Active Site Targeting of Hedgehog Precursor Protein with Phenylarsine Oxide

Owen, Timothy S.; Xie, Xie Jian; Laraway, Benjamin; Ngoje, George; Wang, Chunyu; Callahan, Brian P.

doi:10.1002/cbic.201402421

Cited by 16 publications

(35 citation statements)

References 22 publications

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“…No evidence for decomposition of C-H-Y in the presence of ST044643 was found by SDS-PAGE analysis ( Supporting Figure 6B ). We have observed similar quenching behavior with C-H-Y in the presence of phenylarsine oxide, an active-site covalent inhibitor of cholesterolysis [24]. To explore the specificity of this effect, ST044643 was added to the control construct, C-Y, in which the HhC segment is replaced with a glycine-serine linker; no significant change in the FRET ratio of C-Y was apparent with added ST044643 (p= 0.276, n= 15) ( Supporting Figure 6C ).…”

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confidence: 76%

“…The key construct, termed C - H - Y , has cyan ( C ) and yellow ( Y ) fluorescent proteins [23] fused to the amino and carboxyl termini of H hC. Recently we reported that C-H-Y could be used to detect binding of HhC by an irreversible inhibitor of cholesterolysis [24]. Here we pursue the expectation that cholesterolysis of C-H-Y will yield cholesterol-modified CFP and HhC-YFP, with a relatively large change in the FRET ratio (i.e.…”

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confidence: 99%

“…Prior to cholesterol addition, samples were equilibrated at 30 °C in the plate reader. FRET values calculated during this preincubation proved useful in identifying compounds that interfered optically with the assay output, as well as compounds that were not optically active but could alter the FRET signal from C-H-Y presumably through direct binding [24]. Reactions were initiated with cholesterol (50 µM, or 250 µM, final) and monitored continuously for a period of 1 to 3 h.…”

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confidence: 99%

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Förster resonance energy transfer-based cholesterolysis assay identifies a novel hedgehog inhibitor

Owen

Ngoje

Lageman

et al. 2015

Analytical Biochemistry

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Hedgehog (Hh) proteins function in cell/cell signaling processes linked to human embryo development and the progression of several types of cancer. Here we describe an optical assay of hedgehog cholesterolysis, a unique autoprocessing event critical for Hh function. The assay uses a recombinant FRET-active hedgehog precursor whose cholesterolysis can be monitored continuously in multi-well plates (dynamic range, 4; Z’, 0.7), offering advantages in throughput over conventional SDS-PAGE assays. Application of the optical assay in a pilot small molecule screen produced a novel cholesterolysis inhibitor (apparent IC50, 5×10−6 M) that appears to inactivate hedgehog covalently by a SNAr mechanism.

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confidence: 76%

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confidence: 99%

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confidence: 99%

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Förster resonance energy transfer-based cholesterolysis assay identifies a novel hedgehog inhibitor

Owen

Ngoje

Lageman

et al. 2015

Analytical Biochemistry

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“…We used a construct composed of human HhN and Drosophila HhC as the precursor (SHhN-DHhC) (Fig. 1B), which has been previously used to characterize inhibitors of Hh autoprocessing (28). The autoprocessing reaction was carried out at pH 7.1, the physiologic pH in endoplasmic reticulum where Hh autoprocessing occurs (29).…”

Section: Zinc Inhibits Hh Autoprocessingmentioning

confidence: 99%

Zinc Inhibits Hedgehog Autoprocessing

Xie

Owen

Xia³

et al. 2015

Journal of Biological Chemistry

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Background: In many types of cancers zinc deficiency and overproduction of Hedgehog (Hh) ligand co-exist. Results: Zinc binds to the active site of the Hedgehog-intein (Hint) domain and inhibits Hh ligand production both in vitro and in cell culture. Conclusion: Zinc influences the Hh autoprocessing.Significance: This study uncovers a novel mechanistic link between zinc and the Hh signaling pathway.

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“…14 These include cyclopamine 15 and vismodegib 16 , which correspond to the archetypal member and the first FDA-approved drug, respectively, belonging to this class of Hh pathway antagonists. Compounds that target downstream components of this pathway 14 or processes involved in Shh maturation 17 have also been reported. In stark contrast, potent inhibitors of the Shh/Patched protein-protein interaction have remained elusive.…”

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Design and Evolution of a Macrocyclic Peptide Inhibitor of the Sonic Hedgehog/Patched Interaction

et al. 2017

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The Hedgehog (Hh) signaling pathway plays a central role during embryonic development and its aberrant activation has been implicated in the development and progression of several human cancers. Major efforts toward the identification of chemical modulators of the Hedgehog pathway has yielded several antagonists of the GPCR-like Smoothened receptor. In contrast, potent inhibitors of the Sonic Hedgehog/Patched interaction, the most upstream event in ligand-induced activation of this signaling pathway, have been elusive. To address this gap, a genetically encoded cyclic peptide was designed based on the Shh-binding loop of Hedgehog-Interacting Protein (HHIP) and subjected to multiple rounds of affinity maturation through the screening of macrocyclic peptide libraries produced in E. coli cells. Using this approach, an optimized macrocyclic peptide inhibitor (HL2-m5) was obtained that binds Shh with a KD of 170 nM, which corresponds to a 120-fold affinity improvement compared to the parent molecule. Importantly, HL2-m5 is able to effectively suppress Shh-mediated Hedgehog signaling and Gli-controlled gene transcription in living cells (IC50 = 250 nM), providing the most potent inhibitor of the Sonic Hedgehog/Patched interaction reported to date. This first-in-class macrocyclic peptide modulator of the Hedgehog pathway is expected to provide a valuable probe for investigating and targeting ligand-dependent Hedgehog pathway activation in cancer and other pathologies. This work also introduces a general strategy for the development of cyclopeptide inhibitors of protein-protein interactions.

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Active Site Targeting of Hedgehog Precursor Protein with Phenylarsine Oxide

Cited by 16 publications

References 22 publications

Förster resonance energy transfer-based cholesterolysis assay identifies a novel hedgehog inhibitor

Förster resonance energy transfer-based cholesterolysis assay identifies a novel hedgehog inhibitor

Zinc Inhibits Hedgehog Autoprocessing

Design and Evolution of a Macrocyclic Peptide Inhibitor of the Sonic Hedgehog/Patched Interaction

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