Postsynaptic N-methyl-D-aspartate receptors (NMDARs) phasically activated by presynaptically released glutamate are critical for synaptic transmission and plasticity. However, under pathological conditions, excessive activation of NMDARs by tonically increased ambient glutamate contributes to excitotoxicity associated with various acute and chronic neurological disorders. Here, using heterologously expressed GluN1/GluN2A and GluN1/GluN2B receptors and rat autaptic hippocampal microisland cultures, we show that pregnanolone sulfate inhibits NMDAR currents induced by a prolonged glutamate application with a higher potency than the NMDAR component of EPSCs. For synthetic pregnanolone derivatives substituted with a carboxylic acid moiety at the end of an aliphatic chain of varying length and attached to the steroid skeleton at C3, the difference in potency between tonic and phasic inhibition increased with the length of the residue. The steroid with the longest substituent, pregnanolone hemipimelate, had no effect on phasically activated receptors while inhibiting tonically activated receptors. In behavioral tests, pregnanolone hemipimelate showed neuroprotective activity without psychomimetic symptoms. These results provide insight into the influence of steroids on neuronal function and stress their potential use in the development of novel therapeutics with neuroprotective action.
N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play a crucial role in excitatory synaptic transmission. However, the overactivation of NMDARs can lead to excitotoxic cell damage/death, and as such, they play a role in numerous neuropathological conditions. The activity of NMDARs is known to be influenced by a wide variety of allosteric modulators, including neurosteroids, which in turn makes them promising therapeutic targets. In this study, we describe a new class of neurosteroid analogues which possess structural modifications in the steroid D-ring region. These analogues were tested on recombinant GluN1/GluN2B receptors to evaluate the structure-activity relationship. Our results demonstrate that there is a strong correlation between this new structural feature and the in vitro activity, as all tested compounds were evaluated as more potent inhibitors of NMDA-induced currents (IC50 values varying from 90 nM to 5.4 μM) than the known endogeneous neurosteroid-pregnanolone sulfate (IC50 = 24.6 μM).
Cyclin-dependent kinases are therapeutic
targets frequently deregulated
in various cancers. By convenient alkylation of the 5-sulfanyl group,
we synthesized 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidines with various
substitutions at position 5 with potent antiproliferative activity
in non-Hodgkin lymphoma cell lines. The most potent derivative 4.35 also displayed activities across more than 60 cancer
cell lines. The kinase profiling confirmed high selectivity of 4.35 toward cyclin-dependent kinases (CDKs) 2, 5, and 9, and
the cocrystal with CDK2/cyclin A2 revealed its binding in the active
site. Cultured lymphoma cell lines treated with 4.35 showed
dephosphorylation of CDK substrates, cleavage of PARP-1, downregulation
of XIAP and MCL-1, and activation of caspases, which collectively
confirmed ongoing apoptosis. Moreover, 4.35 demonstrated
significant activity in various cell line xenograft and patient-derived
xenograft mouse models in vivo both as a monotherapy and as a combination
therapy with the BCL2-targeting venetoclax. These findings support
further studies of combinatorial treatment based on CDK inhibitors.
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