Development and Validation of a Robust and Sensitive Assay for the Discovery of Selective Inhibitors for Serine/Threonine Protein Phosphatases PP1α (<i>PPP1C</i>) and PP5 (<i>PPP5C</i>)

Swingle, Mark R.; Honkanen, Richard E.

doi:10.1089/adt.2014.603

Cited by 13 publications

(19 citation statements)

References 64 publications

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“…Cantharidin (2,3-dimethyl-7-oxabicyclo[2.2.1]heptane-2,3-dica rboxylic anhydride) is known to strongly inhibit the catalytic activity of PP1C, PP2AC, PP5C and PP6C (IC 50 ~ 0.2–8 µM), while having little or no inhibitory activity against PP2BC/calcineurin (PP3C) or PP7C (IC 50 > 1 mM) [18,20,52–54]. Determining the activity of inhibitors against PP4C has proved difficult, because to date the successful heterologous expression of active human PP4C in prokaryotes has not been successful.…”

Section: Resultsmentioning

confidence: 99%

Crystal structures and mutagenesis of PPP-family ser/thr protein phosphatases elucidate the selectivity of cantharidin and novel norcantharidin-based inhibitors of PP5C

Chattopadhyay

Swingle

Salter

et al. 2016

Biochemical Pharmacology

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Cantharidin is a natural toxin and an active constituent in a traditional Chinese medicine used to treat tumors. Cantharidin acts as a semi-selective inhibitor of PPP-family ser/thr protein phosphatases. Despite sharing a common catalytic mechanism and marked structural similarity with PP1C, PP2AC and PP5C, human PP4C was found to be insensitive to the inhibitory activity of cantharidin. To explore the molecular basis for this selectivity, we synthesized and tested novel C5/C6-derivatives designed from quantum-based modeling of the interactions revealed in the co-crystal structures of PP5C in complex with cantharidin. Structure–activity relationship studies and analysis of high-resolution (1.25 Å) PP5C-inhibitor co-crystal structures reveal close contacts between the inhibitor bridgehead oxygen and both a catalytic metal ion and a non-catalytic phenylalanine residue, the latter of which is substituted by tryptophan in PP4C. Quantum chemistry calculations predicted that steric clashes with the bulkier tryptophan side chain in PP4C would force all cantharidin-based inhibitors into an unfavorable binding mode, disrupting the strong coordination of active site metal ions observed in the PP5C co-crystal structures, thereby rendering PP4C insensitive to the inhibitors. This prediction was confirmed by inhibition studies employing native human PP4C. Mutation of PP5C (F446W) and PP1C (F257W), to mimic the PP4C active site, resulted in markedly suppressed sensitivity to cantharidin. These observations provide insight into the structural basis for the natural selectivity of cantharidin and provide an avenue for PP4C deselection. The novel crystal structures also provide insight into interactions that provide increased selectivity of the C5/C6 modifications for PP5C versus other PPP-family phosphatases.

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Section: Resultsmentioning

confidence: 99%

Crystal structures and mutagenesis of PPP-family ser/thr protein phosphatases elucidate the selectivity of cantharidin and novel norcantharidin-based inhibitors of PP5C

Chattopadhyay

Swingle

Salter

et al. 2016

Biochemical Pharmacology

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“…The coding sequence of human PPP1CA was expressed as a maltose binding protein fusion in a BL-21 strain of Escherichia coli and purified as previously described (50). Phosphohistone phosphatase assays were performed as previously described (50,51).…”

Section: Determination Of the Effect Of Lb-100 On Ppp1c Phosphatase Amentioning

confidence: 99%

The phosphatase inhibitor LB-100 acts synergistically with the NPR2 agonist BMN-111 to improve bone growth

Shuhaibar

Kaci

Egbert

et al. 2020

Preprint

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Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP (cGMP) in chondrocytes and severe short stature, causing achondroplasia (ACH) and acrosomelic dysplasia type Maroteaux, respectively. Previously we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3Y367C/+), and that in control growth plate chondrocytes, FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein. Here we tested whether a phosphatase inhibitor (LB-100) could enhance bone growth in ACH. In ex vivo imaging experiments using a FRET sensor to measure cGMP production in chondrocytes of living tibias from newborn mice, LB-100 counteracts the FGF-induced dephosphorylation and inactivation of NPR2. In ex vivo experiments with Fgfr3Y367C/+ mice, LB-100 in combination with BMN-111 increases the rate of femur growth by ∼25% vs BMN-111 alone, restores chondrocyte terminal differentiation, increases the proliferative growth plate area of the femur, and reduces the activity of the MAP kinase pathway. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH.GRAPHICAL ABSTRACT

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“…[43,87] In many more cases, detergents are included in the assay buffer for the primary screen right away, [56] which obviates the need for a separate counterscreen, even if this motivation is rarely mentioned explicitly. [30,88] In contrast, demonstrating stoichiometric inhibition by measuring potency at different concentrations of enzyme [25] typically is used later in the flowchart, [52] where the limited number of compounds allows for dose-response experiments. The same holds for direct measurements of compound aggregation which often are used in conjunction with other experimental methods, e.g.…”

Section: Experimental Identification Of Nonstoichiometric Inhibitors mentioning

confidence: 99%

Non-stoichiometric inhibition in integrated lead finding – a literature review

Klumpp

2015

Expert Opinion on Drug Discovery

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Over the last few years, awareness of non-stoichiometric inhibitors within screening libraries and HTS hit lists has considerably increased, not only in the pharmaceutical industry but also in the academic drug discovery community. This has resulted in a variety of methods to detect and handle such compounds. These range from in silico approaches to flag suspicious compounds, and counterassays to measure non-stoichiometric inhibition, to biophysical methods that positively demonstrate stoichiometric binding. In addition, novel technologies to verify target engagement within cells are becoming available. While still a time- and resource-consuming nuisance, non-stoichiometric inhibitors therefore do not fundamentally jeopardize the discovery of low molecular weight lead and drug candidates. Rather, they should be viewed as a manageable issue that with appropriate expertise can be overcome through integration of the above-mentioned approaches.

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Development and Validation of a Robust and Sensitive Assay for the Discovery of Selective Inhibitors for Serine/Threonine Protein Phosphatases PP1α (PPP1C) and PP5 (PPP5C)

Cited by 13 publications

References 64 publications

Crystal structures and mutagenesis of PPP-family ser/thr protein phosphatases elucidate the selectivity of cantharidin and novel norcantharidin-based inhibitors of PP5C

Crystal structures and mutagenesis of PPP-family ser/thr protein phosphatases elucidate the selectivity of cantharidin and novel norcantharidin-based inhibitors of PP5C

The phosphatase inhibitor LB-100 acts synergistically with the NPR2 agonist BMN-111 to improve bone growth

Non-stoichiometric inhibition in integrated lead finding – a literature review

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