Site‐Selective Cysteine‐Cyclooctyne Conjugation

Bradley Pentelute  chi zhang  peng dai  Alexander Vinogradov  zak gates

Angew. Chem. Int. Ed. 2018

doi: 10.1002/anie.201800860

We report a site‐selective cysteine‐cyclooctyne conjugation reaction between a seven‐residue peptide tag (DBCO‐tag, Leu‐Cys‐Tyr‐Pro‐Trp‐Val‐Tyr), at the N or C‐terminus of a peptide or protein, and various aza‐dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO‐tag increases the rate of the thiol‐yne reaction by 220‐fold, enabling selective conjugation of DBCO‐tag to DBCO‐linked fluorescent probes, affinity tags, and cytotoxic drug molecules. Fusion of DBCO‐tag with the protein of interest enables regioselective cysteine modification on proteins that contain multiple endogenous cysteines; these examples include green fluorescent protein and trastuzumab antibody. This study demonstrates short peptide tags could aid in accelerating bond forming reactions that are often slow to non‐existent in water.

Probing the Mechanism of Thermally Driven thiol-Michael Dynamic Covalent Chemistry

Borui Zhang,  Progyateg Chakma,  Max Shulman,  Jun Ke,  Zachary Digby  and  Dominik Konkolewicz

Org. Biomol. Chem., 2018
doi: 10.1039/C8OB00397A

The kinetics and mechanism of the thermally activated dynamic covalent exchange of thiol-Michael adducts is investigated. A model system of thiol-Michael adducts between thiophenol and phenylvinylketone derivatives and adducts between 2-mercaptoethanol phenylvinylketone derivatives in N,N-dimethylformamide (DMF) at elevated temperatures is used to probe the underlying exchange mechanism. The kinetic data show negligible free Michael acceptor which is consistent with the highly efficient thiol-Michael reaction being a “click”-like reaction that significantly favors the adduct form. At elevated temperatures of 90 °C in DMF the thiol-Michael adducts reach equilibrium after 24 h, although equilibration did not occur within 24 h at 60 °C or 75 °C, and negligible exchange occurs under ambient conditions. A kinetic model was developed to describe the dynamic covalent exchange and equilibration. The experimental and simulation kinetic data of dynamic covalent exchange are consistent with the thiol-Michael adducts undergoing a retro-Michael reaction, followed by subsequent addition of a free thiol to the liberated Michael acceptor. Kinetic analysis is consistent with the fragmentation, or retro-Michael reaction, being the rate-determining step in the dynamic covalent exchange. This suggests that the key step in dynamic covalent exchange is not enhanced by addition of free thiol or free Michael acceptor, since the addition reaction is much faster than the retro-Michael reaction. This fundamental study will guide the design of organic compounds, materials, and bioconjugates that utilize the thermally activated dynamic covalent thiol-Michael bonds.

YL143, a novel mutant selective irreversible EGFR inhibitor, overcomes EGFRL858R, T790M‐mutant resistance in vitro and in vivo

Zhang Zhang, Jian Zou, Lei Yu  Jinfeng Luo, Yan Li  Zhengchao Tu, Xiaomei Ren  Hongcheng Wei, Liyan Song, Xiaoyun Lu,  Ke Ding

Cancer Medicine, 2018
doi: 10.1002/cam4.1392

YL143 was identified as a novel wild‐type sparing EGFRT790M inhibitor with good pharmacokinetic properties. It potently suppresses EGFRL858R/T790M with an 50% inhibitory concentration (IC50) value of 2.0 ± 0.3 nmol/L, but is approximately 92‐folds less potent against EGFRWT kinase. YL143 suppresses cellular proliferation and induces G0/G1 phase arrest and apoptosis in H1975 cells with EGFRL858R/T790M mutation at 30 nmol/L. It also exhibits acceptable pharmacokinetics (PK) parameters with an oral bioavailability value of 25.0% after oral administration in rats and exhibits promising antitumor efficacy in a gefitinib‐resistant human H1975 xenografted model after oral administration of 30 mg/kg/day. These data supported that YL143 could be a promising lead compound for overcoming clinical EGFRT790M resistance of patients with non‐small‐cell lung cancer (NSCLC).

Discovery of a 29-Amino-Acid Reactive Abiotic Peptide for Selective Cysteine Arylation

Ethan D. Evans  and Bradley L. Pentelute

ACS Chem. Biol., 2018, 13 (3), pp 527–532

DOI: 10.1021/acschembio.7b00520

The regio- and chemoselective modification of proteins or peptides with chemical reagents is often challenging. One approach to overcome this problem involves identifying abiotic polypeptide sequences that react with specific small molecules. Toward this goal, we profiled ∼5 × 1013 randomized 30-mer peptides using mRNA display and high-throughput sequencing in search of polypeptides that can undergo cysteine arylation with a water-soluble perfluoroarene. Within this vast chemical space, we discovered a cysteine-containing sequence with a second-order rate constant of 0.29 M–1 s–1 for arylation. An N- and C-terminal truncation reduced the reaction rate, as did the addition of denaturants. When the reactive peptide was covalently fused to the enzyme Sortase A, we observed regiospecific arylation at a single cysteine site, leaving the enzyme’s active site cysteine unchanged. Taken together, these results demonstrate that long polypeptides of defined sequence, when matched with the appropriate reactive group, can be used for selective arylation of cysteine in water.

BTKCys481Ser drives ibrutinib resistance via ERK1/2, and protects BTKWild-Type MYD88 mutated cells by a paracrine mechanism

Jiaji G. Chen, Xia Liu, Manit Munshi, Lian Xu, Nicholas Tsakmaklis, Maria G. Demos, Amanda Kofides, Maria Luisa Guerrera, Gloria G. Chan, Christopher J. Patterson, Kirsten E. Meid, Joshua Gustine, Toni Dubeau, Patricia Severns, Jorge J. Castillo, Zachary R. Hunter, Jinhua Wang, Sara J. Buhrlage, Nathanael S. Gray, Steven P. Treon and Guang Yang

Blood 2018 doi: https://doi.org/10.1182/blood-2017-10-811752

Acquired ibrutinib resistance due to BTKCys481 mutations occurs in B-cell malignancies, including those with MYD88 mutations. BTKCys481 mutations are usually sub-clonal, and their relevance to clinical progression remains unclear. Moreover, the signaling pathways that promote ibrutinib resistance remain to be clarified. We therefore engineered BTKCys481Ser and BTKWT expressing MYD88 mutated WM and ABC DLBCL cells, and observed re-activation of BTK–PLCγ2–ERK1/2 signaling in the presence of ibrutinib in only the former. Use of ERK1/2 inhibitors triggered apoptosis in BTKCys481Ser expressing cells, and showed synergistic cytotoxicity with ibrutinib. ERK1/2 re-activation in ibrutinib treated BTKCys481Ser cells was accompanied by release of many pro-survival and inflammatory cytokines, including IL-6 and IL-10 that were also blocked by ERK1/2 inhibition. To clarify if cytokine release by ibrutinib treated BTKCys481Ser cells could protect BTKWT MYD88 mutated malignant cells, we used a TranswellTM co-culture system, and showed that non-transduced BTKWT MYD88 mutated WM or ABC DLBCL cells were rescued from ibrutinib induced killing when co-cultured with BTKCys481Ser but not their BTKWT expressing counterparts. Use of IL-6 and/or IL-10 blocking antibodies abolished the protective effect conferred on non-transduced BTKWT by co-culture with BTKCys481Ser expressing WM or ABC DLBCL cell counterparts. Rebound of IL-6 and IL-10 serum levels also accompanied disease progression in WM patients with acquired BTKCys481 mutations. Our findings show that the BTKCys481Ser mutation drives ibrutinib resistance in MYD88 mutated WM and ABC DLBCL cells through re-activation of ERK1/2 activation, and can confer a protective effect on BTKWT cells through a paracrine mechanism.

High-Throughput Kinetic Analysis for Target-Directed Covalent Ligand Discovery

Gregory Craven, Dominic Affron, Charlotte Allen, Stefan Matthies, Joe Greener,
Rhodri Morgan, Edward Tate, Alan Armstrong, David J. Mann

Angew. Chem. Int. Ed., 2018
doi: 10.1002/anie.201711825

Cysteine-reactive small molecules are used as chemical probes of biological systems and as medicines. Identifying high-quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan-reactive compounds. Here we describe quantitative irreversible tethering (qIT), a general method for screening cysteine-reactive small molecules based upon the maximization of kinetic selectivity. We apply this method prospectively to discover covalent fragments that target the clinically important cell cycle regulator Cdk2. Crystal structures of the inhibitor complexes validate the approach and guide further optimization. The power of this technique is highlighted by the identification of a Cdk2-selective allosteric (type IV) kinase inhibitor whose novel mode-of-action could be exploited therapeutically.