With >800 members in humans, the G protein-coupled receptor (GPCR) super-family is the target for more than 30% of the marketed drugs. The recent boom in GPCR crystallography has enabled the solution of ∼30 different GPCR structures, which boosted the identification and optimization of novel modulators and new chemical entities through structure-based strategies. However, the number of available structures represents a small part of the human GPCR druggable target space, and its complete coverage in the near future seems unlikely. Homology modelling represents a reliable tool to fill this gap, and hence to vastly expand the horizons of structure-based drug discovery and design. In this Feature Article, we show from a wealth of retrospective and prospective studies that in spite of the pitfalls of and standing challenges in homology modelling, structural models have been critical for the blossoming and success of GPCR structure-based lead discovery and optimization endeavours; in addition, they have also been instrumental in characterizing receptor-ligand interaction, guiding the design of site-directed mutagenesis and SAR studies, and assessing off-target effects. Considering though their current limitations, we also discuss the most pressing issues to develop more accurate homology modelling strategies, with a special focus on the integration of computational tools with biochemical, biophysical and QSAR data, highlighting methodological aspects and recent progress. The teachings of the three GPCR Dock community-wide assessments and the fresh developments in GPCR classes B, C and F are commented. This is a fast growing and highly promising field of research, and in the coming years, the use of high-quality models should enable the discovery of a growing number of potent, selective and efficient GPCR drug leads with high therapeutic potential through receptor structure-based strategies.
Our work is focused in the broad area of strategies and efforts to inhibit protein-protein interactions. The possible strategies in this field are definitely much more varied than in the case of ATP-pocket inhibitors. In our previous work (10), we reported that a retro-inverso (RI) form of Helix1 (H1) of c-Myc, linked to an RI-internalization sequence arising from the third alpha-helix of Antennapedia (Int) was endowed with an antiproliferative and proapoptotic activity toward the cancer cell lines MCF-7 and HCT-116. The activity apparently was dependent upon the presence of the Myc motif. In this work, by ala-scan mapping of the H1 portion of our molecules with D-aa, we found two amino acids necessary for antiproliferative activity: D-Lys in 4 and D-Arg in 5 (numbers refer to L-forms). In the natural hetero-dimer, these two side chains project to the outside of the four alpha-helix bundle. Moreover, we were able to obtain three peptides more active than the original lead. They strongly reduced cell proliferation and survival (RI-Int-VV-H1-E2A,S6A,F8A; RI-Int-VV-H1-S6A,F8A,R11A; RI-Int-VV-H1-S6A,F8A,Q13A): after 8 days at 10 muM total cell number was approximately 1% of the number of cells initially seeded. In these more potent molecules, the ablated side chains project to the inside in the corresponding natural four alpha-helix bundle. In the present work, we also investigated the behavior of our molecules at the biochemical level. Using both a circular dichroism (CD) and a fluorescence anisotropy approach, we noted that side chains projecting at the interior of the four alpha-helix bundle are needed for inducing the partial unfolding of Myc-H2, without an opening of the leucine zipper. Side chains projecting at the outside are not required for this biochemical effect. However, antiproliferative activity had the opposite requirements: side chains projecting at the outside of the bundle were essential, and, on the contrary, ablation of one side chain at a time projecting at the inside increased rather than decreased biological activity. We conclude that our active molecules probably interfere at the level of a protein-protein interaction between Myc-Max and a third protein of the transcription complex. Finally, CD and nuclear magnetic resonance (NMR) data, plus dynamic simulations, suggest a prevalent random coil conformation of the H1 portion of our molecules, at least in diluted solutions. The introduction of a kink (substitution with proline in positions 5 or 7) led to an important reduction of biological activity. We have also synthesized a longer peptido-mimetic molecule (RI-Int-H1-S6A,F8A-loop-H2) with the intent of obtaining a wider zone of interaction and a stronger interference at the level of the higher-order structure (enhanceosome). RI-Int-H1-S6A,F8A-loop-H2 was less active rather than more active in respect to RI-Int-VV-H1-S6A,F8A, apparently because it has a clear bent to form a beta-sheet (CD and NMR data).
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