By binding to the spliceosomal protein Snu66, the human ubiquitin-like protein Hub1 is a modulator of the spliceosome performance and facilitates alternative splicing. Small molecules that bind to Hub1 would be of interest to study the protein-protein interaction of Hub1/Snu66, which is linked to several human pathologies, such as hypercholesterolemia, premature aging, neurodegenerative diseases, and cancer. To identify small molecule ligands for Hub1, we used the interface analysis, peptide modeling of the Hub1/Snu66 interaction and the fragment-based NMR screening. Fragment-based NMR screening has not proven sufficient to unambiguously search for fragments that bind to the Hub1 protein. This was because the Snu66 binding pocket of Hub1 is occupied by pH-sensitive residues, making it difficult to distinguish between pH-induced NMR shifts and actual binding events. The NMR analyses were therefore verified experimentally by microscale thermophoresis and by NMR pH titration experiments. Our study found two small peptides that showed binding to Hub1. These peptides are the first small-molecule ligands reported to interact with the Hub1 protein.

Department of Drug Design University of Groningen A Deusinglaan 1 9713 AV Groningen The Netherlands

Department of Innovative Chemistry Palackӯ University CATRIN Šlechtitelů 241 27 779 00 Olomouc Czech Republic

Department of Organic Chemistry Faculty of Chemistry Jagiellonian University Gronostajowa 2 30 387 Krakow Poland

Molecules. 2024 Jun 20;29(12):2926. doi: 10.3390/molecules29122926. PubMed

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Friedman J.S., Koop B.F., Raymond V., Walter M.A. Isolation of a Ubiquitin-like (UBL5) Gene from a Screen Identifying Highly Expressed and Conserved Iris Genes. Genomics. 2001;255:252–255. doi: 10.1006/geno.2000.6439. PubMed DOI

Hochstrasser M. Origin and Function of Ubiquitin-like Proteins. Nature. 2009;458:422–429. doi: 10.1038/nature07958. PubMed DOI PMC

Jentsch S., Pyrowolakis G. Ubiquitin and Its Kin: How Close Are the Family Ties? Trends Cell Biol. 2000;10:335–342. doi: 10.1016/S0962-8924(00)01785-2. PubMed DOI

McNally T., Huang Q., Janis R.S., Liu Z., Olejniczak E.T., Reilly R.M. Structural Analysis of UBL5, a Novel Ubiquitin-like Modifier. Protein Sci. 2003;12:1562–1566. doi: 10.1110/ps.0382803. PubMed DOI PMC

Mishra S.K., Ammon T., Popowicz G.M., Krajewski M., Nagel R.J., Ares M., Holak T.A., Jentsch S. Role of the Ubiquitin-like Protein Hub1 in Splice-Site Usage and Alternative Splicing. Nature. 2011;474:173–180. doi: 10.1038/nature10143. PubMed DOI PMC

Ammon T., Mishra S.K., Kowalska K., Popowicz G.M., Holak T.A., Jentsch S. The Conserved Ubiquitin-like Protein Hub 1 Plays a Critical Role in Splicing in Human Cells. J. Mol. Cell Biol. 2014;6:312–323. doi: 10.1093/jmcb/mju026. PubMed DOI PMC

Masopust M., Weisz F., Bartenschlager H., Knoll A., Vykoukalova Z., Geldermann H., Cepica S. Porcine Ubiquitin-like 5 (UBL5) Gene: Genomic Organization, Polymorphisms, MRNA Cloning, Splicing Variants and Association Study. Mol. Biol. Rep. 2014;41:2353–2362. doi: 10.1007/s11033-014-3089-6. PubMed DOI

Bedford L., Lowe J., Dick L.R., Mayer R.J., Brownell J.E. Ubiquitin-like Protein Conjugation and the Ubiquiting-Proteasome System as Drug Targets. Nat. Rev. Drug Discov. 2011;10:29–46. doi: 10.1038/nrd3321. PubMed DOI PMC

Wells J.A., McClendon C.L. Reaching for High-Hanging Fruit in Drug Discovery at Protein-Protein Interfaces. Nature. 2007;450:1001–1009. doi: 10.1038/nature06526. PubMed DOI

Jones S., Thornton J.M. Review Principles of Protein-Protein Interactions. Proc. Natl. Acad. Sci. USA. 1996;93:13–20. doi: 10.1073/pnas.93.1.13. PubMed DOI PMC

Hummel G., Reineke U., Reimer U. Translating Peptides into Small Molecules. Mol. Biosyst. 2006;2:499–508. doi: 10.1039/b611791k. PubMed DOI

de Esch I.J.P., Erlanson D.A., Jahnke W., Johnson C.N., Walsh L. Fragment-to-Lead Medicinal Chemistry Publications in 2020. J. Med. Chem. 2022;65:84–99. doi: 10.1021/acs.jmedchem.1c01803. PubMed DOI PMC

Li Y., Kang C. Solution NMR Spectroscopy in Target-Based Drug Discovery. Molecules. 2017;22:1399. doi: 10.3390/molecules22091399. PubMed DOI PMC

Musielak B., Janczyk W., Rodriguez I., Plewka J., Sala D., Magiera-Mularz K., Holak T. Competition NMR for Detection of Hit/Lead Inhibitors of Protein-Protein Interactions. Molecules. 2020;25:3017. doi: 10.3390/molecules25133017. PubMed DOI PMC

Perry E., Mills J.J., Zhao B., Wang F., Sun Q., Christov P.P., Tarr J.C., Rietz T.A., Olejniczak E.T., Lee T., et al. Fragment-Based Screening of Programmed Death Ligand 1 (PD-L1) Bioorg. Med. Chem. Lett. 2019;29:786–790. doi: 10.1016/j.bmcl.2019.01.028. PubMed DOI PMC

Krissinel E., Henrick K. Inference of Macromolecular Assemblies from Crystalline State. J. Mol. Biol. 2007;372:774–797. doi: 10.1016/j.jmb.2007.05.022. PubMed DOI

Antes I. DynaDock: A Now Molecular Dynamics-Based Algorithm for Protein-Peptide Docking Including Receptor Flexibility. Proteins. 2010;78:1084–1104. doi: 10.1002/prot.22629. PubMed DOI

Dominguez C., Boelens R., Bonvin A.M.J.J. HADDOCK: A Protein-Protein Docking Approach Based on Biochemical or Biophysical Information. J. Am. Chem. Soc. 2003;125:1731–1737. doi: 10.1021/ja026939x. PubMed DOI

Alam N., Goldstein O., Xia B., Porter K.A., Kozakov D., Schueler-Furman O. High-Resolution Global Peptide-Protein Docking Using Fragments-Based PIPER-FlexPepDock. PLoS Comput. Biol. 2017;13:e1005905. doi: 10.1371/journal.pcbi.1005905. PubMed DOI PMC

Kurcinski M., Pawel Ciemny M., Oleniecki T., Kuriata A., Badaczewska-Dawid A.E., Kolinski A., Kmiecik S. CABS-Dock Standalone: A Toolbox for Flexible Protein-Peptide Docking. Bioinformatics. 2019;35:4170–4172. doi: 10.1093/bioinformatics/btz185. PubMed DOI PMC

Porter K.A., Xia B., Beglov D., Bohnuud T., Alam N., Schueler-Furman O., Kozakov D. ClusPro PeptiDock: Efficient Global Docking of Peptide Recognition Motifs Using FFT. Bioinformatics. 2017;33:3299–3301. doi: 10.1093/bioinformatics/btx216. PubMed DOI PMC

Lee H., Heo L., Lee M.S., Seok C. GalaxyPepDock: A Protein-Peptide Docking Tool Based on Interaction Similarity and Energy Optimization. Nucl. Acids. Res. 2015;43:W431–W435. doi: 10.1093/nar/gkv495. PubMed DOI PMC

Obarska-Kosinska A., Iacoangeli A., Lepore R., Tramontano A. PepComposer: Computational Design of Peptides Binding to a given Protein Surface. Nucl. Acids. Res. 2016;44:W522–W528. doi: 10.1093/nar/gkw366. PubMed DOI PMC

Raveh B., London N., Schueler-Furman O. Sub-Angstrom Modeling of Complexes between Flexible Peptides and Globular Proteins. Proteins. 2010;78:2029–2040. doi: 10.1002/prot.22716. PubMed DOI

Lavi A., Ngan C.H., Movshovitz-Attias D., Bohnuud T., Yueh C., Beglov D., Schueler-Furman O., Kozakov D. Detection of Peptide-Binding Sites on Protein Surfaces: The First Step toward the Modeling and Targeting of Peptide-Mediated Interactions. Protein. 2013;81:2096–2105. doi: 10.1002/prot.24422. PubMed DOI PMC

Hajduk P.J., Greer J. A Decade of Fragment-Based Drug Design: Strategic Advances and Lessons Learned. Nat. Rev. Drug Discov. 2007;6:211–219. doi: 10.1038/nrd2220. PubMed DOI

Mashalidis E.H., Sledz P., Lang S., Abell C. A Three-Stage Biophysical Screening Cascade for Fragment-Based Drug Discovery. Nat. Protoc. 2013;8:2309–2324. doi: 10.1038/nprot.2013.130. PubMed DOI

Fielding L. NMR Methods for the Determination of Protein-Ligand Dissociation Constants. Prog. Nucl. Magn. Reson. Spectrosc. 2007;51:219–242. doi: 10.1016/j.pnmrs.2007.04.001. DOI

Harner M.J., Frank A.O., Fesik S.W. Fragment-Based Drug Discovery Using NMR Spectroscopy. J. Biomol. NMR. 2013;56:65–75. doi: 10.1007/s10858-013-9740-z. PubMed DOI PMC

Williamson M.P. Using Chemical Shift Perturbation to Characterise Ligand Binding. Prog. Nucl. Magn. Reson. Spectrosc. 2013;73:1–16. doi: 10.1016/j.pnmrs.2013.02.001. PubMed DOI

Babaoglu K., Shoichet B.K. Deconstructing Fragment-Based Inhibitor Discovery. Nat. Chem. Biol. 2006;2:720–723. doi: 10.1038/nchembio831. PubMed DOI

Dalvit C., Fogliatto G., Stewart A., Veronesi M., Stockman B. WaterLOGSY as a Method for Primary NMR Screening: Practical Aspects and Range of Applicability. J. Biomol. NMR. 2001;21:349–359. doi: 10.1023/A:1013302231549. PubMed DOI

Kitel R., Rodríguez I., del Corte X., Atmaj J., Żarnik M., Surmiak E., Muszak D., Magiera-Mularz K., Popowicz G.M., Holak T.A., et al. Exploring the Surface of the Ectodomain of the PD-L1 Immune Checkpoint with Small-Molecule Fragments. ACS Chem. Biol. 2022;17:2655–2663. doi: 10.1021/acschembio.2c00583. PubMed DOI PMC

Czisch M., Schleicher M., Horger S., Voelter W., Holak T.A. Conformation of Thymosin Β4 in Water Determined by NMR Spectroscopy. Eur. J. Biochem. 1993;218:335–344. doi: 10.1111/j.1432-1033.1993.tb18382.x. PubMed DOI

D’Silva L., Ozdowy P., Krajewski M., Rothweiler U., Singh M., Holak T.A. Monitoring the Effects of Antagonists on Protein-Protein Interactions with NMR Spectroscopy. J. Am. Chem. Soc. 2005;127:13220–13226. doi: 10.1021/ja052143x. PubMed DOI

Shuker S.B., Hajduk P.J., Meadows R.P., Fesik S.W. Discovering High-Affinity Ligands for Proteins: SAR by NMR. Science. 1996;274:1531–1534. doi: 10.1126/science.274.5292.1531. PubMed DOI

Kolinski A. Protein Modeling and Structure Prediction with a Reduced Representation. Acta Biochim. Pol. 2004;51:349–371. doi: 10.18388/abp.2004_3575. PubMed DOI

Kurcinski M., Blaszczyk M., Ciemny M.P., Kolinski A., Kmiecik S. A Protocol for CABS-Dock Protein-Peptide Docking Driven by Side-Chain Contact Information. Biomed. Eng. Online. 2017;16:73. doi: 10.1186/s12938-017-0363-6. PubMed DOI PMC

Badaczewska-Dawid A.E., Kmiecik S., Koliński M. Docking of Peptides to GPCRs Using a Combination of CABS-Dock with FlexPepDock Refinement. Brief. Bioinform. 2021;22:bbaa109. doi: 10.1093/bib/bbaa109. PubMed DOI PMC

Ciemny M.P., Kurcinski M., Blaszczyk M., Kolinski A., Kmiecik S. Modeling EphB4-EphrinB2 Protein-Protein Interaction Using Flexible Docking of a Short Linear Motif. Biomed. Eng. Online. 2017;16:71. doi: 10.1186/s12938-017-0362-7. PubMed DOI PMC

Kmiecik S., Gront D., Kolinski M., Wieteska L., Dawid A.E., Kolinski A. Coarse-Grained Protein Models and Their Applications. Chem. Rev. 2016;116:7898–7936. doi: 10.1021/acs.chemrev.6b00163. PubMed DOI

Kabsch W., Sander C. Routine Follow up of Patients with Treated Pulmonary Tuberculosis. Biopolymers. 1983;22:2577–2637. doi: 10.1002/bip.360221211. PubMed DOI

Touw W.G., Baakman C., Black J., te Beek T.A.H., Krieger E., Joosten R.P., Vriend G. A Series of PDB-Related Databanks for Everyday Needs. Nucl. Acids Res. 2015;43:D364–D368. doi: 10.1093/nar/gku1028. PubMed DOI PMC

Piotto M., Saudek V., Sklenar V. Gradient-Tailored Excitation for Single-Quantum NMR Spectroscopy of Aqueous Solutions. J. Biomol. NMR. 1992;2:661–665. doi: 10.1007/BF02192855. PubMed DOI

Brutscher B., Schanda P. SOFAST-HMQC Experiments for Recording Two-Dimensional Heteronuclear Correlation Spectra of Proteins within a Few Seconds. J. Biomol. NMR. 2005;33:199–211. doi: 10.1007/s10858-005-4425-x. PubMed DOI