5.4 Enhancement of maximal ATP effect
5.4.3 Enhancing hits at the human P2X7 receptor
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-170- Continuation of Table 5.14.
Cephradine n. c.a (-142 %) -
Medroxy-progesterone
acetate
1.06 ± 0.56 39 ± 1
Methyl-testosterone
n. c.a (-92 %) -
Amiodarone-HCl n. c.a (-183 %) -
Carvedilol n. c.a (-102 %) -
a: no convergence in 2 independent experiments
Cefaclor and cefatrizin showed a concentration-dependent enhancement of maximal ATP effect, but the EC50Enhancement concentration was calculated to be higher than 100 µM, which was the highest concentration used. The experiment was successful for amphotericin B, benzathine-benzylpenicillin, cephaloridine and medroxyprogesterone acetate. The respective concentration-response curves are presented in Figure 5.14. When compared to the enhancing hits at the other receptor subtypes, a common structural feature could be detected. Cefaclor, cephaloridine, cephradine and cefatrizine are broad-spectrum antibiotics of the cephalosporin class.
Benzylpenicillin, also known as penicillin G, the first known antibiotic, bears close structural relation to cephalosporins. Both classes belong to the so-called β-lactam antibiotics. Cephradine, cefatrizine and cephaloridine are part of the first generation cephalosporines. Cephradine is
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orally available, same as the second generation drug cefaclor. Cephaloridine is unique among the cephalosporin antibiotics due to its zwitterionic nature. Like the penicillin drugs, cephalosporines act bactericidal by inhibition of penicillin-binding transpeptidases. This disrupts the bacterial cell wall synthesis, which leads to cell lysis. The diverging antibacterial activity of cephalosporins is attributed to side chain substitutions at position C7 in the lactam-ring. The bioavailability of oral cephalosporins is higher than 95 %. The drugs are eliminated rapidly from the blood in the space of two hours by renal excretion in their unchanged form.335 Penicillin G was available in combination with benzathine, building a water soluble depot complex that is administered intramuscularly. A single dose of 1.2 million units leads to a maximal plasma concentration of 0.154 µM in adults (0.14 mg/l).336 Since the available stock solution contained the water-soluble depot complex, any effect could not be clearly attributed to benzathine or benzylpenicillin, respectively. The common structural feature indicated that the cephalosporin core could be a possible scaffold for positive modulators for purinergic P2X7 receptors. Since cephalosporins and penicillin are present in the blood, interaction with P2X7 receptors expressed in macrophages is possible, but the low plasma concentrations reached by intramuscular depot injection would not be high enough to cause any effect.
The EC50Enhancement concentrations of cephalosporins were comparable, but the magnitude of enhancement showed considerable standard error for depot penicillin combination benzathine-benzylpenicillin and cephaloridine, and also varied between compounds. The highest enhancement of maximal ATP effect was determined for cephaloridine (105 ±56 %), while the concentration-dependent enhancement measured for cefaclor was barely above the normal ATP-induced fluorescence signal (19 ± 6 %). Medroxyprogesterone acetate showed the best reproducibility, the lowest EC50Enhancement concentration and the least standard error of all hits.
None of the other steroidal drugs showed enhancement of maximal ATP effect to this extent.
They are prone to show inhibition of calcium influx in stably transfected cells (see chapter 5.3.1.1).
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Figure 5.14: Mean dose-response curves for determination of enhancement of maximal ATP effect of (A) amphotericin B, (B) benzathine-benzylpenicillin, (C) cefaclor, (D) cefatrizine, (E) cephaloridine and (F) medroxyprogesterone acetate at the P2X7 receptor. Data is presented as mean ± SEM from 3 to 4 independent experiments.
10-11 10-9 10-7 10-5 10-3 0
25 50 75 100 125 150
[cefaclor], M
Fluorescence increase (%)
10-11 10-9 10-7 10-5 10-3 0
25 50 75 100 125 150
[medroxyprogesterone], M
Fluorescence increase (%)
10-11 10-9 10-7 10-5 10-3 0
25 50 75 100 125 150 175
[cefatrizine], M
Fluorescence increase (%)
10-11 10-9 10-7 10-5 10-3 0
50 100 150 200 250 300
[cephaloridine], M
Fluorescence increase (%)
10-11 10-9 10-7 10-5 10-3 0
50 100 150 200 250
[benzathine-benzylpenicillin], M
Fluorescence increase (%)
10-11 10-9 10-7 10-5 10-3 0
25 50 75 100 125 150 175 200
[amphotericin B], M
Fluorescence increase (%)
(A) amphotericin B (B) benzathine-benzylpenicillin
(C) cefaclor (D) cefatrizine
(E) cephaloridine (F) medroxyprogesterone acetate
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Medroxyprogesterone acetate is a synthetic derivative of the female sexual hormone progesterone. It structurally differs from progesterone by introduction of a methyl residue at carbon 6 and an acetate group at carbon 17, which enhances oral bioavailability and progestational potency. It is approved as a contraceptive and for postmenopausal hormone replacement therapy. In the Women’s Health Initiative trial, the use of postmenopausal medroxyprogesterone acetate was associated with increased risk for the development of breast cancer and cardiovascular diseases. This led to a downturn of its use in postmenopausal osteoporosis prevention therapy. Medroxyprogesterone acetate is potent at both the androgen and the glucocorticoid receptor. The application of a 10 mg oral dose leads to peak serum concentrations between 0.0088 µM and 0.011 µM (3.4-4.4 ng/ml).337, 338 These serum concentrations would be insufficient for any enhancing effect on the P2X7 receptor in vivo.
Despite the high structural similarity to medroxyprogesterone acetate, the enhancing effect of methyltestosterone was not quantifiable. This indicates that the acetyl residue in position 17 plays an important role for the positive modulating effect.
An experiment for testing whether the observed enhancement is due to agonistic activity of the drug itself was conducted for amphotericin B, benzathine-benzylpenicillin, cephaloridine and medroxyprogesterone acetate by injecting various concentrations of the respective drugs instead of ATP. No fluorescence signal increase for any compound was observed. The enhancing effect therefore cannot be attributed to any agonistic activity at the P2X7 receptor.
The influence of several single concentrations of the most active enhancers amphotericin B, benzathine-benzylpenicillin, cephaloridine and medroxyprogesterone acetate (1 µM, 10 µM and 100 µM of all compounds, respectively, medroxyprogesterone acetate was additionally tested in a final concentration of 50 µM), on the EC50 concentration of ATP at the P2X7 receptor were tested. The respective ATP dose-response curves are presented in Figure 5.15 and Figure 5.16.
Amphotericin B, benzathine-benzylpenicillin and cephaloridine caused a shift of the ATP EC50
values to the left. The shift was prominent upon incubation with the highest concentration (100 µM). This indicates an increase of ATP affinity as a possible cause for the observed enhancement of receptor activity. The conclusion was supported by the marginal and non-significant effect of the different compound concentration on the maximal ATP effect, determined from the highest ATP concentration used in the experiment (5 mM).
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Figure 5.15: (1) Mean ATP dose response curves, (2) ATP pEC50 values and (3) enhancement of maximal ATP effect characterizing the influence of (A) amphotericin B and (B) benzathine-benzylpenicillin at the human P2X7 receptor. Cells were stimulated with ATP 10 µM-5000 µM after preincubation with 1 µM ( ), 10 µM ( ), 50 µM ( ) or 100 µM ( ), respectively.
Enhancement of fluorescence increase was calculated of cells stimulated with the highest ATP concentration (5 mM), with ATP control ( ) based as 0 %. The unpaired Student’s t-test for determination of significant differences of means was conducted for pEC50 and enhanced fluorescence increase. Significant differences (p < 0.05) are marked with (*) and very significant differences (p < 0.01) are marked with (**). Data is presented as mean ± SEM from 3-4 independent experiments.
A slight shift of the EC50 value to the left could be observed upon incubation with increasing concentrations of medroxyprogesterone acetate. The maximal ATP effect also increased. No ATP EC50 value and no fluorescence signal increase could be determined upon incubation with 100 µM medroxyprogesterone acetate. The mismatch to the other three compounds tested indicates a different mechanism of enhancement that increases both the affinity of the
10-4.5 10-4.0 10-3.5 10-3.0 10-2.5 10-2.0 0
20 40 60 80 100 120 140 160
[ATP], M
Fluorescence increase (%)
con trol
+ 10 µM + 50 µM
+ 100 µM 2.8
2.9 3.0 3.1 3.2
pEC50of ATP
* *
*
con trol
+ 10 µM
+ 50 µM
+ 100 µM 0
5 10 15 20 25
enhanced max. ATP effect (%)
10-4.5 10-4.0 10-3.5 10-3.0 10-2.5 10-2.0 0
20 40 60 80 100 120 140
[ATP], M
Fluorescence increase (%)
control + 1 µM
+ 10 µM + 100 µM 2.8
2.9 3.0 3.1 3.2 3.3 3.4
pEC50 of ATP
**
control + 1 µM
+ 10 µM
+ 100 µM 0
10 20 30 40
*
enhanced max. ATP effect (%)
(A) Amphotericin B at human P2X7
(A1) (A2) (A3)
(B) Benzathine-benzylpenicillin at human P2X7
(B1) (B2) (B3)
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physiological ligand ATP and its effect. A different binding site or a variable conformational change upon the binding of the ligand could be the reason for this effect.
Figure 5.16: (1) Mean ATP dose response curves, (2) ATP pEC50 values and (3) enhancement of maximal ATP effect characterizing the influence of (A) cephaloridine and (B) medroxyprogesterone acetate at the human P2X7 receptor. Cells were stimulated with ATP 10 µM-5000 µM after preincubation with 1 µM ( ), 10 µM ( ), 50 µM ( ) or 100 µM ( ), respectively. Enhancement of fluorescence increase was calculated of cells stimulated with the highest ATP concentration (5 mM), with ATP control ( ) based as 0 %. The unpaired Student’s t-test for determination of significant differences of means was conducted for pEC50 and enhanced fluorescence increase. Significant differences (p < 0.05) are marked with (*), very significant differences (p < 0.01) are marked with (**) and highly significant differences (p < 0.001) are marked with (***). Data is presented as mean ± SEM from 3-4 independent experiments.
10-4.5 10-4.0 10-3.5 10-3.0 10-2.5 10-2.0 0
20 40 60 80 100 120 140
[ATP], M
Fluorescence increase (%)
con trol
+ 1 µM + 10 µM
+ 100 µM 2.80
2.85 2.90 2.95 3.00 3.05 3.10 3.15 3.20
pEC50of ATP
**
con trol
+ 1 µM + 10 µM
+ 100 µM -15
-5 5 15
25 **
enhanced max. ATP effect (%)
10-4.5 10-4.0 10-3.5 10-3.0 10-2.5 10-2.0 0
20 40 60 80 100 120 140 160
[ATP], M
Fluorescence increase (%)
control + 1 µM
+ 10 µM
+ 50 µM
+ 100 µM 2.8
2.9 3.0 3.1
pEC50of ATP
control + 1 µM
+ 10 µM + 50 µM
+ 100 µM -100
-80 -60 -40 -20 0 20 40
60 **
**
***
enhanced max. ATP effect (%)
(A) Cephaloridine at human P2X7
(A1) (A2) (A3)
(B) Medroxyprogesterone acetate at human P2X7
(B1) (B2) (B3)
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