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1.  Mechanistic similarity and diversity among the guanidine-modifying members of the pentein superfamily 
Biochimica et biophysica acta  2010;1804(10):1943-1953.
The pentein superfamily is a mechanistically diverse superfamily encompassing both noncatalytic proteins and enzymes that catalyze hydrolase, dihydrolase and amidinotransfer reactions on guanidine substrates. Despite generally low sequence identity, they possess a conserved structural fold and display common mechanistic themes in catalysis. The structurally characterized catalytic penteins possess a conserved core of residues that include a Cys, His and two polar, guanidine-binding residues. All known catalytic penteins use the core Cys to attack the substrate’s guanidine moiety to form a covalent thiouronium adduct and all cleave one or more of the guanidine C–N bonds. The mechanistic information compiled to date supports the hypothesis that this superfamily may have evolved divergently from a catalytically promiscuous ancestor.
doi:10.1016/j.bbapap.2010.07.016
PMCID: PMC4104755  PMID: 20654741
Pentein; Guanidine; Arginine deiminase; Dimethylarginine dimethylaminohydrolase; Agmatine deiminase; Peptidylarginine deiminase; Arginine:glycine amidinotransferase; Arginine:inosamine phosphate; amidinotransferase; Nα-succinylarginine dihydrolase; Guanidino-modifying superfamily
2.  Discovery of structurally-diverse inhibitor scaffolds by high-throughput screening of a fragment library with dimethylarginine dimethylaminohydrolase 
Bioorganic & medicinal chemistry  2012;20(18):5550-5558.
Potent and selective inhibitors of the enzyme dimethylarginine dimethylaminohydrolase (DDAH) are useful as molecular probes to better understand cellular regulation of nitric oxide. Inhibitors are also potential therapeutic agents for treatment of pathological states associated with the inappropriate overproduction of nitric oxide, such as septic shock, selected types of cancer, and other conditions. Inhibitors with structures dissimilar to substrate may overcome limitations inherent to substrate analogs. Therefore, to identify structurally-diverse inhibitor scaffolds, high-throughput screening (HTS) of a 4000-member library of fragment-sized molecules was completed using the Pseudomonas aeruginosa DDAH and human DDAH-1 isoforms. Use of a substrate concentration equal to its KM value during the primary screen allowed for the detection of inhibitors with different modes of inhibition. A series of validation tests were designed and implemented in the identification of four inhibitors of human DDAH-1 that were unknown prior to the screen. Two inhibitors share a 4-halopyridine scaffold and act as quiescent affinity labels that selectively and covalently modify the active-site Cys residue. Two inhibitors are benzimidazole-like compounds that reversibly and competitively inhibit human DDAH-1 with Ligand Efficiency values ≥ 0.3 kcal / mol / heavy (non-hydrogen) atom, indicating their suitability for further development. Both inhibitor scaffolds have available sites to derivatize for further optimization. Therefore, use of this fragment-based HTS approach is demonstrated to successfully identify two novel scaffolds for development of DDAH-1 inhibitors.
doi:10.1016/j.bmc.2012.07.022
PMCID: PMC3444674  PMID: 22921743
Fragment library; High-throughput screen; Inhibitor discovery; Dimethylarginine dimethylaminohydrolase; Nitric oxide
3.  A Continuous, Fluorescent, High-Throughput Assay for Human Dimethylarginine Dimethylaminohydrolase-1 
Journal of biomolecular screening  2011;16(9):1089-1097.
Inhibitors of human dimethylarginine dimethylaminohydrolase-1 (DDAH-1) are of therapeutic interest for controlling pathological nitric oxide production. Only a limited number of biologically useful inhibitors have been identified, so structurally diverse lead compounds are desired. In contrast with previous assays that do not possesses adequate sensitivity for optimal screening, herein is reported a high-throughput assay that uses an alternative thiol-releasing substrate, S-methyl-L-thiocitrulline, and a thiol-reactive fluorophore, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin, to enable continuous detection of product formation by DDAH-1. The assay is applied to query two commercial libraries totaling 4,446 compounds and two representative hits are described, including a known DDAH-1 inhibitor. This is the most sensitive DDAH-1 assay reported to date, and enables screening of compound libraries using [S] =KM conditions, while displaying Z′ factors from 0.6 – 0.8. Therefore, this strategy now makes possible high-throughput screening for human DDAH-1 inhibitors in pursuit of molecular probes and drugs to control excessive nitric oxide production.
doi:10.1177/1087057111417712
PMCID: PMC3248755  PMID: 21921133
Dimethylarginine dimethylaminohydrolase; high-throughput screening; nitric oxide; CPM
4.  Screening for Dimethylarginine Dimethylaminohydrolase Inhibitors Reveals Ebselen as a Bioavailable Inactivator 
ACS Medicinal Chemistry Letters  2011;2(8):592-596.
Dimethylarginine dimethylaminohydrolase (DDAH) is an endogenous regulator of nitric oxide production and represents a potential therapeutic target. However, only a small number of biologically useful inhibitors have been reported, and many of these are substrate analogues. To seek more diverse scaffolds, we developed a high-throughput screening (HTS) assay and queried two small libraries totaling 2446 compounds. The HTS assay proved to be robust, reproducible, and scalable, with Z′ factors ≥ 0.78. One inhibitor, ebselen, is structurally divergent from substrate and was characterized in detail. This selenazole covalently inactivates DDAH in vitro and in cultured cells. The rate constant for inactivation of DDAH (44000 ± 2400 M–1 s–1) is greater than those reported for any other target, suggesting that this pathway is an important aspect of ebselen's total pharmacological effects.
doi:10.1021/ml2000824
PMCID: PMC3171734  PMID: 21927644
Dimethylarginine dimethylaminohydrolase; high-throughput screening; ebselen; covalent inhibitors; nitric oxide
5.  On the Mechanism of Dimethylarginine Dimethylaminohydrolase Inactivation by 4-Halopyridines 
Journal of the American Chemical Society  2011;133(28):10951-10959.
Small molecules capable of selective covalent protein modification are of significant interest for the development of biological probes and therapeutics. We recently reported that 2-methyl-4-bromopyridine is a quiescent affinity label for the nitric oxide controlling enzyme dimethylarginine dimethylaminohydrolase (DDAH) [Johnson, C.M., Linsky, T.W., Yoon, D.W., Person, M.D. & Fast, W. (2011) J. Am. Chem. Soc. 133, 1553-1562]. Discovery of this novel protein modifier raised the possibility that the 4-halopyridine motif may be suitable for wider application. Therefore, the inactivation mechanism of the related compound 2-hydroxymethyl-4-chloropyridine is probed here in more detail. Solution studies support an inactivation mechanism in which the active-site Asp66 residue stabilizes the pyridinium form of the inactivator, which has enhanced reactivity toward the active site Cys, resulting in covalent bond formation, loss of the halide, and irreversible inactivation. A 2.18 Å resolution X-ray crystal structure of the inactivated complex elucidates the orientation of the inactivator and its covalent attachment to the active-site Cys, but the structural model does not show an interaction between the inactivator and Asp66. Molecular modeling is used to investigate inactivator binding, reaction, and also a final pyridinium deprotonation step that accounts for the apparent differences between the solution-based and structural studies with respect to the role of Asp66. This work integrates multiple approaches to elucidate the inactivation mechanism of a novel 4-halopyridine “warhead,” emphasizing the strategy of using pyridinium formation as a “switch” to enhance reactivity when bound to the target protein.
doi:10.1021/ja2033684
PMCID: PMC3135753  PMID: 21630706
6.  Discovery of Halopyridines as Quiescent Affinity Labels: Inactivation of Dimethylarginine Dimethylaminohydrolase 
In an effort to develop novel covalent modifiers of dimethylarginine dimethylaminohydrolase (DDAH) that are useful for biological applications, a set of “fragment”-sized inhibitors that were identified using a high-throughput screen are tested for time-dependent inhibition. One structural class of inactivators, 4-halopyridines, show time- and concentration-dependent inactivation of DDAH and the inactivation mechanism of one example, 4-bromo-2-methylpyridine (1), is characterized in detail. The neutral form of halopyridines is not very reactive with excess glutathione. However, 1 readily reacts, with loss of its halide, in a selective, covalent and irreversible manner with the active-site Cys249 of DDAH. This active-site Cys is not particularly reactive (pKa ca. 8.8) and 1 does not inactivate papain (Cys pKa ca. ≤ 4), suggesting that, unlike many reagents, Cys nucleophilicity is not a predominating factor in selectivity. Rather, binding and stabilization of the more reactive pyridinium form of the inactivator by a second moiety, Asp66, is required for facile reaction. This constraint imparts a unique selectivity profile to these inactivators. To our knowledge, halopyridines have not previously been reported as protein modifiers, and therefore represent a first-in-class example of a novel type of quiescent affinity label.
doi:10.1021/ja109207m
PMCID: PMC3038607  PMID: 21222447
7.  Screening for dimethylarginine dimethylaminohydrolase inhibitors reveals ebselen as a bioavailable inactivator 
ACS medicinal chemistry letters  2011;2(8):592-596.
Dimethylarginine dimethylaminohydrolase (DDAH) is an endogenous regulator of nitric oxide production and represents a potential therapeutic target. However, only a small number of biologically useful inhibitors have been reported, and many of these are substrate analogs. To seek more diverse scaffolds, we developed a high-throughput screening (HTS) assay and queried two small libraries totaling 2446 compounds. The HTS assay proved to be robust, reproducible and scalable, with Z' factors ≥ 0.78. One inhibitor, ebselen, is structurally divergent from substrate and was characterized in detail. This selenazole covalently inactivates DDAH in vitro and in cultured cells. The rate constant for inactivation of DDAH (44,000 ± 2,400 M−1s−1) is greater than those reported for any other target, suggesting this pathway is an important aspect of ebselen's total pharmacological effects.
doi:10.1021/ml2000824
PMCID: PMC3171734  PMID: 21927644
Dimethylarginine dimethylaminohydrolase; high-throughput screening; ebselen; covalent inhibitors; nitric oxide
8.  Crystal Structure and Promiscuous Partitioning of a Covalent Intermediate Common in the Pentein Superfamily 
Chemistry & biology  2008;15(5):467-475.
Summary
Many enzymes in the pentein superfamily use a transient covalent intermediate in their catalytic mechanisms. Here, we use a mutant (H162G) dimethylarginine dimethylaminohydrolase from Pseudomonas aeruginosa and an alternative substrate, S-methyl-L-thiocitrulline, to trap, crystallize and determine the 2.8 Å resolution structure of a stable covalent adduct which mimics this reaction intermediate. Observed interactions between the trapped adduct and active site residues along with comparison to a previously known product-bound structure provide insight into the normal catalytic mechanism. The plane of the trapped thiouronium intermediate is angled away from that seen in the product and substrate complexes, allowing for an altered angle of attack between the nucleophiles of the first and second half reactions. The stable covalent adduct is also capable of further reaction. Addition of exogenous imidazole can rescue the original hydrolytic activity. Notably, addition of other exogenous amines can instead yield substituted arginine products. These alternative products arise from partitioning of the trapped intermediate into the evolutionarily related amidinotransferase reaction pathway. The enzyme scaffold provides both selectivity and catalysis for the amidinotransferase reaction, underscoring commonalities between different reaction pathways found in this mechanistically diverse enzyme superfamily. The promiscuous partitioning of this covalent intermediate may also help to illuminate the evolutionary history of these enzymes.
doi:10.1016/j.chembiol.2008.03.012
PMCID: PMC2601531  PMID: 18482699

Results 1-8 (8)