High-Affinity Naloxone Binding to Filamin A Prevents Mu Opioid Receptor–Gs Coupling Underlying Opioid Tolerance and Dependence
Authors: Lindsay H Burns
Maya Frankfurt
Hoau-Yan Wang
Journal: PLoS ONE (2008)
Discipline: Multidisciplinary
Keywords: ultra-low-dose opioid antagonists; opioid analgesia; analgesic tolerance; dependence; G protein coupling switch
Affiliations: Department of Physiology and Pharmacology, City University of New York Medical School, New York, New York, USA. 

Abstract:  Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs) by the mu opioid receptor (MOR), although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [(3)H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [(3)H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A(2561-2565) as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A(2561-2565) abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor-Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence. 

Retracted: Yes

PubPeer comments:
 A figure in this paper was discussed in a report mentioned on Twitter - https: twitter.com twhitfill status 1430296059843170305 - and found here: https: www.regulations.gov docket FDA-2021-P-0930 document Figure 7A: * Red boxes: In thee MOR row, the NLX and FLNA panels appear to look identical. * Blue arrows: Some sharp background transitions around bands, suggestive of splicing or patching. Could the authors show us the original uncropped blots? The resolution shown in the paper is not very high, and maybe some of these artifacts might be caused by compression issues. Showing the original blots might take away any concerns. ![file] , 
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 As noted by Dr Elizabeth Bik in her comment, she looked into these 13 year old images after she was carbon-copied by Travis Whitfill on Twitter. Though Dr Elizabeth Bik s observations may be factual and unbiased, it may be useful to note the following: **1**. The tweet that Travis Whitfill was bringing to the attention of Dr Elizabeth Bik was a link to a Citizen s Petition filed by Labaton Sucharow on August 23, 2021, the petition asks that the drug trials of Cassava Science s experimental drug be paused in order to perform a thorough audit. Source: https: twitter.com MSollender status 1430288115990626307?s=20 and https: www.regulations.gov document FDA-2021-P-0930-0001 **2**. Travis Whitfill s Twitter Bio states that he is a partner at Bios Partners Source: https: twitter.com twhitfill ![file] **3**. Bios Partners web site states that one of their investments is Cognition Therapeutics Source: https: biospartners.com portfolio **4**. Four days before (i.e. 8 20 2021) Travis Whitfill asked Dr. Bik about these 13 year old images, Cognition Therapeutics described the following risk in an SEC filing:  We face significant competition in an environment of rapid technological and scientific change, and there is a possibility that our competitors may achieve regulatory approval before us or develop therapies that are safer, more advanced or more effective than ours, which may negatively impact our ability to successfully market or commercialize any product candidates we may develop and ultimately harm our financial condition. Source: https: www.sec.gov Archives edgar data 0001455365 000110465921108070 tm2113659-13_s1a.htm **5**. Cognition Therapeutics develops drugs for Alzheimer s Disease and is a competitor of Cassava Sciences. Source: https: cogrx.com pipeline alzheimers-disease **6**. Cognition Therapeutics had an initial public stock offering (IPO) on October 4, 2021 Although the timing may be suspicious to some, it may be entirely coincidental. Cognition Therapeutics, Bios Partners, and Travis Whitfill may not have engaged in any illegal or unethical behavior, and these types of events may be common in what they note is a an environment with  significant competition and  rapid technological and scientific change . , 
 #4 The arguments made by the Cassava supporters are illuminating. They concern only the possible motivations of some of the actors. It would hardly be surprising for competitors to be amongst those most closely scrutinizing Cassava s scientific basis. And if you discover a problem, what then should you do? The boilerplate quote from the SEC filing (point 4) is astonishingly weak. Anyway, the implicit suggestion that the success of somebody s IPO would depend on Cassava being made less attractive for investment is also weak. I m sure a LOT of investment would be available for any plausible treatment for Alzheimer s disease. Indeed, casting doubt on the field might even have a negative effect on any IPO. More importantly, the arguments do not address any scientific substance. , 
 Published: March 30, 2022 https: doi.org 10.1371 journal.pone.0266627  Following the publication of this article [1], concerns were raised regarding results presented in Figs 1 and 7. Specifically, There appear to be horizontal and vertical irregularities suggestive of splice lines in the following panels: u25cb Between lanes between lanes 4u20135 of the Fig 1A left and right FLNA panels, right MOR panel, and left and right Gu03b1 panels. u25cb Between the 92.3kDa and the 50.4kDa marker of the Fig 1A left MOR panel. u25cb Between lanes 2u20133 of the Fig 7A Morphine + NLX +FLNA2550-2560 panel. u25cb Around multiple bands presented in the Fig 7A MOR and Gu03b1 panels In Fig 1C, neither the published panels nor the underlying data provided in follow-up discussions include a positive control sample. The absence of a positive control calls into question the reliability of the results presented in Fig 1C. The Fig 7A NLX and FLNA2550-2560 panels appear similar. The corresponding author noted that the Fig 7A NLX and FLNA2550-2560 panels were inadvertently duplicated and provided a replacement panel for the FLNA2550-2560 panel. However, the corresponding author disagreed with the Fig 1A concerns, stating that the observations are likely the result of image compression artifacts. The corresponding author provided image data to support the contested western blot results in this [1] and other PLOS ONE articles [2u20135]. Per PLOSu2019 assessment of the data files, the pixel patterns in background areas of blot images provided for multiple panels in [1u20135] appear more similar than would be expected for data obtained in independent experiments. Furthermore, the supporting data files did not contain positive controls as needed to verify the reliability of the results. In response to these concerns, the corresponding author stated that the repetitive features in the background noise of the image data are likely the result of scanner artifacts. The explanation given for the background image similarities does not resolve the journalu2019s concerns in light of PLOSu2019 assessment of the data files. The data and comments provided did not resolve the concerns about the integrity and reliability of data presented in this article. In light of these issues, the PLOS ONE Editors retract this article. HYW and LB did not agree with the retraction. MF either did not respond directly or could not be reached. HYW stands by the articleu2019s findings. (sic) References 1. Wang H-Y, Frankfurt M, Burns LH (2008) High-Affinity Naloxone Binding to Filamin A Prevents Mu Opioid Receptoru2013Gs Coupling Underlying Opioid Tolerance and Dependence. PLoS ONE 3(2): e1554. https: doi.org 10.1371 journal.pone.0001554 PMID: 18253501 2. Wang H-Y, Burns LH (2009) Naloxoneu2019s Pentapeptide Binding Site on Filamin A Blocks Mu Opioid Receptoru2013Gs Coupling and CREB Activation of Acute Morphine. PLoS ONE 4(1): e4282. https: doi. org 10.1371 journal.pone.0004282 PMID: 19172190 3. Bakshi K, Kosciuk M, Nagele RG, Friedman E, Wang H-Y (2011) Prenatal Cocaine Exposure Increases Synaptic Localization of a Neuronal RasGEF, GRASP-1 via Hyperphosphorylation of AMPAR Anchoring Protein, GRIP. PLoS ONE 6(9): e25019. https: doi.org 10.1371 journal.pone.0025019 PMID: 21980374 4. Bakshi K, Parihar R, Goswami SK, Walsh M, Friedman E, Wang H-Y (2014) Prenatal Cocaine Exposure Uncouples mGluR1 from Homer1 and Gq Proteins. PLoS ONE 9(3): e91671. https: doi.org 10. 1371 journal.pone.0091671 PMID: 24626340 5. Stucky A, Bakshi KP, Friedman E, Wang H-Y (2016) Prenatal Cocaine Exposure Upregulates BDNFTrkB Signaling. PLoS ONE 11(8): e0160585. https: doi.org 10.1371 journal.pone.0160585 PMID: 27494324 ,