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1.
Annu Rev Pharmacol Toxicol ; 62: 465-482, 2022 01 06.
Article in English | MEDLINE | ID: mdl-34499524

ABSTRACT

Drug target deconvolution can accelerate the drug discovery process by identifying a drug's targets (facilitating medicinal chemistry efforts) and off-targets (anticipating toxicity effects or adverse drug reactions). Multiple mass spectrometry-based approaches have been developed for this purpose, but thermal proteome profiling (TPP) remains to date the only one that does not require compound modification and can be used to identify intracellular targets in living cells. TPP is based on the principle that the thermal stability of a protein can be affected by its interactions. Recent developments of this approach have expanded its applications beyond drugs and cell cultures to studying protein-drug interactions and biological phenomena in tissues. These developments open up the possibility of studying drug treatment or mechanisms of disease in a holistic fashion, which can result in the design of better drugs and lead to a better understanding of fundamental biology.


Subject(s)
Drug Discovery , Proteome , Humans , Molecular Targeted Therapy , Proteome/analysis , Proteome/antagonists & inhibitors , Proteome/metabolism
2.
Nat Biotechnol ; 38(3): 303-308, 2020 03.
Article in English | MEDLINE | ID: mdl-31959954

ABSTRACT

Monitoring drug-target interactions with methods such as the cellular thermal-shift assay (CETSA) is well established for simple cell systems but remains challenging in vivo. Here we introduce tissue thermal proteome profiling (tissue-TPP), which measures binding of small-molecule drugs to proteins in tissue samples from drug-treated animals by detecting changes in protein thermal stability using quantitative mass spectrometry. We report organ-specific, proteome-wide thermal stability maps and derive target profiles of the non-covalent histone deacetylase inhibitor panobinostat in rat liver, lung, kidney and spleen and of the B-Raf inhibitor vemurafenib in mouse testis. In addition, we devised blood-CETSA and blood-TPP and applied it to measure target and off-target engagement of panobinostat and the BET family inhibitor JQ1 directly in whole blood. Blood-TPP analysis of panobinostat confirmed its binding to known targets and also revealed thermal stabilization of the zinc-finger transcription factor ZNF512. These methods will help to elucidate the mechanisms of drug action in vivo.


Subject(s)
Blood/metabolism , Proteome/chemistry , Proteome/metabolism , Small Molecule Libraries/administration & dosage , Animals , Azepines/administration & dosage , Azepines/pharmacology , Hep G2 Cells , Humans , Kidney/chemistry , Kidney/metabolism , Liver/chemistry , Liver/metabolism , Lung/chemistry , Lung/metabolism , Male , Mass Spectrometry , Mice , Organ Specificity , Panobinostat/administration & dosage , Panobinostat/pharmacology , Protein Stability , Rats , Small Molecule Libraries/pharmacology , Spleen/chemistry , Spleen/metabolism , Testis/chemistry , Testis/metabolism , Thermodynamics , Triazoles/administration & dosage , Triazoles/pharmacology , Vemurafenib/administration & dosage , Vemurafenib/pharmacology
3.
ACS Med Chem Lett ; 10(5): 780-785, 2019 May 09.
Article in English | MEDLINE | ID: mdl-31097999

ABSTRACT

The serine/threonine protein kinase TBK1 (Tank-binding Kinase-1) is a noncanonical member of the IkB kinase (IKK) family. This kinase regulates signaling pathways in innate immunity, oncogenesis, energy homeostasis, autophagy, and neuroinflammation. Herein, we report the discovery and characterization of a novel potent and highly selective TBK1 inhibitor, GSK8612. In cellular assays, this small molecule inhibited toll-like receptor (TLR)3-induced interferon regulatory factor (IRF)3 phosphorylation in Ramos cells and type I interferon (IFN) secretion in primary human mononuclear cells. In THP1 cells, GSK8612 was able to inhibit secretion of interferon beta (IFNß) in response to dsDNA and cGAMP, the natural ligand for STING. GSK8612 is a TBK1 small molecule inhibitor displaying an excellent selectivity profile and therefore represents an ideal probe to further dissect the biology of TBK1 in models of immunity, neuroinflammation, obesity, or cancer.

4.
ACS Chem Biol ; 11(5): 1245-54, 2016 05 20.
Article in English | MEDLINE | ID: mdl-26863403

ABSTRACT

Many protein kinases are valid drug targets in oncology because they are key components of signal transduction pathways. The number of clinical kinase inhibitors is on the rise, but these molecules often exhibit polypharmacology, potentially eliciting desired and toxic effects. Therefore, a comprehensive assessment of a compound's target space is desirable for a better understanding of its biological effects. The enzyme ferrochelatase (FECH) catalyzes the conversion of protoporphyrin IX into heme and was recently found to be an off-target of the BRAF inhibitor Vemurafenib, likely explaining the phototoxicity associated with this drug in melanoma patients. This raises the question of whether FECH binding is a more general feature of kinase inhibitors. To address this, we applied a chemical proteomics approach using kinobeads to evaluate 226 clinical kinase inhibitors for their ability to bind FECH. Surprisingly, low or submicromolar FECH binding was detected for 29 of all compounds tested and isothermal dose response measurements confirmed target engagement in cells. We also show that Vemurafenib, Linsitinib, Neratinib, and MK-2461 reduce heme levels in K562 cells, verifying that drug binding leads to a loss of FECH activity. Further biochemical and docking experiments identified the protoporphyrin pocket in FECH as one major drug binding site. Since the genetic loss of FECH activity leads to photosensitivity in humans, our data strongly suggest that FECH inhibition by kinase inhibitors is the molecular mechanism triggering photosensitivity in patients. We therefore suggest that a FECH assay should generally be part of the preclinical molecular toxicology package for the development of kinase inhibitors.


Subject(s)
Ferrochelatase/antagonists & inhibitors , Ferrochelatase/metabolism , Protein Kinase Inhibitors/pharmacology , Benzocycloheptenes/pharmacology , Cell Line, Tumor , Ferrochelatase/chemistry , Heme/metabolism , Humans , Imidazoles/pharmacology , Indoles/pharmacology , Molecular Docking Simulation , Protein Binding , Proteomics , Pyrazines/pharmacology , Pyridines/pharmacology , Quinolines/pharmacology , Sulfonamides/pharmacology , Vemurafenib
5.
Nat Chem Biol ; 8(6): 576-82, 2012 Apr 29.
Article in English | MEDLINE | ID: mdl-22544264

ABSTRACT

We devised a high-throughput chemoproteomics method that enabled multiplexed screening of 16,000 compounds against native protein and lipid kinases in cell extracts. Optimization of one chemical series resulted in CZC24832, which is to our knowledge the first selective inhibitor of phosphoinositide 3-kinase γ (PI3Kγ) with efficacy in in vitro and in vivo models of inflammation. Extensive target- and cell-based profiling of CZC24832 revealed regulation of interleukin-17-producing T helper cell (T(H)17) differentiation by PI3Kγ, thus reinforcing selective inhibition of PI3Kγ as a potential treatment for inflammatory and autoimmune diseases.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cell Differentiation/drug effects , Enzyme Inhibitors/pharmacology , Interleukin-17/immunology , Phosphoinositide-3 Kinase Inhibitors , Small Molecule Libraries/pharmacology , T-Lymphocytes, Helper-Inducer/drug effects , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Arthritis, Experimental/drug therapy , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Binding, Competitive , Cell Line , Cell Movement/drug effects , Class Ib Phosphatidylinositol 3-Kinase , Drug Discovery , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/therapeutic use , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Molecular Structure , Rats , Rats, Wistar , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacokinetics , Small Molecule Libraries/therapeutic use , Structure-Activity Relationship , T-Lymphocytes, Helper-Inducer/cytology , T-Lymphocytes, Helper-Inducer/enzymology , T-Lymphocytes, Helper-Inducer/immunology
6.
ACS Chem Biol ; 6(10): 1021-8, 2011 Oct 21.
Article in English | MEDLINE | ID: mdl-21812418

ABSTRACT

Leucine-rich repeat kinase-2 (LRRK2) mutations are the most important cause of familial Parkinson's disease, and non-selective inhibitors are protective in rodent disease models. Because of their poor potency and selectivity, the neuroprotective mechanism of these tool compounds has remained elusive so far, and it is still unknown whether selective LRRK2 inhibition can attenuate mutant LRRK2-dependent toxicity in human neurons. Here, we employ a chemoproteomics strategy to identify potent, selective, and metabolically stable LRRK2 inhibitors. We demonstrate that CZC-25146 prevents mutant LRRK2-induced injury of cultured rodent and human neurons with mid-nanomolar potency. These precise chemical probes further validate this emerging therapeutic strategy. They will enable more detailed studies of LRRK2-dependent signaling and pathogenesis and accelerate drug discovery.


Subject(s)
Drug Design , Neurons/drug effects , Parkinson Disease/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proteomics/methods , Animals , Cells, Cultured , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mutation , Neurons/metabolism , Neurons/pathology , Parkinson Disease/genetics , Parkinson Disease/pathology , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/genetics , Rats
7.
Nat Biotechnol ; 25(9): 1035-44, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17721511

ABSTRACT

We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.


Subject(s)
Protein Kinase Inhibitors/pharmacology , Proteomics/methods , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Benzamides , Cell Extracts , Chromatography, Affinity , Discoidin Domain Receptor 1 , Enzymes, Immobilized/antagonists & inhibitors , HeLa Cells , Humans , Imatinib Mesylate , Inhibitory Concentration 50 , K562 Cells , Pharmaceutical Preparations , Phosphorylation/drug effects , Piperazines/pharmacology , Pyrimidines/pharmacology , Quinone Reductases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Signal Transduction/drug effects
8.
J Ocul Pharmacol Ther ; 23(1): 54-6, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17341151

ABSTRACT

OBJECTIVE: The aim of this study was to report on a new technique for examining aqueous humor. PARTICIPANTS: Aqueous humor samples obtained from 198 patients (cataract: n=66 eyes; age-related macular degeneration [AMD]: n=66; diabetic macular edema: n=66) were in vitro examined by mid infrared spectroscopy. After training and validating the technique, 66 samples were taken for the final independent prediction. RESULTS: In computerized analysis of the spectrometric data, 22 (100%) cataract eyes were diagnosed as the cataract group, 17 (77%) diabetic macular edema eyes were diagnosed as the diabetic macular edema group, and 14 (64%) eyes with AMD were diagnosed as the AMD group. Mid infrared spectrometry could statistically significantly (P=0.004) differentiate between the 3 study groups. CONCLUSIONS: Mid infrared spectroscopic examination of aqueous humor may be able to differentiate eyes with cataract, AMD, or diabetic macular edema.


Subject(s)
Aqueous Humor/chemistry , Cataract/diagnosis , Diabetic Retinopathy/diagnosis , Macular Degeneration/diagnosis , Macular Edema/diagnosis , Spectrophotometry, Infrared , Humans , Pilot Projects
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