Cyplecksins Are Covalent Inhibitors of the Pleckstrin Homology Domain of Cytohesin

Hussein, Mohamed M. A.; Bettio, Martina; Schmitz, Anton; Hannam, Jeffrey S.; Theis, Julian; Mayer, Günter; Dosa, Stefan; Gütschow, Michael; Famulok, Michael

doi:10.1002/anie.201302207

Cited by 9 publications

(9 citation statements)

References 35 publications

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“…of lipid-transfer proteins. Our data are also able to reveal the consequences of disease-associated mutations on the binding specificity of PH domains, which may contribute to current efforts in the design of small-molecule inhibitors of PH domains (Hussein et al, 2013).…”

Section: Targeting Of Ph Domains To Organelle and Pm Phosphoinositidesmentioning

confidence: 92%

“…These lipids have a role in virtually all biological processes (van Meer, 2005) through their extensive, regulated association with other lipids and proteins. The significance of these regulatory circuits is evident from the variety of human disorders arising from altered protein-lipid interactions (Bayascas et al, 2008;Lindhurst et al, 2011;Zü chner et al, 2005;Kö berlin et al, 2015), which constitute attractive targets for pharmaceutical drug development (Hussein et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Lipid Cooperativity as a General Membrane-Recruitment Principle for PH Domains

et al. 2015

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Many cellular processes involve the recruitment of proteins to specific membranes, which are decorated with distinctive lipids that act as docking sites. The phosphoinositides form signaling hubs, and we examine mechanisms underlying recruitment. We applied a physiological, quantitative, liposome microarray-based assay to measure the membrane-binding properties of 91 pleckstrin homology (PH) domains, the most common phosphoinositide-binding target. 10,514 experiments quantified the role of phosphoinositides in membrane recruitment. For most domains examined, the observed binding specificity implied cooperativity with additional signaling lipids. Analyses of PH domains with similar lipid-binding profiles identified a conserved motif, mutations in which-including some found in human cancers-induced discrete changes in binding affinities in vitro and protein mislocalization in vivo. The data set reveals cooperativity as a key mechanism for membrane recruitment and, by enabling the interpretation of disease-associated mutations, suggests avenues for the design of small molecules targeting PH domains.

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Section: Targeting Of Ph Domains To Organelle and Pm Phosphoinositidesmentioning

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Lipid Cooperativity as a General Membrane-Recruitment Principle for PH Domains

et al. 2015

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“…The change in thermophoresis can be used to derive dissociation constants (K d ) within minutes by sequentially scanning capillaries with varying ligand concentrations. MST has been shown to be well suited to detect binding of small molecules, fragments, or even ions to biomolecules, 6 – 8 thus making this technique applicable to FBLD. Moreover, since movement of fluorescent molecules through a detection volume is monitored over time, additional information about protein aggregation and denaturation can be derived from the shape of MST traces.…”

Section: Introductionmentioning

confidence: 99%

An Automated Microscale Thermophoresis Screening Approach for Fragment-Based Lead Discovery

et al. 2016

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Fragment-based lead discovery has proved to be an effective alternative to high-throughput screenings in identifying chemical matter that can be developed into robust lead compounds. The search for optimal combinations of biophysical techniques that can correctly and efficiently identify and quantify binding can be challenging due to the physicochemical properties of fragments. In order to minimize the time and costs of screening, optimal combinations of biophysical techniques with maximal information content, sensitivity, and robustness are needed. Here we describe an approach utilizing automated microscale thermophoresis (MST) affinity screening to identify fragments active against MEK1 kinase. MST identified multiple hits that were confirmed by X-ray crystallography but not detected by orthogonal methods. Furthermore, MST also provided information about ligand-induced aggregation and protein denaturation. The technique delivered a large number of binders while reducing experimentation time and sample consumption, demonstrating the potential of MST to execute and maximize the efficacy of fragment screening campaigns.

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“…However, targeting the catalytic domain of these enzymes to develop inhibitor remained quite challenging. For example, several potent inhibitors of AKT1 enzyme turn out to be relatively toxic, presumably due to the inhibition of other ser/threonine kinases [20] , [43] On the other hand, small molecules such as DPIEL and perifosine developed for the inhibition of PIP/PH-domain interaction are comparatively nontoxic and offer a better therapeutic strategy than inhibitors for ATP-site [21] , [41] In recent years, studies have been carried out to identify phosphate or nonphosphate containing small molecules as being inhibitors for PIP-binding domains [8] , [20] , [44] , [45] …”

Section: Resultsmentioning

confidence: 99%

Insights into the inhibitory mechanism of triazole-based small molecules on phosphatidylinositol-4,5-bisphosphate binding pleckstrin homology domain

Gorai

Bagdi

Borah

et al. 2015

Biochemistry and Biophysics Reports

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BackgroundPhosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important regulator of several cellular processes and a precursor for other second messengers which are involved in cell signaling pathways. Signaling proteins preferably interact with PI(4,5)P2 through its pleckstrin homology (PH) domain. Efforts are underway to design small molecule-based antagonist, which can specifically inhibit the PI(4,5)P2/PH-domain interaction to establish an alternate strategy for the development of drug(s) for phosphoinositide signaling pathways.MethodsSurface plasmon resonance, molecular docking, circular dichroism, competitive Förster resonance energy transfer, isothermal titration calorimetric analyses and liposome pull down assay were used.ResultsIn this study, we employed 1,2,3-triazol-4-yl methanol containing small molecule (CIPs) as antagonists for PI(4,5)P2/PH-domain interaction and determined their inhibitory effect by using competitive-surface plasmon resonance analysis (IC50 ranges from 53 to 159 nM for PI(4,5)P2/PLCδ1-PH domain binding assay). We also used phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], PI(4,5)P2 specific PH-domains to determine binding selectivity of the compounds. Various physicochemical analyses showed that the compounds have weak affect on fluidity of the model membrane but, strongly interact with the phospholipase C δ1 (PLCδ1)-PH domains. The 1,2,3-triazol-4-yl methanol moiety and nitro group of the compounds are essential for their exothermic interaction with the PH-domains. Potent compound can efficiently displace PLCδ1-PH domain from plasma membrane to cytosol in A549 cells.ConclusionsOverall, our studies demonstrate that these compounds interact with the PIP-binding PH-domains and inhibit their membrane recruitment.General significanceThese results suggest specific but differential binding of these compounds to the PLCδ1-PH domain and emphasize the role of their structural differences in binding parameters. These triazole-based compounds could be directly used/further developed as potential inhibitor for PH domain-dependent enzyme activity.

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Cyplecksins Are Covalent Inhibitors of the Pleckstrin Homology Domain of Cytohesin

Cited by 9 publications

References 35 publications

Lipid Cooperativity as a General Membrane-Recruitment Principle for PH Domains

Lipid Cooperativity as a General Membrane-Recruitment Principle for PH Domains

An Automated Microscale Thermophoresis Screening Approach for Fragment-Based Lead Discovery

Insights into the inhibitory mechanism of triazole-based small molecules on phosphatidylinositol-4,5-bisphosphate binding pleckstrin homology domain

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