Knowledge-based prediction of DNA hydration using hydrated dinucleotides as building blocks.

Water plays an important role in stabilizing the structure of DNA and mediating its interactions. Here, the hydration of DNA was analyzed in terms of dinucleotide fragments from an ensemble of 2727 nonredundant DNA chains containing 41 853 dinucleotides and 316 265 associated first-shell water molecules. The dinucleotides were classified into categories based on their 16 sequences and the previously determined structural classes known as nucleotide conformers (NtCs). The construction of hydrated dinucleotide building blocks allowed dinucleotide hydration to be calculated as the probability of water density distributions. Peaks in the water densities, known as hydration sites (HSs), uncovered the interplay between base and sugar-phosphate hydration in the context of sequence and structure. To demonstrate the predictive power of hydrated DNA building blocks, they were then used to predict hydration in an independent set of crystal and NMR structures. In ten tested crystal structures, the positions of predicted HSs and experimental waters were in good agreement (more than 40% were within 0.5 Å) and correctly reproduced the known features of DNA hydration, for example the `spine of hydration' in B-DNA. Therefore, it is proposed that hydrated building blocks can be used to predict DNA hydration in structures solved by NMR and cryo-EM, thus providing a guide to the interpretation of experimental data and computer models. The data for the hydrated building blocks and the predictions are available for browsing and visualization at the website https://watlas.datmos.org/watna/.

Synthetic testosterone derivatives modulate rat P2X2 and P2X4 receptor channel gating.

P2X receptors (P2XRs) are ATP-gated cationic channels that are allosterically modulated by numerous compounds, including steroids and neurosteroids. These compounds may both inhibit and potentiate the activity of P2XRs, but sex steroids such as 17ß-estradiol or progesterone are reported to be inactive. Here, we tested a hypothesis that testosterone, another sex hormone, modulates activity of P2XRs. We examined actions of native testosterone and a series of testosterone derivatives on the gating of recombinant P2X2R, P2X4R and P2X7R and native channels expressed in pituitary cells and hypothalamic neurons. The 17ß-ester derivatives of testosterone rapidly and positively modulate the 1 µM ATP-evoked currents in P2X2R- and P2X4R-expressing cells, but not agonist-evoked currents in P2X7R-expressing cells. In general, most of the tested testosterone derivatives are more potent modulators than endogenous testosterone. The comparison of chemical structures and whole-cell recordings revealed that their interactions with P2XRs depend on the lipophilicity and length of the alkyl chain at position C-17. Pre-treatment with testosterone butyrate or valerate increases the sensitivity of P2X2R and P2X4R to ATP by several fold, reduces the rate of P2X4R desensitization, accelerates resensitization, and enhances ethidium uptake by P2X4R. Native channels are also potentiated by testosterone derivatives, while endogenously expressed GABA receptors type A are inhibited. The effect of ivermectin, a P2X4R-specific allosteric modulator, on deactivation is antagonized by testosterone derivatives in a concentration-dependent manner. Together, our results provide evidence for potentiation of particular subtypes of P2XRs by testosterone derivatives and suggest a potential role of ivermectin binding site for steroid-induced modulation. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

3,5,7-Substituted Pyrazolo[4,3- d]pyrimidine Inhibitors of Cyclin-Dependent Kinases and Their Evaluation in Lymphoma Models.

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.

Strong Inhibitory Effect, Low Cytotoxicity and High Plasma Stability of Steroidal Inhibitors of N-Methyl-D-Aspartate Receptors With C-3 Amide Structural Motif.

Herein, we report the synthesis, structure-activity relationship study, and biological evaluation of neurosteroid inhibitors of N-methyl-D-aspartate receptors (NMDARs) receptors that employ an amide structural motif, relative to pregnanolone glutamate (PAG) - a compound with neuroprotective properties. All compounds were found to be more potent NMDAR inhibitors (IC50 values varying from 1.4 to 21.7 µM) than PAG (IC50 = 51.7 µM). Selected compound 6 was evaluated for its NMDAR subtype selectivity and its ability to inhibit AMPAR/GABAR responses. Compound 6 inhibits the NMDARs (8.3 receptors (8.3 ± 2.1 µM) more strongly than it does at the GABAR and AMPARs (17.0 receptors (17.0 ± 0.2 µM and 276.4 ± 178.7 µM, respectively). In addition, compound 6 (10 µM) decreases the frequency of action potentials recorded in cultured hippocampal neurons. Next, compounds 3, 5-7, 9, and 10 were not associated with mitotoxicity, hepatotoxicity nor ROS induction. Lastly, we were able to show that all compounds have improved rat and human plasma stability over PAG.

Positive Modulators of the N-Methyl-d-aspartate Receptor: Structure-Activity Relationship Study of Steroidal 3-Hemiesters.

Here, we report the synthesis of pregn-5-ene and androst-5-ene dicarboxylic acid esters and explore the structure-activity relationship (SAR) for their modulation of N-methyl-d-aspartate receptors (NMDARs). All compounds were positive modulators of recombinant GluN1/GluN2B receptors (EC50 varying from 1.8 to 151.4 µM and Emax varying from 48% to 452%). Moreover, 10 compounds were found to be more potent GluN1/GluN2B receptor modulators than endogenous pregnenolone sulfate (EC50 = 21.7 µM). The SAR study revealed a relationship between the length of the residues at carbon C-3 of the steroid molecule and the positive modulatory effect at GluN1/GluN2B receptors for various D-ring modifications. A selected compound, 20-oxo-pregnenolone hemiadipate, potentiated native NMDARs to a similar extent as GluN1/GluN2A-D receptors and inhibited AMPARs and GABAAR responses. These results provide a unique opportunity for the development of new steroid based drugs with potential use in the treatment of neuropsychiatric disorders involving hypofunction of NMDARs.

Structural Basis of the Interaction of Cyclin-Dependent Kinase†2 with Roscovitine and Its Analogues Having Bioisosteric Central Heterocycles.

The structural basis for the interaction of roscovitine and analogues containing 13 different bioisosteric central heterocycles with the enzyme cyclin-dependent kinase 2 (CDK2) is elucidated. Although all the central scaffolds are very similar to the purine core of roscovitine, the experimentally determined IC50 values of the inhibitors span three orders of magnitude. By using an extensive computational chemistry approach, the affinities of the inhibitors to CDK2 are determined as calculated binding scores of complexes of the inhibitors with the protein. The interactions of the inhibitors with CDK2 are computationally described by using a hybrid quantum mechanics/semi-empirical quantum mechanics method (QM/SQM), which combines the DFT-D method for the QM part and the PM6-D3H4X method for the SQM part. The solvent effect is described by the COSMO implicit solvation model at the SQM level for the whole system. The contributions of the scaffolds and the individual substituents, quantified and evaluated in relation to conformations of optimized protein-inhibitor complexes, are found not to be simply additive. The inhibitory activity of the selected candidates, including two newly prepared compounds, is tested against CDK2. The results of the calculations are in close agreement with the experimental data.

Distributions of therapeutically promising neurosteroids in cellular membranes.

Interactions of two neurosteroids, inhibiting membrane-bound N-Methyl-d-aspartate receptors, with phospholipid membranes are studied. Namely, endogenous pregnanolone sulfate is compared with pregnanolone glutamate, the latter being a novel synthetic steroidal inhibitor of these receptors with potential pharmaceutical use. Molecular-level details of steroid-phospholipid membranes interactions are scrutinized employing molecular dynamics simulations supported by quantum chemical calculations to assess steroid lipophilicity. Moreover, permeability of both species across membranes is experimentally evaluated by immobilized artificial membrane chromatography. We demonstrate that while there is no significant difference of lipophilicity and membrane permeability between the two steroids, they differ significantly regarding detailed localization in phospholipid membranes. The bulky glutamate moiety of pregnanolone glutamate is flexible and well exposed to the water phase while the sulfate group of pregnanolone sulfate is hidden in the membrane headgroup region. This dissimilarity of behavior in membranes can potentially account for the observed different activities of the two steroids toward membrane-bound N-Methyl-d-aspartate receptors.

Physicochemical and biological properties of novel amide-based steroidal inhibitors of NMDA receptors.

Herein, we report a new class of amide-based inhibitors (1-4) of N-methyl-d-aspartate receptors (NMDARs) that were prepared as analogues of pregnanolone sulfate (PAS) and pregnanolone glutamate (PAG) - the steroidal neuroprotective NMDAR inhibitors. A series of experiments were conducted to evaluate their physicochemical and biological properties: (i) the inhibitory effect of compounds 3 and 4 on NMDARs was significantly improved (IC50=1.0 and 1.4µM, respectively) as compared with endogenous inhibitor - pregnanolone sulfate (IC50=24.6µM) and pregnanolone glutamate (IC50=51.7µM); (ii) physicochemical properties (logP and logD) were calculated; (iii) Caco-2 assay revealed that the permeability properties of compounds 2 and 4 are comparable with pregnanolone glutamate; (iv) compounds 1-4 have minimal or no adverse hepatic effect; (v) compounds 1-4 cross blood-brain-barrier.

Neurosteroid-like Inhibitors of N-Methyl-d-aspartate Receptor: Substituted 2-Sulfates and 2-Hemisuccinates of Perhydrophenanthrene.

N-Methyl-d-aspartate receptors (NMDARs) display a critical role in various diseases of the central nervous system. The activity of NMDARs can be modulated by neurosteroids. Herein, we report a structure-activity relationship study for perhydrophenanthrene analogues possessing a framework that mimics the steroidal ring system. This study comprises the design, synthesis, and assessment of the biological activity of a library of perhydrophenanthrene 2-sulfates and 2-hemisuccinates (1-10). Their ability to modulate NMDAR-induced currents was tested on recombinant GluN1/GluN2B receptors. Our results demonstrate that such structural optimization leads to compounds that are inhibitors of NMDARs. Notably, compound 9 (IC50 = 15.6 µM) was assessed as a more potent inhibitor of NMDAR-induced currents than the known endogenous neurosteroid, pregnanolone sulfate (IC50 = 24.6 µM).

Preferential Inhibition of Tonically over Phasically Activated NMDA Receptors by Pregnane Derivatives.

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. SIGNIFICANCE STATEMENT: Synaptic activation of N-methyl-d-aspartate receptors (NMDARs) plays a key role in synaptic plasticity, but excessive tonic NMDAR activation mediates excitotoxicity associated with many neurological disorders. Therefore, there is much interest in pharmacological agents capable of selectively blocking tonically activated NMDARs while leaving synaptically activated NMDARs intact. Here, we show that an endogenous neurosteroid pregnanolone sulfate is more potent at inhibiting tonically than synaptically activated NMDARs. Further, we report that a novel synthetic analog of pregnanolone sulfate, pregnanolone hemipimelate, inhibits tonic NMDAR currents without inhibiting the NMDAR component of the EPSC and shows neuroprotective activity in vivo without inducing psychomimetic side effects. These results suggest steroids may have a clinical advantage over other known classes of NMDAR inhibitors.

A New Class of Potent N-Methyl-D-Aspartate Receptor Inhibitors: Sulfated Neuroactive Steroids with Lipophilic D-Ring Modifications.

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).