Recombinant Escherichia coli O81 Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase (arnC)

What is the precise function of arnC in Escherichia coli O81?

The arnC protein in E. coli O81 functions as an essential transferase enzyme (EC 2.4.2.53) that catalyzes the transfer of 4-deoxy-4-formamido-L-arabinose (Ara4FN) from UDP to undecaprenyl phosphate. This modification is a critical step in the Ara4FN-lipid A pathway. The modified arabinose is subsequently attached to lipid A, a component of the bacterial outer membrane. This enzymatic activity is part of a complex biochemical cascade that ultimately results in altered surface charge of the bacterial cell envelope, which reduces interactions with positively charged antimicrobial molecules . In experimental models, arnC-dependent lipid A modification has been shown to significantly increase bacterial survival rates in the presence of polymyxin antibiotics and various host-derived antimicrobial peptides.

How does the amino acid sequence of arnC relate to its functional domains?

The arnC protein consists of 322 amino acids (full length 1-322) with a specific sequence that can be divided into distinct functional domains . Analysis of the amino acid sequence (MFEIHPVKKVSVVIPVYNEQESLPELIRRTTAACESLGKEYEILLIDDGSSDNSAHMLVEASQAEGSHIVSILLNRNYGQHSAIMAGFSHVTGDLIITLDADLQNPPEEIPRLVAKADEGYDVVGTVRQNRQDSWFRKTASKMINRLIQRTTGKAMGDYGCMLRAYRRHIVDAMLHCHERSTFIPILANIFARRAIEIPVHHAEREFGESKYSFMRLINLMYDLVTCLTTTPLRMLSLLGSIIAIGGFSIAVLLVILRLTFGPQWAAEGVFMLFAVLFTFIGAQFIGMGLLGEYIGRIYTDVRARPRYFVQQVIRPSSKENE) reveals:

• An N-terminal catalytic domain (approximately residues 1-180) containing conserved motifs for nucleotide binding and glycosyltransferase activity

• A central hydrophobic region (approximately residues 180-250) involved in substrate recognition

• C-terminal membrane-associated domains (approximately residues 250-322) that facilitate interaction with the inner membrane where undecaprenyl phosphate is anchored

The protein structure includes multiple transmembrane segments that anchor it to the bacterial inner membrane, positioning the enzyme to efficiently access its membrane-associated substrate .

What evolutionary significance does arnC hold in bacterial antimicrobial resistance?

From an evolutionary perspective, arnC represents a fascinating example of bacterial adaptation to environmental pressures. The gene encoding this protein is part of the arnBCADTEF operon, which is highly conserved across many Gram-negative bacterial species. Phylogenetic analysis suggests that the acquisition and retention of functional arnC confers significant selective advantage in environments where cationic antimicrobial compounds are present.

The presence of arnC-mediated lipid A modifications appears to have evolved as a response to naturally occurring antimicrobial peptides in various ecological niches. This modification system predates human use of polymyxin antibiotics but has become increasingly relevant in clinical settings where these antibiotics are employed as last-resort treatments. Research indicates that upregulation of arnC expression occurs rapidly following exposure to sub-lethal concentrations of polymyxins, demonstrating the dynamic nature of this resistance mechanism .

What are the optimal conditions for expressing soluble recombinant arnC in E. coli expression systems?

Expressing soluble recombinant arnC presents significant challenges due to its membrane-associated nature and hydrophobic domains. Based on experimental data, the following conditions have been optimized for maximizing soluble expression:

Table 1: Optimized Conditions for Soluble arnC Expression

The addition of molecular chaperones (GroEL/GroES) via co-expression strategies has been demonstrated to increase soluble arnC yield by approximately 2.5-fold. Additionally, using the Lemo21(DE3) strain, which allows precise tuning of expression levels, can reduce inclusion body formation significantly compared to conventional BL21(DE3) .

How can researchers overcome inclusion body formation when expressing recombinant arnC?

Inclusion body formation is a common challenge when expressing arnC due to its hydrophobic domains and membrane association. Several strategies have proven effective in minimizing inclusion body formation:

• Expression Parameter Optimization:

  • Reduce expression temperature to 16°C

  • Lower inducer concentration to 0.1 mM IPTG

  • Use auto-induction media with gradual protein expression

• Genetic Modifications:

  • Fusion with solubility-enhancing tags (MBP or SUMO)

  • Co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

  • Use of specialized E. coli strains with enhanced folding capacity

• Media and Buffer Optimization:

  • Supplement with osmolytes (betaine, sorbitol)

  • Add low concentrations of non-denaturing detergents during expression

  • Include specific metal ions that stabilize protein folding

In cases where inclusion bodies still form despite these measures, protocols for refolding from inclusion bodies have been developed. These typically involve solubilization in 8M urea or 6M guanidine hydrochloride, followed by step-wise dialysis in the presence of appropriate detergents and lipids to facilitate proper folding .

What purification strategy yields the highest activity for recombinant His-tagged arnC?

A multi-step purification strategy has been developed that preserves arnC enzymatic activity while achieving >90% purity:

Step 1: Membrane Extraction

• Harvest cells and resuspend in buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol

• Add lysozyme (1 mg/mL) and DNase I (10 μg/mL)

• Lyse cells via sonication or French press

• Centrifuge at low speed to remove unbroken cells

• Ultracentrifuge supernatant (100,000 × g, 1 hour) to isolate membrane fraction

Step 2: Detergent Solubilization

• Resuspend membrane pellet in solubilization buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and 1% n-dodecyl-β-D-maltoside (DDM)

• Incubate with gentle rotation at 4°C for 2 hours

• Ultracentrifuge (100,000 × g, 30 minutes) to remove insoluble material

Step 3: IMAC Purification

• Load solubilized protein onto Ni-NTA resin equilibrated with buffer containing 50 mM Tris-HCl pH a8.0, 300 mM NaCl, 10% glycerol, 0.05% DDM

• Wash with increasing imidazole concentrations (20 mM, 40 mM)

• Elute protein with 250 mM imidazole

Step 4: Size Exclusion Chromatography

• Apply eluted protein to Superdex 200 column equilibrated with 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, 0.05% DDM

• Collect fractions corresponding to monomeric arnC

This protocol typically yields 2-5 mg of purified arnC per liter of bacterial culture with >90% purity as determined by SDS-PAGE . For long-term storage, the addition of 6% trehalose in the final buffer significantly improves protein stability during freeze-thaw cycles. The purified enzyme should be stored at -80°C in small aliquots to avoid repeated freeze-thaw cycles .

What are the established methods for assessing arnC enzymatic activity in vitro?

Several complementary approaches have been developed to measure arnC transferase activity:

• Radiometric Assay:
  This high-sensitivity method utilizes UDP-[14C]-4-deoxy-4-formamido-L-arabinose as substrate. The reaction mixture typically contains:

  • 50 mM HEPES buffer (pH 7.5)

  • 10 mM MgCl2

  • 0.5% DDM or another suitable detergent

  • 50-100 μM undecaprenyl phosphate (acceptor substrate)

  • 10-50 μM UDP-[14C]-4-deoxy-4-formamido-L-arabinose (donor substrate)

  • 0.1-1 μg purified arnC

  After incubation at 30°C for 15-30 minutes, the reaction is stopped with chloroform:methanol (2:1, v/v). The organic phase containing the radiolabeled undecaprenyl phosphate-Ara4FN is separated, washed, and quantified by liquid scintillation counting.

• HPLC-based Assay:
  This non-radioactive method monitors the formation of UDP as a reaction product:

  • Reaction components similar to the radiometric assay but with non-labeled substrates

  • After reaction termination, UDP is quantified by anion-exchange HPLC with UV detection at 262 nm

  • This method allows for kinetic parameter determination with detection limits in the low micromolar range

• Coupled Enzyme Assay:
  This continuous spectrophotometric method links UDP formation to NADH oxidation:

  • Standard reaction mixture supplemented with pyruvate kinase, lactate dehydrogenase, phosphoenolpyruvate, and NADH

  • UDP production is coupled to NADH oxidation, measured at 340 nm

  • Allows real-time monitoring of reaction kinetics

Each method offers distinct advantages depending on the specific research question. The radiometric assay provides highest sensitivity, the HPLC method offers direct product analysis, while the coupled assay enables continuous monitoring of reaction kinetics .

How do structural modifications of arnC impact its catalytic efficiency?

Structure-function studies have revealed several regions critical for arnC catalytic activity:

Table 2: Impact of Key Residues on arnC Function

Deletion studies further indicate that removal of the C-terminal transmembrane region (residues 280-322) results in a soluble protein variant that retains approximately 30% of wild-type activity when assayed in the presence of appropriate detergents. This truncated form may be valuable for structural studies where membrane proteins present significant challenges .

What is the relationship between arnC activity and polymyxin resistance in bacteria?

The relationship between arnC enzymatic activity and polymyxin resistance has been established through multiple complementary approaches:

• Genetic Correlation Studies:

  • Deletion of arnC in E. coli results in 16-32 fold reduction in polymyxin MIC values

  • Complementation with functional arnC restores resistance

  • Point mutations affecting catalytic activity show proportional effects on resistance levels

• Biochemical Correlation:
  Quantitative analysis reveals a strong positive correlation (r = 0.89) between:

  • In vitro transferase activity of arnC variants

  • Levels of Ara4FN-modified lipid A in cellular membranes (measured by mass spectrometry)

  • Polymyxin B minimum inhibitory concentrations (MICs)

• Direct Measurement of Membrane Modification:
  Mass spectrometric analysis of lipid A from strains with varying levels of arnC expression shows:

  • Wild-type E. coli under PmrA/PmrB-activating conditions: 60-85% Ara4FN-modified lipid A

  • arnC deletion mutants: <5% modified lipid A

  • arnC overexpression strains: >90% modified lipid A

These findings collectively demonstrate that arnC activity is rate-limiting in the Ara4FN-modification pathway and directly proportional to the level of polymyxin resistance. The data suggest that inhibition of arnC could potentially restore polymyxin sensitivity in resistant bacteria, making it an attractive target for adjuvant therapy development .

How can arnC be utilized as a target for developing novel antimicrobial adjuvants?

The critical role of arnC in polymyxin resistance makes it an attractive target for developing adjuvant compounds that could restore sensitivity to these last-resort antibiotics. Several approaches have shown promise in research settings:

• Structure-Based Inhibitor Design:
  Using homology models and molecular docking approaches, several classes of potential inhibitors have been identified:

  • UDP-Ara4FN analogs that compete for the donor substrate binding site

  • Undecaprenyl phosphate mimetics that block acceptor substrate binding

  • Allosteric inhibitors that stabilize inactive conformations

• High-Throughput Screening Approaches:
  Assay systems have been developed for screening compound libraries:

  • Fluorescence-based assays using FRET-labeled substrates

  • Cell-based reporter systems that couple arnC inhibition to fluorescent protein expression

  • Phenotypic screens measuring polymyxin sensitivity restoration

• Peptide-Based Inhibitors:
  Peptides derived from interacting regions of arnC and related proteins have shown inhibitory potential:

  • Peptides corresponding to the interaction interface between arnC and other arn pathway proteins

  • Transmembrane peptides that disrupt proper membrane localization of arnC

Preliminary data indicate that compounds inhibiting arnC with IC50 values in the low micromolar range can reduce polymyxin MICs by 4-8 fold in resistant E. coli strains. This suggests significant potential for clinical application, particularly as these compounds would not be directly bactericidal but would rather restore effectiveness of existing antibiotics .

What techniques are most effective for studying arnC-substrate interactions?

Understanding arnC-substrate interactions requires specialized techniques due to the membrane-associated nature of the enzyme and its lipid substrates:

• Surface Plasmon Resonance (SPR) with Lipid Capture:

  • Immobilization of His-tagged arnC on Ni-NTA sensor chips

  • Flowing undecaprenyl phosphate incorporated into nanodiscs or liposomes

  • Real-time binding analysis providing association/dissociation kinetics

  • Typical KD values for undecaprenyl phosphate: 2-5 μM

• Microscale Thermophoresis (MST):

  • Fluorescently labeled arnC mixed with varying concentrations of detergent-solubilized substrates

  • Detection of binding through changes in thermal migration behavior

  • Advantage of low sample consumption and compatibility with detergent systems

  • Useful for comparing binding affinities of substrate analogs

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Monitors substrate-induced changes in protein dynamics and solvent accessibility

  • Identifies specific regions involved in substrate binding

  • Studies reveal that UDP-Ara4FN binding causes significant protection in the N-terminal domain (residues 25-80)

  • Undecaprenyl phosphate binding primarily affects regions in the C-terminal domain (residues 240-290)

• Saturation Transfer Difference NMR (STD-NMR):

  • Non-destructive technique for mapping substrate binding epitopes

  • Identifies specific substrate moieties in close contact with the protein

  • Studies indicate that both the sugar and nucleotide portions of UDP-Ara4FN interact with arnC

These complementary approaches have revealed that arnC follows an ordered binding mechanism where UDP-Ara4FN binding induces conformational changes that enhance affinity for undecaprenyl phosphate .

How does arnC interact with other proteins in the Ara4FN modification pathway?

The Ara4FN modification pathway involves multiple proteins encoded by the arnBCADTEF operon. ArnC functions within a complex enzymatic cascade, with specific protein-protein interactions that enhance pathway efficiency:

• Interaction with ArnA (Bifunctional deformylase/formyltransferase):

  • Co-immunoprecipitation studies demonstrate direct interaction

  • Bacterial two-hybrid assays map interaction to the C-terminal domain of ArnA and N-terminal domain of ArnC

  • Functional significance: Facilitates channeling of UDP-Ara4FN from ArnA to ArnC

  • Interaction is enhanced under low pH conditions that activate the pathway

• Interaction with ArnT (Transferase that attaches Ara4FN to lipid A):

  • Fluorescence resonance energy transfer (FRET) experiments indicate proximity in cell membranes

  • Cross-linking studies capture transient interactions

  • Functional significance: Enables efficient transfer of undecaprenyl phosphate-Ara4FN from ArnC to ArnT

  • Interaction depends on presence of both enzymes' substrates

• Complex Formation with Other Pathway Components:

  • Blue native PAGE and size exclusion chromatography suggest formation of a multi-protein complex

  • Complex includes ArnC, ArnA, ArnB, and ArnT

  • Estimated molecular weight of complex: 220-250 kDa

  • Complex formation enhanced under pathway-inducing conditions (low Mg2+, presence of antimicrobial peptides)

These interactions suggest a highly coordinated enzyme system that functions as a metabolon, increasing pathway efficiency through substrate channeling and localized concentration of enzymatic activities. Disrupting these protein-protein interactions represents an alternative strategy for targeting the Ara4FN modification pathway .

How can researchers address solubility and stability issues with recombinant arnC?

Researchers frequently encounter solubility and stability challenges when working with recombinant arnC. The following strategies have proven effective in addressing these issues:

For Improving Initial Solubility:

• Optimization of Lysis Conditions:

  • Use specialized lysis buffers containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, and 1% DDM

  • Add 5 mM β-mercaptoethanol or 1 mM DTT to prevent oxidation of cysteine residues

  • Include protease inhibitor cocktail to prevent degradation during extraction

• Detergent Screening:
  Systematic testing of different detergents has revealed effectiveness in this order:

  • DDM (n-dodecyl-β-D-maltoside): Most effective, preserves activity

  • LMNG (lauryl maltose neopentyl glycol): Good alternative, stabilizes during purification

  • CHAPS: Moderate effectiveness, may be preferable for certain applications

  • Triton X-100: Less effective, may affect activity

• Fusion Tag Selection:

  • MBP (maltose-binding protein) fusion: Increases solubility by ~3-fold compared to His-tag alone

  • SUMO fusion: Improves folding and can be removed with specific proteases

  • Thioredoxin fusion: Helps maintain proper disulfide bond formation

For Enhancing Long-term Stability:

• Storage Buffer Optimization:

  • Addition of 6% trehalose significantly improves freeze-thaw stability

  • pH maintenance between 7.5-8.0 is critical for enzyme stability

  • Inclusion of 10% glycerol reduces protein aggregation during freezing

• Strategic Mutagenesis:

  • Identification and mutation of surface-exposed hydrophobic residues that contribute to aggregation

  • Introduction of disulfide bonds in relevant positions to stabilize tertiary structure

  • Removal of unnecessary flexible loops that are prone to proteolysis

• Storage Recommendations:

  • Store at -80°C in small aliquots (50-100 μL) to minimize freeze-thaw cycles

  • For working stocks, maintain at 4°C with 0.02% sodium azide for up to 1 week

  • Avoid repeated freeze-thaw cycles which reduce activity by approximately 15-20% per cycle

These approaches can increase the working lifetime of purified arnC preparations by 3-5 fold compared to standard methods, enabling more consistent and reproducible experimental results.

What are common pitfalls in arnC activity assays and how can they be avoided?

Several common pitfalls can compromise the accuracy and reproducibility of arnC activity assays:

• Substrate Availability Issues:

  • Pitfall: Limited commercial availability of UDP-Ara4FN and undecaprenyl phosphate

  • Solution: Establish in-house synthesis protocols using ArnA and ArnB enzymes to generate UDP-Ara4FN; use shorter-chain (C55) analogs of undecaprenyl phosphate (C95) for routine assays

• Detergent Interference:

  • Pitfall: Excessive detergent can form mixed micelles that sequester substrates

  • Solution: Carefully optimize detergent concentration; maintain detergent above CMC but below levels that interfere with substrate accessibility (typically 0.03-0.05% for DDM)

• Metal Ion Dependence:

  • Pitfall: Inconsistent results due to variable metal content in buffers

  • Solution: Include 5-10 mM MgCl2 in all reaction buffers; treat buffers with Chelex resin to remove trace metals before adding back defined concentrations

• Product Detection Challenges:

  • Pitfall: Difficulty in detecting undecaprenyl phosphate-Ara4FN due to hydrophobicity

  • Solution: Use thin-layer chromatography with phosphorimaging for radioactive assays; employ LC-MS/MS with appropriate HILIC columns for non-radioactive detection

• Enzyme Instability:

  • Pitfall: Rapid activity loss during assay setup and incubation

  • Solution: Keep enzyme on ice until immediately before assay; include stabilizing agents like glycerol (10%) and BSA (0.1 mg/mL) in reaction mixtures

Standardization Protocol for Reliable Activity Measurements:

To ensure consistency across experiments, a standardized activity assay has been developed:

Table 3: Standardized arnC Activity Assay Components

This standardized approach typically yields coefficient of variation below 10% across multiple batches and operators .

How can researchers effectively study arnC in the context of bacterial antimicrobial resistance mechanisms?

Studying arnC in the context of bacterial antimicrobial resistance requires specialized approaches that bridge biochemistry, molecular biology, and microbiology:

• Genetic Manipulation Strategies:

  • Controlled expression systems: Use of inducible promoters (PBAD, Ptet) to titrate arnC expression levels

  • Chromosomal tagging approaches: Addition of epitope or fluorescent tags at the C-terminus preserves function while enabling localization and interaction studies

  • CRISPR-Cas9 genome editing: Precise introduction of point mutations to assess structure-function relationships

• Physiological Relevance Assessment:

  • MIC determination under varying conditions: Test polymyxin resistance with standard broth microdilution assays supplemented with pathway activators (low Mg2+, high Fe3+)

  • Combination with efflux pump inhibitors: Distinguish arnC-mediated resistance from efflux-based mechanisms

  • Competition assays: Measure fitness costs of arnC mutations in mixed cultures with and without antimicrobial pressure

• Pathway Integration Analysis:

  • RNA-seq to identify co-regulated genes under different stress conditions

  • Phosphoproteomics to map signaling pathways controlling arnC expression

  • Metabolomics approaches to track flux through the Ara4FN-modification pathway

• Translational Applications:

  • Development of reporter strains with arnC promoter fused to luciferase or fluorescent proteins

  • High-throughput screening systems to identify inhibitors of the pathway

  • Animal infection models to validate the role of arnC in in vivo resistance

Case Study Approach for Comprehensive Analysis:

A particularly effective approach involves parallel analysis of clinical isolates with different resistance phenotypes:

• Characterize polymyxin MICs and perform whole genome sequencing

• Quantify arnC expression levels by qRT-PCR and western blotting

• Analyze lipid A modifications by mass spectrometry

• Complement susceptible strains with functional arnC and test for restored resistance

• Purify arnC from resistant isolates and compare enzymatic properties with reference strains

This integrated approach has revealed that polymyxin-resistant clinical isolates frequently contain mutations in regulatory pathways (PmrAB, PhoPQ) that result in constitutive arnC expression, rather than mutations in arnC itself. This suggests that targeting regulatory systems may be an alternative approach to overcoming this resistance mechanism .

What are emerging technologies that could advance arnC structure-function studies?

Several cutting-edge technologies show promise for deepening our understanding of arnC structure and function:

• Cryo-Electron Microscopy (Cryo-EM):
  Recent advances in single-particle cryo-EM now enable structure determination of membrane proteins without crystallization. This approach could:

  • Resolve the full-length arnC structure, including transmembrane domains

  • Visualize arnC in complex with its substrates and other pathway proteins

  • Capture different conformational states during the catalytic cycle

• Native Mass Spectrometry:
  This emerging technique preserves non-covalent interactions during ionization and can:

  • Determine stoichiometry of arnC complexes with interacting partners

  • Identify binding modes of substrates and inhibitors

  • Monitor conformational changes upon substrate binding

• Advanced Computational Methods:

  • AlphaFold2 and RoseTTAFold can predict arnC structure with increasing accuracy

  • Molecular dynamics simulations with specialized membrane protein force fields can model substrate interactions

  • Machine learning approaches can predict functional effects of mutations

• In-cell NMR Spectroscopy:
  This technique allows observation of proteins in their natural cellular environment:

  • Monitor arnC dynamics and interactions within intact bacterial cells

  • Assess structural changes in response to environmental conditions that activate the pathway

  • Validate structural insights from in vitro studies

• Single-Molecule Studies:

  • Single-molecule FRET to track conformational changes during catalysis

  • Optical tweezers to measure forces involved in substrate binding

  • Super-resolution microscopy to visualize arnC localization and clustering in bacterial membranes

These technologies, especially when used in combination, have the potential to transform our understanding of arnC function and guide the development of targeted inhibitors with therapeutic potential.

How might arnC research contribute to understanding broader mechanisms of bacterial adaptation?

Research on arnC has implications that extend beyond antibiotic resistance to broader aspects of bacterial adaptation:

• Membrane Remodeling as Stress Response:
  The arnC-mediated pathway represents a model system for studying how bacteria dynamically remodel their outer membrane in response to environmental challenges. This pathway illustrates:

  • Integration of multiple stress signals (pH, divalent cations, antimicrobial peptides)

  • Coordinated transcriptional and post-translational regulation

  • Energetic costs and fitness trade-offs of membrane modifications

• Lipid-Protein Interactions in Bacterial Membranes:
  Studies of arnC and its interaction with lipid substrates provide insights into:

  • How integral membrane proteins recognize specific lipids

  • The role of lipid microdomains in organizing bacterial membrane processes

  • Mechanisms of protein-facilitated lipid transport between membrane leaflets

• Evolution of Resistance Mechanisms:
  Comparative genomics approaches examining arnC across bacterial species reveal:

  • Conservation patterns suggesting functional constraints

  • Evidence of horizontal gene transfer events spreading resistance mechanisms

  • Adaptive mutations in response to clinical use of polymyxins

• Host-Pathogen Interactions:
  The arnC pathway's role in resistance to host antimicrobial peptides illuminates:

  • Bacterial strategies for evading innate immune defenses

  • Co-evolutionary dynamics between host defense peptides and bacterial resistance mechanisms

  • Potential trade-offs between resistance and virulence

By exploring these broader implications, arnC research contributes to fundamental understanding of bacterial physiology while also informing practical approaches to combating antimicrobial resistance.

What interdisciplinary approaches might accelerate development of arnC-targeted therapeutics?

Developing therapeutics targeting arnC requires integration of multiple scientific disciplines:

• Structural Biology and Medicinal Chemistry:

  • Structure-based design of small molecule inhibitors targeting catalytic site or allosteric sites

  • Fragment-based drug discovery approaches to identify initial hit compounds

  • Structure-activity relationship studies to optimize potency and specificity

• Synthetic Biology and Enzyme Engineering:

  • Development of enzymatic assays suitable for high-throughput screening

  • Creation of bacterial biosensors that report on arnC inhibition in vivo

  • Engineering of pathway enzymes to produce needed substrates for inhibitor testing

• Nanomedicine and Drug Delivery:

  • Design of nanoparticle systems to deliver hydrophobic inhibitors across the bacterial outer membrane

  • Development of polymyxin-inhibitor conjugates for targeted delivery

  • Creation of prodrug approaches that leverage bacterial enzymes for activation

• Systems Biology and Network Pharmacology:

  • Identification of synergistic drug combinations that enhance arnC inhibitor efficacy

  • Mapping of resistance evolution pathways to anticipate and counter resistance to new inhibitors

  • Understanding of pathway cross-talk to identify vulnerable nodes for intervention

• Translational Research and Clinical Development:

  • Optimization of lead compounds for pharmacokinetic properties and safety

  • Development of diagnostic tools to identify patients with polymyxin-resistant infections

  • Design of clinical trials specifically targeting infections with documented resistance mechanisms

This interdisciplinary approach has already yielded promising early-stage inhibitors with IC50 values in the low micromolar range. Several of these compounds show synergistic effects when combined with polymyxin antibiotics, reducing the effective concentration needed for bacterial killing by 8-16 fold .