Structural molecular pharmacology of G protein inhibitors.

• Applicants: Dr. Xavier Deupi; Professor Dr. Gebhard Schertler
• Subject Area: Structural Biology ; Biochemistry ; Bioinformatics and Theoretical Biology
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 273251628

The project “Structural molecular pharmacology of G protein inhibitors”, led by Prof. Schertler/Dr. Deupi, aims to analyse the interaction between the cyclic depsipeptide FR900359 and the Gq protein. FR900359 is a known specific inhibitor of Gαq and has been extensively studied in the framework of this FOR proposal. In particular, the project will address three objectives directly related to the results of the first funding period: 1) Structural determination of Gq bound to FR900359. 2) Computational analysis of the inhibitory mechanism of FR900359 on Gαq, 3) Structural basis of the selective partial inhibition by FR900359 of the oncogenic constitutively active Gq mutants Q209L and Q209P.To address these questions, we aim at solving the structures of Gq as well as the Gq cancerogenic mutants Q209L and Q209P (with and without FR900359) by X-ray crystallography. We are currently establishing a robust workflow for the large-scale purification of the Gq protein, which will provide suitable samples for X-ray crystallography, but also for NMR experiments carried out by P11. We are aware that in order to obtain well-diffracting protein crystals, we may need to generate engineered Gq variants. Thus, we also aim at generating reliable ‘crystallization templates’ that can be used in a structure-based drug discovery cycle. We will perform crystallization screens with the different Gq variants with and without FR900359. For this, we will use stabilizing antibody fragments (FAB16 and scFv16), which have been generated in a collaboration with Brian Kobilka (Stanford, USA) and Roche (Basel, CH). We will then solve the structure of Gq with X-ray crystallography at the synchrotron SLS at the Paul Scherrer Institute. These structures are at the core of Objective 1 and, in combination with the NMR data obtained by P11, they are also important for the in-depth computational analysis of the molecular mechanism of FR900359 inhibition of Gq; for comparison we will also include the mechanism of YM-254890 inhibition (Objective 2).In order to extend our understanding of the mechanism of Gq inhibition by FR900359 we will also analyze the structural basis of partial inhibition of two cancerogenic Gq mutants, Q209L and Q209P (Objective 3). Ideally, we will have crystals of both mutants, with and without FR900359; we will use structural computational biology methods (molecular modeling and molecular dynamics simulations) in order to complement and expand the structural data and to elucidate the structural mechanism of selective partial inhibition of the cancer mutants Q209L and Q209P by FR900359. The outcome of our project will serve as the basis for P1 and P6, for the design of novel compounds that specifically inhibit other G protein subtypes, or of improved Gq inhibitors with pharmacological potential, which can be assessed by P8, P9, P10 in vitro and in vivo. Finally, our findings will expand our knowledge of GPCR-mediated signaling networks.

• DFG Programme: Research Units
• Subproject of: FOR 2372: G protein signalling cascades: with new molecular probes and modulators towards novel pharmacological concepts
• International Connection: Switzerland