Identification of Promiscuous Small Molecule Activators in High-Throughput Enzyme Activation Screens

Goode, David R; Totten, Ryan K.; Heeres, James T.; Hergenrother, Paul J.

doi:10.1021/jm701583b

Cited by 29 publications

(29 citation statements)

References 20 publications

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“…Compound 1 has reduced effectiveness at the highest enzyme concentration tested, so we further investigated potential non-specific effects by testing it against unrelated enzymes. While general mechanisms for non-specific activation have not been thoroughly investigated, it has been suggested that non-specific activators may act like detergents by interfering with adsorption of protein to surfaces [25]. If this were the mechanism for compound 1, or for our other hit compounds, then it should have similar effects on many other types of enzymes.…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…These values tracked closely the concentrations required for half-maximal effect on catalytic activity (EC 50 values)-7.1 and 7.2 μM for 2A2 and 1F8, respectively. None of the most activating or inhibiting disulfide fragments had a significant impact on the activity of wild-type PDK1 and all were approximately equally effective toward mutants in the absence and presence of the detergent CHAPS, which is known to disperse small molecule aggregates that can lead to spurious enzyme activation or inhibition (15)(16)(17) (Fig. 3B and Fig.…”

Section: Resultsmentioning

confidence: 99%

Turning a protein kinase on or off from a single allosteric site via disulfide trapping

Sadowsky

Burlingame²,

Wolan³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

146

135

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There is significant interest in identifying and characterizing allosteric sites in enzymes such as protein kinases both for understanding allosteric mechanisms as well as for drug discovery. Here, we apply a site-directed technology, disulfide trapping, to interrogate structurally and functionally how an allosteric site on the Ser/Thr kinase, 3-phosphoinositide-dependent kinase 1 (PDK1)-the PDK1-interacting-fragment (PIF) pocket-is engaged by an activating peptide motif on downstream substrate kinases (PIFtides) and by small molecule fragments. By monitoring pairwise disulfide conjugation between PIFtide and PDK1 cysteine mutants, we defined the PIFtide binding orientation in the PIF pocket of PDK1 and assessed subtle relationships between PIFtide positioning and kinase activation. We also discovered a variety of small molecule fragment disulfides (<300 Da) that could either activate or inhibit PDK1 by conjugation to the PIF pocket, thus displaying greater functional diversity than is displayed by PIFtides conjugated to the same sites. Biochemical data and three crystal structures provided insight into the mechanism of action of the best fragment activators and inhibitors. These studies show that disulfide trapping is useful for characterizing allosteric sites on kinases and that a single allosteric site on a protein kinase can be exploited for both activation and inhibition by small molecules.fragment-based drug discovery | kinase allostery

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“…These aggregates are readily identified by diagnostic experiments, including detergent sensitivity, β-lactamase inhibition, and sensitivity to bovine serum albumin (BSA) 14-16 . Furthermore, others have reported that detergent sensitive molecules in screening libraries can also promote enzyme activity 17 . We performed these diagnostic tests on 1541 with mixed results.…”

mentioning

confidence: 99%

Self-Assembling Small Molecules Form Nanofibrils That Bind Procaspase-3 To Promote Activation

et al. 2011

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Modulating enzyme function with small molecule activators, as opposed to inhibitors, offers new opportunities for drug discovery and allosteric regulation. Previously, we identified a compound, called 1541, from a high through-put screen (HTS) that stimulates activation of a proenzyme, procaspase-3, to generate mature caspase-3. Here, we further investigate the mechanism of activation and report the surprising finding that 1541 self-assembles into nanofibrils, exceeding 1 μm in length. These particles are an unanticipated outcome from a HTS with properties distinct from standard globular protein aggregators. Moreover, 1541 nanofibrils function as a unique biocatalytic material, which activate procaspase-3 via induced proximity. These studies demonstrate a novel approach for proenzyme activation through binding to fibrils, which may mimic how procaspases are naturally processed on protein scaffolds.

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Identification of Promiscuous Small Molecule Activators in High-Throughput Enzyme Activation Screens

Cited by 29 publications

References 20 publications

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

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

Turning a protein kinase on or off from a single allosteric site via disulfide trapping

Self-Assembling Small Molecules Form Nanofibrils That Bind Procaspase-3 To Promote Activation

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