High-throughput assays for promiscuous inhibitors

Feng, Brian Y.; Shelat, Anang A.; Doman, Thompson N.; Guy, R. Kiplin; Shoichet, Brian K.

doi:10.1038/nchembio718

Cited by 320 publications

(388 citation statements)

References 14 publications

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“…The inhibition proved to be specific as the compounds did not affect neither CaM nor the pyrophosphatase used in the assay (see SI Text). The presence of detergent in the assay also prevented promiscuous inhibitory effects (46). These results showed that modeling transition paths is a useful tool for drug design to enlarge the search for inhibitors to new chemical families.…”

Section: Discussionmentioning

confidence: 71%

Use of allostery to identify inhibitors of calmodulin-induced activation of Bacillus anthracis edema factor

Laine

Gonçalves

Karst

et al. 2010

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

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Allostery plays a key role in the regulation of the activity and function of many biomolecules. And although many ligands act through allostery, no systematic use is made of it in drug design strategies. Here we describe a procedure for identifying the regions of a protein that can be used to control its activity through allostery. This procedure is based on the construction of a plausible conformational path, which describes protein transition between known active and inactive conformations. The path is calculated by using a framework approach that steers and markedly improves the conjugate peak refinement method. The evolution of conformations along this path was used to identify a putative allosteric site that could regulate activation of Bacillus anthracis adenylyl cyclase toxin (EF) by calmodulin. Conformations of the allosteric site at different steps along the path from the inactive (free) to the active (bound to calmodulin) forms of EF were used to perform virtual screenings and propose candidate EF inhibitors. Several candidates then proved to inhibit calmodulin-induced activation in an in vitro assay. The most potent compound fully inhibited EF at a concentration of 10 μM. The compounds also inhibited the related adenylyl cyclase toxin from Bordetella pertussis (CyaA). The specific homology between the putative allosteric sites in both toxins supports that these pockets are the actual binding sites of the selected inhibitors.anthrax | transition path calculation | molecular dynamics | drug discovery | inhibition of protein-protein association

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Section: Discussionmentioning

confidence: 71%

Use of allostery to identify inhibitors of calmodulin-induced activation of Bacillus anthracis edema factor

Laine

Gonçalves

Karst

et al. 2010

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

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“…These include the sensitivity of inhibition to the presence of a large amount of secondary protein, such as bovine serum albumin (BSA) 12,13 , time-dependence of inhibition and sensitivity to the concentration of the target protein 8,9 . Inhibition of Sup35 polymerization displayed all of these traits.…”

Section: Online)mentioning

confidence: 99%

Small-molecule aggregates inhibit amyloid polymerization

et al. 2008

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Many amyloid inhibitors resemble molecules that form chemical aggregates, which are known to inhibit many proteins. Eight known chemical aggregators inhibited amyloid formation of the yeast and mouse prion proteins Sup35 and recMoPrP in a manner characteristic of colloidal inhibition. Similarly, three known anti-amyloid molecules inhibited β-lactamase in a detergent-dependent manner, which suggests that they too form colloidal aggregates. The colloids localized to preformed fibers and prevented new fiber formation in electron micrographs. They also blocked infection of yeast cells with Sup35 prions, which suggests that colloidal inhibition may be relevant in more biological milieus.The aggregation of proteins into amyloid fibers is associated with a growing list of diseases, including diabetes, Alzheimer's, Parkinson's, Huntington's and the prion diseases. In these disorders, proteins aggregate into long, unbranched fibers after misfolding into a β-sheet-rich conformation 1 . Though there are no approved therapies targeting amyloid formation directly, many organic molecules inhibit fibrillization in vitro [2][3][4][5][6][7] . Some, such as the chelator clioquinol (1), even have activity in vivo 4 . These results have inspired the hope of therapeutic applications for some molecules 3-5 . Curiously, many fibrillization inhibitors resemble molecules known to form promiscuous chemical aggregates. These colloidal particles are composed of small organic molecules and range in size from 50 to over 600 nm 8 . Once formed, they physically sequester proteins and inhibit enzymes nonspecifically 8,9 . Like many inhibitors of amyloid polymerization, these colloidal inhibitors are typically highly conjugated, hydrophobic and dye-like (Supplementary Table 1 online) 8,9 . A good example is the amyloid inhibitor Congo red (2), a dye that was one of the first molecules observed to exhibit colloidal inhibition 8 . The flavonoid baicalein (3), an inhibitor of α-synuclein polymerization 6 , resembles the known chemical aggregator quercetin (4), and 4,5-dianilinophthalimide (DAPH, 5), an inhibitor of Alzheimer's amyloid formation 2 , resembles the aggregator bisindoylmaleimide (6 ; Supplementary Fig. 1 online).Given that chemical aggregates function through enzyme sequestration, we wondered whether they might also sequester protein molecules from each other, thereby preventing amyloid polymerization. Here, we investigate this hypothesis in two classic amyloid-forming proteins: the yeast prion protein Sup35 (ref. 10 ) and the recombinant mouse prion protein recMoPrP 89-230 (ref. 11 ). We ask whether known chemical aggregators can inhibit amyloid fiber formation, whether known fibrillization inhibitors form colloidal aggregates and whether amyloid inhibition by these molecules is in fact mediated via colloidal aggregation.Eight known chemical aggregators and two known nonaggregators 8,9 were tested for inhibition of Sup35 fibrillization in a thioflavin T (ThT, 7) fluorescence assay. All eight inhibited Sup35 fibrillization b...

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“…The assay was miniaturized to a final volume of 3 μL in 1536-well format by direct volume reduction. Additionally, in order to prevent peptide and protein absorption to the polystyrene wells due to the increased surface-to-volume ratio and to minimize the interfering effect of promiscuous inhibitors acting via colloidal aggregate formation [4,5], we included detergent (0.01 % Tween-20) in the assay buffer.…”

Section: Assay Principle Miniaturization and Optimizationmentioning

confidence: 99%

“…In addition to the expansiveness and low definition of the interacting protein surfaces, technical issues pertaining to assay design and screening artifacts further complicate the identification of true PPI disrupters [3]. For example, colloidal aggregates spontaneously formed by certain compounds might reach the size and topology sufficient to perturb protein-protein interactions in a reproducible yet non-specific and biologically irrelevant manner [2,4,5].…”

Section: Introductionmentioning

confidence: 99%

Dual-fluorophore quantitative high-throughput screen for inhibitors of BRCT–phosphoprotein interaction

Simeonov

Yasgar

Jadhav

et al. 2008

Analytical Biochemistry

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Finding specific small-molecule inhibitors of protein-protein interactions remains a significant challenge. Recently, attention has grown toward "hot-spot" interactions where binding is dominated by a limited number of amino acid contacts, theoretically offering an increased opportunity for disruption by small molecules. Inhibitors of the interaction between BRCT (Cterminal portion of BRCA1, a key tumor suppressor protein with various functions), and phosphorylated protein (Abraxas, BACH1, CtIP) implicated in DNA damage response and repair pathways, should prove useful in studies of BRCA1's role in cancer and to potentially sensitize tumors to chemotherapeutic agents. We developed and miniaturized to 1536-well format and 3 μL final volume a pair of fluorescence polarization (FP) assays utilizing fluorescein-and rhodaminelabeled pBACH1 fragment. In order to minimize the effect of fluorescence artifacts and to increase the overall robustness of the screen, the 75,552 compound library members were each assayed against both the fluorescein-and rhodamine-labeled probe-protein complexes in separate but interleaved reactions. In addition, every library compound was tested over a range of concentrations, following the qHTS paradigm (Inglese et al, PNAS, 103, 1147(2006). Analyses of the screening results led to the selection and subsequent confirmation of 16 compounds active in both assays. Faced with a traditionally difficult protein-protein interaction assay, by performing two-fluorophore qHTS we were able to confidently select a number of actives for further studies.

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High-throughput assays for promiscuous inhibitors

Cited by 320 publications

References 14 publications

Use of allostery to identify inhibitors of calmodulin-induced activation of Bacillus anthracis edema factor

Use of allostery to identify inhibitors of calmodulin-induced activation of Bacillus anthracis edema factor

Small-molecule aggregates inhibit amyloid polymerization

Dual-fluorophore quantitative high-throughput screen for inhibitors of BRCT–phosphoprotein interaction

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