Stephan M. Hacker

Synthesis and stability of phosphate modified ATP analogues.

Nucleotides modified at the phosphate have numerous applications. Nevertheless, the number of attachment modes is limited and little is known about their stability. Here, we present results on the elaboration of the synthesis of five classes of ATP analogues and studies concerning their stability. We show that the nitrogen-linked ATP analogue is less stable, whereas the oxygen- and novel carbon-linked adenosine tri- and tetraphosphate analogues are stable from pH 3 to 12 rendering them interesting for further applications and designs.

Global profiling of lysine reactivity and ligandability in the human proteome

Nucleophilic amino acids make important contributions to protein function, including performing key roles in catalysis and serving as sites for post-translational modification. Electrophilic groups that target amino-acid nucleophiles have been used to create covalent ligands and drugs, but have, so far, been mainly limited to cysteine and serine. Here, we report a chemical proteomic platform for the global and quantitative analysis of lysine residues in native biological systems. We have quantified, in total, more than 9,000 lysines in human cell proteomes and have identified several hundred residues with heightened reactivity that are enriched at protein functional sites and can frequently be targeted by electrophilic small molecules. We have also discovered lysine-reactive fragment electrophiles that inhibit enzymes by active site and allosteric mechanisms, as well as disrupt protein-protein interactions in transcriptional regulatory complexes, emphasizing the broad potential and diverse functional consequences of liganding lysine residues throughout the human proteome.

Fingerprinting differential active site constraints of ATPases

The free energy provided by adenosine triphosphate (ATP) hydrolysis is central to many cellular processes and, therefore, the number of enzymes utilizing ATP as a substrate is almost innumerable. Modified analogues of ATP are a valuable means to understand the biological function of ATPases. Although these enzymes have evolved towards binding to ATP, large differences in active site architectures were found. In order to systematically access the specific active site constraints of different ATPases suitable tools are required. Here, we present the synthesis of six new ATP-based ATPase probes modified at three different positions of the nucleobase and the ribose, respectively. Subsequently, we studied the ATPases focal adhesion kinase FAK, the ubiquitin-activating protein UBA1 and the kinesin Eg5 as examples for ATPases that process ATP by different mechanisms. We find that for each of these enzymes at least one position in ATP can be modified without loss of acceptance by the enzyme. However, the positions at which modifications are tolerated significantly differ between the studied enzymes allowing fingerprints to be drawn for reactivity. The introduced ATP analogues may form the basis for the design of tailored probes with increased affinity and specificity for a specific ATPase of interest.

Fluorogenic ATP Analogues for Online Monitoring of ATP Consumption: Observing Ubiquitin Activation in Real Time†

The conjugation of ubiquitin to proteins plays an important role in the regulation of numerous cellular processes. Deregulation of this pathway has been associated with different human disorders including cancer and neurodege nerative diseases. For ubiquitylation, ubiquitin is initially activated by a ubiquitin activating enzyme (E1) at the expense of adenosine triphosphate (ATP; see Figure 1a) to form a thioester bond between the C terminal glycine of ubiquitin and the catalytic cysteine residue of E1. Subse quent transfer to a cysteine residue of a ubiquitin conjugating enzyme (E2) initiates the conjugation of ubiquitin to a target protein (mostly to a lysine residue by means of an isopeptide bond), a process which in many cases requires the help of a ubiquitin ligase (E3). As UBA1 is one of only two known human E1 enzymes for ubiquitin, modulation of its activity may prove beneficial in the treatment of certain disorders. Hence, assays for studying the activation of ubiquitin by UBA1 directly and without the interfering effects of the downstream enzymatic cascade are important tools to analyze UBA1 activity and identify modulators of this enzyme. So far, only a few assays that directly measure E1 activity have been described. However, these are laborious and not suitable for the continuous monitoring of E1 activity. In fact, until now no assay is available that allows the direct detection of ubiquitin activation in real time and that, for example, can be easily used to screen for E1 effectors. We, therefore, decided to elaborate on a conceptually novel assay: real time detection of ubiquitin activation by monitoring the cleavage of an ATP analogue (Figure 1b) that harbors two different fluorophores with the potential to undergo Fçrster resonance energy transfer (FRET). In the envisioned doubly labeled ATP analogue, excitation of the fluorescence donor (D) leads to transfer of the excitation energy to the fluorescence acceptor (A) whose fluorescence is monitored. Upon cleavage of the a/b anhydride bond of ATP, FRET is no longer possible and, thus, direct emission of the fluorescence donor can be detected. In this way, E1 activity results in a large change of fluorescence characteristics of the ATP analogue. Similar approaches based on the cleavage of FRET cassettes have been used for studying other hydro lyzing enzymes like proteases. For the envisaged time resolved ATPase sensor (TRASE) approach two fluorophores have to be attached to ATP. As the N6 position of ATP has been proven to be an attractive site for modifications without compromising UBA1 activity, the second modification has to be placed at the phosphoanhydride chain so that the two fluorophores are spatially separated upon cleavage. As earlier studies revealed that phosphate esters of ATP are stable, we tested g modified triphosphate II and d modified tetraphosphate III in promoting the autoubiquity lation of E6AP, a reaction that involves UBA1 and the E2 enzyme UbcH5b (Figure 2a,b). E6AP is an E3 ligase that has been causally associated with the development of three different human disorders: cervical cancer, Angelman syn drome, and autism spectrum disorders. As only the UBA1 reaction is ATP dependent, the appearance of polyubiquity lated E6AP (E6AP Ub), which does not significantly migrate in the applied gel electrophoresis, and consequently Figure 1. a) Mechanism of the activation of ubiquitin by UBA1. Ubiquitin is loaded onto UBA1 through the formation of a thioester bond with consumption of ATP. AMP: adenosine monophosphate, PP: pyrophosphate. b) Concept of signaling ATP consumption. The intact ATP analogue shows fluorescence of the acceptor (A) upon excitation of the donor (D) due to FRET. Upon cleavage by UBA1 direct donor fluorescence can be observed.

Selective Monitoring of the Enzymatic Activity of the Tumor Suppressor Fhit

Cancer is a leading cause of death worldwide. Functional inactivation of tumor suppressor proteins, mainly by mutations in the corresponding genes, is a key event in cancer development. The fragile histidine triade protein (Fhit) is a tumor suppressor that is frequently affected in different cancer types. Fhit possesses diadenosine triphosphate hydrolase activity, but although reduction of its enzymatic activity appears to be important for exerting its tumor suppressor function, the regulation of Fhit activity is poorly understood. Here, we introduce a novel fluorogenic probe that is suited to selectively analyze the enzymatic activity of Fhit in extracts derived from human cells. This novel method will allow in-depth insight into the mechanisms involved in Fhit regulation in biologically relevant setups and, thus, into its role in the development of cancer.

Thiamine Pyrophosphate Stimulates Acetone Activation by Desulfococcus biacutus As Monitored by a Fluorogenic ATP Analogue

Acetone can be degraded by aerobic and anaerobic microorganisms. Studies with the strictly anaerobic sulfate-reducing bacterium Desulfococcus biacutus indicate that acetone degradation by these bacteria starts with an ATP-dependent carbonylation reaction leading to acetoacetaldehyde as the first reaction product. The reaction represents the second example of a carbonylation reaction in the biochemistry of strictly anaerobic bacteria, but the exact mechanism and dependence on cofactors are still unclear. Here, we use a novel fluorogenic ATP analogue to investigate its mechanism. We find that thiamine pyrophosphate is a cofactor of this ATP-dependent reaction. The products of ATP cleavage are AMP and pyrophosphate, providing first insights into the reaction mechanism by indicating that the reaction proceeds without intermediate formation of acetone enol phosphate.

Direct Monitoring of Nucleotide Turnover in Human Cell Extracts and Cells by Fluorogenic ATP Analogs

Nucleotides containing adenosine play pivotal roles in every living cell. Adenosine triphosphate (ATP), for example, is the universal energy currency, and ATP-consuming processes also contribute to posttranslational protein modifications. Nevertheless, detecting the turnover of adenosine nucleotides in the complex setting of a cell remains challenging. Here, we demonstrate the use of fluorogenic analogs of ATP and adenosine tetraphosphate to study nucleotide hydrolysis in lysates of human cell lines and in intact human cells. We found that the adenosine triphosphate analog is completely stable in lysates of human cell lines, whereas the adenosine tetraphosphate analog is rapidly turned over. The observed activity in human cell lysates can be assigned to a single enzyme, namely, the human diadenosine tetraphosphate hydrolase NudT2. Since NudT2 has been shown to be a prognostic factor for breast cancer, the adenosine tetraphosphate analog might contribute to a better understanding of its involvement in cancerogenesis and allow the straightforward screening for inhibitors. Studying hydrolysis of the analogs in intact cells, we found that electroporation is a suitable method to deliver nucleotide analogs into the cytoplasm and show that high FRET efficiencies can be detected directly after internalization. Time-dependent experiments reveal that adenosine triphosphate and tetraphosphate analogs are both processed in the cellular environment. This study demonstrates that these nucleotide analogs indeed bear the potential to be powerful tools for the exploration of nucleotide turnover in the context of whole cells.

Phosphate-Modified Nucleotides for Monitoring Enzyme Activity

Nucleotides modified at the terminal phosphate position have been proven to be interesting entities to study the activity of a variety of different protein classes. In this chapter, we present various types of modifications that were attached as reporter molecules to the phosphate chain of nucleotides and briefly describe the chemical reactions that are frequently used to synthesize them. Furthermore, we discuss a variety of applications of these molecules. Kinase activity, for instance, was studied by transfer of a phosphate modified with a reporter group to the target proteins. This allows not only studying the activity of kinases, but also identifying their target proteins. Moreover, kinases can also be directly labeled with a reporter at a conserved lysine using acyl-phosphate probes. Another important application for phosphate-modified nucleotides is the study of RNA and DNA polymerases. In this context, single-molecule sequencing is made possible using detection in zero-mode waveguides, nanopores or by a Förster resonance energy transfer (FRET)-based mechanism between the polymerase and a fluorophore-labeled nucleotide. Additionally, fluorogenic nucleotides that utilize an intramolecular interaction between a fluorophore and the nucleobase or an intramolecular FRET effect have been successfully developed to study a variety of different enzymes. Finally, also some novel techniques applying electron paramagnetic resonance (EPR)-based detection of nucleotide cleavage or the detection of the cleavage of fluorophosphates are discussed. Taken together, nucleotides modified at the terminal phosphate position have been applied to study the activity of a large diversity of proteins and are valuable tools to enhance the knowledge of biological systems.

Synthesis of γ‐Phosphate‐Labeled and Doubly Labeled Adenosine Triphosphate Analogs

This unit describes the synthesis of γ‐phosphate‐labeled and doubly labeled adenosine triphosphate (ATP) analogs and their characterization using the phosphodiesterase I from Crotalus adamanteus (snake venom phosphodiesterase; SVPD). In the key step of the synthesis, ATP or an ATP analog, bearing a linker containing a trifluoroacetamide group attached to the nucleoside, are modified with an azide‐containing linker at the terminal phosphate using an alkylation reaction. Subsequently, different labels are introduced to the linkers by transformation of one functional group to an amine and coupling to an N‐hydroxysuccinimide ester. Specifically, the Staudinger reaction of the azide is employed as a straightforward means to obtain an amine in the presence of various labels. Furthermore, the fluorescence characteristics of a fluorogenic, doubly labeled ATP analog are investigated following enzymatic cleavage by SVPD.

Small-Molecule Inhibitors of the Tumor Suppressor Fhit

The tumor suppressor Fhit and its substrate diadenosine triphosphate (Ap3A) are important factors in cancer development and progression. Fhit has Ap3A hydrolase activity and cleaves Ap3A into adenosine monophosphate (AMP) and adenosine diphosphate (ADP); this is believed to terminate Fhit‐mediated signaling. How the catalytic activity of Fhit is regulated and how the Fhit⋅Ap3A complex might exert its growth‐suppressive function remain to be discovered. Small‐molecule inhibitors of the enzymatic activity of Fhit would provide valuable tools for the elucidation of its tumor‐suppressive functions. Here we describe the development of a high‐throughput screen for the identification of such small‐molecule inhibitors of Fhit. Two clusters of inhibitors that decreased the activity of Fhit by at least 90 % were identified. Several derivatives were synthesized and exhibited in vitro IC50 values in the nanomolar range.