Thilo Werner

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

INTRODUCTION Understanding drug mechanism poses the daunting challenge of determining the affinity of the drug for all potential targets. Drug target engagement can be assessed by means of a cellular thermal shift assay (CETSA) based on ligand-induced changes in protein thermal stability. We combined the CETSA method with quantitative mass spectrometry to study the effect of drugs on the thermal profile of a cellular proteome comprising more than 7000 proteins. The approach enabled the monitoring of drug targets and downstream effectors. Tracking drugs in living cells. Drugs alter the thermal stability of proteins directly through compound binding or indirectly through changes in overall protein state. Thermal proteome profiling determines melting curves for thousands of proteins and tracks drug action in cells. RATIONALE We devised a method for the thermal profiling of cellular proteomes. Cells were cultured with or without drugs and heated to different temperatures so as to induce protein denaturation, and remaining soluble proteins were extracted with buffer. At each temperature, soluble proteins were quantified by means of high-resolution mass spectrometry, yielding denaturation curves. This allowed determination of thermal stability and the identification of ligand-induced shifts. To rank binding affinities among multiple targets, we determined stability profiles across a range of compound concentrations at a defined temperature. Comparison of the thermal profiles obtained after drug treatment of intact cells versus cell extract allowed us to distinguish effects induced by ligand binding from those induced by downstream modifications. RESULTS We performed thermal proteome profiling (TPP) on human K562 cells by heating intact cells or cell extracts and observed marked differences in melting properties between the two settings, with a trend toward increased protein stability in cell extract. Adenosine triphosphatase (ATP)–binding proteins showed a significant trend toward increased stability in intact cells, suggesting stabilization by the endogenous ligand. This was confirmed with the addition of ATP to cell extract, which resulted in increased stability for this protein group. The ability of TPP to identify target binding was validated by using the broad-specificity inhibitors staurosporine and GSK3182571, which induced shifts in the melting temperatures of many kinase targets and also affected the thermal profiles of other proteins, including regulatory subunits of kinase complexes. We identified the heme biosynthesis enzyme ferrochelatase (FECH) as an off-target of several kinase inhibitors and showed that the drug vemurafenib reaches full target occupancy of its cognate target BRAF and the off-target FECH within a narrow concentration window. FECH deficiency is genetically linked to protoporphyria, suggesting that the photosensitivity induced by vemurafenib and other drugs is mediated by FECH. Drug treatment of live cells affected not only direct target proteins but also downstream effectors. The ABL inhibitor dasatinib induced thermal shifts in several proteins downstream of BCR-ABL, including CRKL, and at concentrations in good agreement with the effect on cell growth. CONCLUSION Thermal profiling of cellular proteomes enables the differential assessment of protein ligand binding and other protein modifications, providing an unbiased measure of drug-target occupancy for multiple targets and facilitating the identification of markers for drug efficacy and toxicity. Mapping human drug targets in the cell To understand both the beneficial and the side effects of a drug, one would need to know its full binding profile to all cellular proteins. Savitski et al. take significant steps toward meeting this daunting challenge. They monitored the unfolding or “melting” of over 7000 human proteins and measured how small-molecule binding changes individual melting profiles. As a proof of principle, over 50 targets were identified for an inhibitor known to bind a broad spectrum of kinases. Two cancer drugs, vemurafib and Alectinib, are known to have a side effect of photosensitivity. The thermal profiling approach identified drug-protein interactions responsible for these side effects. Science, this issue 10.1126/science.1255784 Monitoring drug effects on the thermal profile of a cell’s proteins identifies drug targets and off-targets. 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.

High-Resolution Enabled TMT 8-plexing

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation ((15)N, (13)C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between (15)N- and (13)C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, (12)C(8)H(16)(15)N(1)(+); TMT127H, (12)C(7)(13)C(1)H(16)(14)N(1)(+); TMT129L, (12)C(6)(13)C(2)H(16)(15)N(1)(+); and TMT129H, (12)C(5)(13)C(3)H(16)(14)N(1)(+)). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays.

A selective inhibitor reveals PI3Kγ dependence of T H 17 cell differentiation

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.

Ion Coalescence of Neutron Encoded TMT 10-Plex Reporter Ions

Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low mass range of tandem MS spectra for relative quantification. The recent extension of TMT multiplexing to 10 conditions has been enabled by utilizing neutron encoded tags with reporter ion m/z differences of 6 mDa. The baseline resolution of these closely spaced tags is possible due to the high resolving power of current day mass spectrometers. In this work we evaluated the performance of the TMT10 isobaric mass tags on the Q Exactive Orbitrap mass spectrometers for the first time and demonstrated comparable quantification accuracy and precision to what can be achieved on the Orbitrap Elite mass spectrometers. However, we discovered, upon analysis of complex proteomics samples on the Q Exactive Orbitrap mass spectrometers, that the proximate TMT10 reporter ion pairs become prone to coalescence. The fusion of the different reporter ion signals into a single measurable entity has a detrimental effect on peptide and protein quantification. We established that the main reason for coalescence is the commonly accepted maximum ion target for MS2 spectra of 1e6 on the Q Exactive instruments. The coalescence artifact was completely removed by lowering the maximum ion target for MS2 spectra from 1e6 to 2e5 without any losses in identification depth or quantification quality of proteins.

The Commonly Used PI3-Kinase Probe LY294002 Is an Inhibitor of BET Bromodomains

A commonly used small-molecule probe in cell-signaling research is the phosphoinositide 3-kinase inhibitor LY294002. Quantitative chemoproteomic profiling shows that LY294002 and LY303511, a close analogue devoid of PI3K activity, inhibit the BET bromodomain proteins BRD2, BRD3, and BRD4 that comprise a family of targets structurally unrelated to PI3K. Both compounds competitively inhibit acetyl-lysine binding of the first but not the second bromodomain of BET proteins in cell extracts. X-ray crystallography shows that the chromen-4-one scaffold represents a new bromodomain pharmacophore and establishes LY294002 as a dual kinase and BET-bromodomain inhibitor, whereas LY303511 exhibits anti-inflammatory and antiproliferative effects similar to the recently discovered BET inhibitors.

Discovery and Characterization of GSK2801, a Selective Chemical Probe for the Bromodomains BAZ2A and BAZ2B

Bromodomains are acetyl-lysine specific protein interaction domains that have recently emerged as a new target class for the development of inhibitors that modulate gene transcription. The two closely related bromodomain containing proteins BAZ2A and BAZ2B constitute the central scaffolding protein of the nucleolar remodeling complex (NoRC) that regulates the expression of noncoding RNAs. However, BAZ2 bromodomains have low predicted druggability and so far no selective inhibitors have been published. Here we report the development of GSK2801, a potent, selective and cell active acetyl-lysine competitive inhibitor of BAZ2A and BAZ2B bromodomains as well as the inactive control compound GSK8573. GSK2801 binds to BAZ2 bromodomains with dissociation constants (KD) of 136 and 257 nM for BAZ2B and BAZ2A, respectively. Crystal structures demonstrated a canonical acetyl-lysine competitive binding mode. Cellular activity was demonstrated using fluorescent recovery after photobleaching (FRAP) monitoring displacement of GFP-BAZ2A from acetylated chromatin. A pharmacokinetic study in mice showed that GSK2801 had reasonable in vivo exposure after oral dosing, with modest clearance and reasonable plasma stability. Thus, GSK2801 represents a versatile tool compound for cellular and in vivo studies to understand the role of BAZ2 bromodomains in chromatin biology.

Thermal proteome profiling for unbiased identification of direct and indirect drug targets using multiplexed quantitative mass spectrometry

The direct detection of drug-protein interactions in living cells is a major challenge in drug discovery research. Recently, we introduced an approach termed thermal proteome profiling (TPP), which enables the monitoring of changes in protein thermal stability across the proteome using quantitative mass spectrometry. We determined the intracellular thermal profiles for up to 7,000 proteins, and by comparing profiles derived from cultured mammalian cells in the presence or absence of a drug we showed that it was possible to identify direct and indirect targets of drugs in living cells in an unbiased manner. Here we demonstrate the complete workflow using the histone deacetylase inhibitor panobinostat. The key to this approach is the use of isobaric tandem mass tag 10-plex (TMT10) reagents to label digested protein samples corresponding to each temperature point in the melting curve so that the samples can be analyzed by multiplexed quantitative mass spectrometry. Important steps in the bioinformatic analysis include data normalization, melting curve fitting and statistical significance determination of compound concentration-dependent changes in protein stability. All analysis tools are made freely available as R and Python packages. The workflow can be completed in 2 weeks.

Thermal proteome profiling monitors ligand interactions with cellular membrane proteins.

We extended thermal proteome profiling to detect transmembrane protein–small molecule interactions in cultured human cells. When we assessed the effects of detergents on ATP-binding profiles, we observed shifts in denaturation temperature for ATP-binding transmembrane proteins. We also observed cellular thermal shifts in pervanadate-induced T cell–receptor signaling, delineating the membrane target CD45 and components of the downstream pathway, and with drugs affecting the transmembrane transporters ATP1A1 and MDR1.

Structure-Based Optimization of Naphthyridones into Potent ATAD2 Bromodomain Inhibitors

ATAD2 is a bromodomain-containing protein whose overexpression is linked to poor outcomes in a number of different cancer types. To date, no potent and selective inhibitors of the bromodomain have been reported. This article describes the structure-based optimization of a series of naphthyridones from micromolar leads with no selectivity over the BET bromodomains to inhibitors with sub-100 nM ATAD2 potency and 100-fold BET selectivity.

Discovery of 5-(2-amino-[1,2,4]triazolo[1,5-a]pyridin-7-yl)-N-(tert-butyl)pyridine-3-sulfonamide (CZC24758), as a potent, orally bioavailable and selective inhibitor of PI3K for the treatment of inflammatory disease

Herein, we disclose the discovery of a series of 7-substituted triazolopyridines which culminated in the identification of 14 (CZC24758), a potent, orally bioavailable small-molecule inhibitor of PI3Kγ, an attractive drug target for inflammatory and autoimmune disorders. Compound 14 has excellent selectivity across the kinome, demonstrates good potency in cell based assays and furthermore exhibits in vivo efficacy in a collagen induced arthritis model in mouse after oral dosing.