Screening-based design, synthesis and testing of potential Gα protein inhibitors as chemical probes for GPCR signaling
Final Report Abstract
The Gαβγ (guanine nucleotide-binding) heterotrimeric proteins commonly operate as molecular switches to activate (“turn on”) or inactivate (“turn off”) various intracellular signalling pathways in response to stimulated G protein-coupled receptors (GPCRs). Modulation of these G proteins can be exhorted via chemical tools, such as bioactive peptides, which are competent in inhibiting protein-protein interactions (PPIs) upon interactions with distinct binding sites within the protein. Although GPCRs are important targets for the development of pharmaceutical tools (approx. 30% of FDA-approved drugs), notable studies have introduced the major difficulty in drugging the “undruggable” G proteins. The depsipeptides FR900359 (FR) and YM- 254890 (YM) are the only known specific inhibitors described for the G proteins targeting specific the Gαq subunits, highlighting the acute necessity of developing such compounds for the rest subfamilies. In this project, we initially focused on producing, functionally characterizing, and modulating the guanine-nucleotide exchange ability of the Gαi/s subunits. All proteins (Gαi1, Gαs(short) and Gαs(long)) were recombinantly expressed in a prokaryotic system via in-house established protocols which result to the highest reported protein yields. Two novel non-radioactive and fluorescently labelled guanine nucleotide analogues were discovered as high affinity binders to the Gαi1 protein according to fluorescence anisotropy-based assays. Unravelling the fundamental biochemical differences between the two isoforms of the Gαs protein, precedes the future analysis of further Gα subunits. As a second scope of this funded project, two different combinatorial peptide libraries were screened against Gαi/s proteins. Peptides derived from a one-bead-one compound (OBOC) combinatorial library, such as linear the lead compound GPM-1 (and derivatives) indicated a GEM (guanine exchange modulator)-like activity for the Gαi/s∙GDP (the inactive protein state). A set of bicyclic compounds emanated from a one-bead-two compound (OBTC) peptide library revealed GPM-3 as the first peptide described as a class- and state-specific GAP (GTPaseactivating protein)-like modulator of Gαi1∙GMPPNP, and GPM-2 as a potential GEF (guanine nucleotide exchange factor) modulator of Gαi1∙GDP. In conclusion, the aforementioned hits are suggested to serve as possible prodrugs or precursors of compounds exhibiting improved pharmacological properties.
Publications
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Strategies towards Targeting Gαi/s Proteins: Scanning of Protein‐Protein Interaction Sites To Overcome Inaccessibility. ChemMedChem, 16(11), 1697-1716.
Nubbemeyer, Britta; Pepanian, Anna; Paul, George Ajay Abisheck & Imhof, Diana
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Targeting of Gαi/s Protein by Peptidic Guanine Nucleotide Exchange Modulators. Scientific poster, Virtual conference entitled Advances in Chemical Biology (Frankfurt am Main, Germany, January 2021)
Nubbemeyer, B., Pepanian, A., Paul George, A. A., Kühl, Τ., Pei, D., Ammer, H. & Imhof, D.
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Fluorescence Anisotropy Assay with Guanine Nucleotides Provides Access to Functional Analysis of Gαi1 Proteins. Analytical Chemistry, 94(41), 14410-14418.
Pepanian, Anna; Sommerfeld, Paul; Kasprzyk, Renata; Kühl, Toni; Binbay, F. Ayberk; Hauser, Christoph; Löser, Reik; Wodtke, Robert; Bednarczyk, Marcelina; Chrominski, Mikolaj; Kowalska, Joanna; Jemielity, Jacek; Imhof, Diana & Pietsch, Markus
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Investigation of potent Gα protein modulators.” Voluntary talk for the graduate school BIGS DrugS of the University of Bonn (Bonn, Germany, July 2022)
Pepanian, A.
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Modulation of Gαi1/s proteins with molecules targeting the guanine nucleotide binding pocket. Scientific poster and invited short talk, Gordon Research Conference (Southbridge, USA, June 2022)
Pepanian, A., Paul George, A. A., Kühl, Τ., Sommerfeld, P., Pietsch, M. & Imhof, D.
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Targeting Gαi/s Proteins with Peptidyl Nucleotide Exchange Modulators. ACS Chemical Biology, 17(2), 463-473.
Nubbemeyer, Britta; Paul, George Ajay Abisheck; Kühl, Toni; Pepanian, Anna; Beck, Maximilian Steve; Maghraby, Rahma; Shetab, Boushehri Maryam; Muehlhaupt, Maximilian; Pfeil, Eva Marie; Annala, Suvi Katariina; Ammer, Hermann; Imhof, Diana & Pei, Dehua
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Targeting Gαi/s Proteins With Peptidyl Nucleotide Exchange Modulators. Scientific poster, 36th European Peptide Symposium and 12th International Peptide Symposium (Sitges, Spain, August 2022, 2nd Poster Prize Award)
Pepanian, A., Nubbemeyer, B., Paul George, A. A., Kühl, Τ., Ammer, H., Pei, D. & Imhof, D.
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Bicyclic Peptide Library Screening for the Identification of Gαi Protein Modulators. Journal of Medicinal Chemistry, 66(17), 12396-12406.
Pepanian, Anna; Binbay, Furkan Ayberk; Roy, Suchismita; Nubbemeyer, Britta; Koley, Amritendu; Rhodes, Curran A.; Ammer, Hermann; Pei, Dehua; Ghosh, Pradipta & Imhof, Diana
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Nucleotide exchange modulation of Gαi/s proteins with linear and macrocyclic peptides. Scientific poster, German Peptide Symposium (Jena, Germany, August 2023)
Pepanian, A., Binbay, F. A., Ammer, H., Ghosh, P., Pei, D. & Imhof, D.
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Quality Assessment of Selected Protein Structures Derived from Homology Modeling and AlphaFold. Pharmaceuticals, 16(12), 1662.
Binbay, Furkan Ayberk; Rathod, Dhruv Chetanbhai; George, Ajay Abisheck Paul & Imhof, Diana
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Design, synthesis, and analysis of macrobicyclic peptides for targeting the Gαi protein. Journal of Peptide Science, 30(6).
Pepanian, Anna; Binbay, F. Ayberk; Pei, Dehua & Imhof, Diana
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In-depth analysis of Gαs protein activity by probing different fluorescently labeled guanine nucleotides. Biological Chemistry, 405(5), 297-309.
Pepanian, Anna; Sommerfeld, Paul; Binbay, Furkan Ayberk; Fischer, Dietmar; Pietsch, Markus & Imhof, Diana
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Linear and macrocyclic peptides including nucleotide exchange modulation of Gαi/s proteins. Invited talk, European Peptide Synthesis Conference (Prague, Czech Republic, April 2024)
Imhof D.
