Sequence: cell division cycle 42 (GTP binding protein, 25kDa) [Homo sapiens]
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AID	Name	Type	Comment
1772	Project utilizing multiplex HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases	summary	Summary for GTPase

University of New Mexico Assay Overview:
Assay Support: NIH I RO3 MH081231-01
HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases
PI: Angela Wandinger-Ness, Ph.D.
Co-PI: Larry Sklar, Ph.D.
Assay Development: Zurab Surviladze, Ph.D.
Assay Implementation: Zurab Surviladze, Ph.D., Anna Waller, Ph.D.

Chemistry: University of Kansas Specialized Chemistry Center
KU Specialized Chemistry Center PI: Jeff Aube, Ph.D.
KU SCC Project Manager: Jennifer E. Golden. Ph.D.
KU SCC Chemists on this project: Chad Schroeder, M.S., Denise Simpson, Ph.D., Julica Noeth, B.S.

Dose Response Assay Background and Significance:

Ras and related small molecular weight GTPases function in the regulation of signaling and cell growth, and collectively serve to control cell proliferation, differentiation and apoptosis [Tekai et al. 2001; Wennerberg et al. 2005]. The Ras-related GTPases are divided into four subfamilies with the Rab proteins regulating membrane transport, Rho proteins (including Rac and Cdc 42) regulating cytoskeletal rearrangements and responses to signaling, Arf/Sar proteins regulating membrane and microtubule dynamics as well as protein transport, and Ran proteins controlling nucleocytoplasmic transport. This project focuses on representative Ras, Rho, and Rab family members to validate the approach for the identification of new chemical compounds with novel therapeutic potential in cell signaling and growth control.

Ras and Ras-related GTPase functions are tightly regulated, and dysregulation is causal in a wide variety of human diseases. Ras mutations resulting in impaired GTP hydrolysis and plasma membrane hyperactivation are linked to many human cancers [Farnsworth et al. 1991; Sukumar et al. 1983; Taparowsky et al. 1982; Boylan et al. 1990; Hruban et al. 2004; Abrams et al. 1996]. Point mutations in the Rab and Rho GTPases are also causal in diverse human diseases affecting pigmentation, immune, and neurologic functions [Houlden et al. 2004; Verhoeven et al 2003; Williams et al. 2000; Bahaderan et al. 2003; and preliminary findings]. Rab and Rho mutants identified in human disease act as dominant negatives either due to a failure to bind GTP or due to inappropriate coupling of the active proteins with downstream effectors. To date, inhibition of Ras and Ras-related proteins has relied largely on altering membrane recruitment with various drugs affecting prenylation [Morgillo F and Lee HY, 2006; Russell RG, 2006; Park, et al. 2002]. Generally, Ras proteins must be farnesylated for proper membrane localization, while Rab and Rho proteins are geranylated. Such strategies lack specificity and are problematic because each of these prenylation machineries is required for the proper function of many Ras superfamily members. Rational drug design has only recently been applied to identify the first two small molecule inhibitors of Rho GTPase family members [Gao, et al. 2004; Nassar et al. 2006]. Therefore, broadly testing the Ras-related GTPases as targets for small molecule inhibitors and activators is expected to identify new classes of compounds that may be useful in the treatment of human disease, as well as in unraveling the molecular details of how Ras-related GTPases function.

The primary HTS screen for compounds effecting the binding of fluorscent GTP to various Ras-related GTPases yielded a number of interesting compounds. In this assay a number of compounds and their effect on different target proteins were assessed in a dose response manner under different conditions than the primary screen. For this assay, the binding buffer has 1 milliM MgCl2 buffer (the primary screen were carried out in 1 milliM EDTA buffer).

Multiplex Dose Response of Test compounds - Six different target proteins binding with fluorescent GTP under the condition of varying test compound concentrations

Data Table(All)

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PID§		Name	Substance		Panel Targets	Description
			Active	Inactive
1		Rac1ACT		32		GTPase activating protein 1 activated mutant
2		Rac1WT	3	29		GTPase activating protein 1 wildtype
3		Cdc42ACT	16	16		Cell Division Cycle 42 activated mutant
4		Cdc42WT	17	15		Cell Division Cycle 42 wildtype
5		RhoACT		32		Rho GTPase activating protein 1 activated mutant
6		RhoWT	1	31		Rho GTPase activating protein 1 wildtype

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§ Panel component ID.

Individual protein targets (4 microM) are bound to glutathione beads overnight at 4 degrees C. Binding assays are performed by incubating 50 microL of GST-target protein-GSH-bead suspension for 2 min with 1 milliM MgCl2 and either DMSO or test compound (6 point 10-fold dilution series 100 microM to 0.02 microM) and subsequently adding 50 microL of a fixed concentration (1.5 nanoM) ice cold BODIPY-GTP. Association of the fluorescent nucleotide is measured using a FacSCAN flow cytometer. The flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nanometer (FL1)are analyzed by IDLQuery software to determine the median fluorescence per bead population.

Calculations:

The binding measurements for each protein target were normalized to the amount of binding in DMSO alone:
%Response = 100*(MCF/MCFwDMSO)
where MCF is the binding of fluorescent GTP at different concentrations of test compound and MCFwDMSO is the binding of fluorescent GTP in the presense of DMSO alone.

The different %Response values were fit to 4-parameter sigmoidal dose-response curve with variable slope:
%Response= Bottom + (Top - Bottom)/(1 +10^((LogEC50-X)*HillSlope))
where Bottom and Top are estimates of minimum and maximum of %Response over the concentration range of test compounds, LogEC50 is the log of the Effective Concentration of test compound yielding 50% change in the %Response, and HillSlope is variable slope of the dose response curve.

Target_SCORE is based on the comparison of the estimated EC50 to the least amount of acceptable EC50, 20 microM, for compounds that had a magnitude of change greater than 15%. Thus Target_SCORE = 100*(1 - (EC50inMicroM/20) where EC50inMicroM is the calculated estimate of EC50 in microM. Active compounds have Target_SCORE greater than 0. The desired outcome was to find compounds with specific Cdc42 activity, however many compounds also affected other protein targets, namely Rac1. Thus the overall PUBCHEM_SCORE was left at 0 and compounds were designated 'inconclusive' due to this mixture of activity.

Abbreviations: microM for micromolar, milliM for millimolar, nanoM for nanomolar, milliL for milliliter

Grant Number: NIH I RO3 MH081231-01