, T., Buchholz, K.R., Li, Q., Verschueren, E., Liu, P., Sangaraju, D., Park, S., Noland, C.L., Storek, K.M., Nickerson, N.N. and Martin, L. Nature 584, no. 7821 (2020): 479-483.
- Departments of Structural Biology, Infectious Diseases, Microchemistry, Proteomics & Lipidomics, Drug Metabolism & Pharmacokinetics, Translational Immunology, BioMolecular Resources, Biochemical & Cellular Pharmacology, Bioinformatics & Computational Biology, Discovery Chemistry Departments, Genentech Inc., South San Francisco, CA, USA.
LAB peptides ([..], CPC Scientific, [..] l, standard solid-phase peptide synthesis) at 10 mM in 50 mM Tris, pH 8, and 100 mM NaCl were diluted in MHB II cation adjusted broth (800 μM top concentration) or LB. Where indicated, EDTA was added to a final concentration of 0.5 mM.
Chan, Leslie W., Melodi N. Anahtar, Ta-Hsuan Ong, Kelsey E. Hern, Roderick R. Kunz, and Sangeeta N. Bhatia. Nature Nanotechnology (2020): 1-9.
HFA-modifed peptides were synthesized by CPC Scientifc (>95% purity). Briefy, the peptide substrate, Ac-CKKK(Cy5)-PEG4-Nle(O-Bzl)-Met(O)2-Oic-Abu-OH, was synthesized on Fmoc-Abu-CTC resin via standard Fmoc solid phase peptide synthesis.