Bibliografía utilizada y consultada

1.4.1 High-Throughput Screening

Development of novel therapeutic agents is an expensive and time consuming process. Since the early 1900s, pharmaceutical companies have used large numbers of small molecules in laborious experimentation to investigate new therapeutic options (118). Small molecules are usually described as chemical compounds that have a low molecular weight. Small molecule libraries are maintained and may contain hundreds of thousands of compounds. Traditional laboratory methods require a significant time and resource commitment to run tests on an entire library. Coupled with the high cost of testing for regulatory clearance, the cost of drug development becomes quickly prohibitive.

In an effort to alter the cost-to-benefit ratio, the need for new testing methodologies was realized (118,119). HTS refers to the ability to quickly test hundreds or thousands of compounds using specially designed assays to look at cellular events of interest. One method of HTS involves the use of 96-, 384-, or 1536-well microwell plates to quickly examine compounds against highly sensitive chemical or cellular assays. This is done using any combination of manned workstations and robotic equipment, such as the HyperCyt®, developed by University of New Mexico Center for Molecular Discovery (UNM-CMD). These assays can be cell based or in vitro assays, but are designed by the investigator.

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The National Institutes of Health realized the benefit of having a small molecule library available to principal investigators who are not working in industry. To fill this need, the NIH Molecular Libraries Program (MLP) maintained HTS centers at

institutions across the United States. A central resource of the MLP are small molecule libraries of thousands of small molecule compounds to be used in assays. The Prestwick library of Food and Drug Administration (FDA) approved compounds is a commercially available library which can be used for primary screening (119). The Prestwick library contains 1,280 compounds that are FDA approved for human use. This library allows for an investigator to rapidly translate active compounds from the screen to a clinical setting.

1.4.2 Identification of Inhibitors of the Rho Family GTPases

The UNM-Center for Molecular Discovery was a Molecular Library Program funded center. Previously, the center, in a collaboration between and Drs. Wandinger- Ness and Sklar, developed a cell-free multiplex polystyrene bead array that could be performed in a 384-well format (120–122). This array uses glutathione beads of different fluorescent intensities bound to GTPases as a quantitative measurement of GTPase activation. Ras family GTPases fused to glutathione-S-transferase were bound to the glutathione beads. Compounds within the small molecule libraries at UNM were tested for activation or inhibition of GTPase nucleotide binding activity using this system. Compounds were loaded into wells containing GTPase conjugated beads and then incubated with BODIPY-conjugated-GTP. Compounds were considered potentially active if there was a 20% change in fluorescence from baseline. This study identified 2 compounds, a Rho family selective inhibitor and a Cdc42 specific inhibitor (120–122).

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1.4.3 Identification of Cdc42 and Rac1 Inhibitors

Drs. Wandinger-Ness and Sklar utilized the described small molecule screen to examine the Prestwick library for GTPase inhibition (120,123). This version of the Prestwick library contained 1208 compounds which have all been previously approved by the FDA and qualify for off patent use. The Prestwick library contained 24

compounds classified as non-steroidal anti-inflammatory drugs which had activity in the primary screen (NSAIDs) (123). Of these, 11 NSAIDs were tested in a confirmatory dose response screen to test for activity against 8 GTPases: Cdc42 wild-type, Cdc42 activated mutant, Rab2, Rab7, Rac1 wild-type, Rac1 activated mutant, Ras wild-type, and Ras activated mutant (123). There were 4 NSAIDs confirmed to have activity against the selected GTPases: R-naproxen, S-ibuprofen, S-naproxen, and sulindac sulfide. R- naproxen was the only identified NSAID with an EC50 less than 3µM and selectively

inhibited GTP-binding of Cdc42 and Rac1 (123). This effect was enantiomer specific, as S-naproxen did not exhibit the same inhibitory effect. This activity is notable because the anti-inflammatory activity of naproxen is due to the S-enantiomer, not the R-enantiomer (124). R-naproxen was previously thought to be largely inert, with no known

pharmacologic activity (124). But further testing showed inhibition of Cdc42 and Rac1 activation in NIH 3T3 cells and Cdc42 and Rac1 regulated cytoskeletal events in OVCA429 and OVCA433, two ovarian cancer cell lines (123). Because the Cdc42 and Rac1 inhibition was enantiomer specific, the UNM investigators postulated that the aryl ring structure and rotational constraints around the chiral center of R-naproxen, place it within a hydrophobic pocket on the GTPase surface (124). Using R-naproxen as the basis for an in silico query, another enantiomer specific compound, R-ketorolac, but not S-

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ketorolac was identified (123). Further docking studies suggested that mechanism of Cdc42 and Rac1 inhibition by R-ketorolac and R-naproxen is through the neutralization of a magnesium ion necessary for nucleotide release within the nucleotide binding pocket.

HTS is an important tool that can be extremely beneficial (118). Using HTS, it was possible to identify FDA approved enantiomer specific NSAIDs which have

previously unknown activity against Cdc42 and Rac1. NSAIDs can be protective in some cancers, suggested to be due to the anti-inflammatory effects (125–128)(153-156).

However, NSAIDs may have COX independent effects, such as disruption of cell adhesion, cytotoxic or anti-proliferative effects which need to be explored. The

enantiomer selective binding of R-ketorolac to Cdc42 and Rac1 shows that NSAIDs can affect proteins not related to inflammation (123).