Serendipitous discovery of light-induced (In Situ) formation of an Azo-bridged dimeric sulfonated naphthol as a potent PTP1B inhibitor.

BACKGROUND: Protein tyrosine phosphatases (PTPs) like dual specificity phosphatase 5 (DUSP5) and protein tyrosine phosphatase 1B (PTP1B) are drug targets for diseases that include cancer, diabetes, and vascular disorders such as hemangiomas. The PTPs are also known to be notoriously difficult targets for designing inihibitors that become viable drug leads. Therefore, the pipeline for approved drugs in this class is minimal. Furthermore, drug screening for targets like PTPs often produce false positive and false negative results. RESULTS: Studies presented herein provide important insights into: (a) how to detect such artifacts, (b) the importance of compound re-synthesis and verification, and (c) how in situ chemical reactivity of compounds, when diagnosed and characterized, can actually lead to serendipitous discovery of valuable new lead molecules. Initial docking of compounds from the National Cancer Institute (NCI), followed by experimental testing in enzyme inhibition assays, identified an inhibitor of DUSP5. Subsequent control experiments revealed that this compound demonstrated time-dependent inhibition, and also a time-dependent change in color of the inhibitor that correlated with potency of inhibition. In addition, the compound activity varied depending on vendor source. We hypothesized, and then confirmed by synthesis of the compound, that the actual inhibitor of DUSP5 was a dimeric form of the original inhibitor compound, formed upon exposure to light and oxygen. This compound has an IC50 of 36 µM for DUSP5, and is a competitive inhibitor. Testing against PTP1B, for selectivity, demonstrated the dimeric compound was actually a more potent inhibitor of PTP1B, with an IC50 of 2.1 µM. The compound, an azo-bridged dimer of sulfonated naphthol rings, resembles previously reported PTP inhibitors, but with 18-fold selectivity for PTP1B versus DUSP5. CONCLUSION: We report the identification of a potent PTP1B inhibitor that was initially identified in a screen for DUSP5, implying common mechanism of inhibitory action for these scaffolds.

Downregulation of lysosomal trafficking in ARPE19 cells leads to decreased transfection efficiency at high passage.

PURPOSE: ARPE19 cells are a commonly used cell culture model for the study of retinal pigment epithelial cell biology and pathologies. However, numerous studies have demonstrated that ARPE19 undergo morphologic, transcriptomic and genomic alterations over time and with increasing passage number. Herein, we explore the mechanisms underlying increased resistance to the delivery of exogenous genetic material via transfection in ARPE19 cells using mass spectrometry. METHODS: ARPE19 cells (N=5 wells/reagent) were seeded in 6-well plates at passages 24 through 30. At 70% confluency an mCherry reporter construct was delivered via transfection using Lipofectamine 3000, Lipofectamine LTX, Lipofectamine Stem, or PEI (polyethylenimine) reagents. After 72 hours, transfection efficiency was quantified by fluorescence microscopy and flow cytometry. Mass spectrometry and immunofluorescence of ARPE19 cells were performed at passages 24 and 30 to evaluate altered protein synthesis and localization between passage numbers. RESULTS: ARPE19 transfection showed a maximum transfection efficiency of 32.4% at P26 using Lipofectamine 3000 reagent. All lipofectamine based reagents demonstrated statistically significant decreases in transfection efficiency between passages 24 and 30. Mass spectrometry analysis revealed 18 differentially expressed proteins, including down-regulation of clathrin light chain B (CLTB) and legumain (LGMN) that was confirmed via immunofluorescence imaging, which indicated altered intracellular localization. CONCLUSIONS: ARPE19 cells demonstrate passage number dependent changes in lipofectamine-based transfection efficiency. Mass spectrometry and immunofluorescence indicates the observed decrease in transfection efficiency involves the dysregulation of endocytosis and intracellular endolysosomal trafficking at later passages. TRANSLATIONAL RELEVANCE: This study contributes to mounting evidence for changes in ARPE19 cell physiology with increasing passage number. This information is of value for the continued use of ARPE19 cells as a model system for RPE biology and the development of therapeutics.

Dual Specificity Phosphatase 5-Substrate Interaction: A Mechanistic Perspective.

The mammalian genome contains approximately 200 phosphatases that are responsible for catalytically removing phosphate groups from proteins. In this review, we discuss dual specificity phosphatase 5 (DUSP5). DUSP5 belongs to the dual specificity phosphatase (DUSP) family, so named after the family members' abilities to remove phosphate groups from serine/threonine and tyrosine residues. We provide a comparison of DUSP5's structure to other DUSPs and, using molecular modeling studies, provide an explanation for DUSP5's mechanistic interaction and specificity toward phospho-extracellular regulated kinase, its only known substrate. We also discuss new insights from molecular modeling studies that will influence our current thinking of mitogen-activated protein kinase signaling. Finally, we discuss the lessons learned from identifying small molecules that target DUSP5, which might benefit targeting efforts for other phosphatases. © 2017 American Physiological Society. Compr Physiol 7:1449-1461, 2017.