Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • In experiments using D to displace binding

    2022-06-29

    In experiments using D22 to displace binding of specific high-affinity radioligands for DAT, NET, and SERT in mouse selinexor tissue, we observed a ranking with D22 displacement efficiency in the order of DAT > SERT > NET (IC50 values being ~ 11 (DAT), 26 (SERT), and 101 (NET) µM; Fig. 1, Fig. 2, Fig. 3, Table 2). Although to a significantly lower extent than the high-affinity blocker cocaine, our data in heterologous, transiently expressed cells lines, show that D22, in contrast to corticosterone, can inhibit [3H]MPP+ uptake in mDAT and mNET, and [3H]5-HT uptake through mSERT, with a similar binding potency rank order of SERT > DAT > NET (IC50 values being 8 (SERT), 13 (DAT), and 35 (NET) µM; Fig. 4, Table 3). However, given that 1) the affinity of psychotherapeutic drugs used clinically to target SERT, NET, and DAT (e.g. SSRIs, selective NET inhibitors) is in the nanomolar range (Frazer, 2001), 2) plasma levels of these drugs in patients are typically in the ng/ml range (Boulton et al., 2010, Kaye et al., 1989), and 3) doses of D22 required to produce behavioral effects in rodents are in the sub-milligram/kg range (Baganz et al., 2008, Horton et al., 2013), it is highly unlikely that behaviorally relevant concentrations of D22 in extracellular fluid would reach the micromolar range required to block SERT, NET or DAT. In general, most of the D22 analogs were more potent inhibitors of SERT and NET than D22. As depicted by dividing the IC50 values for [3H]citalopram by the values for [3H]nisoxetine binding (Fig. 7), all 7 analogs had a potency ratio below 1 and thus favor binding to mSERT over mNET. Compounds 3 (P = 0.0019), 4, 6, and 7 (P < 0.0001) each have a markedly lower IC50 value to inhibit SERT (mean IC50 range 0.75–10 µM) than the parent D22, with the latter three compounds (4, 6, and 7, P < 0.0001) also being statistically more potent at SERT binding than the native substrate, serotonin (Table 2). Compounds 4 (P < 0.001), 6 (P = 0.01), and 7 (P < 0.02) showed a significantly greater affinity to displace binding at DAT when compared to dopamine, as well. Based on having potency ratio values above 1 (Fig. 7), parent D22, along with compounds 2 and 4 prefer binding at mDAT over binding to mSERT. All analogs either shared a similar or had a higher IC50 value (thus, were less potent) than D22 to displace nisoxetine binding at NET. Earlier work established D22 as a potent inhibitor of low-affinity/high-capacity transporters, including OCT2, OCT3, and PMAT (Gasser et al., 2006 ; Hayer‐Zillgen et al., 2002; Russ et al., 1992; Russ et al., 1993a, Russ et al., 1993b, Russ et al., 1996; Schomig et al., 1993; Shirasaka et al., 2016; Sun et al., 2014; Wang et al., 2014; Zhang et al., 1998). In line with prior D22 action at hOCT3 (Hayer‐Zillgen et al., 2002), we found that D22 was a more potent inhibitor of [3H]MPP+ uptake at hOCT3 (IC50 ≈ 0.2 µM) than hOCT2 (IC50 ≈ 10 µM) and extended this finding to hPMAT (IC50 ≈ 1 µM). The glucocorticoid corticosterone also inhibits OCT3-mediated transport and is extremely useful to probe OCT3 function. Our corticosterone competition data (Fig. 5C) are similar to prior studies (Hayer‐Zillgen et al., 2002), where corticosterone preferentially bound to hOCT3 (IC50 = 0.6 µM) relative to hOCT2 (IC50 ≈ 80 µM). Corticosterone is also a poor blocker of hPMAT-driven transport (Engel et al., 2004, Russ et al., 1993a, Russ et al., 1993b), as supported by its observed IC50 ≈ 1 mM in our experiments, and in the literature (Engel et al., 2004, Miura et al., 2017). The consistency selinexor of our results with the literature establishes an important platform on which to perform comparative analyses of D22 analogs in human transporter expressing cell lines. As seen in concentration-response analyses (Fig. 6) and summarized IC50 data (Table 4), all D22 analogs favored hOCT3 binding in comparison to hPMAT or hOCT2, apart from compound 2 where there was not a significant difference between the IC50 values for hOCT2, hOCT3, or hPMAT. The inhibition potency at hOCT2 and hPMAT was equal for most analogs, except compound 3, which had a significantly higher IC50 value, thus lower potency, to inhibit [3H]MPP+ uptake at hOCT2 (P = 0.0001 vs. hPMAT). Therefore, structural changes among many of the analogs do not appear to impart differences in binding affinities for hOCT2 or hPMAT. Taken together with data for D22 (IC50 = 0.2 µM) and corticosterone (IC50 = 0.6 µM), D22 analogs are potent hOCT3-prefering inhibitors with IC50 values ranging from ~0.4–2.6 µM (Table 4). Analogs with IC50 values under or near 1 µM (compounds 3–7) were not statistically different from corticosterone; nor did the lowest of those, being compound 5, have a lower IC50 value than the parent D22 compound. Therefore, even the most potent of D22 analogs was not as potent as the parent compound in blocking hOCT3-mediated [3H]MPP+ uptake.