GPCRs play a pivotal role in transmitting signals at the cellular level and structural insights can be exploited to support structure-based drug discovery endeavors. Despite advances in GPCR crystallography, it remains challenging to obtain crystal structures particularly of active states. Molecular dynamics (MD) simulations have been used to explore the conformational landscape of GPCRs. Nevertheless, the search for physiologically relevant conformations, allosteric pockets and ligand binding events using classical MD simulations is still impractical.

The work I will present shows how by applying adaptive MD it is possible to computationally access, in a rigorous and timely manner, key functional states of a GPCR without a priori structural information of the active state [1]. Moreover, I will show that this cutting-edge MD methodology can help elucidating GPCRs regulatory mechanisms and opening avenues for the study of ligands binding to elusive yet pharmacologically important GPCR states, providing key insights into ligands mode of action [2].

[1] S. Lovera, A. Cuzzolin, S. Kelm, G. De Fabritiis, Z. A. Sands. Reconstruction of apo A2A receptor activation pathways reveal ligand-competent intermediates and state-dependent cholesterol hotspots. Sci Rep 9, 14199 (2019)

[2] S. Lovera, E.J.B. Landin, G. De Fabritiis, S. Kelm, J. Mercier, D. McMillan, R.B. Sessions, R.J. Taylor, Z. A. Sands, L. Joedicke, M.P. Crump. The Aminotriazole Antagonist Cmpd-1 Stabilises a Distinct Inactive State of the Adenosine 2A Receptor. Angewandte Chemie Int. Ed. 58, 1-6 (2019)

A successful approach for design and preparation of peptide based recognition molecules that mimic the activity of antibodies in terms of specificity and affinity is presented. The technology exploits a structurally stabilized β-hairpin scaffold termed a β-body. The scaffold was derived from a combination of observations in crystal structures and NMR-studies on threonine rich mucin glyco-peptides. The structure of the β-body may be further stabilized using the versatile CuAAC click reaction to bridge the C- and N-terminal of the scaffold.

The structural stability of the β-body scaffold allows virtual combinatorial chemistry in the design and development. To this end we have developed an MD approach guided by dipolar electrostatics between interacting electron densities at the interface of the two molecules. A crystal or NMR structure of the target protein is used for the combinatorial approach which within few days can provide several β-bodies with nano-molar affinities in water.

Diagnostic nM assay for Interleukin 6 comprising two designed β-bodies in a sandwich with IL-6.

The versatility of the β-body approach to molecular recognition and inhibitors of protein-protein interaction (PPI’s) will be illustrated with biologically relevant examples. The process is not so effective when PBS buffers are used in binding assays. This will be discussed in detail.