Reversible activators of enzymes

Sebastian, John F.

doi:10.1021/ed064p1031

Cited by 11 publications

(10 citation statements)

References 4 publications

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“…The kinetics of BtGH84 activation are consistent with a nonessential type activation model (Scheme 1 ),[ 20 ] similar to reversible mixed inhibition. In this model, the dissociation constant of the substrate is modified in the presence of the activator (and vice versa); this modifier is denoted by the constant α .…”

supporting

confidence: 62%

Discovery of Selective Small‐Molecule Activators of a Bacterial Glycoside Hydrolase

et al. 2014

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Fragment-based approaches are used routinely to discover enzyme inhibitors as cellular tools and potential therapeutic agents. There have been few reports, however, of the discovery of small-molecule enzyme activators. Herein, we describe the discovery and characterization of small-molecule activators of a glycoside hydrolase (a bacterial O-GlcNAc hydrolase). A ligand-observed NMR screen of a library of commercially available fragments identified an enzyme activator which yielded an approximate 90 % increase in kcat/KM values (kcat=catalytic rate constant; KM=Michaelis constant). This compound binds to the enzyme in close proximity to the catalytic center. Evolution of the initial hits led to improved compounds that behave as nonessential activators effecting both KM and Vmax values (Vmax=maximum rate of reaction). The compounds appear to stabilize an active “closed” form of the enzyme. Such activators could offer an orthogonal alternative to enzyme inhibitors for perturbation of enzyme activity in vivo, and could also be used for glycoside hydrolase activation in many industrial processes.

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supporting

confidence: 62%

Discovery of Selective Small‐Molecule Activators of a Bacterial Glycoside Hydrolase

et al. 2014

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“…Kinetic parameters ( k cat and K m ) were determined by the Michaelis-Menten equation using initial velocity non-linear regression analysis in Kaleidagraph (Synergy Software, v 4.5). Data for compound 1 was fit by a two-parameter activation model (Eq 1), fixing K m to the value from data taken in the absence of compound, to extract K act [19]. …”

Section: Methodsmentioning

confidence: 99%

Designing small molecules to target cryptic pockets yields both positive and negative allosteric modulators

Hart

Moeder

et al. 2017

PLoS ONE

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Allosteric drugs, which bind to proteins in regions other than their main ligand-binding or active sites, make it possible to target proteins considered “undruggable” and to develop new therapies that circumvent existing resistance. Despite growing interest in allosteric drug discovery, rational design is limited by a lack of sufficient structural information about alternative binding sites in proteins. Previously, we used Markov State Models (MSMs) to identify such “cryptic pockets,” and here we describe a method for identifying compounds that bind in these cryptic pockets and modulate enzyme activity. Experimental tests validate our approach by revealing both an inhibitor and two activators of TEM β-lactamase (TEM). To identify hits, a library of compounds is first virtually screened against either the crystal structure of a known cryptic pocket or an ensemble of structures containing the same cryptic pocket that is extracted from an MSM. Hit compounds are then screened experimentally and characterized kinetically in individual assays. We identify three hits, one inhibitor and two activators, demonstrating that screening for binding to allosteric sites can result in both positive and negative modulation. The hit compounds have modest effects on TEM activity, but all have higher affinities than previously identified inhibitors, which bind the same cryptic pocket but were found, by chance, via a computational screen targeting the active site. Site-directed mutagenesis of key contact residues predicted by the docking models is used to confirm that the compounds bind in the cryptic pocket as intended. Because hit compounds are identified from docking against both the crystal structure and structures from the MSM, this platform should prove suitable for many proteins, particularly targets whose crystal structures lack obvious druggable pockets, and for identifying both inhibitory and activating small-molecule modulators.

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“…According to the Botts and Morales general modifier mechanism (Fig. 2e ) 23 , 24 , the presence of a modifier (either an inhibitor or an activator) might have an effect on the dissociation constant of the ternary enzyme-substrate-modifier complex, this effect is given by the constant α. In addition, the modifier can have an effect on k cat , which is described by the coefficient β. Non-essential activation is generally characterized by a ligand that can bind and activate an enzyme, not being essential for enzymatic activity.…”

Section: Resultsmentioning

confidence: 99%

Uncoupling conformational states from activity in an allosteric enzyme

et al. 2017

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ATP-phosphoribosyltransferase (ATP-PRT) is a hexameric enzyme in conformational equilibrium between an open and seemingly active state and a closed and presumably inhibited form. The structure-function relationship of allosteric regulation in this system is still not fully understood. Here, we develop a screening strategy for modulators of ATP-PRT and identify 3-(2-thienyl)-l-alanine (TIH) as an allosteric activator of this enzyme. Kinetic analysis reveals co-occupancy of the allosteric sites by TIH and l-histidine. Crystallographic and native ion-mobility mass spectrometry data show that the TIH-bound activated form of the enzyme closely resembles the inhibited l-histidine-bound closed conformation, revealing the uncoupling between ATP-PRT open and closed conformations and its functional state. These findings suggest that dynamic processes are responsible for ATP-PRT allosteric regulation and that similar mechanisms might also be found in other enzymes bearing a ferredoxin-like allosteric domain.

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Reversible activators of enzymes

Abstract: Describes the kinetic mechanisms of reversible activators of enzymes that normally follow Michaelis-Menten kinetics.

Cited by 11 publications

References 4 publications

Discovery of Selective Small‐Molecule Activators of a Bacterial Glycoside Hydrolase

Discovery of Selective Small‐Molecule Activators of a Bacterial Glycoside Hydrolase

Designing small molecules to target cryptic pockets yields both positive and negative allosteric modulators

Uncoupling conformational states from activity in an allosteric enzyme

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