Selective Small Molecule Activators of TREK‑2 Channels Stimulate Dorsal Root Ganglion c‑Fiber Nociceptor Two-Pore-Domain Potassium Channel Currents and Limit Calcium Influx

The two-pore-domain potassium (K2P) channel TREK-2 serves to modulate plasma membrane potential in dorsal root ganglia c-fiber nociceptors, which tunes electrical excitability and nociception. Thus, TREK-2 channels are considered a potential therapeutic target for treating pain; however, there are currently no selective pharmacological tools for TREK-2 channels. Here we report the identification of the first TREK-2 selective activators using a high-throughput fluorescence-based thallium (Tl⁺) flux screen (HTS). An initial pilot screen with a bioactive lipid library identified 11-deoxy prostaglandin F2α as a potent activator of TREK-2 channels (EC₅₀ ≈ 0.294 μM), which was utilized to optimize the TREK-2 Tl⁺ flux assay (<i>Z</i>′ = 0.752). A HTS was then performed with 76 575 structurally diverse small molecules. Many small molecules that selectively activate TREK-2 were discovered. As these molecules were able to activate single TREK-2 channels in excised membrane patches, they are likely direct TREK-2 activators. Furthermore, TREK-2 activators reduced primary dorsal root ganglion (DRG) c-fiber Ca²⁺ influx. Interestingly, some of the selective TREK-2 activators such as 11-deoxy prostaglandin F2α were found to inhibit the K2P channel TREK-1. Utilizing chimeric channels containing portions of TREK-1 and TREK-2, the region of the TREK channels that allows for either small molecule activation or inhibition was identified. This region lies within the second pore domain containing extracellular loop and is predicted to play an important role in modulating TREK channel activity. Moreover, the selective TREK-2 activators identified in this HTS provide important tools for assessing human TREK-2 channel function and investigating their therapeutic potential for treating chronic pain

Behavior of established compounds in VSD assay.

<p><b>(A1)</b> Normalized fluorescence data for VSD assay on A2R-stg cells with 50 μM fluorowillardiine (black) with maxGLU (red) and NBQX (pink) controls, all normalized to EC₅₀GLU (blue). <b>(A2)</b> CRC curves for FW against A2-stg and (<b>A3)</b> A2-C3 cell lines calculated from the CMPDslope window. %max GLUslope = (CMPDslope—mean VHLslope)/(mean maxGLUslope—mean VHLslope) is further described in methods. (<b>B1)</b> Normalized fluorescence data for VSD assay on A2R-stg cells with 1 mM CX-546 (black) with maxGLU (red) and NBQX (pink) controls, all normalized to EC₅₀GLU (blue). <b>(B2)</b> CRC curves for CX-546 against A2R-stg and (<b>B3)</b> A2R-C3 cell lines calculated from the GLUslope window. %max GLUslope = (GLUslope—mean VHLslope)/(mean maxGLUslope—mean VHLslope) <b>(C1)</b> Normalized fluorescence data for VSD assay on A2R-stg cells with 500 μM cyclothiazide (CTZ, black) with maxGLU (red) and NBQX (pink), all normalized to EC₅₀GLU (blue). <b>(C2)</b> CRC curves for CTZ against A2-stg and <b>(C3)</b> A2-C3 cell lines calculated from the GLUslope window. Compound EC₅₀ values that could be reliably calculated are in the top left corner of each graph.</p

Characterization of VU0539491.

<p><b>(A)</b> Raw data for compound CRCs in the VSD assay. A2R-stg (orange) and A2R-C3 (blue). EC₅₀GLU traces are represented by dashed lines. Compound concentrations are indicated in the top left corner. <b>(B)</b> CRCs calculated from GLUslope in VSD assay for A2R-stg and A2R-C3. <b>(C)</b> Raw data for compound CRCs in the calcium flux assay. A2Q (red), A2Q-stg (orange), A2Q-GSG (green), and A2Q-C3 (blue). Traces obtained from applying 1 mM glutamate are represented by dashed lines. <b>(D)</b> Compound CRCs calculated from the GLUslope1 window in our calcium flux assay for A2Q-stg, A2Q-C3, A2Q-GSG, and A2Q cell lines. These show negative and positive trends but no curve fits. <b>(E)</b> Compound CRCs calculated from the AUC in the GLUmaxmin window of the calcium flux assay plotted as %max AUC vs. log [compound] as in (7E).</p

Screening for AMPA receptor auxiliary subunit specific modulators

<div><p>AMPA receptors (AMPAR) are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP <i>γ</i>-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD) to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM), respectively. They both have lower IC₅₀/EC₅₀s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R)-CNIH3 and GluA2(Q) complexes and PAM activity in GluA2(Q)-GSG1L complexes.</p></div

Table of results from VSD and calcium flux assays.

<p>EC₅₀ or IC₅₀ values determined by CRC fits from GLUslope. Boxes highlighted green indicate an EC₅₀ or a positive trend, red indicate an IC₅₀ or a negative trend. Estimated EC₅₀ values are added in italics, but are estimated due to incomplete CRC curves or insufficient differences in %max GLUslope across the CRC. Glutamate potency fold-shift assays indicate how much fold-change occurred in the glutamate EC₅₀ when cells were pretreated with 30 μM compound. Values greater than 2 indicate PAM activity and less than 1 indicate NAM activity.</p

Characterization of VU0627849.

<p><b>(A)</b> Raw data for compound CRCs in the VSD assay. A2R-stg (orange) and A2R-C3 (blue). EC₅₀GLU traces are represented by dashed lines. Concentrations of compound are indicated in the top left corner. <b>(B)</b> CRCs calculated from GLUslope in the VSD assay for A2R-stg and A2R-C3. <b>(C)</b> Raw data for compound CRCs in the calcium flux assay. A2Q (red), A2Q-stg (orange), A2Q-GSG (green), and A2Q-C3 (blue). 1 mM glutamate traces are represented by dashed lines. <b>(D)</b> CRCs calculated from GLUslope1 in calcium flux assay for A2Q-stg, A2Q-C3, A2Q-GSG, and A2Q cell lines. Plotted as %max GLUslope vs. log [compound].</p

Electrophysiology with VU0627849.

<p><b>(A</b>) Whole-cell recordings of A2R cell line with (blue) and without (red) VU0627849 (40μM). <b>(B)</b> Whole-cell recordings of A2R-stg cell line with (blue) and without (red) VU0627849 (40μM). Recording after washout of drug is in black. In these experiments, glutamate (1mM) is applied for 100 ms and 20 ms pulses with or without VU0627849.</p

Inhibitors of 7‑Dehydrocholesterol Reductase: Screening of a Collection of Pharmacologically Active Compounds in Neuro2a Cells

A small library of pharmacologically active compounds (the NIH Clinical Collection) was assayed in Neuro2a cells to determine their effect on the last step in the biosynthesis of cholesterol, the transformation of 7-dehydrocholesterol (7-DHC) to cholesterol promoted by 7-dehydrocholesterol reductase, <i>DHCR7.</i> Of some 727 compounds in the NIH Clinical Collection, over 30 compounds significantly increased 7-DHC in Neuro2a cells when assayed at 1 μM. Active compounds that increased 7-DHC with a <i>Z-</i>score of +3 or greater generally gave rise to modest decreases in desmosterol and increases in lanosterol levels. Among the most active compounds identified in the library were the antipsychotic, antidepressant, and anxiolytic compounds that included perospirone, nefazodone, haloperidol, aripiprazole, trazodone, and buspirone. Fluoxetine and risperidone were also active at 1 μM, and another 10 compounds in this class of pharmaceuticals were identified in the screen at concentrations of 10 μM. Increased levels of 7-DHC are associated with Smith-Lemli-Opitz syndrome (SLOS), a human condition that results from a mutation in the gene that encodes <i>DHCR7.</i> The SLOS phenotype includes neurological deficits and congenital malformations, and it is linked to a higher incidence of autism spectrum disorder. The significance of the current study is that it identifies common pharmacological compounds that may induce a biochemical presentation similar to SLOS. Little is known about the side effects of elevated 7-DHC postdevelopmentally, and the elevated 7-DHC that results from exposure to these compounds may also be a confounder in the diagnosis of SLOS

Workflow for identifying AMPAR-auxiliary subunit modulators.

<p><b>(A)</b> 39,202 compounds were initially screened using the VSD assay against A2R-stg cells. <b>(B)</b> 1,184 hits from (A) were counter-screened against A2R, TetON, and A2R-C3 cells. <b>(C)</b> 116 compounds were identified from counter-screening in (B) as being stargazin or auxiliary subunit specific (i.e. they did not hit on A2R or TetON cells). These were tested for full compound CRCs against A2R-stg and A2R-C3 cells using the VSD assay. These CRCs identified 90 hits that fit to sigmoidal dose response curves with potency under 10 μM. <b>(D)</b> We identified 39 stargazin specific PAMs, 2 CNIH3 specific PAMs, and 36 PAMs that had activity in both A2R-stg and A2R-C3 cells. We also found 1 stargazin specific NAM and 9 compounds with NAM activity on both cell lines. Three compounds gave opposite effects in the two cell lines. Hits were discarded for reorder if they showed activity in the compound only window. Hits with activity in the CMPD only windows were discarded. <b>(E)</b> 57 of the 90 compounds in (D) were re-screened with new batch samples as compound CRCs in the VSD assay. <b>(F)</b> 57 hits were tested in the glutamate potency fold-shift calcium flux assay and 28 were subjected to a full compound CRC calcium flux assay to study their effects using an orthogonal approach.</p

Chemical structures of our candidate hits.

<p><b>(A)</b> Structure of VU0612951 highlighting the 1,3-triazole group in red. <b>(B)</b> Structure of VU0627849 highlighting the isoxazole group in red. <b>(C)</b> Structure of VU0539491 highlighting the 1,2,4-oxadiazole group in red.</p