Evidence for effective structure-based neuromodulatory effects of new analogues of neurosteroid allopregnanolone

The neurosteroid allopregnanolone (AP) modulates neuroendocrine/neurobiological processes, including hypothalamic-­pituitary-­adrenocortical activities, pain, anxiety, neurogenesis and neuroprotection. These observations raised the hope of developing AP-­based therapies against neuroendocrine and/or neurodegenerative disorders. However, the pleiotropic actions of AP, particularly its cell-­proliferation-­promoting effects, hamper the development of selective/targeted therapies. For example, although AP-­induced neurogenesis may serve to compensate neuronal loss in degenerative brains, AP-­evoked cell-­proliferation is contraindicated for steroid-­sensitive cancer patients. To foster progress, we synthesised 4 novel AP analogues of neurosteroids (ANS) designated BR053 (12-o­ xo-e­ pi-­AP), BR297 (O-­allyl-­epi-­AP), BR351 (O-­allyl-­AP) and BR338 (12-­oxo-­AP). First, because AP is well-­known as allosteric modulator of GABAA receptors (GABAA-­R), we used the electrophysiological patch-­clamp technique to determine the structure-­activity relationship of our ANS on GABAA-­activated current in NCB20 cells expressing functional GABAA-­R. We found that the addition of 12-o­ xo-g­ roup did not significantly change the respective positive or negative allosteric effects of 3α-­AP or 3β-(­epi)-­AP analogues. Importantly, substitution of the 3α-h­ ydroxyl-­ group by 3α-O­ -­allyl highly modified the ANS activities. Unlike AP, BR351 induced a long-l­asting desensitisation/inhibition of GABAA-­R. Interestingly, replacement of the 3β-­hydroxyl by 3β-O­ -­allyl (BR297) completely reversed the activity from negative to positive allosteric action. In a second step, we compared the actions of AP and ANS on SH-­SY5Y neuronal cell viability/proliferation using MTT-­reduction assays. Different dose-r­ esponse curves were demonstrated for AP and the ANS. By contrast to AP, BR297 was totally devoid of cell-­proliferative effect. Finally, we compared AP and ANS abilities to protect against oxidative stress-­induced neuronal death pivotally involved in neurodegenerative diseases. Both BR351 and BR297 had notable advantages over AP in protecting SH-­SY5Y cells against oxidative stress-­induced death. Thus, BR297 appears to be a potent neuroprotective compound devoid of cell-­proliferative activity. Altogether, our results suggest promising perspectives for the development of neurosteroid-­based selective and effective strategies against neuroendocrine and/or neurodegenerative disorders