Kallikrein 5 1536 HTS
Human kallikrein 5 (hK5) is a member of the human tissue kallikrein family, which contains 15 kallikrein-like serine proteases (1). It is synthesized as a 293 amino acid zymogen, and loses a 29 amino acid signal peptide upon secretion, followed by cleavage at the Arg66-Ile67 bond, which releases a 37 amino acid activation peptide, resulting in a 237 amino acid mature enzyme (2). hK5 generally more ..
BioActive Compounds: 1264
Molecular Library Screening Center Network (MLSCN)
Penn Center for Molecular Discovery (PCMD)
Assay Provider: Scott L. Diamond, University of Pennsylvania
MLSCN Grant: X01-MH076406-01
Human kallikrein 5 (hK5) is a member of the human tissue kallikrein family, which contains 15 kallikrein-like serine proteases (1). It is synthesized as a 293 amino acid zymogen, and loses a 29 amino acid signal peptide upon secretion, followed by cleavage at the Arg66-Ile67 bond, which releases a 37 amino acid activation peptide, resulting in a 237 amino acid mature enzyme (2). hK5 generally exhibits a trypsin-like specificity for P1-Arg over P1-Lys residues, and has been observed to digest different extracellular matrix and plasma proteins (3, 4). Protein inhibitors include alpha2-antiplasmin, antithrombin III and alpha2-macroglobulin (4). In addition, Zn2+ and other divalent cations strongly inhibit hK5 (5).
hK5 is highly expressed in skin tissue, specifically the outermost layer of the skin, the stratum corneum, but is found in other tissues, including brain, placenta, and kidney (2). Within the stratum corneum, hK5 is found in the upper spinous and granular layers, where keratinocytes undergo turnover and differentiation into flattened structures called corneocytes that make up the stratum corneum (6, 7). It has also been characterized as a marker for ovarian and breast cancer, having previously been shown to be differentially regulated in several different hormone-dependent malignancies (8, 9).
Recently, hK5 was found to be involved in the common, chronic skin condition rosacea (10). This condition involves dilation of blood vessels near the surface of the skin, resulting in erythema accompanied by visible blood vessels along the middle of the face. Abnormal cutaneous vasculature is often cited as an underlying cause, but immune responses associated with microbes, and the resultant acne, have also been suggested as causes. To this end, Yamasaki et al. report that high levels of the antimicrobial peptide cathelicidin were found in rosacea lesions, suggesting that abnormal innate immune responses are to blame (10). High levels of hK5 expression were also found, and its expression was colocalized with cathilicidin, while lower levels were observed on normal skin. In situ zymography showed that protease activity overall was elevated in rosacea, while absent in normal skin, and the serine protease inhibitors aprotinin and AEBSF suppressed the protease activity in rosacea lesions. Thus, isolation of inhibitors against hK5, as well as other proteases from the skin, could allow for development of a therapeutic topical agent that would alleviate rosacea.
HTS was performed using 214,203 compounds of the MLSCN library individually plated into 10ul 1536 compound plates at a concentration of 2.5 mM each, which were diluted 500-fold into 5 ul 1536 well assay plates (final concentration 5 uM each compound). The assay used to test for percent inhibition was a fluorescence assay utilizing hydrolysis of Boc-Vsl-Pro-Arg-AMC, as first described by Michael et al. (4).
1.E. P. Diamandis et al., Clin Chem 46, 1855 (Nov, 2000).
2.M. Brattsand, T. Egelrud, J Biol Chem 274, 30033 (Oct 15, 1999).
3.M. Debela et al., J Biol Chem 281, 25678 (Sep 1, 2006).
4.I. P. Michael et al., J Biol Chem 280, 14628 (Apr 15, 2005).
5.I. P. Michael et al., J Biol Chem 281, 12743 (May 5, 2006).
6.A. Ishida-Yamamoto et al., J Invest Dermatol 124, 360 (Feb, 2005).
7.B. Sondell, L. E. Thornell, T. Egelrud, J Invest Dermatol 104, 819 (May, 1995).
8.E. P. Diamandis et al., Tumour Biol 24, 299 (Nov-Dec, 2003).
9.G. M. Yousef et al., Cancer Res 63, 3958 (Jul 15, 2003).
10.K. Yamasaki et al., Nat Med 13, 975 (Aug, 2007).
Human kallikrein 5 (hK5) was purchased from R&D Systems (Cat #1108-SE). Substrate Boc-Val-Pro-Arg-AMC was from Bachem (Cat #I-1120.0050). Assay buffer consisted of 50 mM Tris, pH 8.0, 0.05% Tween 20. 1536-well black plates were from Corning (Item #3728).
hK5 (0.60 ug/mL) was incubated with Boc-Phe-Ser-Arg-AMC substrate (15 uM) in 5 uL of assay buffer (see above) for 2 hr at room temperature. HTS was performed using 5 uM compound.
1.Fill 1536 well plate with 4 uL of Boc-Glu-Ala-Arg-AMC substrate (18.75 uM in 1x assay buffer) using Aquamax DW4
2.Add 1 uL assay buffer to columns 1, 2, 45, and 46 using Aquamax DW4
3.Add 10 nL of compound (0.25 mM in DMSO) using 3 transfers of 3.3nl with the Evolution 1536 pintool (washed with water and isopropanol after each transfer)
4.Add 1 uL enzyme (1.35 ug/mL in assay buffer) using Aquamax DW4 to all columns except 1, 2, 45, and 46
5.Incubate for 2 hr at room temperature
6.Read fluorescence (excitation 355, emission 460) on Envision reader
Data were analyzed in IDBS ActivityBase. Each HTS plate a single test compound (5 uM in 0.2% DMSO) in columns 5-44, controls (enzyme, no compound) in columns 3, 4, 47, and 48, and blanks (no enzyme) in columns 1, 2, 45, and 46. HTS percent inhibition was calculated for each compound from the signal in fluorescence units (FU) and the mean of the plate controls and the mean of the plate blanks using the following equation:
% Inhibition = 100*(1-((signal-blank mean)/(control mean-blank mean)))
Activity scores were calculated as follows:
For positive percent inhibition, score = 0.4 x Percent inhibition
For negative percent inhibition, score = 0
Activity outcome is reported as follows:
(1) Percent inhibition >= 40 = active
(2) Percent inhibition < 40 = inactive
Analysis of screening results
HTS plate statistics were as follows:
Number of plates = 184
Median Z-factor = 0.74
Median control percent CV = 6.27
Maximum control percent CV = 15.85
A hit cut-off of 40% inhibition was selected. Based on this cutoff, a hit rate of 0.59% was observed.
This assay was submitted to the PCMD by Scott Diamond, assay development and HTS were conducted by Paul Riley and Sahil Batta, and data were submitted by Paul Riley, Sahil Batta, and Andrew Napper all of the University of Pennsylvania.
** Test Concentration.
Data Table (Concise)