For structure-guided development of subtype-selective ligands, knowledge of their binding modes in the pocket is required. However, the exact molecular interactions of benzodiazepines at the GABA A receptor subunits have not been well characterized and remain under debate in the literature.
In silico studies, however, led to controversial hypotheses about the exact nature of this common binding mode. In the study presented here, we aimed to find experimental evidence that favored one binding mode or the other.
We therefore selected three scaffolds for docking and three corresponding pairs of substituted enantiomers for experimental testing.
This in silico docking confirmed the crucial position at the methyl group of the seven-membered diazepine ring. We investigated a set of three stereoisomeric pairs in radioligand displacement assays and with the two-electrode voltage clamp method. Interestingly, our data gave diverging results for the different chemical scaffolds, suggesting that different benzodiazepine ligands use distinct binding modes rather than a common binding mode.
In the accompanying manuscript, 13 we also present evidence of different binding mode usage based on an orthogonal mutational approach. Results and Discussion. We reasoned that this fact could possibly be used to find experimental evidence supporting one binding mode or the other.
High Resolution Image. In this study, we therefore selected three pairs of stereoisomers for our binding and electrophysiological studies, each with a chiral methyl group at position 3 of the seven-membered diazepine ring in the R or S configuration. To correlate the computational results with the binding studies, we updated and revisited the in silico approach for the prediction and validation of binding modes.
For the docking procedure, we employed the three achiral scaffolds from the six molecules, which we studied experimentally, and added the respective R - and S -methyl groups to the candidate binding modes. This procedure avoids excessive false positive in silico docking solutions for the experimentally tested compounds. In addition, docking the unsubstituted parent compounds of the chiral ligands facilitates direct comparison with the previous docking studies in which mainly achiral compounds have been studied.
Subsequent in silico derivatization of the docked ligands into the six test compounds led to predictions that were tested in the experimental part of this study. Careful analysis of the two binding modes supported our initial hypothesis that the introduction of a methyl group into the seven-membered diazepine ring could serve as tool for examining binding mode orientations of different benzodiazepine ligands.
In detail, the methyl group at the diazepine ring leads to two distinct isomers, namely, an R isomer and an S isomer. According to our model, the methyl groups of both isomers are pointing in the direction of the more flexible loop C if the ligands bind in a BM I orientation, and thus, both isomers should be active see panels a and b of Figure 3. On the contrary, if the ligands use a BM II orientation, methyl groups of the two isomers are pointing in substantially different directions.
Hence, in silico docking leads to the prediction that for ligands using BM I, R and S isomer analogues should both be binding with high or at least intermediate affinity and differ only mildly in affinity, while for BM II binding scaffolds, the R isomer should be inactive or display a marked drop in affinity compared to that of the S isomer. While for the S isomers compounds 1-S , 2-S , and 3-S intermediate to high binding affinities for the recombinant receptors were observed, which is in accordance with the results of our docking studies, the R isomers showed diverse binding behavior.
Interestingly, only imidazobenzodiazepine compound 1-R retained its potential to displace the radioligand [ 3 H]flunitrazepam, whereas compounds 2-R and 3-R were inactive, suggesting nonbinding. Generally, the S isomers display affinities higher than those of their R analogues. The obtained K i values are listed in Table 1. Table 1. Compounds 1-S , 2-S , 3-S , and 1-R potently replaced [ 3 H]flunitrazepam in all receptor subtypes, while compounds 2-R and 3-R failed to displace the radioligand in all receptors tested see Table 1.
However, we did observe differences in the potency rank order. Diazepam-like compound 3-S showed low subtype selectivity. Compounds 2-R and 3-R failed to displace [ 3 H]flunitrazepam binding at all GABA A receptor subtypes investigated even at high micromolar concentrations.
Radioligand displacement assays provide information about the binding of a compound to only a specific binding site. Electrophysiological experiments supported our radioligand binding assay findings. Two-voltage clamp recordings provide us with the additional knowledge that those drugs behaved as positive allosteric modulators as they potentiated the GABA-induced chloride ion flux.
In accordance with the previous data, compounds 1-S , 2-S , 3-S , and 1-R displayed a positive modulation in all receptor subtypes, while compounds 2-R and 3-R remained mainly modulatory silent. In this study, we used a combined approach comprising computational ligand docking, radioligand binding assays, and functional two-electrode voltage clamp assays to revisit the common binding mode hypothesis for diazepam-derived ligands versus imidazobenzodiazepines with different substituents on the imidazole ring.
Overall, we found that our chiral benzodiazepine ligand 1-R , representing an ester-substituted imidazobenzodiazepine, binds to the GABA A receptor benzodiazepine binding site. In this study, imidazobenzodiazepines interacted with the artificial site in a manner completely different from that of diazepam, which was also investigated with computational models and in turn interpreted as a sign of different binding site usage.
Our results are in line with previous experiments, in which it was demonstrated that the binding affinity, efficacy, and in vivo activity of a series of ligands with a chiral S-CH3 group at C-4 are different from those of the R-CH3 group. The compounds tested have a rather low affinity for the benzodiazepine binding site of GABA A receptors.
In addition, if we compare the substitutions on the basic benzodiazepine core scaffold , the pairs of compounds differ at several positions. In Figure 2 , the top row scaffold 1 and the middle row scaffold 2 differ in three substituents and are thus not a systematic variation on the scaffold see also Figure S4.
The middle row scaffold 2 differs from the bottom row scaffold 3 in two positions, which again is not a systematic variation. It still would not be appropriate to generalize to other similar compounds and look for structure—activity relationships SARs based on these compounds; our results do indicate in fact that chemically similar compounds can display strikingly dissimilar binding properties and ligand similarity-based SAR extrapolations might fail. The flunitrazepam-like compounds possess a 1,4-benzodiazepine nucleus with a 5-phenyl substituent, the midazolam-like compounds an imidazo ring with a 5-phenyl substituent, and the flumazenil-like compounds such as Ro and Ro an imidazo ring without a 5-phenyl substituent.
It is therefore not surprising that some experimental evidence points to the fact that some drugs interact with GABA A receptors differently than others. To ensure convergence of the sampling, genetic algorithm runs were performed. The ligands were built in MOE using the M conformation of the seven-membered ring that is supported by experimental studies.
Before the docking run, the ligand was energetically minimized using the MMFF94 force field. MOE Builder was used to derivatize the docked parent compounds with an S - or R -methyl at position 3. Thesis of S. Supporting Information. Author Information. Alshaimaa A. David C. James M. The authors declare no competing financial interest. Elsevier Inc. A review. The extrasynaptic GABA A R clusters monitored at the same time, which were significantly smaller in size, but larger in number than synaptic clusters, remained unaffected by diazepam Supplementary Figure 1a and b.
The total number of immunolabelled GABAergic or glutamatergic neurones remained unchanged data not shown. Consistent with the observed structural changes in synapses was a prominent reduction in the frequency and amplitude Fig.
When Ro flumazenil , a specific competitive antagonist at benzodiazepine binding site on the GABA A R, was applied together with diazepam, the decrease in size Fig. However, Ro alone caused a decrease in the number of GABAergic terminals colocalised with the postsynaptic GABA A Rs, suggesting that it may have additional presynaptic effects that remain to be elucidated.
Quantification was done using ImageJ. Crucially, the diazepam-dependent decrease in GABA A Rs surface levels in neurones was abolished in the presence of dynamin-inhibitory peptide Fig.
This indicates that the underlying cause of reduction at the cell surface was indeed dynamin-dependent endocytosis of GABA A Rs rather than inhibition in protein synthesis or reduced insertion into the plasma membrane. Consistent with this was the loss of cell surface receptors Fig. To establish which of these phosphatases are involved in diazepam-triggered endocytosis of GABA A Rs, treatments of cultured neurones were carried out in the presence of either low 0.
The decrease in size Fig. Moreover, cyclosporine A and diazepam, when applied together, caused a significant increase in the size of GABA A R clusters in comparison with the control, diazepam or cyclosporine A alone Fig. Furthermore, a functional link between prolonged diazepam-dependent stimulation of GABA A Rs and disassembly of GABAergic synapses, was assessed in experiments in which structural elements of synapses were analysed in the presence of bicuculline Fig. Bicuculline significantly attenuated the diazepam-dependent decrease in size Fig.
Diazepam and bicuculline added together also caused a significant increase in the size of GABA A R clusters in comparison with control, diazepam or bicuculline treatments alone Fig. Collectively, these data evince a cascade of signalling events triggered by prolonged stimulation of synaptic GABA A Rs which, in turn, leads to a calcineurin-mediated dephosphorylation and endocytosis of these receptors, and a consequent disassembly of GABAergic synapses.
Importantly, this increase was completely abolished when diazepam was applied in the presence of Ro Fig. The prevalence of stress-related psychiatric disorders, particularly anxiety mixed with depression, panic attacks or insomnia, leads to an estimated 12 million prescriptions of benzodiazepines every year in the UK UK Addiction Treatment Centres.
However, our current understanding of the long-term effects of benzodiazepines on cellular and molecular processes in the brain remains limited. Although studied in vitro, these processes are closely correlated in time to in vivo downregulation of GABA A Rs and the onset of tolerance to benzodiazepines in rodents [ 36 ]. These processes are however in sharp contrast with the initial diazepam-dependent facilitation of GABA A R channel gating activity [ 25 ], increased mobilisation of GABA A Rs to synapses [ 37 , 38 ], and enhanced inhibitory synaptic transmission [ 39 ], possibly representing a form of neuronal adaptation in order to maintain a critical balance between the excitation and inhibition in the brain.
A key role of calcineurin in diazepam-dependent endocytosis of GABA A Rs is in agreement with the previously reported effects on GABA A R migration out of the synaptic contacts [ 31 , 40 , 41 ], and internalisation from the cell surface [ 42 ]. Subsequent loss of inhibitory synapses indicates that depletion of postynaptic GABA A Rs destabilises synaptic contacts, an observation consistent with their activity-dependent regulation reported previously [ 43 ], and also with a direct structural role of GABA A Rs in the formation of these synapses [ 17 , 44 , 45 ].
As changes in cell surface GABA A Rs are known to precede changes in the postsynaptic gephyrin scaffold [ 46 ], our findings are also in agreement with the previously observed diazepam-dependent reduction in the size of gephyrin clusters [ 47 ].
This diazepam-induced breakdown of inhibitory GABAergic synapses not only correlates well in time with the development of tolerance but also provides a likely explanation for the severe withdrawal symptoms, increased anxiety and even seizures, observed in animal models and patients following sudden termination of chronic benzodiazepine treatment [ 60 ], possibly due to uncontrolled excitatory drive in the absence of functional inhibition.
This signalling mechanism offers a new spectrum of possible molecular interventions that could be tailored towards extending the initial highly beneficial clinical outcomes of benzodiazepines, while preventing the subsequent disruption of GABAergic synapses and development of pharmacological and behavioural tolerance to these widely prescribed drugs.
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Dopamine appeared very early in the course of evolution and is involved in many functions that are essential for survival of the organism, such as motricity, attentiveness, motivation, learning, and memorization. But most of all, dopamine is a key element in identifying natural rewards for the organism.
These natural stimuli such as food and water cause individuals to engage in approach behaviours. Dopamine is also involved in unconscious memorization of signs associated with these rewards.
It has now been established that all substances that trigger dependencies in human beings increase the release of a neuromediator, dopamine, in a specific area of the brain: the nucleus accumbens. Click on the names of each of the following drugs to read about how they work and what effects they have.
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