mitochondrial respiration, negative allosteric modulation of CB1
was investigated and identification of binding pocket was carried
out with Forced-Biased Metropolis Monte Carlo simulated annealing calculation [4]. The binding pocket of pregnenolone was localized at the CB1 receptor lipid interface and this pocket faces the
TMH1/TMH7/Hx8 region of the receptor (Fig. 6). Because
E1.49 was identified as the key residue for pregnenolone by
Monte Carlo simulations, the pregnenolone pocket was validated
using a mutant hCB1 receptor where an aspartate residue in the
helix 1 (E1.49) was mutated. Then, pregnenolone lost its effects as
NAM of CB1, when cells expressing hCB1 mutant receptors were
treated with THC [4] (see Note 2).
2.2.2 Metadynamics
In the recent paper of Wang et al. [22] a new allosteric pocket was
identified in the purinergic channel receptor P2X3, which is different from the one identified in the P2X7 receptor [24]. Figure 7
shows the superimposition of the P2X3 receptor bound to a negative allosteric ligand, AF-219 (magenta cartoon and sticks), with
the P2X7 receptor model [11]. In Fig. 7, the P2X7 receptor is
represented as a ribbon and residues are colored as function of
H
H
H
O
HO
Fig. 5 Pregnenolone (pregn-5-en-3β-ol-20-one) structure
Fig. 6 Binding pocket of pregnenolone in the CB1 receptor
Molecular Dynamics and Drug Discovery
251
was investigated and identification of binding pocket was carried
out with Forced-Biased Metropolis Monte Carlo simulated annealing calculation [4]. The binding pocket of pregnenolone was localized at the CB1 receptor lipid interface and this pocket faces the
TMH1/TMH7/Hx8 region of the receptor (Fig. 6). Because
E1.49 was identified as the key residue for pregnenolone by
Monte Carlo simulations, the pregnenolone pocket was validated
using a mutant hCB1 receptor where an aspartate residue in the
helix 1 (E1.49) was mutated. Then, pregnenolone lost its effects as
NAM of CB1, when cells expressing hCB1 mutant receptors were
treated with THC [4] (see Note 2).
2.2.2 Metadynamics
In the recent paper of Wang et al. [22] a new allosteric pocket was
identified in the purinergic channel receptor P2X3, which is different from the one identified in the P2X7 receptor [24]. Figure 7
shows the superimposition of the P2X3 receptor bound to a negative allosteric ligand, AF-219 (magenta cartoon and sticks), with
the P2X7 receptor model [11]. In Fig. 7, the P2X7 receptor is
represented as a ribbon and residues are colored as function of
H
H
H
O
HO
Fig. 5 Pregnenolone (pregn-5-en-3β-ol-20-one) structure
Fig. 6 Binding pocket of pregnenolone in the CB1 receptor
Molecular Dynamics and Drug Discovery
251