In the present study, a small library of bisphenol Z (BPZ) derivatives was synthesized and investigated for anti-proliferative effects in cultured breast and glioblastoma cell lines. Synthesized BPZ derivatives varied in molecular size, polarity, and lipophilicity. Of the 8 derivatives tested, compounds 4 and 6, both of which displayed the highest degree of lipophilicity, were most active at inducing cell death as determined by the XTT assay. Cell membranes were interrogated using trypan blue staining and were shown to remain intact during treatments with 4 and 6. Activation of caspase enzymes (3 and/or 7) was noted to occur following treatment with compound 4.Polar BPZ derivatives, those with a substituted amine or alcohol, were devoid of any inhibitory or proliferative effects. The remaining derivatives seem to lack sufficient lipophilicity to execute an overt toxic effect. Our results suggest that increasing the lipophilic character of BPZ enhances the cytotoxic effects. K E Y W O R D Sapoptosis, bisphenol Z, cytotoxicity, derivative synthesis, XTT | 1575 STITZLEIN ET aL.

Dopamine (DA) receptors (DARs) regulate diverse physiological functions and are classified as either D1‐like (D1R and D5R) or D2‐like (D2R, D3R, and D4R) based on structural homology and pharmacological properties. DARs can signal through two main pathways: activation of G proteins and recruitment of β‐arrestin, a multivalent scaffolding protein that coordinates downstream signaling events. Upon agonist binding to the receptor, an active state is formed that activates G proteins and facilitates receptor phosphorylation by one or more members of the G protein‐coupled receptor kinase (GRK) family. GRK‐mediated receptor phosphorylation is widely believed to enhance GPCR‐β‐arrestin interactions, but the precise mechanism remains unclear for the D2R. We recently identified a single amino acid (F189 (5.38 using Ballesteros‐Weinstein numbering)) within the ligand binding site of the D2R that acts as a micro‐switch for regulating D2R interactions with β‐arrestin. When F189 is mutated to alanine, the mutant D2R (D2R‐F1895.38A) is capable of activating G proteins, but is unable to recruit β‐arrestin, i.e., D2R‐F1895.38A is a G protein‐biased mutant. To investigate the mechanisms underlying the biased signaling of D2R‐F1895.38A, its interactions with GRK2 were explored using a variety of bioluminescence resonance energy transfer (BRET) assays. GRK2 has been shown to phosphorylate the D2R‐WT in response to agonist stimulation and its overexpression can enhance the ability of DA to promote β‐arrestin recruitment to the receptor. Importantly, we found that the G protein‐biased D2R‐F1895.38A mutant does not recruit GRK2 upon agonist stimulation. This finding suggests that the absence of D2R‐F1895.38A ‐GRK2 interactions may underlie the receptor’s inability to recruit β‐arrestin. To test this hypothesis, we examined DA‐stimulated β‐arrestin and GRK2 recruitment using a phosphorylation‐defective D2R mutant (Namkung et. al., JBC 284:34103, 2009), a catalytically inactive GRK2 mutant (GRK2‐K220R), and compound 101, a GRK2/3 inhibitor. Strikingly, the phosphorylation‐defective D2R mutant was not impaired in its ability to recruit either GRK2 or β‐arrestin. In addition, overexpression of GRK2‐K220R, or treatment with compound 101, resulted in a decrease, but not elimination of DA‐stimulated β‐arrestin recruitment to the D2R, suggesting that, while GRK2 kinase activity is required for enhancement of β‐arrestin recruitment, phosphorylation of the D2R is not involved. We further examined the role of GRK2 using a cell line in which GRK2 was knocked out and found that, while β‐arrestin recruitment to the D2R was diminished, it was not eliminated. Taken together, these data suggest that while GRK2 plays a facilitatory role in regulating β‐arrestin recruitment to the D2R, and thus can potentially mediate receptor signaling bias, receptor phosphorylation itself does not appear to be involved. Support or Funding Information NINDS Intramural Research Program

Dopamine (DA) receptors (DARs) are G protein‐coupled receptors (GPCRs) that regulate diverse physiological functions including movement, cognition, mood, and reward‐related behaviors, as well as cardiovascular and renal physiology. Multiple diseases are linked to dysregulated dopaminergic functioning including Parkinson's disease, schizophrenia, substance use disorder, and hypertension. DARs are classified as either D1‐like (D1R and D5R) or D2‐like (D2R, D3R, and D4R) based on structural homology and pharmacological properties. The D1‐like DARs (D1R and D5R) increase cAMP, while the D2‐like DARs (D2R, D3R, D4R) decrease cAMP. All DARs also recruit β‐arrestin which activates separate signaling cascades and also initiates receptor desensitization and internalization. Generally, agonist activation of GPCRs, including DARs, rapidly leads to receptor desensitization and a return to basal levels of signaling. This occurs even in the continued presence of agonist, ensuring homeostasis. This desensitization process is intimately linked with receptor phosphorylation. The D1R is highly phosphorylated, with 32 intracellular serine and threonine residues, and is known to be phosphorylated by several kinases including protein kinase A (PKA), protein kinase C (PKC), and G protein‐coupled receptor kinases (GRKs). Previous studies by our lab indicate that the D1R is phosphorylated on its third intracellular loop (ICL3) and C‐terminus in a hierarchical fashion, in that phosphorylation must first occur on the C‐terminus before the ICL3 can be phosphorylated. Using systematic mutational analyses, we previously identified the PKC‐mediated D1R phosphorylation sites. We have now extended these studies to completely identify the DA‐induced, GRK‐mediated phosphorylation sites on the D1R. We found that GRK‐mediated phosphorylation involves several serine and threonine residues on the C‐terminus and ICL3. Mutation of these residues to alanine or valine, respectively, abolishes DA‐induced D1R phosphorylation and severely impairs β‐arrestin recruitment, but causes little effect on G protein‐mediated signaling. Our results indicate that a large fraction of DA‐induced D1R phosphorylation occurs on residues T360 and S362 in the proximal C‐terminus, and that these residues are also responsible for the majority of DA‐induced β‐arrestin recruitment to the D1R. Using HEK cells that have had their endogenous GRKs knocked out via CRISPR, we found that DA‐induced β‐arrestin recruitment to the D1R is severely impaired. However, β‐arrestin recruitment can be restored by expressing exogenous GRKs in these cells, further suggesting that DA‐induced β‐arrestin recruitment to the D1R is highly dependent on GRK phosphorylation. As GRK distribution varies by tissue and brain region, it is intriguing to postulate that D1R phosphorylation by different GRKs can add layers of regulatory fine‐tuning through differential effects on D1R signaling or trafficking outcomes.

Much of the research surrounding bisphenol derivatives focuses on their use as polymers and plasticizers for industry. Research has demonstrated that bisphenol‐A (BPA) adversely affects humans via endocrine disruption through hormonal modulation. Despite the recent explosion in BPA research, little effort has been devoted to researching the biological activities of other bisphenol derivatives, including bisphenol‐Z (BPZ). Initial screening of BPZ revealed moderate in vitro anticancer activity against four diverse human cancer cell lines (A375 malignant melanoma, A‐172 glioblastoma, HT‐29 colorectal adenocarcinoma, and MCF7 breast adenocarcinoma) with IC50 values of approximately 65 μM following 24 hour treatments. In an attempt to enhance the activity of BPZ, seven analogs with varying degrees of polarity, lipophilicity, and sterics on the cyclohexane ring were synthesized. Screening of these analogs revealed that polar substitutions resulted in a loss of activity, while a simple methyl substitution had similar activity to BPZ. Anticancer activity was enhanced relative to BPZ following substitution with an ethyl, isopropyl, or N‐palmitoyl group at the 4‐ position. The most active compounds from this structure‐activity study were the N‐palmitoyl (LS4) and isopropyl (LS6) derivatives with 24 hour IC50 values ranging from 25 to 53 μM and 33 to 42 μM respectively.Given the increased activity of LS4 and LS6, additional studies were performed including a trypan blue exclusion assay and a Caspase‐Glo® 3/7 assay. The results of these studies revealed exclusion of trypan blue at supra‐IC50 concentrations six hours after treatments, suggestive of a non‐necrotic mechanism of death. To determine if caspase‐dependent apoptosis was involved, we performed a Caspase‐Glo® 3/7 assay. Functional screening of executioner caspase‐3 and ‐7 revealed significant increases in activity following a 12 hour treatment with LS4 in all cells lines. This effect was least evident in the MCF7 cells, potentially due to a mutation wherein these cells lack expression of caspase‐3. In contrast, LS6 failed to activate executioner caspases‐3 and ‐7 in these same cell lines.In conclusion, this structure‐activity study revealed that nonpolar substitution at the 4‐ position of BPZ enhanced anticancer activity. Additionally, membrane integrity remained intact following exposure to LS4 and LS6. While LS4 activated executioner caspases‐3/7, LS6 failed to activate this hallmark pathway of apoptotic death. Further investigations are ongoing to elucidate the mechanisms of cell death, identify putative cellular targets, and determine potential selectivity against normal human cell lines.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Poster Board 281Dopamine receptors (DARs) are G-protein coupled receptors (GPCRs) that regulate diverse physiological functions including cognition, mood, movement, and reward-related behaviors, and are involved in the treatment or etiology of many neuropsychiatric disorders including schizophrenia and substance use disorder (SUD). DARs are classified as either D1-like (D1R and D5R) or D2like (D2R, D3R, and D4R) based on structural homology and pharmacological profiles. Antagonists of D2-like DARs are currently used in the therapies for many neuropsychiatric disorders, but D3R-selective antagonists may be better therapeutics for schizophrenia or SUD as they could attenuate psychotic or drug craving symptoms without the motor side effects frequently produced by D2R-preferring antagonists due to limited distribution of the D3R in the brain. However, discovery of D3R-selective compounds is challenging due to high sequence homology of the D2R and D3R within their orthosteric binding sites, leading to the potential for off-target side effects produced by currently available compounds due to simultaneous antagonism of both subtypes or other closely related receptors. Our lab has endeavored to overcome the selectivity challenges posed by orthosteric antagonists by utilizing a D3R-mediated b-arrestin recruitment assay to screen the NIH Molecular Libraries Program 400,000+ small molecule library for compounds that inhibit the D3R via binding to less conserved allosteric sites. The most potent hit compound, MLS6357, was selective for the D3R versus the D2R and D4R in several functional outputs including b-arrestin recruitment and G-protein activation. Radioligand binding and functional assays using closely related GPCRs revealed that MLS6357 has very limited cross-reactivity with other GPCRs. Additionally, Schild-type functional assays showed that MLS6357 acts as a purely non-competitive negative allosteric modulator (NAM) of the D3R. We synthesized and characterized > 70 analogs of MLS6357 using iterative medicinal chemistry approaches which produced analogs that are 100-fold and 60-fold more potent than the parent compound in D3R-mediated b-arrestin recruitment and G-protein activation assays, respectively, and revealed structure-activity relationships for further optimization of the scaffold. Moreover, some analogs appear to display functional selectivity for inhibition of G-protein activation versus inhibition of b-arrestin recruitment, and vice versa, and some also display inverse agonist activity in G-protein signaling assays. Using in vivo pharmacokinetic experiments in mice via i.p. administration, one of the lead analogs was found to be brain penetrant and achieved sufficient concentrations to occupy the D3R in vivo. To identify the allosteric binding site for the MLS6357 scaffold on the D3R, we utilized various D3R/D2R chimeras, receptor mutants, and molecular modeling techniques to reveal and characterize receptor regions necessary for compound efficacy. Further refinement of the binding pocket for MLS6357 will ...