1.2 Purinergic receptors
1.2.9 The P2X4 receptor subtype
-24- 1.2.8.2 P2X3 receptor allosteric modulators
Cibacron Blue 3GA, the purified ortho-isomer of RB-2 (see Figure 1.10), was identified as a PAM of human and rat P2X3 receptor by Alexander et al. in 1999.127 It enhanced the ATP-induced calcium influx and transmembrane currents three- to seven times higher than the normal level with low micromolar potency (EC50Enhancement 1.4 ± 0.5 µM). The enhancing effect is not dependent on the agonist, since it is also observed when the receptor is stimulated with the ATP analogs BzATP, 2-meSATP or α,β-me-ATP.
-25- 1.2.9.1 P2X4 receptor antagonists
The rat P2X4 receptor is not inhibited by classic standard antagonists such as suramin and PPADS in concentrations up to 300 µM. In contrast, the mouse and human orthologues have a weak sensitivity to PPADS, and Coomassie Brilliant Blue G (BBG) is also a weak antagonist, as well as RB-2.28 TNP-ATP is the most potent competitive, though non-selective antagonist (see Table 1.1).
Since antidepressants have been proven to be effective against neuropathic pain states, a selection of antidepressant compounds have been tested for inhibitory potency at the P2X4 receptor. Paroxetine was identified as a potent allosteric antagonist of rat and human P2X4 receptors with low micromolar potency. Furthermore, it was demonstrated to be an effective drug in a neuropathic pain model. The antinociceptive effect could not be attributed to the serotonin receptor pathway.135 Amitriptyline, a tricyclic antidepressant clinically approved for the treatment of neuropathic pain, is only a weak inhibitor of P2X4 receptors. Due to its lack of potency, P2X4 receptor inhibition is not considered as the defining factor for efficacy against neuropathic pain.136
Figure 1.16: Structure and potency of P2X4 antagonists BBG, paroxetine, BX430, PSB-12054, PSB-12062, N,N-diisopropylcarbamazepine and 5-BDBD. The potency of BBG at the P2X7 receptor is further discussed in chapter 1.2.12.1.64, 135, 137-140
BBG
IC50 (P2X4) 3.16 ± 0.75 µM IC50 (P2X7) 0.01-0.265 µM
Paroxetine IC50 1.87-2.45 µM
BX430 IC50 0.54 µM
PSB-12054 IC50 0.189-2.10 µM
PSB-12062 IC50 0.928-1.76 µM
N,N-Diisopropyl-carbamazepine IC50 3.44-54.6 µM
5-BDBD IC50 1.2 µM
-26-
Benzodiazepine derivative 5-BDBD is a selective antagonist of the P2X4 receptor. The information of its mode of action is inconclusive. References for competitive and allosteric binding of 5-BDBD are known.137, 141 Another lead structure for successful and selective P2X4 antagonism is phenoxazine. A compound library of effective N-substituted derivatives was synthesized. They enabled structure-activity relationships at the P2X4 receptor for the first time.
The structure of the most active compound PSB-12054 inhibited the P2X4 receptor with nanomolar potency and was at least tenfold selective towards human P2X1, P2X2, P2X3, and P2X7 receptors. One disadvantage of the compound is its poor water solubility. The closely related derivative 12062 is more water-soluble with potency and selectivity similar to PSB-12054.64 Structurally distantly related anticonvulsant drug carbamazepine and derivatives were identified as potent P2X4 receptor antagonists as well. The most effective compound of the series was N,N-diisopropylcarbamazepine. The compound was fairly selective towards human P2X2 and P2X7, but not P2X1 and P2X3 receptors (IC50 P2X1 5.32 ± 1.91 µM and P2X7 7.18 ± 2.86 µM, respectively).138 The newest P2X4 receptor-selective phenylurea antagonist BX430 was recently discovered by screening the Canadian Chemical Biology Network library using calcium imaging and whole cell patch clamp experiments. BX430 inhibited the P2X4 receptor with high nanomolar potency. The compound is described to be soluble in physiological solutions and stable at room temperature, and to be selective towards human P2X1-3, P2X5 and P2X7 receptors.139
1.2.9.2 P2X4 receptor allosteric modulators
Ethanol was identified as an allosteric antagonist of the P2X4 receptor. The inhibitory effect can only be seen at high concentrations (100 mM), which makes ethanol not very interesting as a P2X4 receptor antagonist. However, there are several genomic findings including the decrease of P2X4 receptor mRNA expression in the brain of alcohol-preferring rats indicating that P2X4 receptor activation may play a role as a reducer of alcohol intake and preference.142 Therefore, the activated P2X4 receptors may contribute to sobriety and reduction of alcohol use disorders.
The broad-spectrum antiparasitic drug ivermectin has been identified as a positive allosteric modulator of the P2X4 receptor with high nanomolar to low micromolar potency in 1999 (see Figure 1.17).143 Ivermectin is a macrolide drug isolated from Streptomyces avermectinius as a mixture of two compounds Ivermectin H2B1a and ivermectin H2B1b, and is approved for the treatment of onchocerciasis and in a topical form for rosacea. Further investigations identified ivermectin as an effective drug to reduce alcohol intake and preference by antagonizing ethanol-induced inhibition of P2X4 receptors, which could be verified in an animal model.144-146 The
-27-
positive allosteric modulation can be observed at other ion channels like the glutamate-gated chloride channel, γ-aminobutyric acid type-A receptor, glycine receptor or neuronal α7-nicotinic receptor.147 Ivermectin is perorally bioavailable, widely distributed in the body, especially in cell membranes due to its high lipophilicity. It also penetrates past the blood brain barrier into the CNS.148 Low concentrations of ivermectin increase the ATP-induced signal at the P2X4 receptor, higher concentrations retard channel closing and receptor desensitization during sustained ATP presence and thereby temporarily induce pore dilation.147, 149 Those different effects were explained by the existence of two different binding sites for ivermectin at the P2X4 receptor, one with high affinity in the high nanomolar and one with lower affinity in the lower micromolar range.147, 150 This theory is not proven since binding site of ivermectin has not yet been fully identified. There is evidence that the transmembrane domains are involved in ivermectin recognition. Several residues apparently involved in ivermectin recognition could be identified by side-directed mutagenesis.151
Other PAMs of the P2X4 receptor are d-tubocurarine, a muscle relaxant first isolated from Chondrodendron tormentosum, RB-2, the standard antagonist at the P2X2 receptor, and i.v.
anesthetic propofol. The allosteric effects of d-tubocurarine were identified while characterizing the rat P2X4 receptor using the radioligand [35S]ATPγS. D-tubocurarine increased radioligand binding when used in concentrations above 10 µM.152 Cibacron Blue 3GA is a weak inhibitor especially at the P2X2 receptor. The compound is derived from anthraquinone, and was selected as the lead structure for the development of a broad variety of P2X receptor ligands (see chapter 3.1). The positive allosteric modulation is expressed in increased responses of rat P2X4 receptors to ATP. Cells were preincubated with 3-30 µM Cibacron Blue 3GA prior to ATP stimulation. The increase could be attributed to enhanced ATP potency without affecting the maximum signal.153 Propofol is known as an allosteric enhancer at the GABAA receptor, which is considered responsible for its general anesthetic and anticonvulsant effect. It was found to enhance ATP-evoked signal at rat P2X4 receptors when used in concentrations higher than 50 µM, and was selective towards rat P2X2 and P2X2/3 receptors.154 Hasaka et al. confirmed this observation, but enhancement of ATP-induced signal was observed at much lower propofol concentrations (0.3-3 µM). Higher concentrations were observed to inhibit ATP-induced P2X4 receptor currents in mouse microglia cells.155 The activity of propofol at the P2X4 receptor was deemed responsible for excitatory side effects of propofol administration.154
-28-
Figure 1.17: Structure and potency of positive allosteric P2X4 modulators. The potency of ivermectin and propofol at the P2X7 receptor is discussed in chapter 1.2.12.2.143, 152, 154-156