uHTS identification of modulators of interaction between CendR and NRP-1 using Fluorescence Polarization assay
Neuropilin-1 (NRP-1) is a a pleiotrophic cell surface receptor with multiple ligands that plays an essential role in angiogenesis, cardiovascular development, regulation of vascular permeability and development of the nervous system [1-4]. NRP-1 is required for induction of vascular permeability by VEGF  and semaphorin 3A , which interact with the b1 domain of NRP-1 via their C-terminal basic domains. ..more
BioActive Compounds: 3086
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford Burnham Medical Research Institute, La Jolla, CA
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1 R03 MH096589-01
Assay Provider: Dr. Erkki Ruoslahti, Sanford Burnham Medical Research Institute, La Jolla, CA
Neuropilin-1 (NRP-1) is a a pleiotrophic cell surface receptor with multiple ligands that plays an essential role in angiogenesis, cardiovascular development, regulation of vascular permeability and development of the nervous system [1-4]. NRP-1 is required for induction of vascular permeability by VEGF  and semaphorin 3A , which interact with the b1 domain of NRP-1 via their C-terminal basic domains.
A recently described class of synthetic "C-end Rule" (CendR) peptides share a R/K/XXR/K motif with the C-terminal domains of VEGF-A165 and some semaphorins, and upon binding to NRP-1 induce cell internalization and tissue penetration [7-9]. It was shown that CendR peptides are able to take cargo up to the size of a nanoparticle deep into extravascular tissue [4, 10-12].
The aim of this lead discovery campaign is to find small molecule modulators of the CendR transport pathway. CendR agonists can potentially be useful as tools for introducing compounds into cells and tissues, and in probing the workings of the CendR pathway. Antagonists could potentially be useful in preventing tissue penetration of inflammatory mediators, toxins, and infectious agents that use the CendR pathway.
This biochemical uHTS assay is based on the ability of a FAM-labeled CendR peptide to bind to the b1b2 domain of the NRP-1 receptor, resulting in an increase in fluorescence polarization.
1. Takashima S, Kitakaze M, Asakura M, Asanuma H, Sanada S, Tashiro F, Niwa H, Miyazaki Ji J, Hirota S, Kitamura Y, Kitsukawa T, Fujisawa H, Klagsbrun M, Hori M. (2002) Targeting of both mouseneuropilin-1 and neuropilin-2 genes severely impairs developmental yolk sac and embryonic angiogenesis. Proc Natl Acad Sci USA 99:3657-3662.
2. Staton CA, Kumar I, Reed MWR, Brown NJ (2007) Neuropilins in physiological and
pathological angiogenesis. J Pathol 212:237-248.
3. Sternlicht, M. D. and Werb, Z., How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 17: 463-516, 2001.
4. Sugahara, K.N., Teesalu, T., Karmali, PP., Kotamraju, VR., Agemy, L., Hanahan, D., and Ruoslahti, E. Tissue-penetrating delivery of compounds and nanoparticles into tumors. Cancer Cell. 2009 Dec 8; 16(6):510-20.
5. Becker PM, Waltenberger J, Yachechko R, Mirzapoiazova T, Sham JS, Lee CG, Elias JA, Verin AD. (2005) Neuropilin-1 regulates vascular endothelial growth factor mediated
endothelial permeability. Circ Res 96:1257-1265.
6. Acevedo LM, Barillas S, Weis SM, Go thert JR, Cheresh DA (2008) Semaphorin 3A
suppresses VEGF-mediated angiogenesis yet acts as a vascular permeability factor.
7. Jia H, Bagherzadeh A, Hartzoulakis B, Jarvis A, Lohr M, Shaikh S, Aqil R, Cheng L, Tickner M, Esposito D, Harris R, Driscoll PC, Selwood DL, Zachary IC. (2006) Characterization of a bicyclic peptide neuropilin-1 (NP-1) antagonist (EG3287) reveals importance of vascular endothelial growth factor exon 8 for NP-1 binding and role of NP-1 in KDR signaling. J Biol Chem 281:13493-13502.
8. Starzec A, et al. (2006) Antiangiogenic and antitumor activities of peptide inhibiting
the vascular endothelial growth factor binding to neuropilin-1. Life Sci
9. Teesalu, T., Sugahara, K. N., Kotamraju, V. R. and Ruoslahti, E. C-end rule peptides mediate neuropilin-1-dependent cell, vascular, and tissue penetration. Proc Natl Acad Sci U S A. 2009 Sep 22; 106(38):16157-62. Epub 2009 Sep 2.
10. Porkka, K., Laakkonen, P., Hoffman, J.A., Bernasconi, M., and Ruoslahti, E. A fragment of the HMGN2 protein homes to the nuclei of tumor cells and tumor endothelial cells in vivo. Proc. Natl. Acad. Sci. USA. 99: 7444-7449, 2002.
11. Laakkonen, P., Akerman, M.E., Biliran, H., Yang, M., Ferrer, F., Karpanen, T., Hoffman, R.M., and Ruoslahti, E. Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells. Proc. Natl. Acad. Sci. USA 101: 9381-9386, 2004.
12. Karmali, P. P., Kotamraju, V.R., Kastantin, M., Black, M., Missirlis, D, Tirrell, M., and Erkki
Ruoslahti. Targeting of albumin-embedded taxol nanoparticles to tumors. Nanomedicine. 2009 Mar 5 (1):73-82. Epub 2008 Oct 1.
1) NRP-1(b1b2) protein was purified by Sanford Burnham Protein Facility
2) FAM-RPARPAR peptide was provided by Dr. Ruoslahti's laboratory.
3) Assay Buffer: 50 mM Bis-TRIS pH 7.4, 0.05% Tween, 1 mM DTT
4) 1536-well solid black plates were obtained from Corning (Cat # 3724)
1) Using LabCyte Echo, transfer 15 nL of test compounds from a 5 mM compound source plate into assay plate Cols. 5-48 (Final concentration of test compounds is 25 uM, 2 % DMSO). Transfer 15 nL of 100% DMSO into assay plate Col. 1-4.
2) Using Thermo Combi nL, dispense 1.5 uL of assay buffer to columns 1-2.
3) Using Thermo Combi nL, dispense 1.5 uL of 7.2 uM NRP1(b1-b2) in assay buffer to columns 3-48.
4) Spin plates at 1500 rpm for 1 minute with Eppendorf centrifuge 5810.
5) Incubate for 30 minutes at room temperature.
6) Using Thermo Combi nL, dispense 1.5 uL of 20 nM FAM-RPARPAR in assay buffer to columns 1-48.
7) Spin plates at 1500 rpm for 1 minute with Eppendorf centrifuge 5810.
8) Incubate for 60 minutes at room temperature.
9) Read plates on BMG Labtech Pherastar at Ex/Em 485/520 nm in fluorescence polarization mode.
Compounds that at 25 uM concentration demonstrated a normalized inhibition of >= 32% or corrected inhibition of >= 25% and F-Ratio in the range of 0.5-1.5 are defined as actives in this assay.
The experimental values were normalized by the difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc. Further information about data correction is available at http://www.genedata.com/products/screener.html.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
** Test Concentration.
Data Table (Concise)