Abstract
Antimicrobial host defense peptides are promising alternatives to resistance prone small molecule antibiotics. To overcome the poor physiologic stability of these therapeutic candidates it is common to prepare proteolytically resistant retro-inverso analogues, where sequence backbone direction and amino acid chirality are reversed. However, in many cases, gains in stability are offset by altered assembly propensities and reduced biologic potency. Here, we show that, contrary to the dogma for non-mycobacterial pathogens, retro-inversion of antimycobacterial host defense peptides improves their potency, specificity and host safety; in some cases by more than an order of magnitude. Biophysical assays suggest that altered mycomembrane thermodynamics, instead of improved proteo…
Abstract
Antimicrobial host defense peptides are promising alternatives to resistance prone small molecule antibiotics. To overcome the poor physiologic stability of these therapeutic candidates it is common to prepare proteolytically resistant retro-inverso analogues, where sequence backbone direction and amino acid chirality are reversed. However, in many cases, gains in stability are offset by altered assembly propensities and reduced biologic potency. Here, we show that, contrary to the dogma for non-mycobacterial pathogens, retro-inversion of antimycobacterial host defense peptides improves their potency, specificity and host safety; in some cases by more than an order of magnitude. Biophysical assays suggest that altered mycomembrane thermodynamics, instead of improved proteolytic stability, plays a causative role in retro-inverso mediated potency gains. Additional bacteriologic assays using a lead retro-inversed candidate, MAD1-RI, demonstrate this analogue rapidly sterilizes replicating cultures of Mycobacterium tuberculosis, is effective towards drug-resistant clinical isolates of the pathogen, and synergistically enhances the activity of co-incubated antibiotics. Transcriptomic studies uncover complementary membrane destabilizing and metabolic mechanisms of antitubercular action for MAD1-RI, and in doing so identify sequence retro-inversion as a simple, but powerful, modality in the de novo design of non-natural antimycobacterial peptides.
Data availability
Source data are provided with this paper. The primary RNA-seq data generated in this study have been deposited in the National Center for Biotechnology Information database under accession codes SRX25995207–SRX25995212 [https://www.ncbi.nlm.nih.gov/sra/PRJNA1157802]. All other data generated in this study are provided in the Supplementary Information/Source Data file. Source data are provided with this paper.
References
Zhang, Y. & Yew, W. Mechanisms of drug resistance in Mycobacterium tuberculosis: update 2015. Int. J. Tuberc. Lung Dis. 19, 1276–1289 (2015).
Rima, M. et al. Antimicrobial peptides: a potent alternative to antibiotics. Antibiotics 10, 1095 (2021). 1.
Landman, D., Georgescu, C., Martin, D. A. & Quale, J. Polymyxins revisited. Clin. Microbiol. Rev. 21, 449–465 (2008).
Li, Z., Cao, Y., Yi, L., Liu, J. H. & Yang, Q. Emergent polymyxin resistance: end of an era? Open Forum Infect. Dis. 6, ofz368 (2019). 1.
Rezaei Javan, R., Van Tonder, A. J., King, J. P., Harrold, C. L. & Brueggemann, A. B. Genome sequencing reveals a large and diverse repertoire of antimicrobial peptides. Front. Microbiol. 9, 2012 (2018).
Lazzaro, B. P., Zasloff, M. & Rolff, J. Antimicrobial peptides: application informed by evolution. Science 368, eaau5480 (2020).
Santos-Júnior, C. D. et al. Discovery of antimicrobial peptides in the global microbiome with machine learning. Cell 187, 3761-3778.e16 (2024). 1.
Maasch, J. R. M. A., Torres, M. D. T., Melo, M. C. R. & de la Fuente-Nunez, C. Molecular de-extinction of ancient antimicrobial peptides enabled by machine learning. Cell Host Microbe 31, 1260–1274.e1266 (2023).
Wan, F., Torres, M. D. T., Peng, J. & de la Fuente-Nunez, C. Deep-learning-enabled antibiotic discovery through molecular de-extinction. Nat. Biomed. Eng. 8, 854–871 (2024). 1.
Chorev, M. & Goodman, M. A dozen years of retro-inverso peptidomimetics. Acc. Chem. Res. 26, 266–273 (1993).
Goodman, M. & Chorev, M. On the concept of linear modified retro-peptide structures. Acc. Chem. Res. 12, 1–7 (1979).
Doti, N., Mardirossian, M., Sandomenico, A., Ruvo, M. & Caporale, A. Recent Applications of Retro-Inverso Peptides. Int. J. Mol. Sci. 22, 8677 (2021). 1.
Li, C. et al. Limitations of peptide retro-inverso isomerization in molecular mimicry. J. Biol. Chem. 285, 19572–19581 (2010).
Preston, G. W. Different directions for retro-inverso peptides. J. Pept. Sci. 28, e3384 (2022).
Al Musaimi, O. Unlocking the potential of retro-inverso (RI) peptides as future drug candidates. Int. J. Pept. Res. Ther. 30, 56 (2024).
Simonson, A. W. et al. Pathogen-specific antimicrobials engineered de novo through membrane-protein biomimicry. Nat. Biomed. Eng. 5, 467–480 (2021).
Biswas, D. et al. Lead informed artificial intelligence mining of antitubercular host defense peptides. Biomacromolecules 26, 3167–3179 (2025).
de Moraes, L. et al. First generation of multifunctional peptides derived from latarcin-3a from Lachesana tarabaevi spider toxin. Front. Microbiol. 13, 965621 (2022).
Rangel, M. et al. Chemical and biological characterization of four new linear cationic α-helical peptides from the venoms of two solitary eumenine wasps. Toxicon 57, 1081–1092 (2011).
Chaparro-Aguirre, E. et al. Antimicrobial activity and mechanism of action of a novel peptide present in the ecdysis process of centipede Scolopendra subspinipes subspinipes. Sci. Rep. 9, 13631 (2019).
Cochran, A. G., Skelton, N. J. & Starovasnik, M. A. Tryptophan zippers: Stable, monomeric β-hairpins. Proc. Natl. Acad. Sci. USA 98, 5578–5583 (2001).
Liu, J., Yong, W., Deng, Y., Kallenbach, N. R. & Lu, M. Atomic structure of a tryptophan-zipper pentamer. Proc. Natl. Acad. Sci. USA 101, 16156–16161 (2004).
Micsonai, A. et al. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc. Natl. Acad. Sci. USA 112, E3095–E3103 (2015).
Micsonai, A. et al. BeStSel: webserver for secondary structure and fold prediction for protein CD spectroscopy. Nucleic Acids Res. 50, W90–W98 (2022).
Micsonai, A. et al. Disordered–ordered protein binary classification by circular dichroism spectroscopy. Front. Mol. Biosci. 9 (2022). 1.
Klinkenberg, L. G., Sutherland, L. A., Bishai, W. R. & Karakousis, P. C. Metronidazole lacks activity against Mycobacterium tuberculosis in an in vivo hypoxic granuloma model of latency. J. Infect. Dis. 198, 275–283 (2008).
Hunt, D. M. et al. Long-range transcriptional control of an operon necessary for virulence-critical ESX-1 secretion in Mycobacterium tuberculosis. J. Bacteriol. 194, 2307–2320 (2012).
Boradia, V., Frando, A. & Grundner, C. The Mycobacterium tuberculosis PE15/PPE20 complex transports calcium across the outer membrane. PLoS Biol. 20, e3001906 (2022).
Pang, X. et al. The β-propeller gene Rv1057 of Mycobacterium tuberculosis has a complex promoter directly regulated by both the MprAB and TrcRS two-component systems. Tuberculosis 91, S142–S149 (2011).
Wiegand, I., Hilpert, K. & Hancock, R. E. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 3, 163–175 (2008).
Tang, S. et al. Structural and functional insight into the Mycobacterium tuberculosis protein PrpR reveals a novel type of transcription factor. Nucleic Acids Res. 47, 9934–9949 (2019).
Kurthkoti, K. et al. The mycobacterial iron-dependent regulator IdeR induces ferritin (bfrB) by alleviating Lsr2 repression. Mol. Microbiol. 98, 864–877 (2015).
Savvi, S. et al. Functional characterization of a vitamin B12-dependent methylmalonyl pathway in Mycobacterium tuberculosis: implications for propionate metabolism during growth on fatty acids. J. Bacteriol. 190, 3886–3895 (2008).
Hancock, R. E. W. & Sahl, H.-G. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat. Biotechnol. 24, 1551–1557 (2006).
Thoma-Uszynski, S. et al. Induction of direct antimicrobial activity through mammalian toll-like receptors. Science 291, 1544–1547 (2001).
Collins, J. M., Porter, K. A., Singh, S. K. & Vanier, G. S. High-Efficiency Solid Phase Peptide Synthesis (HE-SPPS). Org. Lett. 16, 940–943 (2014).
Geberetsadik, G. et al. Lysocin E targeting menaquinone in the membrane of mycobacterium tuberculosis is a promising lead compound for antituberculosis drugs. Antimicrob. Agents Chemother. 66, e0017122 (2022).
Martin, A. et al. Multicenter study of MTT and resazurin assays for testing susceptibility to first-line anti-tuberculosis drugs. Int. J. Tuberc. Lung Dis. 9, 901–906 (2005).
Odds, F. C. Synergy, antagonism, and what the chequerboard puts between them. J. Antimicrob. Chemother. 52, 1 (2003).
Gengenbacher, M., Rao, S. P. S., Pethe, K. & Dick, T. Nutrient-starved, non-replicating Mycobacterium tuberculosis requires respiration, ATP synthase and isocitrate lyase for maintenance of ATP homeostasis and viability. Microbiology 156, 81–87 (2010).
Jeon, A. B. et al. 2-aminoimidazoles potentiate ß-lactam antimicrobial activity against Mycobacterium tuberculosis by reducing ß-lactamase secretion and increasing cell envelope permeability. PLoS One 12, e0180925 (2017).
Peterson, N. D., Rosen, B. C., Dillon, N. A. & Baughn, A. D. Uncoupling environmental pH and intrabacterial acidification from pyrazinamide susceptibility in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 59, 7320–7326 (2015).
Acknowledgements
We thank William R. Jacobs, Jr and Michael Berney of Albert Einstein College of Medicine for gifting the various mycobacterial strains used in this study. We also wish to thank the assistance of Julia Fecko at the X-ray Crystallography core at the Penn State Huck Institutes of the Life Sciences, Missy Hazen at the Penn State Materials Research Institute, and the UMN BioSafety Level 3 Program for facility management. Funding for this work was provided by NIH R01AI165996 to S.H.M., and NIH S10OD032215 and S10OD028589 to N. H. Y.
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Author notes
These authors contributed equally: Hugh D. Glossop, Gebremichal Gebretsadik.
Authors and Affiliations
Department of Biomedical Engineering, Pennsylvania State University, University Park, PA, USA
Hugh D. Glossop, Sabiha Sultana, Diptomit Biswas & Scott H. Medina 1.
Department of Microbiology and Immunology, The University of Minnesota Medical School, Minneapolis, MN, USA
Gebremichal Gebretsadik, Nathan A. Schacht, Muzafar Ahmad Rather & Anthony D. Baughn 1.
Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA
Diptomit Biswas, Neela H. Yennawar & Scott H. Medina
Authors
- Hugh D. Glossop
- Gebremichal Gebretsadik
- Sabiha Sultana
- Diptomit Biswas
- Nathan A. Schacht
- Neela H. Yennawar
- Muzafar Ahmad Rather
- Anthony D. Baughn
- Scott H. Medina
Contributions
H.D.G. and G.G. led compound synthesis, bacteriologic and biophysical assays, and supported manuscript writing/editing. S.S., D.B., and N.A.S. supported the synthetic and bacteriologic work, as well as data analysis and interpretation, and supported manuscript writing/editing. N.H.Y. performed biophysical ultracentrifugation studies and subsequent data analysis. M.A.R. led compound screening in clinical Mtb isolates. A.D.B. supported experimental design and result interpretation, led the supervision of work in BSL-3 facilities, and supported manuscript writing/editing. S.H.M. led the supervision of synthetic, biophysical and BSL-2 bacteriologic work, acquisition of resources, and manuscript writing.
Corresponding author
Correspondence to Scott H. Medina.
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Glossop, H.D., Gebretsadik, G., Sultana, S. et al. Retro-inversion imparts antimycobacterial specificity to host defense peptides. Nat Commun (2025). https://doi.org/10.1038/s41467-025-67162-0
Received: 25 April 2025
Accepted: 24 November 2025
Published: 07 December 2025
DOI: https://doi.org/10.1038/s41467-025-67162-0