Electrophilic Nucleosides: Tools to Explore Protein Kinases

The protein kinase family represents a significant challenge in medicinal chemistry. This important class of enzymes shares a structurally conserved catalytic core, making selective inhibition of individual members difficult. Covalent inhibition offers the potential of additional selectivity based on chemical reactivity and here I have designed and synthesized a panel of electrophilic nucleosides to investigate select protein kinases. These compounds share a nucleoside scaffold that combines elements of the broad spectrum tyrosine kinase inhibitor pyrazolopyrimidine-1 (PP1) and an electrophilic nucleoside, p-fluorosulfonylbenzoyl 5' adenosine. This combination increases the scaffold's reversible binding affinity to target a subset of protein kinases. I tested the effect of several different 5' electrophiles on the activity of Src-kinase family members. A 5'-vinyl sulfonate ester reacted with a cysteine in the "glycine-rich" loop of Src. This cysteine is also found in FGFR tyrosine kinases, but is present in only three of the eleven Src-family kinases (Src, Yes, and Fgr). A pyrrolopyrimidine nucleoside bearing a 5'-vinyl sulfonate ester inhibited autophosphorylation and FGFR3 dependent cell proliferation in a cysteine dependent manner. Kinases of the eukaryotic parasite Trypanosoma brucei were profiled using the electrophilic nucleoside 47, which targets an essential catalytic lysine found in nearly all protein kinases. This compound binds to a subset of the T. brucei kinases, five of which are essential: TbPK50 and homologues of casein kinase 1, Gsk3, NIMA, and Clk protein kinases. I used compound 47 in conjunction with quantitative mass spectrometry as a tool for chemical proteomics. Using this technique, covalently modified T. brucei kinases were profiled for binding with the reversible inhibitor PD0166326. Exchanging the 5' electrophile of 47 for an acrylate ester changes the compound's effect on T. brucei. This new electrophile still preferentially reacts with protein kinases, but with higher selectivity. One unique target of the acrylate is the T. brucei homologue of Bud32 kinase. This atypical kinase may have important functions in T. brucei biology and virulence

Selective Targeting of Distinct Active Site Nucleophiles by Irreversible Src-Family Kinase Inhibitors

Src-family tyrosine kinases play pivotal roles in human physiology and disease, and several drugs that target members of this family are in clinical use. None of these drugs appear to discriminate among closely related kinases. However, assessing their selectivity toward endogenous kinases in living cells remains a significant challenge. Here, we report the design of two Src-directed chemical probes, each consisting of a nucleoside scaffold with a 5′-electrophile. A 5′-fluorosulfonylbenzoate (<b>1</b>) reacts with the conserved catalytic lysine (Lys295) and shows little discrimination among related kinases. By contrast, a 5′-vinylsulfonate (<b>2</b>) reacts with a poorly conserved, proximal cysteine (Cys277) found in three Src-family and six unrelated kinases. Both <b>1</b> and <b>2</b> bear an alkyne tag and efficiently label their respective endogenous kinase targets in intact cells. Using <b>1</b> as a competitive probe, we determined the extent to which ponatinib, a clinical Bcr-Abl inhibitor, targets Src-family kinases. Remarkably, while ponatinib had little effect on endogenous Fyn or Src, it potently blocked the critical T-cell kinase, Lck. Probes <b>1</b> and <b>2</b> thus enable competitive profiling versus distinct kinase subsets in living cells

Hypothemycin, a fungal natural product, identifies therapeutic targets in Trypanosoma brucei [corrected].

Protein kinases are potentially attractive therapeutic targets for neglected parasitic diseases, including African trypanosomiasis caused by the protozoan, Trypanosoma brucei. How to prioritize T. brucei kinases and quantify their intracellular engagement by small-molecule inhibitors remain unsolved problems. Here, we combine chemoproteomics and RNA interference to interrogate trypanosome kinases bearing a Cys-Asp-Xaa-Gly motif (CDXG kinases). We discovered that hypothemycin, a fungal polyketide previously shown to covalently inactivate a subset of human CDXG kinases, kills T. brucei in culture and in infected mice. Quantitative chemoproteomic analysis with a hypothemycin-based probe revealed the relative sensitivity of endogenous CDXG kinases, including TbGSK3short and a previously uncharacterized kinase, TbCLK1. RNAi-mediated knockdown demonstrated that both kinases are essential, but only TbCLK1 is fully engaged by cytotoxic concentrations of hypothemycin in intact cells. Our study identifies TbCLK1 as a therapeutic target for African trypanosomiasis and establishes a new chemoproteomic tool for interrogating CDXG kinases in their native context. DOI:http://dx.doi.org/10.7554/eLife.00712.001

Hypothemycin, a fungal natural product, identifies therapeutic targets in Trypanosoma brucei [corrected].

Protein kinases are potentially attractive therapeutic targets for neglected parasitic diseases, including African trypanosomiasis caused by the protozoan, Trypanosoma brucei. How to prioritize T. brucei kinases and quantify their intracellular engagement by small-molecule inhibitors remain unsolved problems. Here, we combine chemoproteomics and RNA interference to interrogate trypanosome kinases bearing a Cys-Asp-Xaa-Gly motif (CDXG kinases). We discovered that hypothemycin, a fungal polyketide previously shown to covalently inactivate a subset of human CDXG kinases, kills T. brucei in culture and in infected mice. Quantitative chemoproteomic analysis with a hypothemycin-based probe revealed the relative sensitivity of endogenous CDXG kinases, including TbGSK3short and a previously uncharacterized kinase, TbCLK1. RNAi-mediated knockdown demonstrated that both kinases are essential, but only TbCLK1 is fully engaged by cytotoxic concentrations of hypothemycin in intact cells. Our study identifies TbCLK1 as a therapeutic target for African trypanosomiasis and establishes a new chemoproteomic tool for interrogating CDXG kinases in their native context. DOI:http://dx.doi.org/10.7554/eLife.00712.001

Hypothemycin, a fungal natural product, identifies therapeutic targets in Trypanosoma brucei [corrected].

Protein kinases are potentially attractive therapeutic targets for neglected parasitic diseases, including African trypanosomiasis caused by the protozoan, Trypanosoma brucei. How to prioritize T. brucei kinases and quantify their intracellular engagement by small-molecule inhibitors remain unsolved problems. Here, we combine chemoproteomics and RNA interference to interrogate trypanosome kinases bearing a Cys-Asp-Xaa-Gly motif (CDXG kinases). We discovered that hypothemycin, a fungal polyketide previously shown to covalently inactivate a subset of human CDXG kinases, kills T. brucei in culture and in infected mice. Quantitative chemoproteomic analysis with a hypothemycin-based probe revealed the relative sensitivity of endogenous CDXG kinases, including TbGSK3short and a previously uncharacterized kinase, TbCLK1. RNAi-mediated knockdown demonstrated that both kinases are essential, but only TbCLK1 is fully engaged by cytotoxic concentrations of hypothemycin in intact cells. Our study identifies TbCLK1 as a therapeutic target for African trypanosomiasis and establishes a new chemoproteomic tool for interrogating CDXG kinases in their native context. DOI:http://dx.doi.org/10.7554/eLife.00712.001

Thermotolerant Guard Cell Protoplasts of Tree Tobacco Do Not Require Exogenous Hormones to Survive in Culture and Are Blocked from Reentering the Cell Cycle at the G1-to-S Transition.

When guard cell protoplasts (GCPs) of tree tobacco [Nicotiana glauca (Graham)] are cultured at 32°C with an auxin (1-napthaleneacetic acid) and a cytokinin (6-benzylaminopurine), they reenter the cell cycle, dedifferentiate, and divide. GCPs cultured similarly but at 38°C and with 0.1 µM ± -cis,trans-abscisic acid (ABA) remain differentiated. GCPs cultured at 38°C without ABA dedifferentiate partially but do not divide. Cell survival after 1 week is 70% to 80% under all of these conditions. In this study, we show that GCPs cultured for 12 to 24 h at 38°C accumulate heat shock protein 70 and develop a thermotolerance that, upon transfer of cells to 32°C, enhances cell survival but inhibits cell cycle reentry, dedifferentiation, and division. GCPs dedifferentiating at 32°C require both 1-napthaleneacetic acid and 6-benzylaminopurine to survive, but thermotolerant GCPs cultured at 38°C ± ABA do not require either hormone for survival. Pulse-labeling experiments using 5-bromo-2-deoxyuridine indicate that culture at 38°C ± ABA prevents dedifferentiation of GCPs by blocking cell cycle reentry at G1/S. Cell cycle reentry at 32°C is accompanied by loss of a 41-kD polypeptide that cross-reacts with antibodies to rat (Rattus norvegicus) extracellular signal-regulated kinase 1; thermotolerant GCPs retain this polypeptide. A number of polypeptides unique to thermotolerant cells have been uncovered by Boolean analysis of two-dimensional gels and are targets for further analysis. GCPs of tree tobacco can be isolated in sufficient numbers and with the purity required to study plant cell thermotolerance and its relationship to plant cell survival, growth, dedifferentiation, and division in vitro

Selective Targeting of Distinct Active Site Nucleophiles by Irreversible Src-Family Kinase Inhibitors

Src-family tyrosine kinases play pivotal roles in human physiology and disease, and several drugs that target members of this family are in clinical use. None of these drugs appear to discriminate among closely related kinases. However, assessing their selectivity toward endogenous kinases in living cells remains a significant challenge. Here, we report the design of two Src-directed chemical probes, each consisting of a nucleoside scaffold with a 5'- electrophile. A 5'-fluorosulfonylbenzoate (1) reacts with the conserved catalytic lysine (Lys295) and shows little discrimination among related kinases. By contrast, a 5'-vinylsulfonate (2) reacts with a poorly conserved, proximal cysteine (Cys277) found in three Src-family and six unrelated kinases. Both 1 and 2 bear an alkyne tag and efficiently label their respective endogenous kinase targets in intact cells. Using 1 as a competitive probe, we determined the extent to which ponatinib, a clinical Bcr-Abl inhibitor, targets Src-family kinases. Remarkably, while ponatinib had little effect on endogenous Fyn or Src, it potently blocked the critical Tcell kinase, Lck. Probes 1 and 2 thus enable competitive profiling vs. distinct kinase subsets in living cells