Tracking cancer drugs in living cells by thermal profiling of the proteome

Science ◽  
2014 ◽  
Vol 346 (6205) ◽  
pp. 1255784 ◽  
Author(s):  
Mikhail M. Savitski ◽  
Friedrich B. M. Reinhard ◽  
Holger Franken ◽  
Thilo Werner ◽  
Maria Fälth Savitski ◽  
...  
Keyword(s):  
Drug Target ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Drug Efficacy ◽  
Living Cells ◽  
Live Cells ◽  
Downstream Effectors ◽  
Small Molecule Ligands ◽  
Thermal Profiling ◽  
Thermal Stability Of

The thermal stability of proteins can be used to assess ligand binding in living cells. We have generalized this concept by determining the thermal profiles of more than 7000 proteins in human cells by means of mass spectrometry. Monitoring the effects of small-molecule ligands on the profiles delineated more than 50 targets for the kinase inhibitor staurosporine. We identified the heme biosynthesis enzyme ferrochelatase as a target of kinase inhibitors and suggest that its inhibition causes the phototoxicity observed with vemurafenib and alectinib. Thermal shifts were also observed for downstream effectors of drug treatment. In live cells, dasatinib induced shifts in BCR-ABL pathway proteins, including CRK/CRKL. Thermal proteome profiling provides an unbiased measure of drug-target engagement and facilitates identification of markers for drug efficacy and toxicity.

Rapid discovery of drug target engagement by isothermal shift assay

2019 ◽  
Author(s):  
Kristofor J. Webb ◽  
Kerri A. Ball ◽  
Stephen J. Coleman ◽  
Jeremy Jacobsen ◽  
Michael H.B. Stowell ◽  
...  
Keyword(s):  
Drug Target ◽  
Drug Targets ◽  
Kinase Inhibitors ◽  
Rapid Identification ◽  
Living Cells ◽  
Experimental Designs ◽  
Living Systems ◽  
Target Engagement ◽  
Protein Targets ◽  
Drug Molecules

Identifying protein targets directly bound by drug molecules within living systems remains challenging. Here we present the isothermal shift assay, iTSA, for rapid identification of drug targets. Compared with thermal proteome profiling, a prevailing method for target engagement, iTSA offers a simplified workflow, 4-fold higher throughput, and multiplexed experimental designs with higher replication. We demonstrate application of iTSA to identify targets for several kinase inhibitors in lysates and living cells.

scholarly journals Targeting HER3, a Catalytically Defective Receptor Tyrosine Kinase, Prevents Resistance of Lung Cancer to a Third-Generation EGFR Kinase Inhibitor

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2394
Author(s):  
Donatella Romaniello ◽  
Ilaria Marrocco ◽  
Nishanth Belugali Nataraj ◽  
Irene Ferrer ◽  
Diana Drago-Garcia ◽  
...  
Keyword(s):  
Drug Target ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Treatment Options ◽  
Acquired Resistance ◽  
Underlying Mechanism ◽  
Xenograft Models ◽  
Emergence Of Resistance ◽  
Egfr Kinase ◽  
Egfr Antibody

Although two growth factor receptors, EGFR and HER2, are amongst the best targets for cancer treatment, no agents targeting HER3, their kinase-defective family member, have so far been approved. Because emergence of resistance of lung tumors to EGFR kinase inhibitors (EGFRi) associates with compensatory up-regulation of HER3 and several secreted forms, we anticipated that blocking HER3 would prevent resistance. As demonstrated herein, a neutralizing anti-HER3 antibody we generated can clear HER3 from the cell surface, as well as reduce HER3 cleavage by ADAM10, a surface metalloproteinase. When combined with a kinase inhibitor and an anti-EGFR antibody, the antibody completely blocked patient-derived xenograft models that acquired resistance to EGFRi. We found that the underlying mechanism involves posttranslational downregulation of HER3, suppression of MET and AXL upregulation, as well as concomitant inhibition of AKT signaling and upregulation of BIM, which mediates apoptosis. Thus, although HER3 is nearly devoid of kinase activity, it can still serve as an effective drug target in the context of acquired resistance. Because this study simulated in animals the situation of patients who develop resistance to EGFRi and remain with no obvious treatment options, the observations presented herein may warrant clinical testing.

scholarly journals Structure-Unbinding Kinetics Relationship of p38α MAPK Inhibitors

2019 ◽  
Author(s):  
Xiaoxia Ge ◽  
Hepan Tan ◽  
Lei Xie
Keyword(s):  
Residence Time ◽  
Drug Target ◽  
Kinase Inhibitors ◽  
Drug Efficacy ◽  
Distinct Type ◽  
Rational Drug Design ◽  
Average Lifetime ◽  
Kinetics Of

AbstractRational Drug Design still faces a major hurdle for the prediction of drug efficacyin vivosolely based on its binding affinity for the targetin vitro. The traditional perspective has proven to be inadequate as it lacks the consideration of essential aspects such as pharmacokinetics and binding kinetics in determining drug efficacy and toxicity. Residence time, the average lifetime of drug-target complex, has gained broader recognition as a better predictor for lead optimization. Long residence time could contribute to sustained pharmacological effect and may mitigate off-target toxicity as well. To unravel the underlining mechanism for variation of residence time and determine the ligand features governing the unbinding kinetics, unbinding kinetics of two distinct type II inhibitors of p38α MAP kinase were investigated and compared by molecular dynamics and metadynamics simulation approaches. Free energy landscape of key motions associated with unbinding was constructed for both inhibitors. Multiple unbinding pathways and rebinding were revealed during the drug-target dissociation process of faster unbinder Lig3 and slower unbinder Lig8 respectively, suggesting a novel mechanism of unbinding kinetics. This comparative study implies that hydrophobic and hydrogen-bonding interactions in the R1 group of ligands are crucial for slow unbinding. Such kind of structure-kinetics relationship approaches could also be applied to predict unbinding pathways and kinetics of many other small molecules, and facilitate the design of efficient kinase inhibitors.

scholarly journals Chaperones as thermodynamic sensors of drug-target interactions reveal kinase inhibitor specificities in living cells

2013 ◽  
Vol 31 (7) ◽  
pp. 630-637 ◽  
Author(s):  
Mikko Taipale ◽  
Irina Krykbaeva ◽  
Luke Whitesell ◽  
Sandro Santagata ◽  
Jianming Zhang ◽  
...  
Keyword(s):  
Drug Target ◽  
Kinase Inhibitor ◽  
Living Cells

scholarly journals Discovery of catalytic-site-fluorescent probes for tracing phosphodiesterase 5 in living cells

RSC Advances ◽  
2021 ◽  
Vol 11 (51) ◽  
pp. 31967-31971
Author(s):  
Meiying Qiu ◽  
Deyan Wu ◽  
Yi-You Huang ◽  
Yue Huang ◽  
Qian Zhou ◽  
...  
Keyword(s):  
Drug Screening ◽  
Fluorescent Probes ◽  
Drug Target ◽  
Living Cells ◽  
Catalytic Site ◽  
Live Cells ◽  
Tissue Slices ◽  
Phosphodiesterase 5

To enhance the understanding of PDE5 as the drug target. Herein, we designed catalytic-site-fluorescent probes that can be applied to PDE5 visualization in live cells and tissue slices, implying the potential in diagnosis and drug screening.

p19INK4d: More than Just a Cyclin-Dependent Kinase Inhibitor

2019 ◽  
Vol 21 (1) ◽  
pp. 96-102
Author(s):  
Xu Han ◽  
Yijin Kuang ◽  
Huiyong Chen ◽  
Ting Liu ◽  
Ji Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Drug Target ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Cell Cycle Regulators ◽  
Cyclin Dependent Kinase ◽  
Disease Treatment ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Damage Repair

Cyclin-dependent kinase inhibitors (CDKIs) are important cell cycle regulators. The CDKI family is composed of the INK4 family and the CIP/KIP family. p19INK4d belongs to the INK4 gene family and is involved in a series of normal physiological activities and the pathogenesis of diseases. Many factors play regulatory roles in the p19INK4d gene expression at the transcriptional and posttranscriptional levels. p19INK4d not only regulates the cell cycle but also plays regulatory roles in apoptosis, DNA damage repair, cell differentiation of hematopoietic cells, and cellular senescence. In this review, the regulatory network of the p19INK4d gene expression and its biological functions are summarized, which provides a basis for further study of p19INK4d as a drug target for disease treatment.

Abstract C085: A new method to determine drug-target residence time of kinase inhibitors in living cells

2019 ◽  
Author(s):  
Takeomi Inoue ◽  
Aki Emi ◽  
James D Vasta ◽  
Matthew B Robers ◽  
Yusuke Kawase
Keyword(s):  
Residence Time ◽  
Drug Target ◽  
Kinase Inhibitors ◽  
Living Cells ◽  
New Method

4-dimensional, high-resolution imaging of living cells

1992 ◽  
Vol 50 (1) ◽  
pp. 416-417
Author(s):  
Shinya Inoué
Keyword(s):  
Light Microscope ◽  
Living Cells ◽  
Live Cells ◽  
Video Tape ◽  
Active Living ◽  
High Resolution Imaging ◽  
3 Dimensional ◽  
Dynamic Architecture ◽  
Resolution Imaging ◽  
Disk Memory

This paper reports progress of our effort to rapidly capture, and display in time-lapsed mode, the 3-dimensional dynamic architecture of active living cells and developing embryos at the highest resolution of the light microscope. Our approach entails: (A) real-time video tape recording of through-focal, ultrathin optical sections of live cells at the highest resolution of the light microscope; (B) repeat of A at time-lapsed intervals; (C) once each time-lapsed interval, an image at home focus is recorded onto Optical Disk Memory Recorder (OMDR); (D) periods of interest are selected using the OMDR and video tape records; (E) selected stacks of optical sections are converted into plane projections representing different view angles (±4 degrees for stereo view, additional angles when revolving stereos are desired); (F) analysis using A - D.

Sign in / Sign up

Export Citation Format

Share Document