Diazepam is not a direct allosteric modulator of α1‐adrenoceptors, but modulates receptor signaling by inhibiting phosphodiesterase‐4

Abstract α1A‐ and α1B‐adrenoceptors (ARs) are G protein‐coupled receptors (GPCRs) that are activated by adrenaline and noradrenaline to modulate smooth muscle contraction in the periphery, and neuronal outputs in the central nervous system (CNS). α1A‐ and α1B‐AR are clinically targeted with antagonists for hypertension and benign prostatic hyperplasia and are emerging CNS targets for treating neurodegenerative diseases. The benzodiazepines midazolam, diazepam, and lorazepam are proposed to be positive allosteric modulators (PAMs) of α1‐ARs. Here, using thermostabilized, purified, α1A‐ and α1B‐ARs, we sought to identify the benzodiazepine binding site and modulatory mechanism to inform the design of selective PAMs. However, using a combination of biophysical approaches no evidence was found for direct binding of several benzodiazepines to purified, stabilized α1A‐ and α1B‐ARs. Similarly, in cell‐based assays expressing unmodified α1A‐ and α1B‐ARs, benzodiazepine treatment had no effect on fluorescent ligand binding, agonist‐stimulated Ca2+ release, or G protein activation. In contrast, several benzodiazepines positively modulated phenylephrine stimulation of a cAMP response element pathway by α1A‐ and α1B‐ARs; however, this was shown to be caused by off‐target inhibition of phosphodiesterases, known targets of diazepam. This study highlights how purified, stabilized GPCRs are useful for validating allosteric ligand binding and that care needs to be taken before assigning new targets to benzodiazepines.


| INTRODUCTION
Adrenergic receptors, or adrenoceptors (ARs), are a family of G protein-coupled receptors (GPCRs) that bind endogenous adrenaline and noradrenaline and are important for modulating the cardiovascular and nervous systems. GPCRs are seven α-helical transmembrane (TM) proteins that bind ligands on the extracellular face shifting the conformational equilibria of the GPCR to active states and promoting cytoplasmic interactions with heterotrimeric G proteins and other signaling proteins. 1 There are three AR subfamilies, α 1 , α 2 , and β-ARs, each comprising three receptor subtypes. 2 The α 1 -AR subtypes, α 1A -AR, α 1B -AR, and α 1D -AR couple to Gα q/11 G proteins to activate phospholipase C-β (PLCβ) that catalyses the formation of the second messenger, inositol 1,4,5-trisphosphate (IP 3 ), thereby stimulating intracellular calcium mobilization. α 1 -AR signaling stimulates smooth muscle contraction and thus α 1 -AR antagonists are prescribed for hypertension and benign prostatic hyperplasia. 3 α 1A -AR and α 1B -AR are also highly expressed in the brain, and transgenic rodent models have indicated α 1A -AR activation stimulates neurogenesis, while prolonged α 1B -AR stimulation promotes apoptotic neurodegeneration. 3,4 In the failing rodent heart, α 1A -AR stimulation drives adaptive hypertrophy, whereas chronic α 1B -AR activation causes maladaptive hypertrophy as a result of hemodynamic overload. 3 Thus, selective α 1A -AR activation, or α 1B -AR blockade, could be useful therapeutic strategies for certain diseases.
While there are some α 1A -AR and α 1B -AR subtype-selective ligands available, no highly α 1 -AR subtype-selective drugs have been approved for use in the clinic. The highly similar ligand binding sites in closely related AR subtypes makes identifying subtype-selective ligands challenging. Allosteric ligands, on the other hand, bind to distinct sites in the receptor and can modulate the activity of the receptor in response to agonist binding. As allosteric sites are less conserved between receptor subtypes it may be possible that allosteric modulators offer scope for achieving subtype selectivity. Several ligands have been reported to act as allosteric modulators of α 1A -AR and α 1B -AR, including the conotoxin ρ-TIA, 5 9-aminoacridine, 6 and the benzodiazepines diazepam, lorazepam, and midazolam. 7 However, the exact structural mechanisms by which these ligands modulate the receptors are unknown.
Benzodiazepines are positive allosteric modulators (PAMs) of GABA A R ion channels and are widely prescribed as sedatives, anxiolytics, anticonvulsants, and myorelaxants. 8 The benzodiazepines, diazepam and lorazepam, inhibit Ca 2+ oscillations in pulmonary artery smooth muscle cells, suggestive of off-target interactions with ARs. 9 Waugh et al. 7 postulated that benzodiazepines directly bind to We recently engineered thermostabilized mutants of α 1A -AR and α 1B -AR, which enabled binding epitope determination of orthosteric ligands with nuclear magnetic resonance (NMR) spectroscopy. 10 In this work we sought to use these thermostabilized receptors and our NMR approach to further understand how benzodiazepines bind to and modulate α 1 -ARs so as to apply this information as a starting point for developing more selective modulators targeting this allosteric site. Instead we observed no evidence for the direct binding of several benzodiazepines to purified α 1A -AR and α 1B -AR using NMR and fluorescent ligand binding assays. Cell-based binding and calcium signaling assays with wild-type (WT) α 1 -ARs also failed to detect any direct modulatory effects of diazepam on these receptors. While diazepam could positively modulate the stimulation of a cAMP response element (CRE) reporter through α 1 -AR activation, this was found to be driven through the ability of diazepam to inhibit phosphodiesterases (PDEs), a known target of some benzodiazepines. This study highlights how purified GPCRs can be used to directly investigate allosteric modulator mechanisms that have been proposed from cell-based assays, where off-target actions are difficult to control for.

| Benzodiazepine preparation
Nordiazepam and diazepam were synthesized from 2-amino-5-chlorobenzophenone using the method of Sternbarch et al. 11 N-hydroxyethyl-nordiazepam was synthesized from nordiazepam according to the method of Archer et al. 12 Further details of benzodiazepine preparation and chemical synthesis can be found in Supporting Information.

| Protein expression, purification, and binding
assays α 1A -AR variant A4 and α 1B -AR variant #15 were expressed in Escherichia coli and purified as described previously. 10 Stabilized rat neurotensin receptor 1 (enNTS 1 ) was expressed and purified as described by Bumbak et al. 13 BODIPY-FL-prazosin (QAPB [quinazoline piperazine bodipy]) competition binding assays were performed as described previously. 10 Briefly, 2 nmol of purified receptor, with C-terminal mCherry-Avi tag fusion, were incubated with 100 μL pmol of receptor per well. A Kingfisher 96 magnetic particle processor was then used to transfer the beads into plates containing QAPB and various competitors, which was incubated for 2 hours at 22°C with gentle mixing. The beads were then washed for 1 minute in binding buffer then transferred to 100 μL binding buffer in black Greiner nonbinding 96-well plates. QAPB fluorescence was measured using 485/12 nm excitation and 520/10 nm emission filters, while mCherry fluorescence was measured using 544 nm excitation and 590/10 nm emission filters in an Omega POLARstar plate reader (BMG Labtech, Ortenberg, Germany).

| Whole cell QAPB binding and Ca 2+ mobilization assays
Saturation binding of QAPB in the absence or presence of 50 μmol/L diazepam was measured using COS-7 cells stably expressing WT human α 1A -AR and α 1B -AR as previously described. 10  To determine nonspecific binding, cells were exposed to QAPB at the same concentrations as above, but in the presence of 100 μmol/ L phentolamine. The cells were incubated with ligands for 1 hour at 20°C prior to detection of bound QAPB with flow cytometry using CytoFLEX LX flow cytometer (Beckman Coulter, Brea, CA, USA). Intracellular Ca 2+ mobilization assays were performed on nontransfected COS-7 cells and cells stably expressing either α 1A -AR or α 1B -AR. Cells were seeded at 25 000 cells per well into 96-well culture plates and allowed to grow overnight at 37°C, 5% CO 2 . Cells were washed twice with Ca 2+ assay buffer (150 mmol/L NaCl, 2.6 mmol/L KCl, 1.2 mmol/L MgCl 2 , 10 mmol/L D-glucose, 10 mmol/L HEPES, 2.2 mmol/L CaCl 2 , 0.5% [w/v] BSA, and 4 mmol/L probenecid, pH 7.4) and then incubated in Ca 2+ assay buffer containing 1 mmol/L Fluo-4-AM for 1 hour in the dark at 37°C and 5% CO 2 .
After two washes with Ca 2+ assay buffer and the addition of phenylephrine solutions (or co-addition of phenylephrine and benzodiazepines) fluorescence was measured for 1.5 minute in a Flexstation plate reader (Perkin Elmer, Waltham, MA, USA) using an excitation wavelength of 485 nm and emission wavelength of 520 nm. HEK293T cells transiently transfected with GPR68 and pCRE were stimulated with sodium bicarbonate-free, low glucose DMEM, supplemented with 20 mmol/L HEPES and 0.5% FBS at pH 6.8, 7.0, 7.2, 7.4, and 7.8. The pH was adjusted at 37°C with NaOH. Cells were incubated for 6 hours at 37°C, in a room atmosphere incubator. Diazepam or lorazpam were made up in media at pH 7.2 or 7.8, co-added with rolipram and stimulated for 6 hours in above conditions.

| Data analysis
Nuclear magnetic resonance (NMR) data were processed in Topspin 3.5 using squared cosine-bells in both dimensions and zero-filled once, prior to Fourier-transformation. All other data was analyzed  performed in triplicate wells and normalized to the peak response elicited by 3 μmol/L ionomycin. CRE assay was conducted in triplicate, and data were normalized to the response elicited by the vehicle. Curves were fitted with three variable nonlinear regressions. For GPR68 assays, responses were normalized to CRE response at pH 6.8 (100%) and pH 7.8 (0%).

| RESULTS
Previously the benzodiazepines lorazepam,diazepam, and midazolam were shown to behave as positive modulators of α 1 -AR agonists in cell lines overexpressing α 1 -ARs, with low micromolar potencies. 7 However, direct binding of benzodiazepines to purified α 1 -AR proteins has never been demonstrated. Thermostabilized GPCRs are receptors containing mutations that improve the protein stability upon solubilization and purification using detergents. 14 The retention of natural receptor pharmacology enables thermostabilized receptors to be used for structural biology 15 and for probing the mechanisms and kinetics of ligand binding in a purified system. 10,13,16 The thermostabilized variants α 1A -AR A4 and α 1B -AR #15 were recently described and exhibit the stability required to probe the binding of benzodiazepines to the purified receptors. 10 We hypothesized that if benzodiazepines are direct allosteric modulators of α 1 -AR, then they should influence the binding of orthosteric antagonists and/or agonists to α 1 -AR. Biotinylated α 1A -AR A4 or α 1B -AR #15 was immobilized onto streptavidin-coated paramagnetic beads and placed in a 10 nmol/L solution of fluorescent-labeled BODIPY-FL prazosin (QAPB), an approximately K d concentration, with serial dilutions of validated competitors,diazepam or lorazepam, for 2 hours. The K d of QAPB at α 1A -AR A4 and α 1B -AR #15 has been previously reported to be 11.6 and 8.5 nmol/L respectively. 10 The agonist phenylephrine displaced QAPB at both receptor subtypes in an expected dosedependent manner ( Figure 1A,B). Conversely, neither diazepam nor lorazepam displaced QAPB at either receptor, even at concentrations of up to 200 μmol/L ( Figure 1A,B). As diazepam is predicted to bind to an allosteric site distinct from the orthosteric site, where QAPB binds, this discrepancy could be due to noncompetitive binding of the two ligands. However, the ability of diazepam to increase the potency and efficacy of the α 1 -AR agonist phenylephrine suggests that allosteric binding of diazepam influences the affinity of phenylephrine for the receptors. Thus, QAPB competition binding assays were performed at α 1A -AR A4 and α 1B -AR #15 where a sub-IC 50 concentration of phenylephrine (2.5 mmol/L) was included and the ability of increasing concentrations of diazepam to positively modulate QAPB displacement by phenylephrine was monitored. No cooperativity between diazepam and phenylephrine was observed at either receptor subtype. These data suggest that diazepam is either not binding to the thermostabilized α 1 -ARs, has a very low affinity interaction, or is allosterically binding, but not able to modulate phenylephrine binding in this purified system. Saturation transfer difference (STD) NMR is a ligand-observed experiment that is especially sensitive at monitoring ligands that bind weakly to proteins (K d > 1 μmol/L) and does not require labeled F I G U R E 1 Diazepam and lorazepam quinazoline piperazine bodipy (QAPB) competition binding assays. Chemical structures of (A) diazepam and (B) lorazepam. QAPB competition binding at (C) α 1A -AR A4 and (D) α 1B -AR #15. Binding was normalized with 100% representing 10 nmol/ L QAPB without competition and 0% 10 nmol/L QAPB with competition with 10 μmol/L prazosin. Competition was performed with increasing concentrations of phenylephrine, diazepam, and lorazepam, or diazepam and lorazepam in the presence of 2.5 mmol/L phenylephrine. Data are mean ± SD of three independent experiments performed in duplicate ligands or proteins. 17 We recently applied STD NMR to the study of orthosteric agonist binding at α 1A -AR A4 and α 1B -AR #15. 10 Here, STD NMR was applied to determine if diazepam and lorazepam bind to purified α 1A -AR A4. STD NMR signals were observed for diazepam and lorazepam (Figure 2A,E) when incubated with DDM micelles that were not loaded with protein ( Figure 2B,F Figure 3C). To probe positive modulation of this response, α 1A -AR and α 1B -AR expressing cells were treated with an EC 50 concentration of phenylephrine (10 nmol/L) and increasing concentrations of diazepam or lorazepam before measuring the mobilization of intracellular calcium 5 minute after treatment. Treatment with these benzodiazepines (0 to 50 μmol/L), had no effect in the absence ( Figure 3D), or presence of phenylephrine on both receptor expressing cell lines ( Figure 3E,F). To gain a more complete measure of phenylephrineinduced α 1 -AR signaling over a 6-h stimulation period, a CRE-reporter gene assay was employed. In this assay, diazepam positively modulated the potency of phenylephrine at α 1A -AR and α 1B -AR expressing cells and also increased the E max of phenylephrine at α 1B -AR expressing cells ( Figure 4A,B and Table 1). Diazepam modulated the phenylephrine CRE response with potencies of 6.5 ± 4.8 μmol/L on α 1A -AR and 7.8 ± 1.9 μmol/L on α 1B -AR (mean EC 50 ± SD from three experiments, Figure 4C). Critically, diazepam treatment in the absence of phenylephrine did not induce a CRE response (Figure 4C). However, diazepam positively modulated the phenylephrine-induced CRE response on COS-7 cells that do not express α 1A -AR and α 1B -AR ( Figure 4D), suggesting that the action of diazepam on CRE signaling is independent of α 1 -AR stimulation. All α 1 -ARs signal primarily through Gα q/11 G proteins, which activate the effector protein phospholipase C (PLC). PLC catalyses the formation of diacylglycerol (DAG), which then activates phosphokinase C (PKC), and IP 3 causing Ca 2+ release from the endoplasmic reticulum via InsP3R Ca 2+ channels. 18 α 1 -AR activation also has a secondary effect of stimulating cAMP production via calmodulin (CaM)-stimulated adenylate cyclase (AC). 19 PKC and secondary G protein coupling may also play a role in AC activation. 20,21 CaM family kinases I and IV are activated by CaM which phosphorylate the transcription factor cAMP response binding protein, leading to its activation, and upregulation of CRE genes. 22 Thus, there are many molecular targets in the CRE pathway that benzodiazepines may be interacting with to positively modulate the α 1 -AR CRE response. To assess the specificity of this action of diazepam, CRE assays were performed on human embryonic kidney (HEK) 293-T cells, which endogenously express β-ARs, treated with the β 2 -AR agonist isoprenaline. Treatment with diazepam at 50 μmol/L significantly increased the CRE efficacy of isoprenaline at HEK293-T cells ( Figure 4E), suggesting that diazepam modulation of the CRE response is not specific to α 1 -AR stimulation. Furthermore, 50 μmol/L diazepam positively modulated the potency of the AC activator forskolin in HEK293-T cells ( Figure 4F), indicating that diazepam is not modulating CRE activity at the receptor level, but at some other step of the signaling pathway.  Table 1.
Interestingly, the N-methyl group of diazepam, which is lacking in nordiazepam, was found to be critical for positive modulation of the phenylephrine-induced CRE activation in α 1B -AR expressing COS-7 cells and forskolin stimulation of CRE in untransfected HEK cells ( Figure 5 and Table 1). Furthermore, the GABA A R-inactive benzodiazepine, 7-phenyl-diazepam, was able to modulate the phenylephrine-induced CRE activation, although its actions on forskolin failed to reach statistical significance ( Figure 5 and Table 1). This defined SAR indicated that the benzodiazepine mechanism driving this positive modulation of phenylephrine was likely being driven by a specific binding interaction with a target other than GABA A R in the cells rather than nonspecific interactions, for example, with the cell membrane.
Next, we sought to define the mechanism by which diazepam was modulating CRE activation by using inhibitors of various potential targets. Co-addition of the GABA A R antagonist bicuculline had no effect on the positive modulation of phenylephrine or forskolin by diazepam ( Figure 6A,B). Voltage-gated calcium channels, through a CaM-dependent mechanism, are known to play a role in α 1 -ARinduced cAMP production and diazepam is thought to bind to some Ca 2+ channels. 23 Cotreatment of cells with the broad spectrum Ca 2+ channel inhibitor benidipine significantly reduced both the phenylephrine response and the diazepam modulation of the CRE response in α 1A -AR expressing cells but had no effect on the positive modulation of forskolin-stimulated CRE response in the same cells (Figure 6A,B). Similarly, the CaM inhibitor W-7 hydrochloride significantly reduced both the phenylephrine response and the diazepam modulation of the CRE response in α 1A -AR expressing cells, probably by blocking the same pathway as the calcium channel inhibitor but had no significant effect on forskolin stimulation (Figure 6C,D). These results implicate Ca 2+ channels in α 1 -AR-induced CRE activation, however are unlikely to be driving diazepam mediated positive modulation as diazepam modulation of forskolininduced CRE was unaffected by benidipine or W-7 hydrochloride.
Other inhibitors such as the InsP3R antagonist 2-APB, the L-type calcium channel inhibitor (R)-(+)-Bay K 8644, and the Ca 2+ /CaMdependent protein kinase II inhibitor KN-93, had no significant effect on the ability of diazepam to positively modulate the phenylephrineor forskolin-stimulated CRE responses ( Figure 6C,D).
Diazepam is an inhibitor of PDEs, especially PDE-4, 24 and thus should increase the levels of cAMP in cells during the CRE assay, potentially explaining our observations. In this case, we would expect co-addition of the broad-spectrum PDE inhibitor IBMX, or the PDE-4 inhibitor rolipram, to have no additional modulatory effect on top of diazepam treatment in our CRE assays. Indeed, in α 1A -AR expressing COS-7 cells, IBMX and rolipram positively modulated the CRE response of phenylephrine to a similar level as diazepam ( Figure 6E); however, cotreatment using IBMX or rolipram with diazepam had no additional positive modulatory effect on the phenylephrine response ( Figure 6E). Similarly, IBMX and rolipram positively modulated the CRE response of forskolin on these same cells, but no additive effect was observed for either IMBX plus diazepam or rolipram plus diazepam ( Figure 6F). In fact, cotreatment of rolipram and diazepam significantly decreased the forskolin-induced CRE response compared to rolipram alone ( Figure 6F), possibly indicating competition between diazepam and rolipram at the same binding site on PDE-4.
These data strongly suggest that the positive modulation of the phenylephrine-induced α 1 -AR CRE response by diazepam, and other benzodiazepines, is caused through inhibition of PDEs.
Lorazepam was recently reported to be an allosteric modulator of the pH-sensitive GPCR, GPR68. 25 GPR68 has been shown to couple to G q , G s , G 12/13, and G i/o proteins 25

| DISCUSSION
The benzodiazepines diazepam, lorazepam and midazolam have been reported to be PAMs at the α 1 -ARs. 7 Waugh et al. 7 used indirect radioligand binding and signaling assays on receptor-overexpressing cell lines to demonstrate positive modulation of these benzodiazepines on α 1A -AR, α 1B -AR, and α 1D -AR; however, direct binding of the benzodiazepines to α 1 -ARs has never been measured. Here, purified, thermostabilized α 1 -AR variants were used as a tool to probe whether diazepam and lorazepam positively modulate α 1 -AR signaling via direct binding to the receptors. Interestingly, no evidence was obtained that diazepam or lorazepam could bind to either purified α 1 -AR subtype using fluorescent ligand binding assay (Figure 1 preclude binding at allosteric sites. However, diazepam also had no effect on the binding of an orthosteric antagonist (QAPB) to cells stably expressing WT α 1B -AR and did not positively modulate phenylephrine-induced intracellular Ca 2+ release in cells expressing WT α 1A -AR or α 1B -AR (Figure 3), also suggesting that diazepam does not bind to these receptors.
Using a CRE reporter assay, which is responsive to multiple GPCR-stimulated signaling pathways to detect downstream effects, diazepam was found to positively modulate phenylephrine-induced CRE stimulation in WT α 1A -AR and α 1B -AR expressing cells. The ability of diazepam to modulate the CRE response was independent of α 1 -AR stimulation, which was shown using β-AR agonists and cells that natively express β-ARs, 26 and by directly activating AC with forskolin ( Figure 4). This suggests that diazepam acts upon signaling elements downstream of the receptor. Interestingly, while diazepam treatment improved the potency of phenylephrine at activating CRE in both α 1A -AR and α 1B -AR expressing cells, it enhanced the efficacy of phenylephrine only at α 1B -AR expressing cells. This likely reflects differences between how phenylephrine activates CRE at α 1A -AR compared to α 1B -AR.
The SAR governing benzodiazepine action at GABA A receptors is well understood. The substituent at C-7 is of paramount importance; small electron-withdrawing substituents at C-7 generally impart high activity, whereas electron donors or large groups are inactive. 27 Substitutions at N-1 in contrast are generally tolerated, even though Points represent mean values from replicate experiments, horizontal lines the means and error bars the standard deviations. Statistical differences were determined using one-way ANOVA and Sidak multiple comparisons test they can impart significant modulatory effects on GABA A activity. 28 By screening various analogues of diazepam, the SAR governing CRE modulation was found to be different to that at GABA A R (Table 1), with the methyl group at the N-1 position of diazepam shown to be vital for modulating the CRE response, whereas phenyl substitution at C-7 was tolerated. These chemical differences indicate that the effect observed here is through a target other than GABA A R.
Benzodiazepine interactions have been reported against cholecystokinin receptors, 29 α 2 -ARs, 30 HIV-1 reverse transcriptase, 30 κopioid receptors, 30 muscarinic receptors, 30 translocator protein, 31 Ca 2+ channels, 23 Ca 2+ /CaM-dependent protein kinases 32 , and PDE. 24,33 Using inhibitors of several of the other potential target proteins that could be responsible for CRE modulation we were able to conclude that inhibition of PDE, most likely PDE-4, by diazepam causes modulation of the CRE response ( Figure 6). Diazepam inhibits the activity of guinea pig PDE-4 with an IC 50 of 9 μmol/L, 24 which is similar to the potency of diazepam for modulating the CRE response ( Figure 4). Diazepam also competes with 3 H-rolipram at purified guinea pig PDE-4, 24 which potentially explains the ability of diazepam to compete with rolipram ( Figure 6). Collado et al. screened several benzodiazepines for activity at guinea pig PDE-4 and found that clonazepam, nitrazepam, and lorazepam, each of which are unsubstituted at N-1, were less potent than diazepam, 24 which broadly matches the SAR observed here. A similar benzodiazepine, lorazepam, also lacked the ability to modulate the CRE response, but was recently found to positively modulate the pH-sensitive GPCR and GPR68. Here, we confirmed the activity of lorazepam upon GPR68 using the CRE assay and demonstrated that its activity is not due to PDE inhibition. Notably, lorazepam induced a significantly higher GPR68-induced CRE response than diazepam or rolipram treatment.
Interestingly, diazepam did not significantly increase the CRE response of GPR68 at pH 7.2, suggesting that GPR68 stimulates CRE in a different, more robust way to α 1 -ARs. This may be related to the fact that GPR68 can couple to Gs proteins to directly activate cAMP accumulation, and thus CRE, 25 whereas α 1 -ARs stimulate CRE indirectly through Gq.
In summary, this work shows the value of stabilized, purified α 1 -ARs to probe direct molecular interactions, allowing us to show that the modulation of α 1 -AR activity by benzodiazepines in cell-based assays is not a result of direct ligand binding. We further demonstrated that significant modulation of cellular CRE responses by diazepam most likely occurred through inhibition of PDE-4. Given the widespread use of benzodiazepines therapeutically, this off-target effect may contribute to their clinical actions and side effects and warrants further study. In contrast to this, lorazepam was validated as a direct-acting, PAM of the pH sensitive receptor GPR68.

ACKNOWLEDG EMENTS
We thank Sharon Layfield and Tania Ferraro for assistance with cell culture and CRE response assays. We thank Dr Lauren May (Monash