title: The effects of benzylsulfonyl-D-Ser-homoPhe-(4-amidino-benzylamide), a dual plasmin and urokinase inhibitor, on facial skin barrier function in subjects with sensitive skin.
abstract: OBJECTIVE
  The aim of this study was to optimize the synthesis of the plasmin and urokinase (uPA) inhibitor benzylsulfonyl-D-Ser-homoPhe-(4-amidino-benzylamide) (BSFAB), to characterize its activity and mechanism of action and to assess its use to improve stratum corneum (SC) barrier function.
  
  
  METHODS
  Peptide coupling methods were used to synthesize BSFAB, and high-performance liquid chromatography-mass spectrometry (HPLC-MS) together with1H- and13C-nuclear magnetic resonance spectroscopy (NMR) were applied to clarify its structure and determine its purity. Its binding mode was determined by docking studies to the catalytic domains of plasmin and uPA. Inhibition constants (Ki) were determined by enzyme kinetic studies, and the effect of BSFAB on plasmin, uPA and transglutaminase 1 expression was evaluated in non-cytokine and cytokine-stimulated keratinocytes. A vehicle-controlled clinical study on SC barrier function was conducted on facial skin of subjects with self-perceived sensitive skin.
  
  
  RESULTS
  BSFAB was synthesized with high purity (97.3%). In silico studies indicated that the amidine moiety of BSFAB was anchored in the S1 pocket of both enzymes by binding to Asp189, Ser190 and Gly219, whereas the backbone of the D-Ser residue makes an anti-parallel β-sheet interaction with Gly216. BSFAB was shown to be an effective inhibitor of plasmin and uPA with Kivalues of 29 and 25 nM, respectively. BSFAB also inhibited keratinocyte-secreted protease activities in basal (plasmin inhibition 37.7%, P < 0.05 and uPA inhibition 96.6%, P < 0.01) and cytokine-induced conditions (plasmin inhibition 41.1%, P < 0.05 and uPA inhibition 97.0%, P < 0.001) and stimulated the gene expression of transglutaminase 1 in cytokine-stimulated keratinocytes (approximately 4.5 times increased expression, P < 0.01). Clinically, BSFAB was shown to improve SC barrier integrity (P < 0.02 on day 29) and subjective improvements in the perception of healthy skin (P < 0.05 on day 28).
  
  
  CONCLUSIONS
  BSFAB binds as a reversible competitive inhibitor to the active sites of plasmin and uPA. Additionally, BSFAB positively improved keratinocyte differentiation gene expression (transglutaminase 1). These effects were translated into improvements in SC barrier integrity clinically in subjects with dry and sensitive skin and improved their perception of having a healthy skin condition.
authors.affiliation: DSM Nutritional Products Ltd., Wurmisweg 571, 4303, Kaiseraugst, Switzerland.
authors.forename: R
authors.initials: R
authors.lastname: Voegeli
authors.affiliation: DSM Nutritional Products Ltd., Wurmisweg 571, 4303, Kaiseraugst, Switzerland.
authors.forename: P
authors.initials: P
authors.lastname: Wikstroem
authors.affiliation: DSM Nutritional Products Ltd., Wurmisweg 571, 4303, Kaiseraugst, Switzerland.
authors.forename: R
authors.initials: R
authors.lastname: Campiche
authors.affiliation: Philipps University, Department of Pharmacy, Marbacher Weg 6, 35032, Marburg, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Steinmetzer
authors.affiliation: DSM Nutritional Products Ltd., Wurmisweg 571, 4303, Kaiseraugst, Switzerland.
authors.forename: E
authors.initials: E
authors.lastname: Jackson
authors.affiliation: DSM Nutritional Products Ltd., Wurmisweg 571, 4303, Kaiseraugst, Switzerland.
authors.forename: M
authors.initials: M
authors.lastname: Gempeler
authors.affiliation: DSM Nutritional Products Ltd., Wurmisweg 571, 4303, Kaiseraugst, Switzerland.
authors.forename: D
authors.initials: D
authors.lastname: Imfeld
authors.affiliation: AVR Consulting Ltd, 26 Shavington Way, Northwich, Cheshire, UK.
authors.forename: A V
authors.initials: AV
authors.lastname: Rawlings
mesh_terms: D001798:Blood Proteins / Q000494:pharmacology*; D066241:Carbon-13 Magnetic Resonance Spectroscopy; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004151:Dipeptides / Q000494:pharmacology*; D005145:Face; D005341:Fibrinolysin / Q000037:antagonists & inhibitors*; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects; D013058:Mass Spectrometry; D066244:Proton Magnetic Resonance Spectroscopy; D012867:Skin / Q000187:drug effects*; D012879:Skin Physiological Phenomena / Q000187:drug effects*
chemical_list: D001798:Blood Proteins; D004151:Dipeptides; C000626011:benzylsulfonylseryl-homophenylalanine-(4-amidinobenzylamide); C031220:urokinase inhibitor; D005341:Fibrinolysin
keywords: chemical synthesis; computer modelling; plasmin and urokinase inhibitor; skin barrier; stratum corneum and sensitive skin