Posttranslational modifications are used by cells from all kingdoms of life to control enzymatic activity and to regulate protein function. For many cellular processes, including DNA repair, spindle function, and apoptosis, reversible mono-and polyADP-ribosylation constitutes a very important regulatory mechanism. Moreover, many pathogenic bacteria secrete toxins which ADP-ribosylate human proteins, causing diseases such as whooping cough, cholera, and diphtheria. Whereas the 3D structures of numerous ADP-ribosylating toxins and related mammalian enzymes have been elucidated, virtually nothing is known about the structure of protein de-ADP-ribosylating enzymes. Here, we report the 3D structure of human ADP-ribosylhydrolase 3 (hARH3). The molecular architecture of hARH3 constitutes the archetype of an all-␣-helical protein fold and provides insights into the reversibility of protein ADP-ribosylation. Two magnesium ions flanked by highly conserved amino acids pinpoint the active-site crevice. Recombinant hARH3 binds free ADP-ribose with micromolar affinity and efficiently de-ADP-ribosylates poly-but not monoADP-ribosylated proteins. Docking experiments indicate a possible binding mode for ADP-ribose polymers and suggest a reaction mechanism. Our results underscore the importance of endogenous ADP-ribosylation cycles and provide a basis for structure-based design of ADP-ribosylhydrolase inhibitors.protein structure ͉ posttranslational modification ͉ glycohydrolase ͉ docking P osttranslational modifications (PTMs) are covalent modifications of amino acid side chains in proteins that come in many different sizes and shapes, ranging from the simple addition of a phosphate group to complex multistep glycosylations. Enzyme-catalyzed PTMs allow rapid responses to environmental stimuli and play crucial roles in signal transduction. NAD-dependent ADP-ribosylation is a reversible PTM in which mono-and polyADP-ribosyltransferases (ARTs and PARPs) and ADP-ribosylhydrolases (ARHs) and poly(ADP-ribose) glycohydrolases (PARGs) catalyze amino acid-specific ADPribosylation and de-ADP-ribosylation, respectively ( Fig. 1) (1-10). ADP-ribosylation has attracted attention because bacterial virulence factors, including diphtheria, cholera, and pertussis toxin, use it as part of their pathogenic mechanism (2, 11). Mono-and polyADP-ribosylation have been recognized also as regulatory mechanisms in many cellular processes, including DNA-repair, chromatin decondensation, transcription, telomere function, mitotic spindle formation, and apoptosis (5-10, 12).Several enzymes have been cloned that catalyze de-ADPribosylation of mono-or polyADP-ribosylated proteins (13-15). Dinitrogenase-activating glycohydrolase (DRAG), an Arg-specific ARH from the phototrophic bacterium Rhodospirillum rubrum, regulates a key enzyme of nitrogen fixation (16-18). The human genome encodes three DRAG-related proteins designated ARH1, ARH2, and ARH3 (19), which are 357, 354, and 363 residues long, respectively. ARH1, like DRAG, specifically de-ADP-ribosylates prote...

Dedicated to Professor Günther Jung on the occasion of his 70th birthdayThe conventional approach to identify biologically active, druglike small molecules is based on high-throughput screening (HTS) of chemical libraries. However, the composition of large chemical libraries and their screening are time-consuming and expensive endeavors; the success relies heavily on the quality of the available libraries, and even the largest library can span only a minute section of the virtual chemical space. Therefore, over the past decade several strategies have been proposed to facilitate the development process by using the protein target as a template for ligand assembly.[1-3] The binding of low-molecular-weight fragments has been detected "directly" by NMR spectroscopy [2a, b] or X-ray crystallography. [2c, d] These biophysical methods have been demonstrated to provide low-affinity ligands as rational starting points for the iterative development of potent protein binders. Alternatively, protein-binding molecules have been identified from mixtures of compounds formed in dynamic equilibria. In the presence of a protein the equilibrium was shifted, and the best binding products were concentrated in the mixture and could be detected by chromatography, mass spectrometry, or NMR spectroscopy.[3a, b] The reported fragment-based methods have in common that they detect binding, not biological activity. Moreover, all these methods require large amounts of protein and test compounds and suffer from the difficult, timeconsuming, and expensive detection of active compounds.We envisioned that the detection of bioactive ligands should be sensitized considerably if reversibly formed ligation products compete in dynamic equilibrium with a fluorogenic reporter substrate for an enzyme (Figure 1). This approach would combine dynamic, target-assisted formation of inhibitory species and detection by a fluorescence-based screening methodology; thus, we designated it dynamic ligation screening (DLS). In DLS, the application of chemically reactive inhibitors as directing probes should enable the testing of inhibitory fragments for a defined binding site on the protein surface. Using an enzymatic reaction for fragment detection amplifies the signals and thus reduces the required amount of protein drastically. Finally, enzymatic detection with a fluorescent reporter molecule should enable high-throughput screening (HTS) in microtiter plates (MTPs); thus, for the first time conventional HTS methodology could be employed in fragment-based dynamic ligand development.The SARS coronavirus main protease (SARS-CoV M pro ; SARS = severe acute respiratory syndrome) was selected as the protein target to demonstrate the DLS approach. SARSCoV M pro is a cysteine protease that is essential for replication of the virus inside the infected host cell. Thus, it has been proposed as a drug target for SARS and-owing to the reported high homology among coronaviral main proteasesalso for other coronaviral infections.[5] Several irreversible (covalent) peptide-based...

Success in small molecule screening relies heavily on the preselection of compounds. Here, we present a strategy for the enrichment of chemical libraries with potentially bioactive compounds integrating the collected knowledge of medicinal chemistry. Employing a genetic algorithm, substructures typically occurring in bioactive compounds were identified using the World Drug Index. Availability of compounds containing the selected substructures was analysed in vendor libraries, and the substructure-specific sublibraries were assembled. Compounds containing reactive, undesired functional groups were omitted. Using a diversity filter for both physico-chemical properties and the substructure composition, the compounds of all the sublibraries were ranked. Accordingly, a screening collection of 16,671 compounds was selected. Diversity and chemical space coverage of the collection indicate that it is highly diverse and well-placed in the chemical space spanned by bioactive com-

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Protein-templated fragment ligations have been established as a powerful method for the assembly and detection of optimized protein ligands. Initially developed for reversible ligations, the method has been expanded to irreversible reactions enabling the formation of super-additive fragment combinations. Here, protein-induced Mannich ligations are discovered as a biocatalytic reaction furnishing inhibitors of the transcription factor STAT5. STAT5 protein catalyzes multicomponent reactions of a phosphate mimetic, formaldehyde, and 1H-tetrazoles yielding protein ligands with greatly increased binding affinity and ligand efficiency. Reactions are induced under physiological conditions selectively by native STAT5 but not by other proteins. Formation of ligation products and (auto-)inhibition of the reaction are quantified and the mechanism is investigated. Inhibitors assembled by STAT5 block specifically the phosphorylation of this protein in a cellular model of acute myeloid leukemia (AML), DNA-binding of STAT5 dimers, expression of downstream targets of the transcription factor, and the proliferation of cancer cells in mice.