Cryptotanshinone from Salvia miltiorrhiza Roots Reduces Cytokeratin CK1/10 Expression in Keratinocytes by Activation of Peptidyl-prolyl-cis-trans-isomerase FKBP1A

Abstract

Cryptotanshinone (CTS) (1 µM) from the roots of exerts a strong influence on the terminal differentiation of human keratinocytes (HaCaT cell line, primary natural human keratinocytes) and downregulates the expression of differentiation-specific cytokeratins CK1 and CK10 on protein and gene level. Other differentiation specific proteins as involucrin, filaggrin, loricrin, and transglutaminase were not affected to a higher extent. CTS (1 µM) did not influence the cell viability and the proliferation of keratinocytes. Using a combination of drug affinity response target stability assay in combination with a proteomic approach and multivariate statistics for target elucidation, peptidyl-prolyl-cis-trans-isomerase FKBP1A (known target of inhibitors such as tacrolimus or rapamycin) was addressed as potential molecular target of CTS. The interaction of CTS with FKBP1A was additionally shown by thermal shift and enzymatic activity assays. Interestingly, CTS served as an activator of FKBP1A, which led to a reduced activity of the TGF receptor pathway and therefore to a diminished CK1 and CK10 expression. The combination of the FKBP1A activator CTS with the inhibitor tacrolimus neutralized the effects of both compounds. From these data, a potential dermatological use of CTS and CTS-containing plant extracts (e.g., hydroalcoholic extract from the roots ) for keratinopathic ichthyosis, a disease characterized by overexpression of CK1 and CK10, is discussed. This study displays an experimental strategy for combining phytochemical aspects on active natural products with systematic identification of molecular targets on gene, protein, and cell level.

Recent advances in identifying protein targets of bioactive natural products

“…Moreover, DARTS confirmed the targets of polyphyllin D, a natural product effective against NSCLC [ 49 ], ergolide, a sesquiterpene lactone natural product with anti-inflammatory and anticancer activities used to treat inflammatory diseases [ 50 ], aconitine, an active compound exhibiting cardiotoxic effects found in Aconitum species [ 51 ], crellastatin A, a cytotoxic sulfated bis-steroid isolated from the Vanuatu Island marine sponge Crella sp. [ 52 ], cryptotanshinone, isolated from the roots of Salvia miltiorrhiza inhibiting the terminal differentiation of human keratinocytes [ 53 ], and grape seed extract in anti-colorectal cancer applications [ 54 ] ( Table 6 ).…”

Target identification of small molecules: an overview of the current applications in drug discovery

“…Genetic screening is another unique, unbiased method for cellular drug target identification [119,122]. In this method, a knockout library of the selective target(s) of interest was designed (utilizing RNA interference (RNAi) or CRISPR-Cas9) and screened for loss-of-function of the probable drug target(s).…”

An update of label-free protein target identification methods for natural active products

“…Because the method relies on the intuitive recognition of different intensities of protein bands on SDS-PAGE gels, the actual sampled proteome space for DARTS analysis was limited to relatively high abundance proteins. Recently, DARTS samples were directly analysed using MS without gel electrophoresis to increase protein coverage, enabling the identification of protein targets of NAPs such as cryptotashinone and arctigenin 111 , 112 .…”