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Primary Screens

2.3.2.1 RGS4 Protein Target

The GPC spin column/ESI-MS screening methodology was used to identify non-covalent inhibitors of regulator of G protein signaling (RGS4) protein. The RGS4 protein accelerates GTPase activity of the subunit of trimeric G protein and is indicated in central nervous system (CNS) disorders. Compounds were sought that bind to RGS4, interfere with the binding of RGS4 to the G alpha protein, inhibit the endogenous GTPase activity of the G alpha protein (Gia1) and are active in a luciferace yeast pheromone RGS4 response (functional) assay [24].

About 32 000 compounds were screened to identify compounds that bind non-covalently to RGS4 using the GPC spin column/ESI-MS methodology and 1720 compounds were identified to bind (including very weak binders) to RGS4 [15]. The 50 highest scoring compounds in the ESI-MS analyses were each evaluated by 2D 1H-15N HSQC NMR in the presence of RGS4 protein (see Section 2.3.3.2.2). Two compounds were found to be hits by generating RGS4 protein chemical shift perturbations; however, the region of the perturbations were not in the desired RGS4/G alpha interface region. These compounds still exhibited activity and suggested an allosteric binding site that prevented the necessary con-formational change in RGS4 to bind G alpha. Nevertheless, using Lipinski's rules, the list of 1720 compounds was reduced to 743 compounds from which the top 150 candidates were screened in a RGS4/G alpha/GTPase assay. Two of these compounds were found to inhibit the RGS4 function in the GTPase assay. Furthermore, 58 compounds that tested positive in the luciferase phermone RGS4 assay were also present in the hit list of 1720 compounds of the GPC spin col-umn/ESI-MS assay. These results verify the validity of the GPC spin column/ ESI-MS method for drug screening.

As a follow-up to the RGS4 primary screen, the GPC spin column/ESI-MS methodology was applied to a selected series of seven RGS4 drug candidates to identify those compounds that bind non-covalently to RGS4 and not to G alpha [24]. Three ESI-MS experiments were performed with each of the compounds. The first experiment ascertained the response factor for each of the molecular ions formed. The second experiment demonstrated that the drug candidates do not pass through the spin columns in the absence of the proteins. This experiment served as a control to validate the final experiments where the individual protein and drug candidates, after incubation, were passed through a spin column and the eluates analyzed by ESI-MS for residual non-covalently bound drug. Figure 2.16 illustrates the experimental results for compound WY817 (MW 450 Da). Figure 2.16A demonstrates the production of a molecular ion for 250 pg of WY817. Figure 2.16B demonstrates the absence of a molecular ion when @100 mg of WY817 passed through the spin column. Figure 2.16C demonstrates the presence of WY817 when @100 mg of WY817 were incubated in the presence of 25 mL of 125 mM RGS4. Finally, Fig. 2.16D demonstrates the absence ofWY817 when @100 mg of WY817 were incubated in 25 mL of 37 mM G alpha protein. These data demonstrate that WY817 satisfies a condition required for a potential small molecule drug candidate in that it non-covalently binds to RGS4 and does not bind to G alpha protein. Since high concentrations of compound and protein were used, WY817 is a weak non-covalent binder to RGS4. The results for all the seven drug candidates, all analyzed identically using the GPC spin column/ESI-MS screening methodology, are tabulated in Fig. 2.17. The relative affinities of the compounds to the proteins were determined from the observed ion abundances normalized to the response factors for each drug candidate. Four compounds

BG04293 17CP4 13 (0,477) Sm (SG. KM Cm (6 55-1 S.3 000) 100-, J5',27B

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