Discovery of Halopyridines as Quiescent Affinity Labels: Inactivation of Dimethylarginine Dimethylaminohydrolase

Johnson, Corey M.; Linsky, Thomas W.; Yoon, Dae; Person, Maria D.; Fast, Walter

doi:10.1021/ja109207m

Cited by 35 publications

(59 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Biochemically, they are typically stable to co-incubation with thiols under ambient conditions but under enzyme catalysis can be selectively activated to irreversibly inactivate enzymes such as dimethylarginine dimethylaminohydrolase (DDAH). 7, 8 The enzyme mediated mechanism for the glutathione inactivation of 4-chloropyridyl spectinamides can be envisioned as shown in Figure 3, similar to the proposed DDAH mechanism which has been explored in detail. In this study, the modulating effect of chemical substitution at the 5-position was explored in regards to hepatic glutathione mediated conjugation.…”

Section: Resultsmentioning

confidence: 93%

Phase II metabolic pathways of spectinamide antitubercular agents: a comparative study of the reactivity of 4-substituted pyridines to glutathione conjugation

2016

View full text Add to dashboard Cite

Spectinamides are promising new semisynthetic anti-tubercular agents that are modified with a pyridyl side chain, which blocks native efflux from the tuberculosis cell. This study, describes the stability of an advanced panel of spectinamide analogs, with varying substitutions to the pyridyl side chain, to Phase-II conjugative metabolism by glucuronosyl transferase, sulfotransferase and glutathione-S-transferase enzymes using both human and rat S9 enzyme fractions. All solely 5-substituted pyridyl spectinamides exhibited complete stability towards Phase II conjugative enzymes. However, 4-chloro substituted pyridyl spectinamides were susceptible to glutathione conjugation with rates dependent on other substitutions to the pyridine ring. Electron donating 5-substitutions increased the propensity for glutathione conjugation and conversely the introduction of an electron withdrawing 5-fluoro group blocked all observed glutathione conjugation. Based on these Phase II metabolism studies, lead spectinamides 1329, 1445, 1599, 1661 and 1810 were found to have favorable properties for potential lead compounds with no Phase II liabilities.

show abstract

Section: Resultsmentioning

confidence: 93%

Phase II metabolic pathways of spectinamide antitubercular agents: a comparative study of the reactivity of 4-substituted pyridines to glutathione conjugation

2016

View full text Add to dashboard Cite

show abstract

“…[7] This configuration of Cys and Asp/Glu residues is not likely unique to DDAH and different mechanisms to catalyze labeling by 4-chloropyridines may exist. Therefore, we sought to determine whether the naïve soluble proteome of E. coli (which lacks DDAH1) contains other protein targets (Figure 3).…”

Section: Resultsmentioning

confidence: 99%

“…[7a, 20] The observed inactivation rates ( k obs ) were determined by fitting the time-dependence of remaining activity (%) to a single-exponential equation. The resulting k obs values were then plotted against inactivator concentration to determine the second order rate constant for each compound tested using a linear equation.…”

Section: Methodsmentioning

confidence: 99%

“…For one example, we discovered that fragment-sized (< 300 Da) 4-halopyridines are quiescent affinity labels of the enzyme dimethylarginine dimethylaminohydrolase (DDAH). [7] These compounds typically have p K a values < 5 and are not very reactive to nucleophiles in solution at neutral pH, due to the unfavorable formation of two lone electron pairs on the pyridine nitrogen in the σ-complex (Scheme 1). DDAH catalyzes the modification of its active-site Cys residue simply by stabilizing the more reactive protonated pyridinium through interaction with a neighboring Asp residue.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Selective Covalent Protein Modification by 4‐Halopyridines through Catalysis

Schardon

Tuley

et al. 2017

ChemBioChem

Self Cite

View full text Add to dashboard Cite

4-Halopyridines are developed herein as selective, tunable, and “switchable” covalent protein modifiers for use in the development of chemical probes. Non-enzymatic reactivity of 4-chloropyridine with amino acids and thiols is ranked with respect to common covalent protein modifying reagents and found to have reactivity similar to that of acrylamide, but can be switched to a reactivity similar to that of iodoacetamide upon stabilization of the positively charged pyridinium. Diverse fragment-sized 4-halopyridines inactivate human dimethylarginine dimethylaminohydrolase-1 (DDAH1) through covalent modification of the active-site Cys, acting as quiescent affinity labels that require “off pathway” catalysis via a stabilization of the protonated pyridinium by a neighboring Asp residue. A series of 2-fluoromethyl-substituted 4-chloropyridines demonstrate that pKa and kinact/KI can be predictably varied over several orders of magnitude. Covalent labeling of proteins in an E. coli lysate is shown to require folded proteins, indicating that alternative proteins can be targeted and modification is likely to be catalysis-dependent. 4-Halopyridines, and quiescent affinity labels in general, represent an attractive strategy to develop reagents with “switchable” electrophilicity as selective covalent protein modifiers.

show abstract

“…12 Mechanistic analysis determined that this 4-halopyridine is a covalent inactivator that selectively modifies the active-site Cys residue. The inactivation mechanism is most consistent with that described for quiescent affinity labels 13,14 in that the compound is relatively unreactive (quiescent) to most biological nucleophiles, but demonstrates an enhanced reactivity when bound to the active site of the target enzyme.…”

mentioning

confidence: 99%

On the Mechanism of Dimethylarginine Dimethylaminohydrolase Inactivation by 4-Halopyridines

Johnson

Monzingo²,

Ke³

et al. 2011

J. Am. Chem. Soc.

Self Cite

View full text Add to dashboard Cite

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) 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.[Small molecules that are capable of covalently modifying proteins are currently undergoing a type of renaissance as they are newly applied to solve problems in chemical biology and proteomics, as well as in drug design, discovery and development. [1][2][3] There are a number of strategies that incorporate moderately electrophilic groups into the design of affinity-based probes, activity-based probes and activity-based protein profiling reagents. A few noted examples include the use of electrophilic phosphonates to target serine hydrolases, 4 * To whom correspondence should be addressed. W.F.: College of Pharmacy, PHAR-MED CHEM, 1 University Station; C0850, Austin, Texas 78712; Phone: (512) Fax: (512) During an effort to find novel inhibitors of the nitric oxide-controlling enzyme dimethylarginine dimethylaminohydrolase (DDAH), 11 2-methyl-4-bromopyridine was discovered to be a time-dependent inhibitor. 12 Mechanistic analysis determined that this 4-halopyridine is a covalent inactivator that selectively modifies the active-site Cys residue. The inactivation mechanism is most consistent with that described for quiescent affinity labels 13,14 in that the compound is relatively unreactive (quiescent) to most biological nucleophiles, but demonstrates an enhanced reactivity when bound to the active site of the target enzyme. In the specific case of 2-methyl-4-bromopyridine, DDAH was pro...

show abstract

Discovery of Halopyridines as Quiescent Affinity Labels: Inactivation of Dimethylarginine Dimethylaminohydrolase

Cited by 35 publications

References 30 publications

Phase II metabolic pathways of spectinamide antitubercular agents: a comparative study of the reactivity of 4-substituted pyridines to glutathione conjugation

Phase II metabolic pathways of spectinamide antitubercular agents: a comparative study of the reactivity of 4-substituted pyridines to glutathione conjugation

Selective Covalent Protein Modification by 4‐Halopyridines through Catalysis

On the Mechanism of Dimethylarginine Dimethylaminohydrolase Inactivation by 4-Halopyridines

Contact Info

Product

Resources

About