ABSTRACT Novel classes of broad-spectrum antibiotics have been extremely difficult to discover, largely due to the impermeability of the Gram-negative membranes coupled with a poor

understanding of the physicochemical properties a compound should possess to promote its accumulation inside the cell. To address this challenge, numerous methodologies for assessing

intracellular compound accumulation in Gram-negative bacteria have been established, including classic radiometric and fluorescence-based methods. The recent development of accumulation

assays that utilize liquid chromatography–tandem mass spectrometry (LC-MS/MS) have circumvented the requirement for labeled compounds, enabling assessment of a substantially broader range of

developed a complementary web tool, eNTRyway, which calculates relevant properties and predicts compound accumulation. Here we provide a comprehensive protocol for analysis and prediction of

intracellular accumulation of small molecules in _E. coli_ using an LC-MS/MS-based assay (which takes ~2 d) and eNTRyway, a workflow that is readily adoptable by any microbiology,

biochemistry or chemical biology laboratory. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access

through your institution Access Nature and 54 other Nature Portfolio journals Get Nature+, our best-value online-access subscription $29.99 / 30 days cancel any time Learn more Subscribe to

this journal Receive 12 print issues and online access $259.00 per year only $21.58 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy

now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer

support SIMILAR CONTENT BEING VIEWED BY OTHERS COMBINING CRISPRI AND METABOLOMICS FOR FUNCTIONAL ANNOTATION OF COMPOUND LIBRARIES Article 22 February 2022 EXPANDING THE SEARCH FOR

SMALL-MOLECULE ANTIBACTERIALS BY MULTIDIMENSIONAL PROFILING Article 23 May 2022 NANORAPIDS AS AN ANALYTICAL PIPELINE FOR THE DISCOVERY OF NOVEL BIOACTIVE METABOLITES IN COMPLEX CULTURE

EXTRACTS AT THE NANOSCALE Article Open access 01 April 2024 DATA AVAILABILITY The main data discussed in this protocol are available in the supporting primary research papers

(https://doi.org/10.1038/nature22308 and https://doi.org/10.1038/s41564-019-0604-5). Source data are provided with this paper. Additional requests should be addressed to the corresponding

authors. CODE AVAILABILITY Source code for eNTRyway for local use is available on GitHub (https://github.com/HergenrotherLab/entry-cli). REFERENCES * Centers for Disease Control and

Prevention. Antibiotic Resistance Threats in the United States, 2019. https://www.cdc.gov/drugresistance/pdf/threats-report/2019-ar-threats-report-508.pdf (2019). * Tommasi, R., Brown, D.

G., Walkup, G. K., Manchester, J. I. & Miller, A. A. ESKAPEing the labyrinth of antibacterial discovery. _Nat. Rev. Drug. Discov._ 14, 529–542 (2015). Article  CAS  PubMed  Google

Scholar  * Richter, M. F. & Hergenrother, P. J. The challenge of converting Gram-positive-only compounds into broad-spectrum antibiotics. _Ann. NY Acad. Sci._ 1435, 18–38 (2019). Article

  CAS  PubMed  Google Scholar  * Rice, L. B. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. _J. Infect. Dis._ 197, 1079–1081 (2008). Article 

PubMed  Google Scholar  * Lewis, K. Platforms for antibiotic discovery. _Nat. Rev. Drug Discov._ 12, 371–387 (2013). Article  CAS  PubMed  Google Scholar  * Gladki, A., Kaczanowski, S.,

Szczesny, P. & Zielenkiewicz, P. The evolutionary rate of antibacterial drug targets. _BMC Bioinforma._ 14, 36–36 (2013). Article  Google Scholar  * Krishnamoorthy, G. et al. Synergy

between active efflux and outer membrane diffusion defines rules of antibiotic permeation into Gram-negative bacteria. _mBio_ 8, e01172–172017 (2017). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Krishnamoorthy, G. et al. Breaking the permeability barrier of _Escherichia coli_ by controlled hyperporination of the outer membrane. _Antimicrob. Agents Chemother._ 60,

7372–7381 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Bazile, S., Moreau, N., Bouzard, D. & Essiz, M. Relationships among antibacterial activity, inhibition of DNA

gyrase, and intracellular accumulation of 11 fluoroquinolones. _Antimicrob. Agents Chemother._ 36, 2622–2627 (1992). Article  CAS  PubMed  PubMed Central  Google Scholar  * Cai, H., Rose,

K., Liang, L. H., Dunham, S. & Stover, C. Development of a liquid chromatography/mass spectrometry-based drug accumulation assay in _Pseudomonas aeruginosa_. _Anal. Biochem._ 385,

321–325 (2009). Article  CAS  PubMed  Google Scholar  * Capobianco, J. O. & Goldman, R. C. Macrolide transport in _Escherichia coli_ strains having normal and altered OmpC and/or OmpF

porins. _Int. J. Antimicrob. Agents_ 4, 183–189 (1994). Article  CAS  PubMed  Google Scholar  * Chopra, I. Transport of tetracyclines into _Escherichia coli_ requires a carboxamide group at

the C2 position of the molecule. _J. Antimicrob. Chemother._ 18, 661–666 (1986). Article  CAS  PubMed  Google Scholar  * de Cristóbal, R. E., Vincent, P. A. & Salomón, R. A. Multidrug

resistance pump AcrAB-TolC is required for high-level, Tet(A)-mediated tetracycline resistance in _Escherichia coli_. _J. Antimicrob. Chemother._ 58, 31–36 (2006). Article  PubMed  Google

Scholar  * Li, X. Z., Livermore, D. M. & Nikaido, H. Role of efflux pump(s) in intrinsic resistance of _Pseudomonas aeruginosa_: resistance to tetracycline, chloramphenicol, and

norfloxacin. _Antimicrob. Agents Chemother._ 38, 1732–1741 (1994). Article  CAS  PubMed  PubMed Central  Google Scholar  * Piddock, L. J. V., Jin, Y.-F., Ricci, V. & Asuquo, A. E.

Quinolone accumulation by _Pseudomonas aeruginosa_, _Staphylococcus aureus_ and _Escherichia coli_. _J. Antimicrob. Chemother._ 43, 61–70 (1999). Article  CAS  PubMed  Google Scholar  *

Williams, K. J. & Piddock, L. J. Accumulation of rifampicin by _Escherichia coli_ and _Staphylococcus aureus_. _J. Antimicrob. Chemother._ 42, 597–603 (1998). Article  CAS  PubMed 

Google Scholar  * Richter, M. F. et al. Predictive compound accumulation rules yield a broad-spectrum antibiotic. _Nature_ 545, 299–304 (2017). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Motika, S. E. et al. A Gram-negative antibiotic active through inhibition of an essential riboswitch. _J. Am. Chem. Soc._ 142, 10856–10862 (2020). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Parker, E. N. et al. Implementation of permeation rules leads to a FabI inhibitor with activity against Gram-negative pathogens. _Nat. Microbiol._ 5, 67–75 (2020).

Article  CAS  PubMed  Google Scholar  * Li, Y. et al. First-generation structure-activity relationship studies of 2,3,4,9-tetrahydro-1H-carbazol-1-amines as CpxA phosphatase inhibitors.

_Bioorg. Med. Chem. Lett._ 29, 1836–1841 (2019). Article  PubMed  PubMed Central  CAS  Google Scholar  * Lukežič, T. et al. Engineering atypical tetracycline formation in _Amycolatopsis

sulphurea_ for the production of modified chelocardin antibiotics. _ACS Chem. Biol._ 14, 468–477 (2019). Article  PubMed  CAS  Google Scholar  * Masci, D. et al. Switching on the activity of

1,5-diaryl-pyrrole derivatives against drug-resistant ESKAPE bacteria: structure-activity relationships and mode of action studies. _Eur. J. Med. Chem._ 178, 500–514 (2019). Article  CAS 

PubMed  Google Scholar  * Hu, Y. et al. Discovery of pyrido[2,3-b]indole derivatives with gram-negative activity targeting both DNA gyrase and topoisomerase IV. _J. Med. Chem._ 63, 9623–9649

(2020). Article  CAS  PubMed  Google Scholar  * Andrews, L. D. et al. Optimization and mechanistic characterization of pyridopyrimidine inhibitors of bacterial biotin carboxylase. _J. Med.

Chem._ 62, 7489–7505 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Cohen, F. et al. Optimization of LpxC inhibitors for antibacterial activity and cardiovascular safety.

_ChemMedChem_ 14, 1560–1572 (2019). Article  CAS  PubMed  Google Scholar  * Perlmutter, S. J. et al. Compound uptake into _E. coli_ can be facilitated by _N_-alkyl guanidiniums and

pyridiniums. _ACS Infect. Dis._ 7, 162–173 (2021). Article  CAS  PubMed  Google Scholar  * Munoz, K. A. & Hergenrother, P. J. Facilitating compound entry as a means to discover

antibiotics for Gram-negative bacteria. _Acc. Chem. Res._ 54, 1322–1333 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * O’Shea, R. & Moser, H. E. Physicochemical

properties of antibacterial compounds: implications for drug discovery. _J. Med. Chem._ 51, 2871–2878 (2008). Article  PubMed  CAS  Google Scholar  * Thanassi, D. G., Suh, G. S. B. &

Nikaido, H. Role of outer membrane barrier in efflux-mediated tetracycline resistance in _Escherichia coli_. _J. Bacteriol._ 177, 998–1007 (1995). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Leive, L., Telesetsky, S., Coleman, W. G. J. & Carr, D. Tetracyclines of various hydrophobicities as a probe for permeability of _Escherichia coli_ outer membranes.

_Antimicrob. Agents Chemother._ 25, 539–544 (1984). Article  CAS  PubMed  PubMed Central  Google Scholar  * Lindley, E. V., Munske, G. R. & Magnuson, J. A. Kinetic analysis of

tetracycline accumulation by _Streptococcus faecalis_. _J. Bacteriol._ 158, 334–336 (1984). Article  CAS  PubMed  PubMed Central  Google Scholar  * Sigler, A., Schubert, P., Hillen, W. &

Niederweis, M. Permeation of tetracyclines through membranes of liposomes and _Escherichia coli_. _Eur. J. Biochem._ 267, 527–534 (2000). Article  CAS  PubMed  Google Scholar  * Vergalli,

J. et al. Spectrofluorimetric quantification of antibiotic drug concentration in bacterial cells for the characterization of translocation across bacterial membranes. _Nat. Protoc._ 13,

1348–1361 (2018). Article  CAS  PubMed  Google Scholar  * Cinquin, B. et al. Microspectrometric insights on the uptake of antibiotics at the single bacterial cell level. _Sci. Rep._ 5, 17968

(2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Masi, M. et al. Fluorescence enlightens RND pump activity and the intrabacterial concentration of antibiotics. _Res.

Microbiol._ 169, 432–441 (2018). Article  CAS  PubMed  Google Scholar  * Renggli, S., Keck, W., Jenal, U. & Ritz, D. Role of autofluorescence in flow cytometric analysis of _Escherichia

coli_ treated with bactericidal antibiotics. _J. Bacteriol._ 195, 4067–4073 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Six, D. A., Krucker, T. & Leeds, J. A.

Advances and challenges in bacterial compound accumulation assays for drug discovery. _Curr. Opin. Chem. Biol._ 44, 9–15 (2018). Article  CAS  PubMed  Google Scholar  * Davis, T. D., Gerry,

C. J. & Tan, D. S. General platform for systematic quantitative evaluation of small-molecule permeability in bacteria. _ACS Chem. Biol._ 9, 2535–2544 (2014). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Volkmer, B. & Heinemann, M. Condition-dependent cell volume and concentration of _Escherichia coli_ to facilitate data conversion for systems biology

modeling. _PLoS One_ 6, e23126 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Prochnow, H. et al. Subcellular quantification of uptake in Gram-negative bacteria. _Anal.

Chem._ 91, 1863–1872 (2019). Article  CAS  PubMed  Google Scholar  * Iyer, R. et al. Evaluating LC–MS/MS to measure accumulation of compounds within bacteria. _ACS Infect. Dis._ 4, 1336–1345

(2018). Article  CAS  PubMed  Google Scholar  * Smith, P. A. et al. Optimized arylomycins are a new class of Gram-negative antibiotics. _Nature_ 561, 189–194 (2018). Article  CAS  PubMed 

Google Scholar  * Rhomberg, P. R., Sader, H. S. & Jones, R. N. Reproducibility of daptomycin MIC results using dry-form commercial trays with appropriate supplemental calcium content.

_Int. J. Antimicrob. Agents_ 25, 274–275 (2005). Article  CAS  PubMed  Google Scholar  * O’Boyle, N. M. et al. Open Babel: an open chemical toolbox. _J. Cheminformatics_ 3, 33 (2011).

Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank M. Richter for optimization and development of the LC-MS/MS-based accumulation assay, and we thank B. Drown for

the creation of the web tool eNTRyway. This work was supported by the University of Illinois and the NIH (R01AI136773). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Roger Adams Laboratory,

Department of Chemistry, University of Illinois, Urbana, IL, USA Emily J. Geddes & Paul J. Hergenrother * Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana, IL,

USA Emily J. Geddes & Paul J. Hergenrother * Metabolomics Lab, Roy J. Carver Biotechnology Center, University of Illinois, Urbana, IL, USA Zhong Li Authors * Emily J. Geddes View author

publications You can also search for this author inPubMed Google Scholar * Zhong Li View author publications You can also search for this author inPubMed Google Scholar * Paul J.

Hergenrother View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS E.J.G. and Z.L. performed experiments. All authors wrote the manuscript and

were involved in editing of the final manuscript. CORRESPONDING AUTHOR Correspondence to Paul J. Hergenrother. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing

interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Protocols_ thanks Kim Lewis and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. RELATED LINKS KEY REFERENCES USING THIS PROTOCOL:

Richter, M. F. et al. _Nature_ 545, 299–304 (2017): https://doi.org/10.1038/nature22308 Parker, E. N. et al. _Nat. Microbiol_. 5, 67–75 (2020): https://doi.org/10.1038/s41564-019-0604-5

Motika, S. E. et al. _J. Am. Chem. Soc_. 142, 10856–10862 (2020): https://doi.org/10.1021/jacs.0c04427 EXTENDED DATA EXTENDED DATA FIG. 1 IMPORTANCE OF AMINE STERIC ACCESSIBILITY AND

AMPHIPHILIC MOMENT (VSURF_A). A, Primary amines demonstrate higher accumulation than the mono-methyl amine, di-methyl amine, tri-methyl amine and amide comparisons. Primary amines on primary

carbons also show improved accumulation over primary amines on secondary or tertiary carbons. B, Increasing amphiphilic moment trends with increasing accumulation. Accumulation is reported

in nmol/1012 CFUs. Data are taken from Richter et al.17 with permission. EXTENDED DATA FIG. 2 SCREENSHOT OF THE ‘INPUT’ BOX FOR SMILES STRINGS. SMILES strings are canonicalized using Open

Babel GUI. EXTENDED DATA FIG. 3 SCREENSHOTS OF THE PROCESS OF PREDICTING ACCUMULATION USING THE WEB TOOL ENTRYWAY. SMILES strings are submitted to eNTRyway, and compounds are prioritized for

evaluation based on how closely they meet the eNTRy rules. In the example here, both ampicillin and 6-DNM-NH3 meet all of the criteria and are predicted to accumulate, whereas penicillin

and DNM are not. A portion of this figure is taken from Parker et al.19 with permission. SUPPLEMENTARY INFORMATION REPORTING SUMMARY SOURCE DATA SOURCE DATA FIG. 4 Raw accumulation data.

SOURCE DATA FIG. 5 Calibration curve and mass spectrum. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Geddes, E.J., Li, Z. & Hergenrother, P.J. An

LC-MS/MS assay and complementary web-based tool to quantify and predict compound accumulation in _E. coli_. _Nat Protoc_ 16, 4833–4854 (2021). https://doi.org/10.1038/s41596-021-00598-y

Download citation * Received: 22 December 2020 * Accepted: 25 June 2021 * Published: 03 September 2021 * Issue Date: October 2021 * DOI: https://doi.org/10.1038/s41596-021-00598-y SHARE THIS

ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard

Provided by the Springer Nature SharedIt content-sharing initiative