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.
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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
Artikel in diesem Heft
- 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
Artikel in diesem Heft
- 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
