Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands

Abstract

Neuroactive steroids are potent positive allosteric modulators of GABAA receptors (GABAAR), but the locations of their GABAAR binding sites remain poorly defined. To discover these sites, we synthesized two photoreactive analogs of alphaxalone, an anesthetic neurosteroid targeting GABAAR, 11β-(4-azido-2,3,5,6-tetrafluorobenzoyloxy)allopregnanolone, (F4N3Bzoxy-AP) and 11-aziallopregnanolone (11-AziAP). Both photoprobes acted with equal or higher potency than alphaxalone as general anesthetics and potentiators of GABAAR responses, left-shifting the GABA concentration – response curve for human α1β3γ2 GABAARs expressed in Xenopus oocytes, and enhancing [3H]muscimol binding to α1β3γ2 GABAARs expressed in HEK293 cells. With EC50 of 110 nM, 11-AziAP is one the most potent general anesthetics reported. [3H]F4N3Bzoxy-AP and [3H]11-AziAP, at anesthetic concentrations, photoincorporated into α- and β-subunits of purified α1β3γ2 GABAARs, but labeling at the subunit level was not inhibited by alphaxalone (30 μM). The enhancement of photolabeling by 3H-azietomidate and 3H-mTFD-MPAB in the presence of either of the two steroid photoprobes indicates the neurosteroid binding site is different from, but allosterically related to, the etomidate and barbiturate sites. Our observations are consistent with two hypotheses. First, F4N3Bzoxy-AP and 11-aziAP bind to a high affinity site in such a pose that the 11-photoactivatable moiety, that is rigidly attached to the steroid backbone, points away from the protein. Second, F4N3Bzoxy-AP, 11-aziAP and other steroid anesthetics, which are present at very high concentration at the lipid-protein interface due to their high lipophilicity, act via low affinity sites, as proposed by Akk et al. (Psychoneuroendocrinology 2009, 34S1, S59–S66).

Comparison of αβδ and αβγ GABAA receptors: Allosteric modulation and identification of subunit arrangement by site-selective general anesthetics

“…In studies of phasic inhibition associated with αβγ receptors, etomidate [96, 97], propofol [98, 99], the barbiturate pentobarbital [100, 101], and the neurosteroids tetrahydrodeoxycorticosterone (THDOC) [102] and alphaxalone [103] all prolong IPSC decay. Potent general anesthetics also enhance GABA-evoked activation of oocyte-expressed α1β2/3γ2 receptors (Figure 2B), producing large “leftward shifts” (decreased EC 50 ) and a small “upward shift” (increased maximal GABA current response) in concentration-responses [46, 60, 69, 104, 105]. This pattern of anesthetic-induced changes indicates high GABA intrinsic efficacy in αβγ receptors, consistent with previous single channel studies [59, 106, 107].…”

“…SCAMP experiments comparing contacts for propofol, etomidate and alphaxalone on β3-M3 in α1β3γ2 receptors show that alphaxalone protects cysteine substitutions at β3 residues V290, F293, L297 and F301, and some of these mutations also reduce neurosteroid sensitivity [174]. In addition, SCAMP studies confirm that neurosteroids do not interact with established etomidate and R- m TFD-MPAB contacts [105]. Neurosteroid sites are thus adjacent and intracellular to the sites where etomidate binds in β+/α − interfaces, and the effects of mutations in these sites suggest that they account for the majority of neurosteroid effects on channel gating.…”

Photoaffinity labeling identifies an intersubunit steroid-binding site in heteromeric GABA type A (GABAA) receptors

“…Substitution of an 11b-OH further reduced potency by 20-fold (3a5b-P-11b-ol, IC 50 ; 200 mM). In contrast, high affinity binding was retained in the presence at C-11 of either the small azi-group (11-Azi-AP, IC 50 = 0.4 mM) or the bulky azidotetrafluorophenyl-group (11-F4N3Bzoxy-AP, IC 50 = 0.1 mM) in two recently introduced photoreactive 3a5a-P derivatives that act as potent GABA A R PAMs and general anesthetics (45).…”

Multigram Synthesis of Functionalized Spirocyclic Diazirines

“…steroid analogues such as 4 , Boc‐protected photo‐proline 5 , ), synthesis of spirocyclic diazirine‐containing building blocks is discussed in the literature to a lesser extent. The prominent role of spirocyclic diazirines in biomedical research,, , physical organic chemistry and organic synthesis, motivated us to challenge the synthesis of a series of diazirines derived from saturated carbo‐ and heterocycles with different ring size and substitution pattern. Since it was outlined that amide formation was the major reaction for diazirine photoaffinity labelling probes synthesis (43 % of the methods used), we have focused on the preparation of building blocks containing carboxylic acid and amine functions.…”