Periodic Reporting for period 1 - MIDAS (Predictive modelling of GPCR druggable allosteric sites)
Période du rapport: 2017-11-01 au 2019-10-31
1) Identification of suitable probes for MD-based co-solvent mapping
2) Development of a computational procedure to identify ABSs within the helical bundle
3) Development of a computational procedure to identify ABSs between helices and lipids
4) Prospective validation of the computational procedures via site-directed mutagenesis experiments
a) the M2 muscarinic receptor in ternary complex with the agonist iperoxo and the extra-cellular positive allosteric modulator (PAM) LY2119620;
b) the Beta2 adrenergic receptor in complex with the intra-cellular peptide-like negative allosteric modulator (NAM) Cmp-15;
c) the CCR2 chemokine receptor in complex with the intra-cellular small molecule NAM CCR2-RA-[R];
d) the purinergic P2Y1 receptor in complex with the interface NAM BPTU.
MIDAS technology has been applied in conjunction with the conventional co-solvent mapping approach and its performance compared with a novel ad hoc developed strategy that implements in silico FBDD concepts. The novel, alternative approach applies chemo-informatics analyses to identify “privileged” fragments to use in probe mapping MD simulations. After MIDAS successful retrospective validation in the four selected case studies, the ER has applied the methodology prospectively in two case studies by predicting the location of the ABSs in class A bio-aminergic GPCRs – namely the Dopamine D2 and serotonin 5HT2C receptors – with available X-ray structures and known AMs. The ER has tested the computational predictions experimentally through secondment in Dr. Peter McCormick’s lab at Queen Mary University London by performing site-directed mutagenesis experiments and cell-based fluorescent binding assays.