Targeting the zinc-dependent mobilized colistin resistance metalloenzyme to combat clinical resistance towards colistin and β-lactam antibiotics

Antibiotic resistance is an urgent global public health crisis, projected to cause about 50 million annual fatalities by 2050. Bacterial enzymes play a crucial role in promoting this phenomenon, complicating efforts to combat fatal infections. The plasmid-localized mcr-1 gene encodes an enzyme that confers resistance to colistin, a last-resort antibiotic used to treat deadly multidrug-resistant Enterobacteriaceae infections. MCR-1 confers resistance by covalently modifying lipopolysaccharides (LPS) with a positively charged phosphoethanolamine (PEA) moiety, derived from phosphatidylethanolamine (PE). This transformation neutralizes the membrane charge, preventing binding by colistin, a cationic polypeptide antibiotic. MCR-1 catalysis requires two Zn2+ ions as cofactors; one facilitating cleavage of PEA in the first step, and the other involved in its conjugation onto lipid A in the second step. This work sought to identify MCR-1 inhibitors to restore colistin activity against Gram-negative pathogens. Computer-aided virtual screening was used to identify small molecules predicted to bind with high affinity to the substrate- or cofactor-binding regions. Cell-based screens against MCR-1-expressing Escherichia coli cells revealed no independent antibacterial activity by the test compounds. Combinations of colistin with these compounds identified a promising indol-benzohydrazide derivative, JNAL-042, which displayed strong antibiotic-adjuvant properties. This compound restored colistin’s activity in resistant cells expressing MCR-1. Fluorescence-based time-kill reporter assays displayed no MCR-1-mediated resistance to colistin 7 hours post-treatment in the presence of JNAL-042. Computational docking predicted binding of JNAL-042 near the lipid A-binding site, suggesting potential interference with the second catalytic step, involving the Zn2+-dependent transfer of PEA onto LPS. Preliminary in vitro experiments with purified MCR-1 supported this hypothesis, demonstrating that JNAL-042 did not inhibit PE cleavage. Ongoing experiments seek to validate MCR-1 as the biological target of JNAL-042 and elucidate the molecular mechanism of enzyme inhibition using site-directed mutagenesis. Collectively, our findings offer a promising strategy employing antibiotic adjuvants to tackle MCR-1-mediated resistance and secure the clinical use of colistin as a last-resort antibiotic against multidrug-resistant pathogens.

Introduction and Goal

Diagram of the mechanism of colistin action on MCR-1 and the mechanism of MCR-1-mediated colistin resistance

▪Antibiotic resistance is an urgent threat to human health globally. It is estimated that by 2050, drug-resistant microbes will lead to over 50 million deaths per year if this problem is not resolved.

▪In some cases, antibiotic resistance is mediated by enzymes that degrade an antibiotic or chemically modify its cellular target. Genes for these enzymes are located on mobile genetic elements that are easily shared between bacterial cells through horizontal gene transfer.

▪Colistin is a last-resort antibiotic reserved for treating of multi-drug-resistant Gram-negative bacterial infections.

▪mcr-1 is a gene located on a naturally occurring plasmid, originally discovered in Escherichia coli, that has spread to several other species within the Enterobacteriaceae family.

▪The mcr-1 gene encodes an enzyme (MCR-1) that covalently modifies lipid A, a component of lipopolysaccharides and colistin’s target in the cell, rendering colistin unable to bind to and lyse Gram-negative bacterial membranes.

▪MCR-1-M6 is one of multiple recently discovered naturally-occurring variants of MCR-1 that confers resistance to both colistin and β-lactam class antibiotics, including ampicillin, penicillin, and cefoxitin, making potential infections extremely difficult to treat.

▪The goal of this project is to characterize MCR-1-M6 and other MCR-1 variants to better understand the mechanism by which it confers resistance to β-lactam antibiotics, and to identify compounds that can inhibit this enzyme’s activity to resensitize bacteria to colistin and β-lactam antibiotics

Methods and Results

Graph showing MIC values for six MCR-1 variants

Antibiotic susceptibility screens confirm resistance to colistin and cefoxitin mediated by MCR-1 M6

Methods:

▪Cloned variants of mcr-1 gene into pMyBADC-kan vectors and transformed plasmids into E. coli BW25113 ΔtolC for cell-based antibiotic susceptibility assays

▪Performed minimum inhibitory concentration (MIC) assays using E. coli BW25113 ΔtolC cells supplemented with 1% arabinose to express various MCR-1 variants and a vector control against colistin concentrations ranging from 0.005 μg/mL to 10 μg/mL

Results:

▪E. coli cells expressing MCR-1 variants M2, M3, M4, and M6 demonstrated a 5-8 fold increase in MIC relative to the vector control, indicating resistance to colistin

▪E. coli cells overexpressing a MCR-1 triple mutant variant (L165P, P188A, and P195S) demonstrated lower resistance than other mutants.

Plot showing cefoxitin MICs for MCR-1 M6 and VC from 1 to 10 ug/mL.

Methods:

▪Determined MICs using the agar dilution method with E. coli WT BW25113 cells cultured in media containing 1% arabinose for the expression of MCR-1-M6

▪Compared MIC of cefoxitin to a vector control strain lacking the mcr-1-M6 gene

Results:

▪WT E. coli BW25113 expressing MCR-1-M6 displayed an increase in colony forming units (CFU/mL) compared to vector controls, signifying resistance at higher doses of cefoxitin

Potential inhibitors for MCR-1 M6 identified through computer-aided virtual screening

Methods:

▪Evaluated a phenotypic biodiversity library and natural product library (Life Chemicals) against MCR-1 (PDB: 9NWW) using Autodock Vina

▪Screened docked compounds for drug-like properties using SwissADME computation software

Results:

▪Identified compound JNAL-42 as a promising inhibitor predicted to bind with high affinity (-9.7 kcalmol-1) near LPS binding site and Zn2+ coordination center

Plot of checkerboard assay for colistin and JNAL-42.

JNAL-42 restores colistin activity in MCR-1 variant expressing strains

Methods:

▪Performed combinatorial checkerboard assay using colistin (starting concentration 4 μg/mL) and JNAL-42 (starting concentration 48.1 μg/mL) using E. coli BW25113 ΔtolC cells expressing MCR-1 M6

Results:

▪JNAL-42 restores colistin activity at concentrations as low as 12 μg/mL

plot of colistin MICs with or without JNAL-42 for MCR-1 M6

Methods:

▪Performed antibiotic susceptibility assays using E. coli BW25113 ΔtolC cells and wildtype E. coli BW25113 cells expressing MCR-1 variants and a vector control. Assessed colistin concentrations ranging from 0.005 μg/mL to 10 μg/mL, (±) 24 μg/mL JNAL-42 (INH)

Results:

▪JNAL-42 reverts resistance, restoring colistin activity in efflux deficient at WT cells expressing MCR-1 M6 or MCR-1 L165P

Time kill assays for colistin using VC and MCR-1 M6 with and without JNAL42

Method:

▪Conducted time-kill Assays to assess colistin activity with E. coli BW25113 ΔtolC cells ± MCR-1 M6 (± JNAL-42)

Results:

▪MCR-1 M6-expressing cells demonstrate ~4-fold increase in colistin resistance compared to vector controls over 7-hour growth period

▪JNAL-42 (24 µg/mL) restores colistin activity in MCR-1 M6 expressing cells to levels seen in vector controls

Bar plot of cefoxitin MICs for VC and M6 with or without inhibitor.

Method:

▪Conducted antibiotic susceptibility assays using the agar dilution method with E. coli WT BW25113 cells expressing MCR-1-M6 and vector controls with cefoxitin ± JNAL-42 (24 µg/mL)

Results:

▪JNAL-42 (24 µg/mL) reverts MCR-1 M6-mediated resistance against cefoxitin in WT E. coli cells

Bar graph showing substrate and product ratios for different TLC assays.

JNAL-42 does not inhibit MCR-1-M6-mediated PEA cleavage

Method:

▪Purified MCR-1 M6 protein from induced E. coli BL21 (DE3) under native conditions using a Ni-NTA column through Fast Protein Liquid Chromatography

▪Incubated purified full-length MCR-1 M6 with NBD-glycerol-3-PEA, a fluorescently labeled PEA donor, LPS, and Zn2+ +/- JNAL-42 for 16 hours at room temperature, and varied times at 37 C

▪Performed thin-layer chromatography (TLC) to distinguish between remaining NBD-glycerol-3-PEA and NBD-glycerol, a product of generated by MCR-1 mediated cleavage

Results:

▪TLC shows products of MCR-1 M6 catalytic activity towards NBD-glycerol-3-PEA with or without JNAL-42

Mechanism schematic of MCR-1 M6 activity assay.

Conclusions

▪E. coli cells expressing MCR-1 variants demonstrate a 5-8 fold increase in colistin resistance and 2 fold increase in cefoxitin resistance compared to vector controls

▪The identified inhibitor JNAL-42 restores colistin and cefoxitin activity in MCR-1 M6 expressing strains

▪JNAL-42 is predicted to bind in a location potentially interfering with lipid A binding and Zn2+ coordination

▪JNAL-42 competes with LPS for MCR-1 M6 binding, confirming interference with lipid A binding

▪JNAL-42 does not inhibit MCR-1-mediated PEA cleavage, and likely interferes with the lipid A modification step

Future Study

Determination of syngery between JNAL-42 and β-lactam antibiotics

▪Perform checkerboard assays combining JNAL-42 and cefoxitin to determine if JNAL-42 restores β-lactam activity

▪Perform time-kill assays confirming synergy of JNAL-42 and β-lactam antibiotics

In-vitro assays to determine mode of MCR-1 M6 inhibition by JNAL-42

▪Perform binding competition assays using purified MCR-1 M6, lipid A, and JNAL-42 along with kinetics assays to elucidate mechanism of inhibition by JNAL-42

Validation of MCR-1 as biological target of JNAL-42

▪Utilize site-directed mutagenesis to develop MCR-1 M6 mutants with mutations in residues predicted to bind to JNAL-42, and use cell-based antibiotic susceptibility assays and in vitro binding assays to determine whether JNAL-42 activity is disrupted

▪Design analogue of JNAL-42 that reacts using photo-affinity labeling and click chemistry to serve as probe to validate MCR-1 as biological target of JNAL-42

The following is an image of poster presented at the 2026 Undergraduate Research Forum

Research poster presented at 2026 Undergraduate Research Forum (same content as website).

Acknowledgements

▪This research was supported by The U.S. National Science Foundation under Grant No. CHE-1851795 and Miami University Start-Up Funds to JNA

▪Additional support from the Office of Research for Undergraduates (URA) and Department of Chemistry and Biochemistry (NSF-REU Program)

▪Special thanks to Ruth Ndupu, Madelyn Baranowsky, Taylor Barber, and Morgan Price

References

Liang L, et al. 2023. PLoS Biol.
Xu Y, et al. 2018. MBio
Zhang Q, et al. 2022. PNAS
Wei P, et al. 2018. FASEB J
Wang R, et al. 2018. Nat. Commun.
Schematics created with BioRender.com

NACE Career Readiness Competencies

1. Critical Thinking

This research project involved many stages of failures and trouble-shooting. Using setbacks as a way to gain new information and create a new plan of actionhas strengthened my abilities to assess situations and seek out new alternatives to solve problems.

2. Leadership

Working on an independent leadership project has allowed me to greatly develop my leadership skills. Planning out my timelines by semester and planning and conducting experiments independently has helped me develop strong time management and organizational skills, as well as a sense of commitment and personal accountability to my project.

3. Communication

This research project has allowed me to better understand how to effectively and professionally communicate my research findings both written and verbally. Creating this poster for the URF as well as presenting my research in a variety of settings has allowed me to understand how I can communicate complicated scientific concepts from my research to all audiences.

Research Compliance Protocols

Research Compliance Protocols Used

Citi Training courses, including lab chemical safety, OSHA bloodborne pathogens, the initial biosafety training course, and basic introduction to biosafety. Biosafety level 2 protocol IBC#: Miami002_Alumasa_2023_12_12.

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