SYNTHESIS AND APPLICATION
OF THE FIRST SMALL-MOLECULE RADIOLIGAND
TARGETING THE HUMAN CHEMOKINE RECEPTOR CXCR3
V. Bernat†, Prof. Dr. M. Heinrich†, P. Baumeister‡, Prof. Dr. A. Buschauer‡, Dr. N. Tschammer†
†University of Erlangen-Nuremberg, ‡University of Regensburg
Department of Chemistry and Pharmacy
Emil Fischer Center
Friedrich-Alexander-University
Schuhstraße 19, D-91052 Erlangen
www.medchem.uni-erlangen.de
CXCR3
CXCL9
AMG487
GPCR
40.7 kDa
Chemokine
14.0 kDa
Small molecule
0.6 kDa
Mechanism of action: Endogenous ligands (CXCL9, CXCL10 and CXCL11) bind to N-terminus of CXCR3 and activate it with their own flexible N-terminus via transmembrane binding pocket. Synthetic allosteric modulators bind directly to the TM domain without interaction with receptor’s N-terminus.
Involvement of CXCR3 – chemokine signaling system in pathological conditions: Multiple sclerosis Psoriasis Rheumatoid arthritis Breast and colon cancer Transplant rejection
Biological function of chemokine system: Migration and response of immune cells
Chemokine-based binding assays are inconvenient Allosteric radioligand is needed
a) N-Boc-D-alanine, IBCF, NMM, then ANA, DCM, - 20°C … RT, 20 h; b) p-phenetidine, DCM, RT, 24 h; c) IBCF, NMM, DCM, RT, 2 h; d) TFA, DCM, 4–8 h; e) (±)-N-Boc-2-aminobutyric acid, IBCF, NMM, then ANA, DCM, - 20°C … RT, 20 h; f) N-Boc-L-phenylalanine, IBCF, NMM, then ANA, DCM, - 20°C … RT, 20 h; g) 4-methoxybenzylamine, DCM, RT, 20 h; h) N-Boc-β-alanine, IBCF, NMM, then ANA, DCM, - 20°C … RT, 20 h; i) pyridine-3-carbaldehyde, NaBH(OAc)3, DCE, RT, 20 h; j) corresponding substituted phenylacetic acid, HATU, DIPEA, DMF, 45°C, 20 h; k) Ammonium Cerium (IV) Nitrate, CH3CN/H2O, RT, 20 h, 85%.
Binding Characteristics of RAMX3
at the human CXCR3 receptor expressed in HEK-293 cells
Saturation Binding Binding Kinetics
Kd , nM Kd , nM kon , min-1nM-1 koff , min-1 t1/2 , min
1.08±0.18[a] 0.93[b] 0.045±0.003[c] 0.041±0.007[d] 16.9[e]
[a] Equilibrium dissociation constant; values reflect the means ± SEM of 5 independent experiments performed in triplicate. [b] Kinetically derived dissociation constant. [c] Association rate constant ± SEM. [d] Dissociation rate constant ± SEM from linear regression analysis. [e] Half-life, calculated as ln2/koff
Binding data and functional activities
of the 8-azaquinazolinone derivatives
at the human CXCR3 receptor
expressed in HEK-293 cells
Compd pKi[a] pIC50
[b]
CXCL11 7.23±0.09 8.45±0.11[c]
rac-AMG487 7.85±0.10 6.05±0.09
rac-NBI-74330 9.02±0.16 6.97±0.08
“cold” RAMX3 8.85±0.11 7.40±0.13
6b 8.20±0.20 4.99±0.13
6d <5.00 5.54±0.16
6e 6.36±0.14 5.72±0.12
7b 5.05±0.42 5.46±0.09
7c 6.38±0.27 5.68±0.20
7e 6.33±0.17 4.89±0.21
8d <5.00 n.d.
NVP-BDZ824[d] 8.93±0.14 n.d.
RAMX3
Comparison of binding and functional data revealed some unusual correlation between affinity and activity of compounds: compound 6b has higher affinity than AMG487 but lower efficacy than its own truncated analogue 7b. On the contrary, compound 6d is not able to completely displace RAMX3 at a concentration of 10 μM but still inhibits activation of CXCR3 with efficiency similar to other tested compounds. These observations are in accord with the notion that allosteric modulator SAR needs to differentiate the influence of chemical modifications on compound affinity from the cooperativity exhibited toward orthosteric ligands, as the two properties are not correlated. Data for compound 8d imply that a phenyl substituent attached to 8-azaquinazolinone core is essential for affinity and efficacy of this class of CXCR3 receptor negative allosteric modulators. Binding experiments with ergoline derivative NVP-BDZ824 demonstrated that RAMX3 can be used as a tool to interrogate allosteric binding sites. The obtained binding affinity of this compound was found to be five-fold higher than Ki value reported for the assay using [125I]CXCL11 (1.17 nM vs. 6 nM respectively)[2].
NVP-BDZ824
Conclusions
[a] Radioligand binding: determined from the displacement of the radioligand RAMX3 (c = 1 nM) from HEK-293 cell membranes expressing CXCR3. IC50 values were converted into pKi values according to Cheng-Prussoff equation; values reflect the mean ± SEM of two to three experiments performed in triplicate .
[b] Inhibition of CXCL11-mediated activation of CXCR3: determined by the gene reporter (luciferase) assay; values reflect the mean ± SEM of three to five experiments performed in triplicate. All derivatives fully inhibited CXCL11 (20 nM)-mediated activation of the CXCR3 receptor. [c] pEC50 value for the stimulation of CXCR3 by CXCL11; the value reflects the mean ± SEM of three experiments performed in triplicate [d] An ergoline derivative developed by Novartis[2].
References
1. V. Bernat et al. ChemMedChem, 7 (2012), 1481-1489
2. G. Thoma et al. Bioorg. Med. Chem. Lett, 19 (2009), 6185-6188
CHEMBL1077827