In this article we describe the production and screening of a genetically encoded library of 106 lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein–protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product–like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Nature Communications Open Access 23 March 2023
Phylogeny-guided genome mining of roseocin family lantibiotics to generate improved variants of roseocin
AMB Express Open Access 20 March 2023
Signal Transduction and Targeted Therapy Open Access 11 March 2023
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Marsault, E. & Peterson, M. L. Macrocycles are great cycles: applications, opportunities, and challenges of synthetic macrocycles in drug discovery. J. Med. Chem. 54, 1961–2004 (2011).
Driggers, E. M., Hale, S. P., Lee, J. & Terrett, N. K. The exploration of macrocycles for drug discovery—an underexploited structural class. Nat. Rev. Drug Discov. 7, 608–624 (2008).
Cardote, T. A. & Ciulli, A. Cyclic and macrocyclic peptides as chemical tools to recognise protein surfaces and probe protein-protein interactions. Chem. Med. Chem 11, 787–794 (2016).
Gao, M., Cheng, K. & Yin, H. Targeting protein-protein interfaces using macrocyclic peptides. Biopolymers 104, 310–316 (2015).
Lennard, K. R. & Tavassoli, A. Peptides come round: using SICLOPPS libraries for early stage drug discovery. Chemistry 20, 10608–10614 (2014).
Heinis, C., Rutherford, T., Freund, S. & Winter, G. Phage-encoded combinatorial chemical libraries based on bicyclic peptides. Nat. Chem. Biol. 5, 502–507 (2009).
Miranda, E. et al. A cyclic peptide inhibitor of HIF-1 heterodimerization that inhibits hypoxia signaling in cancer cells. J. Am. Chem. Soc. 135, 10418–10425 (2013).
Passioura, T., Katoh, T., Goto, Y. & Suga, H. Selection-based discovery of druglike macrocyclic peptides. Annu. Rev. Biochem. 83, 727–752 (2014).
Heinis, C. & Winter, G. Encoded libraries of chemically modified peptides. Curr. Opin. Chem. Biol. 26, 89–98 (2015).
Birts, C. N. et al. A cyclic peptide inhibitor of C-terminal binding protein dimerization links metabolism with mitotic fidelity in breast cancer cells. Chem. Sci. 4, 3046–3057 (2013).
Tavassoli, A. SICLOPPS cyclic peptide libraries in drug discovery. Curr. Opin. Chem. Biol. 38, 30–35 (2017).
Arnison, P. G. et al. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30, 108–160 (2013).
Ortega, M. A. & van der Donk, W. A. New insights into the biosynthetic logic of ribosomally synthesized and post-translationally modified peptide natural products. Cell Chem. Biol. 23, 31–44 (2016).
Kuipers, O. P. et al. Protein engineering of lantibiotics. Antonie van Leeuwenhoek 69, 161–169 (1996).
Cotter, P. D. et al. Complete alanine scanning of the two-component lantibiotic lacticin 3147: generating a blueprint for rational drug design. Mol. Microbiol. 62, 735–747 (2006).
Pavlova, O., Mukhopadhyay, J., Sineva, E., Ebright, R. H. & Severinov, K. Systematic structure-activity analysis of microcin J25. J. Biol. Chem. 283, 25589–25595 (2008).
Islam, M. R. et al. Evaluation of essential and variable residues of nukacin ISK-1 by NNK scanning. Mol. Microbiol. 72, 1438–1447 (2009).
Pan, S. J. & Link, A. J. Sequence diversity in the lasso peptide framework: discovery of functional microcin J25 variants with multiple amino acid substitutions. J. Am. Chem. Soc. 133, 5016–5023 (2011).
Young, T. S., Dorrestein, P. C. & Walsh, C. T. Codon randomization for rapid exploration of chemical space in thiopeptide antibiotic variants. Chem. Biol. 19, 1600–1610 (2012).
Zhang, F. & Kelly, W. L. In vivo production of thiopeptide variants. Methods Enzymol. 516, 3–24 (2012).
Boakes, S. et al. Generation of an actagardine A variant library through saturation mutagenesis. Appl. Microbiol. Biotechnol. 95, 1509–1517 (2012).
Deane, C. D., Melby, J. O., Molohon, K. J., Susarrey, A. R. & Mitchell, D. A. Engineering unnatural variants of plantazolicin through codon reprogramming. ACS Chem. Biol. 8, 1998–2008 (2013).
Weiz, A. R. et al. Harnessing the evolvability of tricyclic microviridins to dissect protease-inhibitor interactions. Angew. Chem. Int. Ed. Engl. 53, 3735–3738 (2014).
Houssen, W. E. et al. An efficient method for the in vitro production of azol(in)e-based cyclic peptides. Angew. Chem. Int. Ed. Engl. 53, 14171–14174 (2014).
Ruffner, D. E., Schmidt, E. W. & Heemstra, J. R. Assessing the combinatorial potential of the RiPP cyanobactin tru pathway. ACS Synth. Biol. 4, 482–492 (2015).
Repka, L. M., Chekan, J. R., Nair, S. K. & van der Donk, W. A. Mechanistic understanding of lanthipeptide biosynthetic enzymes. Chem. Rev. 117, 5457–5520 (2017).
Li, B. et al. Catalytic promiscuity in the biosynthesis of cyclic peptide secondary metabolites in planktonic marine cyanobacteria. Proc. Natl. Acad. Sci. USA 107, 10430–10435 (2010).
Tang, W. & van der Donk, W. A. Structural characterization of four prochlorosins: a novel class of lantipeptides produced by planktonic marine cyanobacteria. Biochemistry 51, 4271–4279 (2012).
Yu, Y., Mukherjee, S. & van der Donk, W. A. Product formation by the promiscuous lanthipeptide synthetase ProcM is under kinetic control. J. Am. Chem. Soc. 137, 5140–5148 (2015).
Giordanetto, F. & Kihlberg, J. Macrocyclic drugs and clinical candidates: what can medicinal chemists learn from their properties? J. Med. Chem. 57, 278–295 (2014).
Foster, A. D. et al. Methods for the creation of cyclic peptide libraries for use in lead discovery. J. Biomol. Screen. 20, 563–576 (2015).
Quartararo, J. S. et al. A bicyclic peptide scaffold promotes phosphotyrosine mimicry and cellular uptake. Bioorg. Med. Chem. 22, 6387–6391 (2014).
Tavassoli, A. et al. Inhibition of HIV budding by a genetically selected cyclic peptide targeting the Gag-TSG101 interaction. ACS Chem. Biol. 3, 757–764 (2008).
Garrus, J. E. et al. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 107, 55–65 (2001).
Martin-Serrano, J., Zang, T. & Bieniasz, P. D. HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Nat. Med. 7, 1313–1319 (2001).
Demirov, D. G., Ono, A., Orenstein, J. M. & Freed, E. O. Overexpression of the N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late domain function. Proc. Natl. Acad. Sci. USA 99, 955–960 (2002).
Shi, Y., Yang, X., Garg, N. & Van Der Donk, W. A. Production of lantipeptides in Escherichia coli. J. Am. Chem. Soc. 133, 2338–2341 (2011).
VerPlank, L. et al. Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag). Proc. Natl. Acad. Sci. USA 98, 7724–7729 (2001).
Male, A. L. et al. Targeting Bacillus anthracis toxicity with a genetically selected inhibitor of the PA/CMG2 protein-protein interaction. Sci. Rep. 7, 3104 (2017).
Im, Y. J. et al. Crystallographic and functional analysis of the ESCRT-I /HIV-1 Gag PTAP interaction. Structure 18, 1536–1547 (2010).
Pornillos, O., Alam, S. L., Davis, D. R. & Sundquist, W. I. Structure of the Tsg101 UEV domain in complex with the PTAP motif of the HIV-1p6 protein. Nat. Struct. Biol. 9, 812–817 (2002).
Pornillos, O. et al. Structure and functional interactions of the Tsg101 UEV domain. EMBO J. 21, 2397–2406 (2002).
Morris, C. R., Stanton, M. J., Manthey, K. C., Oh, K. B. & Wagner, K. U. A knockout of the Tsg101 gene leads to decreased expression of ErbB receptor tyrosine kinases and induction of autophagy prior to cell death. PLoS One 7, e34308 (2012).
Ruland, J. et al. p53 accumulation, defective cell proliferation, and early embryonic lethality in mice lacking tsg101. Proc. Natl. Acad. Sci. USA 98, 1859–1864 (2001).
Pornillos, O. et al. HIV Gag mimics the Tsg101-recruiting activity of the human Hrs protein. J. Cell Biol. 162, 425–434 (2003).
Lu, Q., Hope, L. W., Brasch, M., Reinhard, C. & Cohen, S. N. TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation. Proc. Natl. Acad. Sci. USA 100, 7626–7631 (2003).
Jencks, W. P. On the attribution and additivity of binding energies. Proc. Natl. Acad. Sci. USA 78, 4046–4050 (1981).
Knappe, T. A. et al. Introducing lasso peptides as molecular scaffolds for drug design: engineering of an integrin antagonist. Angew. Chem. Int. Ed. Engl. 50, 8714–8717 (2011).
Conibear, A. C. et al. Approaches to the stabilization of bioactive epitopes by grafting and peptide cyclization. Biopolymers 106, 89–100 (2016).
Rink, R. et al. To protect peptide pharmaceuticals against peptidases. J. Pharmacol. Toxicol. Methods 61, 210–218 (2010).
Newcombe, R. G. Two-sided confidence intervals for the single proportion: comparison of seven methods. Stat. Med. 17, 857–872 (1998).
The authors thank S. Eyckerman for pMET7-GAG-EGFP (via Addgene, plasmid # 80605), and D. Gomez-Nicola for HEK239T cells. This work was supported by the National Institutes of Health (R37 GM 058822 to W.A.V.; F32 GM0112284 to M.C.W.), Cancer Research UK (A20185 to A.T.), and the Engineering and Physical Sciences Research Council and C4X Drug Discovery (EP/L505067/1, Ph.D. studentship for K.R.L. to A.T.), AstraZeneca (Ph.D. studentship for A.T.B. to A.T.) and the Southampton University Institute for Life Sciences (studentship for C.D. to A.T.).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Yang, X., Lennard, K.R., He, C. et al. A lanthipeptide library used to identify a protein–protein interaction inhibitor. Nat Chem Biol 14, 375–380 (2018). https://doi.org/10.1038/s41589-018-0008-5
This article is cited by
Phylogeny-guided genome mining of roseocin family lantibiotics to generate improved variants of roseocin
AMB Express (2023)
Nature Communications (2023)
Signal Transduction and Targeted Therapy (2023)
Nature Communications (2021)
Exploring structural signatures of the lanthipeptide prochlorosin 2.8 using tandem mass spectrometry and trapped ion mobility-mass spectrometry
Analytical and Bioanalytical Chemistry (2021)