Final Report Summary - CANNABIDIM (Molecular modelling of the cannabinoid CB1 receptor homodimer and its interaction with ligands: the role of membrane cholesterol and the CRIP1a protein)
The cannabinoid CB1 receptor (CB1R), is an attractive molecular target for the treatment of substance abuse, drug addiction, schizophrenia, bipolar disorder, motor dysfunction including Huntington’s disease, as well as cardiometabolic disease and metabolic syndrome. The general aim of the project is to investigate in silico the phenomenon of the homodimerization of the CB1R, the interaction of the CB1R homodimer with ligands, and to study the effect of membrane cholesterol and the cannabinoid receptor interacting protein 1a, CRIP1a, on the functioning of the CB1R homodimer.
The project involves the following detailed objectives:
(1) homology modelling of the CB1R monomer in the active and inactive state;
(2) application of protein-protein docking and surface roughness scoring to construct a model of the CB1R homodimer in the active, inactive and “mixed” state;
(3) all-atom molecular dynamics (MD) simulations of respective dimers and monomers in a lipid bilayer;
(4) all-atom MD simulations of the respective dimers and monomers in a lipid bilayer containing cholesterol;
(5) modelling of CRIP1a;
(6) modelling of the CB1R-CRIP1a complex applying protein-protein docking and MD simulations;
(7) coarse-grained MD to investigate the assembly of CB1R monomers and CB1R-CRIP1a complexes in a lipid bilayer;
(8) coarse-grained MD to investigate the assembly of CB1R monomers and CB1R-CRIP1a complexes in a lipid bilayer containing cholesterol;
(9) application of structure-based drug design methods to elaborate compounds that modulate dimer activity.
The fellow performed the following research regarding the goals mentioned above and regarding further aims not involved in the project:
1. Construction of homology models of CB1R in the active and inactive state. In addition an active model was constructed in complexes with the respective G proteins. Furthermore, homology models of CB2R and GPR55 receptors were constructed in the inactive state and in the active state in complexes with the respective G proteins (100% of realization of objective 1). Additionally the fellow investigated interactions of orthosteric and allosteric ligands (including natural products) with these receptors.
2. Improvement of a protocol for GPCR dimer modelling (including new scoring parameters). In this approach the fellow applied protein-protein docking with Rosetta software to obtain populations of dimers as present in membranes with all possible interfaces (28 interfaces for a homodimer). As the number of possible interfaces is reasonable small, it is possible to carry out detailed local docking to optimize each possible docking pose. At the next stage consensus scoring procedure according to (i) Rosetta score (ii) surface of the dimer interface, (iii) polar contribution to the dimer interface, (iv) fractal dimension of the dimer interface (Kaczor et al., 2012), (v) evolutionary conservation score (Ashkenazy, Erez, Martz, Pupko, & Ben-Tal, 2010), (vi) shape complementarity, (vii) electrostatic complementarity, (viii) potential energy and (ix) free energy of binding is applied. The best models are minimized, and the whole cycle is iteratively repeated until the results converge to a consistent dimer formation. The improved protocol made it possible to construct models of dimer of CB1R in an active, inactive and mixed state (100% of realization of objective 2).
3. Molecular dynamics (all-atom) of dimers and monomers of CB1R in a lipid bilayer. The MD simulations were first performed in POPC membrane using Desmond software and relatively short times of simulations. Then, simulations were performed with Gromacs, using different membrane compositions and longer times of simulations (100% of realization of objective 3). All-atom MD simulations of the respective dimers and monomers in a lipid bilayer containing cholesterol (100% of realization of objective 4). The results of these simulations are currently under analysis and will be published during next six months.
4. Construction of a model of CRIP1A protein (100% of realization of objective 5).
5. Construction of a model of the complex of CB1R and CRIP1A protein. In order to check the effect of CRIP1a protein on the CB1 receptor functioning, MD simulations were performed as described above. It was discovered that CRIP1a destabilizes CB1 receptor active conformation which can lead to reduction of receptor basal activity and which is in agreement with experimental data. Moreover, it was determined that CRIP1a blocks CB1 receptor interactions with Gi and Go proteins. (100% of realization of objective 6). The results of this study will be published within next six months.
6. Coarse-grained MD to investigate the assembly of CB1R monomers and CB1R-CRIP1a complexes in a lipid bilayer (100% of realization of objective 7) and coarse-grained MD to investigate the assembly of CB1R monomers and CB1R-CRIP1a complexes in a lipid bilayer containing cholesterol. (100% of realization of objective 8). It was found that transmembrane helices TM1, TM2, TM4 and TM5 are involved in the CB1 receptor oligomerization which is in accordance with the experimental data. The local membrane deformation appears to be a key factor defining the rate, extent, and orientational preference of protein-protein association. It was also determined that the intracellular loop 3 may interact with the receptor C-terminus during dimer formation. Furthermore, it was found that cholesterol facilitates oligomerization of the CB1 receptor, acting as glue at the oligomerization interface. The obtained results may be generalized for the whole family of rhodopsin-like GPCRs, uncovering the universal mechanism of GPCR oligomerization. The results of this study will be published within next six months.
7. Modeling of interactions of bivalent ligands with CB1 and application of virtual screening to identify novel orthosteric and allosteric ligands of CB2 receptor (100 % of realization of a slightly modified objective 9). After additional training in medicinal chemistry and development of knowledge on the cannabinoid system the fellow decided to focus on orthosteric and allosteric, not bivalent ligands as they are more drug-like and to search for ligands of CB2, not CB1 receptor as they are supposedly devoid of psychoactive activity. The fellow performed virtual screening for agonists and allosteric modulators of CB2 receptor and bought 40 compounds which are currently under experimental verification.
8. Additionally, the research was extended to cover other drug targets in the cannabinoid system and QM/MM studies of reversibility of FAAH inhibitors were performed. Moreover, the fellow was involved in searching for inhibitors of ABHD6 and ABHD12 and modeling of respective inhibitor-enzyme interactions as well as QSAR studies. The fellow also studied GPR55 receptor and its interactions with ligands using molecular dynamics approach.
9. Furthermore, the fellow extended her research on GPCR dimerization to D2 receptor dimers: she constructed the models of D2 dimers and elaborated the mode of interactions of bivalent ligands with the receptor complexes (collaboration with Glaxo-Smith-Kline company). The fellow also performed virtual screening for D2 antagonists and allosteric modulators and bought 39 compounds. The in vitro tests are currently being performed, but partial results are promising: in total 11 compounds are active orthosteric ligands, involving compounds with nanomolar activity. The identified D2 antagonists have beneficial selectivity profiles characteristic for the second and the third generations of neuroleptics.
10. In additional time the fellow was developing her molecular modeling skills, working on other transmembrane receptors (glutamate and nicotinic ion channels) and performing structure-activity studies of novel compounds with a central nervous system activity. Furthermore, she was involved in molecular modeling of novel bioinorganic compounds.
Comparison of the planned and performed work
The fellow performed 100% of planned research activities, achieving all assumed objectives of these activities. Furthermore, the fellow performed several additional research activities which resulted from the extension of the project (see above).
Important achievements during Marie Curie fellowship
The fellow was granted as a principal investigator a computational grant by PRACE: Molecular simulations of transmembrane receptors in native like membranes: focus on allosteric ligands (ALLOTRANS). 7 mln core hours were granted. The project involves three researchers from Medical University of Lublin, Poland and three researchers from GRIB/PRBB, Barcelona, Spain. Furthermore, the fellow was awarded in Inter competition by Polish Science Foundation for her skills of presenting science for general public.