High Throughput screening for modulators of tissue-kallikrein expression Ioannis Prassas, Miltiadis Paliouras and Eleftherios P. Diamandis 1 The Samuel Lunenfeld Research Institute Department of Laboratory Medicine and Pathobiology, University of Toronto 2Samuel Lunenfeld Research Institute, Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada ABSTRACT METHODS RESULTS Human tissue kallikreins (KLKs) comprise a subgroup of 15 homologous secreted serine proteases. A) Initial screening Primarily known for their clinical use as cancer biomarkers (e.g. PSA), KLKs are found to be PRESTWICK SPECTRUM 80 LOPAC 60 implicated in cancer-related processes including invasion, metastasis and angiogenesis. Therefore, 60 70 50 ROBOT modulators of expression of KLKs might help us understand their regulatory pathways and be of 50 60 2X SD 2X SD 50 40 40 40 DMSO therapeutic value. s 30 30 DMSO 2x SD KLK5 (ug/ L) 30 20 20 2X SD DMSO A cell-based high throughput screening of 3 compound libraries (~4000 small molecules) was 2X SD 20 KLK5 (ug/L) KLK5 10 performed under fully automated robotic facilities and sensitive immunoassays (ELISA) were used to 10 10 2x SD 0 0 0 (ug/L) KLK5 levels expression 0 200 400 600 800 1000 1200 1400 0 200 400 600 800 1000 1200 measure KLKs in a panel of different cell lines. The ‘validated leads’ were further assayed in-vitro, to compounds 0 500 1000 1500 2000 2500 measure their effectiveness (IC50s) at protein (ELISA) and mRNA levels (RT-PCR), cytotoxicity (LDH) compounds compounds and cell viability (MTT assay). Fig.1. Initial Screening of the 3 libraries ( ~4000 compounds) identified 66 small molecules as The initial screening resulted in 66 ‘putative hits’ that decreased KLK expression by at least 50% over putative hits. Those were further analysed in the ‘secondary-validation’ step. Step 1. The robot collects and seeds a 50000 cells/well in a 96-well plate. The cells stay in serum containing media for 48 h, in order to control. Secondary screening and mini-dose-response assays reduced this number to 21 ‘leads’. detach to the wells. These were classified into 3 different functional groups. IC50 calculations revealed the different LOPAC 'Leads' Prestwick 'Leads" Spectrum 'Leads' 40 60 Cycloheximide 35 potency of the compounds within the same group and the most potent ones were further analyzed in 35 CYMARIN 50 Anisomycin 30 30 DIGITOXIN g/L) Cephaeline dihydrochloride Brefeldin A µ 40 heptahydrate 25 GENTIAN VIOLET ROBOT vitro. Novel inhibitors of KLK expression, acting at low concentrations (10-50 nM) were identified. 25 Emetine dihydrochloride hydrate Emetine dihydrochloride STROPHANTHIDIN Ouabain Lasalocid sodium salt 20 20 30 Rotenone Strophantine octahydrate PERUVOSIDE Wortmannin 15 Further experiments to elucidate their exact mode of function are ongoing. 15 CONVALLATOXIN 20 Digoxin Thapsigargin 10 Puromycin dihydrochloride CYCLOHEXIMIDE hK5 Concentration (ug/L) hK5 10 KLK5 ExpressionKLK5 ( 10 Benzethonium chloride 5 NERIIFOLIN This HTS-technique proved effective in identifying inhibitors of expression of our target protein 5 KLK5 expression (ug/L) DIGOXIN 0 0 0 1uM 2uM 5uM 1uM 2.5uM 5uM 1uM 2uM 5uM throughout large chemical libraries. The precise pathways that seem to be blocked by these inhibitors Compound Concentrations Concentrations concentration and the therapeutic applications of their use, are currently under investigation. Figure 2. Mini dose-response treatments with the ‘putative hits’ separated the ‘real leads’ from the false positives. A total of 21 lead-compounds were identified and proceeded to the IC50-calculation step. Step 2. The robot washes the wells and changes the medium (serum-free). Then it dispenses the chemical library. We leave 30h for the KLKs to accumulate, and the robot collects the supernatants, INTRODUCTION which are sent to ELISA. OUABAIN Inhibitors of KLK5 expression Relative Cytotoxicity ¾ The KLK family is a subgroup of secreted serine peptidases, comprised of fifteen PUROMY CIN 1 CONV ALLA TOXIN members . 120 12 DIGITOXIN B) Selected compound concentrations n 100 10 BENZ ETHONIUM 80 5uM 8 for IC50s: ¾ Although primarily known for their clinical applicability as cancer biomarkers (eg PSA), 60 GENTIAN V IOLET 2uM 6 40 DIGOXIN 2uM accumulating evidence implicates KLKs in many cancer related processes, such as cell- 20 1uM 4 PERUVOSIDE 20uM 10uM 5uM % inhibitio ROTENONE Relative LDHRelative units 2 0 te 2uM a e e e e DMSO20uM 10uM5uM 2uM5uM 2 r n t n in a rid g EMETINE yd oxin xin lorid o ycin one 0 growth regulation, angiogenesis, invasion and metastasis t to yd r h Violet losid n annin ar ah la h c m ig l gi n Chl sa Digoxin m s pt Neriifolin Di Cymari ro phantidi Ouabaina ro rt CYCLOHEXIMIDE va o m La u Rote o DMSO 20uM 5uM 1uM 500nM 100nM DMSO 2uM he n Peruvosi de Brefeldin A iu P 20uM 10uM5uM 2uM5uM Anisomycin ihyd n W Thap Co Cycloheximid d Str Genti o ide tine Octa STROPHANTIDINE lor n compound concentration ch a etine o h enzeth BREFELDIN A ¾ Given that KLK5 is shown : dr rop Em B St dihy 100nM line 1uM 500nM 200nM e ha • to efficiently cleave the extracellular matrix components, collagens type I, II, III, IV, Cep DMSO1uM 500nM5uM 200nM2uM 100nM IC50 calculation: fibronectin and laminin in-vitro 3 compounds DMSO1uM 500nM5uM 200nM2uM 100nM In 12-well plates seed 500000 cells/well. 50nM Cells only DMSO Following the same protocol (manually), • to be responsible for the degradation of corneodesmosomal cadherin in LEKTI (-/-) collect the supernatant and send to ELISA 4 Fig.3. Mini-dose responses with the 21 lead-compounds (A) and their relative cytotoxicity DMSO50nM Cells5uM only DMSO2uM ELISA reader knockout-mice (+measure LDH, +collect RNA) (LDH measurement) DMSO50nM Cells5uM only DMSO2uM • to represent important signalling molecule through its ability to regulate PARs in physiological conditions5, 3 FUNCTIONAL GROUPS Æ we assumed that it can potentially represent a novel tharapeutic target CARDIAC Fig.4. Classification of the 21 compounds. CONCLUSION ¾ Identification of small molecules as modulators of KLK5-expression would help us GYCOSIDES According to their mode of function these elucidate their regulatory pathways, understand the mechanisms that lead to their aberrant compounds can be grouped to three The HTS worked very efficiently for our assay. Out of a total of 4000 compounds, 21 TRANSLATION categories: molecules (~0.5%) are validated as inhibitors of KLKs (5,6 and 10), which belong to 3 expression in many different cancers and measure their biological impact in cancer related INHIBITORS processes. 1. Ion pump inhibitors (cardiac glycosides) functional-families of compounds. Furthermore there was a very small overlap among the 3 TRAFFICKING libraries and these molecules came up as hits from all different libraries, reflecting the ¾ High-throughput screening (HTS) plays a major role in modern drug discovery, since it INHIBITORS 2. Translation inhibitors allows screening of large compound libraries against your putative target 5 reproducibility & sensitivity of the technique. OTHERS 3. Trafficking inhibitors RT-PCR experiments of treated cells show that these molecules block a certain pathway which regulates the expression of some proteins, including KLKs(data in progress). Western OBJECTIVES CARDIAC GLYCOSIDES IC50s Blot experiments and further quantitative proteomic techniques seem to be the way to Peruvoside Ouabain DMSO ¾ Development of a cell-based high throughput screening assay, suitable for the screening of large 90.00 50nM 50nM further elucidate such pathways. 80.00 chemical libraries 70.00 KLK5 60.00 REFERENCES ¾ Optimization of the following in-vitro validation assays, in order to eliminate the false-positive initial 50.00 KLK6 hits and focus on the ‘actual hits’ 40.00 1). Borgono CA, Michael IP, and Diamandis EP. (2004). Mol. Cancer Res. 2: 257-280 30.00 KLK10 ¾ Measure the potency of these hits, in parallel with their cytototoxic or apoptotic potential 20.00 2). Borgono CA and Diamandis EP. (2004). Nat. Rev. Cancer. 4: 876-890 KLK5 expression (ug/L) 10.00 Actin ¾ Elucidate the pathway that regulates KLK5 expression. Check specificity with the other KLKs. 0.00 3) Michael IP et al. (2005). JBC. 280: 14628-14635 nM 5uM 2uM 1uM 20uM 10uM 50nM 500nM 200 100nM 4). Descarques P., et al (2006) J. Invest Dermatol., 126(7):1622-32 ¾ Measure their therapeutic potential with biological assays. compound concentration ¾ Development of ‘compound-cocktails’ against KLK5, or cocktails of compounds which specifically 5) Oikonomopoulou K., et al. (2006). JBC. 281(43):32095-112 target different KLKs (tissue specificity) Fig.5. Striking differences in the potency Fig.5. Semi-quantitative RT-PCRs show a 6) Brideau C., et al. (2003) J Biomol Screen. 8(6): 634-647 of compounds despite their very high similar decrese in mRNA levels (in non- structural similarities (same family). cytotoxic & non-apoptotic concentrations ACKNOWLEDGEMENTS of the compounds) We would like to thank Alessandro Datti et al from the SLRI/HTS Robotics Facilities for their assistance with high-throughput screening..