R-Ketorolac Targets Cdc42 and Rac1 and Alters Ovarian Cancer Cell Behaviors Critical for Invasion and Metastasis
Authors: Cristina Murray-Krezan
Larry A Sklar
S Ray Kenney
Teresa Rutledge
Yuna Guo
Rytis Prekeris
Angela Wandinger-Ness
Laurie G Hudson
Sarah Adams
Carolyn Y Muller
Elsa Romero
Journal: Molecular Cancer Therapeutics (2015)
Discipline: CLINICAL MEDICINE
Keywords: Cdc42; Rac1; inhibitor; ovarian cancer; GTPases
Affiliations: Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico. Cancer Center, University of New Mexico, Albuquerque, New Mexico., Cancer Center, University of New Mexico, Albuquerque, New Mexico. Department of Pharmaceutical Sciences, University of New Mexico College of Pharmacy, Albuquerque, New Mexico., Cancer Center, University of New Mexico, Albuquerque, New Mexico. Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of New Mexico School of Medicine, Albuquerque, New Mexico., Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico., Division of Epidemiology, Biostatistics and Preventive Medicine, Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, New Mexico., Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, Colorado., Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico. Cancer Center, University of New Mexico, Albuquerque, New Mexico. .

Abstract:  Cdc42 (cell division control protein 42) and Rac1 (Ras-related C3 botulinum toxin substrate 1) are attractive therapeutic targets in ovarian cancer based on established importance in tumor cell migration, adhesion, and invasion. Despite a predicted benefit, targeting GTPases has not yet been translated to clinical practice. We previously established that Cdc42 and constitutively active Rac1b are overexpressed in primary ovarian tumor tissues. Through high-throughput screening and computational shape homology approaches, we identified R-ketorolac as a Cdc42 and Rac1 inhibitor, distinct from the anti-inflammatory, cyclooxygenase inhibitory activity of S-ketorolac. In the present study, we establish R-ketorolac as an allosteric inhibitor of Cdc42 and Rac1. Cell-based assays validate R-ketorolac activity against Cdc42 and Rac1. Studies on immortalized human ovarian adenocarcinoma cells (SKOV3ip) and primary patient-derived ovarian cancer cells show that R-ketorolac is a robust inhibitor of growth factor or serum-dependent Cdc42 and Rac1 activation with a potency and cellular efficacy similar to small-molecule inhibitors of Cdc42 (CID2950007/ML141) and Rac1 (NSC23766). Furthermore, GTPase inhibition by R-ketorolac reduces downstream p21-activated kinases (PAK1/PAK2) effector activation by &gt;80%. Multiple assays of cell behavior using SKOV3ip and primary patient-derived ovarian cancer cells show that R-ketorolac significantly inhibits cell adhesion, migration, and invasion. In summary, we provide evidence for R-ketorolac as a direct inhibitor of Cdc42 and Rac1 that is capable of modulating downstream GTPase-dependent, physiologic responses, which are critical to tumor metastasis. Our findings demonstrate the selective inhibition of Cdc42 and Rac1 GTPases by an FDA-approved drug, racemic ketorolac, that can be used in humans. 

Retracted: No

PubPeer comments:
 The authors claim to have established R-ketorolac as an u201callosteric inhibitoru201d of both the Cdc42 and the Rac1 GTPases. The claim is based on the data presented under Figure 1, and appears to be generated using a bead-based flow cytometry assay. In fact, Figure 1 clearly negates the authors claim as it shows no such inhibitory or allosteric effects of R-ketorolac on both GTPases: 1.tThe authors write u201cThe maximal inhibitory response was ~ 20% for both GTPasesu201d (Figure 1 F G) and they provide EC50 values of 18 uM for CDC42 and 23 uM for Rac1. The authors cheated by showing the percentage normalized intensity on the y-axis from 60 to 120% instead of 0 to 120%. If the plot was to be redrawn, no inhibitory effect would have been observed on visual inspection f the data. This defies the definition of a half-maximal effective concentration which means that the observed activity must reach 50%; in this case, it reached barely 20% at the highest R-ketorolac concentration of 100 uM. This is misleading and constitutes misrepresentation of the observed inhibitory effects of R-ketolorac on both GTPases. This has led for few follow up papers from the same authors with no reproducibility of their data by others. The correct observation to have been reported should have been u2013 no inhibitory activity was observed. 2.tThe authors claim u201cMechanistically, R-ketorolac action is consistent with an allosteric inhibition of GTPase nucleotide bindingu201d. It is rather astonishing that such a claim could have been made based solely on primary plots and without any determination of both Km and Bmax to concretely demonstrate this alleged allosteric effect. Experimentally speaking, there is no difference in the Bmax values for both GTPases (Fig 1 B D) to make such claims. 3.tThese claims were the basis of multiple patents and patent applications; in particular, published application # 10,857,130 claiming R-ketolorac as an inhibitor of both GTPases without showing any new and convincing data. ![file] , 
 Yet another article by the same group of authors keep claiming in Figure 1 that R-ketorolac is an inhibitor of RAC1 and CDC42 with IC50 values of 0.57 and 1.07, respectively. BMC Cancer 2021 Jan 7;21(1):40. doi: 10.1186 s12885-020-07716-1. The R-enantiomer of ketorolac reduces ovarian cancer tumor burden in vivo Martha M Grimes 1 , S Ray Kenney 2 3 , Dayna R Dominguez 2 , Kathryn J Brayer 4 5 , Yuna Guo 6 , Angela Wandinger-Ness 6 , Laurie G Hudson 2 PMID: 33413202; PMCID: PMC7791840 ![file] , 
 UPDATES Few months after concerns appeared on @PubPeer about the questionable inhibition of Rac1 by R-Ketolorac, The Biotech Company Revere Pharmaceuticals drops their lead candidate REV440 (R-Ketolorac) licensed from the University of New Mexico (UNM) and based on this publication. They terminated their long term licensing agreement and returs the asset to UNM. Revere seems to agree with the reported concerns suggest that for over 5+ years, they failed to reproduce data published by UNM researchers. Their decision to terminate the agreement was due to lack of cooperation by UNM on the published studies. https: twitter.com RogerBiotech status 1509526238754267143 , 
 ![file] ![file] ![file] ![file] ![file] ![file] Examination of six papers on the use of cytometry to screen for inhibitors of GTPases incl. Rab7, Rac1, and Cdc42, revealed that with the exception of this MCT paper where the y axis for the IC50 plots were 60 to 120%, all the other five publications were correct with displaying their IC50 plots with the y axis range from 0 to 120 or 125%. It is mathematically impossible to obtain an IC50 value from this publication as there is no 50% data point. Wow they even screened over 300K compounds and found nothing with cell-activity. Both Sklar and last author Wandinger published a high profile paper in 2012 in ACS Chemical Biology where in Fig 5 they defined active vs inactive profile for one of the identified hit from MLPSN screening; the y-axis for the IC50 plots were 0 to 120%. ,