Hit evaluation of an α-helical peptide: Ala-scan, truncation and sidechain-to-sidechain macrocyclization of an RNA polymerase Inhibitor
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Ahmed Ashraf Moustafa Kamal
Abstract
RNA polymerase (RNAP) remains a relatively underexplored target with only rifampicin and fidaxomicin in clinical use. Hence, the concurrent rise in bacterial resistance rate urges the search for novel RNAP inhibitors with a novel mode of action. In this work, we investigated the impact of several systematic modifications including sidechain-to-sidechain macrocylization in the α-helical content and biological activity of a previously identified inhibitory sigma factor fragment. Ala-scan results, peptide truncation from both the N- and C-terminus and modifications inspired by other RNAP inhibitors revealed novel structure activity relationships but did not yield a superior sequence. Additionally, four insertion points for non-natural amino acids bearing side chains required for macrocylization were explored. Linear precursors showed improved stabilization of the α-helical content compared to the original sequence as demonstrated by circular dichroism (CD) spectroscopy. However, this increase in α-helicity did not translate into improved biological activity. Instead, complete abolishment of RNAP inhibitory activity occurred. We hypothesize three possible reasons for such a discrepancy and offer the basis for further optimization efforts for this peptidic RNAP inhibitor.
Acknowledgments
We are grateful for Werner Tegge for managing our in-house peptide platform operations. We are thankful for Richard Ebright for his attempts in obtaining X-ray co-crystals. We thank Jeanine Jung and Tabea Schramm for technical assistance and diligence in performing the biological assays. We are appreciative for Michael Hoffmann’s assistance in obtaining the HRMS spectra.
References
Arkin, M.R., Tang, Y., and Wells, J.A. (2014). Small-molecule inhibitors of protein-protein interactions: progressing toward the reality. Chem. Biol. 21, 1102–1114.10.1016/j.chembiol.2014.09.001Search in Google Scholar PubMed PubMed Central
Avrutina, O., Empting, M., Fabritz, S., Daneschdar, M., Frauendorf, H., Diederichsen, U., and Kolmar, H. (2009). Application of copper(I) catalyzed azide-alkyne 3+2 cycloaddition to the synthesis of template-assembled multivalent peptide conjugates. Org. Biomol. Chem. 7, 4177–4185.10.1039/b908261aSearch in Google Scholar PubMed
Cardote, T.A.F. and Ciulli, A. (2016). Cyclic and macrocyclic peptides as chemical tools to recognise protein surfaces and probe protein-protein interactions. ChemMedChem 11, 787–794.10.1002/cmdc.201500450Search in Google Scholar PubMed PubMed Central
Craik, D.J., Fairlie, D.P., Liras, S., and Price, D. (2013). The future of peptide-based drugs. Chem. Biol. Drug. Des. 81, 136–147.10.1111/cbdd.12055Search in Google Scholar PubMed
Doak, B.C., Over, B., Giordanetto, F., and Kihlberg, J. (2014). Oral druggable space beyond the rule of 5: insights from drugs and clinical candidates. Chem. Biol. 21, 1115–1142.10.1016/j.chembiol.2014.08.013Search in Google Scholar PubMed
Driggers, E.M., Hale, S.P., Lee, J., and Terrett, N.K. (2008). The exploration of macrocycles for drug discovery – an underexploited structural class. Nat. Rev. Drug Discov. 7, 608–624.10.1038/nrd2590Search in Google Scholar PubMed
El-Mowafi, S.A., Sineva, E., Alumasa, J.N., Nicoloff, H., Tomsho, J.W., Ades, S.E., and Keiler, K.C. (2015). Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay. Antimicrob. Agents Chemother. 59, 193–205.10.1128/AAC.03979-14Search in Google Scholar PubMed PubMed Central
Empting, M., Avrutina, O., Meusinger, R., Fabritz, S., Reinwarth, M., Biesalski, M., Voigt, S., Buntkowsky, G., and Kolmar, H. (2011). “Triazole bridge”: disulfide-bond replacement by ruthenium-catalyzed formation of 1,5-disubstituted 1,2,3-triazoles. Angew. Chem. Int. Ed. 50, 5207–5211.10.1002/anie.201008142Search in Google Scholar PubMed
Fittler, H., Avrutina, O., Glotzbach, B., Empting, M., and Kolmar, H. (2013). Combinatorial tuning of peptidic drug candidates: high-affinity matriptase inhibitors through incremental structure-guided optimization. Org. Biomol. Chem. 11, 1848–1857.10.1039/c3ob27469aSearch in Google Scholar PubMed
Fosgerau, K. and Hoffmann, T. (2015). Peptide therapeutics: current status and future directions. Drug Disc. Today 20, 122–128.10.1016/j.drudis.2014.10.003Search in Google Scholar PubMed
Goldflam, M. and Ullman, C.G. (2015). Recent advances toward the discovery of drug-like peptides de novo. Front. Chem. 3, 69.10.3389/fchem.2015.00069Search in Google Scholar PubMed PubMed Central
Hüsecken, K., Negri, M., Fruth, M., Boettcher, S., Hartmann, R.W., and Haupenthal, J. (2013). Peptide-based investigation of the Escherichia coli RNA polymerase σ(70): core interface as target site. ACS Chem. Biol. 8, 758–766.10.1021/cb3005758Search in Google Scholar PubMed
Kuznedelov, K., Korzheva, N., Mustaev, A., and Severinov, K. (2002). Structure-based analysis of RNA polymerase function: the largest subunit’s rudder contributes critically to elongation complex stability and is not involved in the maintenance of RNA-DNA hybrid length. EMBO J. 21, 1369–1378.10.1093/emboj/21.6.1369Search in Google Scholar PubMed PubMed Central
Lau, Y.H., de Andrade, P., Wu, Y., and Spring, D.R. (2015). Peptide stapling techniques based on different macrocyclisation chemistries. Chem. Soc. Rev. 44, 91–102.10.1039/C4CS00246FSearch in Google Scholar
Li, H., Aneja, R., and Chaiken, I. (2013). Click chemistry in peptide-based drug design. Molecules 18, 9797–9817.10.3390/molecules18089797Search in Google Scholar PubMed PubMed Central
Ma, C., Yang, X., Kandemir, H., Mielczarek, M., Johnston, E.B., Griffith, R., Kumar, N., and Lewis, P.J. (2013). Inhibitors of bacterial transcription initiation complex formation. ACS Chem. Biol. 8, 1972–1980.10.1021/cb400231pSearch in Google Scholar PubMed
Marsault, E. and Peterson, M.L. (2011). Macrocycles are great cycles: applications, opportunities, and challenges of synthetic macrocycles in drug discovery. J. Med. Chem. 54, 1961–2004.10.1021/jm1012374Search in Google Scholar PubMed
Martí-Centelles, V., Pandey, M.D., Burguete, M.I., and Luis, S.V. (2015). Macrocyclization reactions: the importance of conformational, configurational, and template-induced preorganization. Chem. Rev. 115, 8736–8834.10.1021/acs.chemrev.5b00056Search in Google Scholar PubMed
Murakami, K.S. (2013). X-ray crystal structure of Escherichia coli RNA polymerase σ70 holoenzyme. J. Biol. Chem. 288, 9126–9134.10.1074/jbc.M112.430900Search in Google Scholar PubMed PubMed Central
Okamoto, T., Zobel, K., Fedorova, A., Quan, C., Yang, H., Fairbrother, W.J., Huang, D.C.S., Smith, B.J., Deshayes, K., and Czabotar, P.E. (2013). Stabilizing the pro-apoptotic BimBH3 helix (BimSAHB) does not necessarily enhance affinity or biological activity. ACS Chem. Biol. 8, 297–302.10.1021/cb3005403Search in Google Scholar PubMed
Otvos, L. and Wade, J.D. (2014). Current challenges in peptide-based drug discovery. Front. Chem. 2, 62.10.3389/fchem.2014.00062Search in Google Scholar PubMed PubMed Central
Pasini, D. (2013). The click reaction as an efficient tool for the construction of macrocyclic structures. Molecules 18, 9512–9530.10.3390/molecules18089512Search in Google Scholar PubMed PubMed Central
Pelay-Gimeno, M., Glas, A., Koch, O., and Grossmann, T.N. (2015). Structure-based design of inhibitors of protein-protein interactions: mimicking peptide binding epitopes. Angew. Chem. Int. Ed. 54, 8896–8927.10.1002/anie.201412070Search in Google Scholar PubMed PubMed Central
Tischler, M., Nasu, D., Empting, M., Schmelz, S., Heinz, D.W., Rottmann, P., Kolmar, H., Buntkowsky, G., Tietze, D., and Avrutina, O. (2012). Braces for the peptide backbone: insights into structure-activity relationships of protease inhibitor mimics with locked amide conformations. Angew. Chem. Int. Ed. 51, 3708–3712.10.1002/anie.201108983Search in Google Scholar PubMed
Toulokhonov, I. and Landick, R. (2006). The role of the lid element in transcription by E. coli RNA polymerase. J. Mol. Biol. 361, 644–658.10.1016/j.jmb.2006.06.071Search in Google Scholar PubMed
Vlieghe, P., Lisowski, V., Martinez, J., and Khrestchatisky, M. (2010). Synthetic therapeutic peptides: science and market. Drug Disc. Today 15, 40–56.10.1016/j.drudis.2009.10.009Search in Google Scholar PubMed
White, C.J. and Yudin, A.K. (2011). Contemporary strategies for peptide macrocyclization. Nat. Chem. 3, 509–524.10.1038/nchem.1062Search in Google Scholar PubMed
Supplementary Material
The online version of this article offers supplementary material (https://doi.org/10.1515/hsz-2018-0333).
©2019 Walter de Gruyter GmbH, Berlin/Boston
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Articles in the same Issue
- Frontmatter
- Protein engineering comes of age
- Microbial transglutaminase for biotechnological and biomedical engineering
- Computational design of structured loops for new protein functions
- Formylglycine-generating enzymes for site-specific bioconjugation
- Chemical modification of neuropeptide Y for human Y1 receptor targeting in health and disease
- Manipulating the stereoselectivity of a thermostable alcohol dehydrogenase by directed evolution for efficient asymmetric synthesis of arylpropanols
- Radiometal-labeled anti-VCAM-1 nanobodies as molecular tracers for atherosclerosis – impact of radiochemistry on pharmacokinetics
- Hit evaluation of an α-helical peptide: Ala-scan, truncation and sidechain-to-sidechain macrocyclization of an RNA polymerase Inhibitor
- Highly flexible, IgG-shaped, trivalent antibodies effectively target tumor cells and induce T cell-mediated killing
- An engineered lipocalin that tightly complexes the plant poison colchicine for use as antidote and in bioanalytical applications
- Sequence selection by FitSS4ASR alleviates ancestral sequence reconstruction as exemplified for geranylgeranylglyceryl phosphate synthase
- Facile generation of antibody heavy and light chain diversities for yeast surface display by Golden Gate Cloning
- Peptide binding affinity redistributes preassembled repeat protein fragments
- Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method
- Rational design of an improved photo-activatable intein for the production of head-to-tail cyclized peptides
- Characterization and engineering of photoactivated adenylyl cyclases
