Recombinant Rana grahami Grahamin-1

What is Grahamin-1 and what is its biological origin?

Grahamin-1 is an antimicrobial peptide originally isolated from the skin secretions of Rana grahami (Yunnanfu frog, also classified as Odorrana grahami). It is one of two related antimicrobial peptides discovered in this species, with Grahamin-1 having the primary structure GLLSGILGAGKHIVCGLSGLC as determined through Edman degradation and mass spectrometry techniques . These peptides are part of the amphibian innate immune system, produced in specialized granular glands of the skin, and demonstrate broad-spectrum antimicrobial activity against various microorganisms .

What is the molecular structure of Grahamin-1?

Grahamin-1 is a 21-amino acid peptide with the sequence GLLSGILGAGKHIVCGLSGLC . It contains a characteristic C-terminal loop region delineated by an intra-disulfide bridge, commonly referred to as the "Rana box" . This structural feature is conserved among many antimicrobial peptides isolated from Rana species. The peptide can be classified into the family of antimicrobial peptides containing a single intra-disulfide bridge, which is critical for its biological activity . When studying recombinant Grahamin-1, researchers typically express the 46-66 region of the precursor protein .

What are the optimal storage conditions for recombinant Grahamin-1?

For short-term storage, recombinant Grahamin-1 should be stored at -20°C. For extended storage and maximum stability, conservation at -80°C is recommended . The peptide should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Addition of glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) before aliquoting is advised for long-term storage at -20°C/-80°C . Repeated freezing and thawing cycles should be avoided as they may compromise protein integrity. Working aliquots can be stored at 4°C for up to one week . The shelf life of the liquid form is typically 6 months at -20°C/-80°C, while the lyophilized form maintains stability for approximately 12 months at the same temperature ranges .

What analytical techniques are used to characterize Grahamin-1 structure?

The structural characterization of Grahamin-1 employs multiple complementary techniques:

• Edman Degradation: Used for primary sequence determination through sequential removal and identification of N-terminal amino acids .

• Mass Spectrometry: Provides precise molecular weight verification and can identify post-translational modifications. This technique complements Edman degradation for complete sequence validation .

• cDNA Cloning and Sequencing: Molecular cloning of the precursor gene from skin cDNA libraries allows deduction of the complete peptide sequence, including signal peptides and pro-regions .

• Circular Dichroism (CD) Spectroscopy: Similar to the analysis of related frog peptides like Temporin-Lb, CD can be employed to determine the secondary structure properties of Grahamin-1 in different environments .

• Disulfide Bridge Analysis: Specific techniques to verify the presence and correct formation of the characteristic intra-disulfide bridge (Rana box) that is critical for biological activity .

For comprehensive characterization, researchers typically integrate these methods, comparing results from Edman degradation with amino acid sequences deduced from cDNA sequences to ensure accurate structural determination .

How can researchers effectively express and purify recombinant Grahamin-1?

Effective expression and purification of recombinant Grahamin-1 involves a multi-step process:

• Expression System Selection: E. coli is the standard expression system for Grahamin-1 production . Researchers should select strains optimized for disulfide bond formation (e.g., Origami, SHuffle) to ensure proper folding of the Rana box structure.

• Vector Design: Design an expression vector containing the coding sequence for the mature peptide (region 46-66 of the precursor) , with appropriate fusion tags (e.g., His-tag, GST) to facilitate purification and enhance solubility.

• Expression Optimization: Optimize culture conditions including temperature (typically lower temperatures like 16-20°C post-induction), IPTG concentration, and induction time to maximize yield while maintaining proper folding.

• Purification Strategy:

  • Initial capture using affinity chromatography based on the fusion tag

  • Tag removal using specific proteases (e.g., TEV, thrombin)

  • Secondary purification using reverse-phase HPLC

  • Verification of purity using SDS-PAGE (target >85% purity)

• Disulfide Bond Formation: Ensure proper oxidative conditions to form the critical disulfide bridge, which may require additional refolding steps.

• Quality Control: Confirm identity and activity through mass spectrometry and antimicrobial activity assays against reference strains.

• Storage: Properly aliquot and store with glycerol (5-50%) at -20°C/-80°C to maintain stability .

This methodological approach maximizes yield, purity, and biological activity of the recombinant peptide.

What antimicrobial assay methodologies are most appropriate for evaluating Grahamin-1 activity?

To comprehensively evaluate the antimicrobial activity of Grahamin-1, researchers should employ multiple complementary methodologies:

• Minimum Inhibitory Concentration (MIC) Determination:

  • Broth microdilution method using 96-well plates

  • Serial dilutions of Grahamin-1 (typically 0.5-256 μg/mL)

  • Standard microbial strains (e.g., E. coli ATCC 25922, S. aureus ATCC 25923)

  • Incubation at 37°C for 16-24 hours

  • MIC determination by visual inspection or spectrophotometric measurement

• Time-Kill Kinetics:

  • Exposure of microorganisms to Grahamin-1 at various concentrations (0.5×, 1×, 2×, 4× MIC)

  • Sampling at defined time points (0, 1, 2, 4, 8, 24 hours)

  • Plating on agar media and colony counting

  • Generation of time-kill curves to assess bactericidal versus bacteriostatic activity

• Membrane Permeabilization Assays:

  • Fluorescent dye uptake (e.g., SYTOX Green, propidium iodide)

  • Membrane potential indicators (e.g., DiSC3(5))

  • Observation of morphological changes using electron microscopy

• Resistance Development Assessment:

  • Serial passage of microorganisms with sub-MIC concentrations

  • Monitoring MIC changes over 20-30 passages

  • Comparison with conventional antibiotics

• Synergy Testing:

  • Checkerboard assays with conventional antibiotics

  • Calculation of Fractional Inhibitory Concentration (FIC) indices

  • Time-kill assays with combination treatments

These methods provide a comprehensive profile of antimicrobial activity, mechanism of action, and potential for clinical application. Given the structural similarity to nigrocins from Rana nigromaculata, comparative antimicrobial profiling with these related peptides can provide valuable insights into structure-activity relationships .

How can structure-activity relationship studies enhance the understanding of Grahamin-1?

Structure-activity relationship (SAR) studies of Grahamin-1 can provide critical insights through systematic methodological approaches:

• Alanine Scanning Mutagenesis:

  • Systematic replacement of each amino acid with alanine

  • Expression and purification of mutant peptides

  • Antimicrobial activity testing to identify essential residues

  • This approach would be particularly informative for understanding the importance of specific residues in the conserved GLLSGILGAGKHIVCGLSGLC sequence

• Disulfide Bridge Modification:

  • Mutation or chemical modification of cysteine residues

  • Assessment of the impact on secondary structure and antimicrobial activity

  • Particularly important given the critical role of the Rana box in peptide function

• Truncation and Extension Analysis:

  • Creation of N-terminal and C-terminal truncated variants

  • Assessment of minimum sequence required for activity

  • Evaluation of the importance of the Rana box region versus the N-terminal hydrophobic region

• Comparative Analysis with Related Peptides:

  • Comparison with Grahamin-2 (GLLSGILGAGKNIVCGLSGLC), which differs by a single amino acid (H/N)

  • Analysis relative to structurally similar nigrocins from Rana nigromaculata

  • This comparative approach can pinpoint how minor sequence variations affect functional properties

• Secondary Structure Modification:

  • Design of peptides with enhanced or disrupted helical propensity

  • CD spectroscopy to confirm structural changes

  • Correlation of structural changes with antimicrobial activity

  • Similar to methods used for other frog peptides, such as those from Rana catesbeiana

These SAR approaches provide mechanistic insights into how Grahamin-1 exerts its antimicrobial effects and can guide the rational design of more potent or selective antimicrobial peptide derivatives.

What are the challenges in scaling up recombinant Grahamin-1 production for research purposes?

Scaling up recombinant Grahamin-1 production for research presents several technical challenges requiring methodological solutions:

• Cytotoxicity to Expression Host:

  • Antimicrobial peptides can be toxic to the bacterial expression host

  • Solution: Use tightly regulated expression systems (e.g., pET with T7 lysozyme co-expression) and fusion partners (e.g., thioredoxin, SUMO) to mitigate toxicity

• Disulfide Bond Formation:

  • Ensuring correct formation of the critical intramolecular disulfide bridge in the Rana box

  • Solution: Employ specialized E. coli strains (SHuffle, Origami) with enhanced disulfide bond formation capability or use periplasmic expression strategies

• Proteolytic Degradation:

  • Susceptibility to host proteases

  • Solution: Use protease-deficient host strains and optimize induction conditions (lower temperature, shorter induction time)

• Inclusion Body Formation:

  • Tendency to form insoluble aggregates

  • Solution: Develop robust refolding protocols from inclusion bodies using controlled oxidation conditions, or optimize soluble expression using solubility-enhancing tags

• Purification Efficiency:

  • Difficulty in separating the target peptide from host cell proteins

  • Solution: Implement multi-step purification strategies combining affinity chromatography, ion exchange, and reverse-phase HPLC

• Activity Verification:

  • Ensuring biological activity comparable to native peptide

  • Solution: Establish standardized antimicrobial activity assays against reference strains to confirm functional integrity

• Batch-to-Batch Consistency:

  • Maintaining consistent quality across production batches

  • Solution: Develop rigorous quality control protocols including mass spectrometry, HPLC profiling, and activity testing

These challenges can be addressed through careful optimization of each production step with an emphasis on maintaining the critical structural features necessary for antimicrobial activity, particularly the proper formation of the disulfide-bonded Rana box .

How does Grahamin-1 compare to other antimicrobial peptides from amphibian skin in terms of mechanism and specificity?

Grahamin-1 shares similarities with other amphibian antimicrobial peptides but also exhibits distinctive features:

• Structural Comparison:

  • Grahamin-1 belongs to the family of antimicrobial peptides containing a single intra-disulfide bridge (Rana box)

  • Most closely related to nigrocins from Rana nigromaculata, sharing significant sequence homology

  • Unlike some amphibian peptides (e.g., magainins, bombinins) that adopt α-helical structures without disulfide bonds

  • Distinct from temporins (like Temporin-Lb from Rana catesbeiana) which are shorter (10-14 amino acids) and lack the disulfide bridge

• Mechanism of Action:

  • Like many amphibian AMPs, Grahamin-1 likely acts primarily through membrane disruption

  • The Rana box structure may confer specific membrane interactions distinct from purely linear α-helical peptides

  • Comparative mechanism studies with related peptides would follow methodologies similar to those used for other frog peptides, employing membrane models and permeabilization assays

• Antimicrobial Spectrum:

  • Exhibits broad-spectrum antimicrobial activity against various microorganisms

  • Specificity profile may differ from other amphibian AMPs due to unique structural elements

  • Nigrocin-2 peptides (structurally related) from Odorrana frogs demonstrate significant antimicrobial potency

• Evolution and Diversity:

  • Part of the remarkable diversity of amphibian skin peptides

  • Distinct evolutionary adaptation compared to other frog AMP families

  • Nigrocin-2 peptides are particularly diverse in structure in the Odorrana group

• Potential Applications:

  • While peptides like temporins from R. catesbeiana have been studied for antitumor effects , Grahamin-1's potential in this area requires further investigation

  • The stable disulfide bridge may confer higher stability in biological environments compared to linear AMPs

The integration of structural, functional, and evolutionary analyses provides a comprehensive understanding of Grahamin-1's unique position within the diverse landscape of amphibian antimicrobial peptides .

What advanced analytical methods can be used to study Grahamin-1 interactions with bacterial membranes?

To elucidate the molecular mechanisms of Grahamin-1's interaction with bacterial membranes, researchers can employ several sophisticated analytical techniques:

• Atomic Force Microscopy (AFM):

  • Direct visualization of peptide-induced membrane disruption

  • Time-lapse imaging of bacterial membrane morphological changes

  • Quantification of membrane roughness, thickness, and nanoscale defects

  • Force spectroscopy to measure peptide-membrane binding forces

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Solution NMR to determine 3D structure in membrane-mimetic environments

  • Solid-state NMR to study peptide orientation and dynamics in lipid bilayers

  • 31P and 2H NMR to monitor lipid headgroup and acyl chain perturbations

  • Determination of specific peptide-lipid interactions, particularly important for the disulfide-bonded Rana box region

• Surface Plasmon Resonance (SPR):

  • Real-time binding kinetics to model membranes with varying lipid compositions

  • Determination of association/dissociation constants

  • Investigation of the impact of the disulfide bond on binding properties

  • Comparison with structurally related peptides like Grahamin-2 and nigrocins

• Fluorescence Techniques:

  • Fluorescence spectroscopy with tryptophan-labeled Grahamin-1 variants

  • Förster resonance energy transfer (FRET) to measure peptide-membrane distances

  • Fluorescence microscopy with labeled peptides to visualize membrane localization

  • Fluorescent dye leakage assays using liposomes to quantify membrane permeabilization

• Molecular Dynamics (MD) Simulations:

  • Atomistic simulations of peptide-membrane interactions

  • Free energy calculations of membrane insertion and pore formation

  • Structure-function relationship analysis of the Rana box contribution

  • Comparison of wild-type and mutant peptide behaviors

• Differential Scanning Calorimetry (DSC) and Isothermal Titration Calorimetry (ITC):

  • Thermodynamic characterization of peptide-membrane interactions

  • Determination of enthalpy, entropy, and binding stoichiometry

  • Evaluation of membrane phase transitions in the presence of Grahamin-1

• Cryo-Electron Microscopy:

  • Visualization of peptide-induced membrane structures at near-atomic resolution

  • Observation of pore formation or other membrane disruption mechanisms

  • Study of bacterial cell envelope structural changes upon peptide exposure

These advanced analytical approaches provide complementary insights into the molecular basis of Grahamin-1's antimicrobial activity, essential for rational design of optimized antimicrobial peptides for potential therapeutic applications.

How can researchers effectively compare the activities of Grahamin-1 and Grahamin-2?

A systematic comparative analysis of Grahamin-1 and Grahamin-2 requires multi-faceted experimental approaches:

• Sequence and Structural Comparison:

  • Alignment analysis highlighting the single amino acid difference (H vs N at position 10: GLLSGILGAGKHIVCGLSGLC vs GLLSGILGAGKNIVCGLSGLC)

  • CD spectroscopy to compare secondary structure profiles in various environments

  • NMR structural analysis to determine if the single residue change alters the 3D conformation

• Antimicrobial Activity Profiling:

  • Parallel MIC determination against a panel of Gram-positive and Gram-negative bacteria

  • Time-kill kinetics under identical conditions

  • Membrane permeabilization assays to assess potential differences in mechanism

  • The following table represents a methodological framework for standardized comparison:

  Test Parameter	Methodology	Organisms	Measurement Endpoints
  MIC Determination	Broth microdilution	E. coli, S. aureus, P. aeruginosa, K. pneumoniae	μg/mL values
  Time-Kill Kinetics	CFU counting at intervals	Same as above	Log reduction over time
  Membrane Permeability	SYTOX Green uptake	Same as above	Fluorescence intensity
  Salt Sensitivity	MIC in varying salt concentrations	E. coli, S. aureus	Fold change in MIC

• Physicochemical Property Analysis:

  • Hydrophobicity profiles and charge distribution comparison

  • pH-dependent activity assessment

  • Stability in serum and in the presence of proteolytic enzymes

  • Salt resistance profiles (particularly important for potential therapeutic applications)

• Functional Specialization Investigation:

  • Testing against expanded panels of microorganisms including fungi and resistant strains

  • Evaluation of anti-biofilm activity

  • Host cell toxicity comparison using hemolysis and mammalian cell viability assays

  • Immunomodulatory property assessment

• Structure-Function Analysis:

  • Creation of hybrid peptides incorporating elements of both sequences

  • Point mutations at position 10 and surrounding residues

  • Correlation of structural features with functional differences

This comprehensive comparison would reveal how the single histidine/asparagine difference between Grahamin-1 and Grahamin-2 influences their biological properties and potential applications .

What potential therapeutic applications have been investigated for antimicrobial peptides similar to Grahamin-1?

Antimicrobial peptides structurally similar to Grahamin-1 have been investigated for various therapeutic applications, providing a roadmap for Grahamin-1 research:

• Topical Antimicrobial Formulations:

  • Treatment of skin infections and wounds

  • Incorporation into antimicrobial coatings for medical devices

  • Development of preservatives for topical pharmaceutical formulations

  • Challenges include stability in formulation and potential for irritation

• Anti-Biofilm Strategies:

  • Targeting bacterial biofilms in chronic infections

  • Combination therapy with conventional antibiotics

  • Peptide modifications to enhance penetration of biofilm matrix

  • Related frog antimicrobial peptides have shown promise in disrupting established biofilms

• Anticancer Applications:

  • Related peptides like Temporin-Lb from Rana catesbeiana have demonstrated antitumor effects on cancer cell lines

  • Potential mechanisms include membrane disruption or apoptosis induction

  • Selective cytotoxicity against cancer cells versus normal cells

  • The cell morphology of cancer cells changes after exposure to some amphibian peptides

• Immunomodulatory Functions:

  • Regulation of innate immune responses

  • Anti-inflammatory activities

  • Enhancement of wound healing processes

  • These effects have been observed with several frog-derived antimicrobial peptides

• Delivery System Development:

  • Nanoparticle encapsulation to improve stability and reduce toxicity

  • Targeted delivery approaches

  • Stimuli-responsive release mechanisms

  • Peptide-antibiotic conjugates for enhanced delivery of conventional antibiotics

• Resistant Infection Treatment:

  • Activity against multidrug-resistant bacteria

  • Novel mechanisms less prone to resistance development

  • Potential for synergistic effects with conventional antibiotics

  • Effectiveness against bacterial persisters

The investigation of Grahamin-1 for these applications would require addressing challenges including stability, potential immunogenicity, and cost-effective production methods . The structural features of Grahamin-1, particularly its disulfide-bonded Rana box, may confer advantages for specific therapeutic applications compared to linear antimicrobial peptides.

What experimental approaches can assess potential synergistic effects between Grahamin-1 and conventional antibiotics?

Investigating synergistic interactions between Grahamin-1 and conventional antibiotics requires systematic methodological approaches:

• Checkerboard Assay:

  • Standard method for quantitative assessment of antimicrobial interactions

  • Serial dilutions of both Grahamin-1 and antibiotics in a 96-well matrix format

  • Calculation of Fractional Inhibitory Concentration Index (FICI):

    • FICI = (MIC of antibiotic in combination/MIC of antibiotic alone) + (MIC of Grahamin-1 in combination/MIC of Grahamin-1 alone)

    • FICI ≤ 0.5: synergy; 0.5 < FICI ≤ 1: additivity; 1 < FICI ≤ 4: indifference; FICI > 4: antagonism

  • Testing against multiple bacterial species including resistant strains

• Time-Kill Kinetics:

  • Assessment of bactericidal activity over time (0, 1, 2, 4, 8, 24 hours)

  • Combinations at sub-inhibitory concentrations (e.g., 0.25× MIC of each agent)

  • Synergy defined as ≥2 log₁₀ decrease in CFU/mL by the combination compared to the most active single agent

  • Graphical representation of killing curves for visual assessment of interactions

• Membrane Permeabilization Studies:

  • Investigation of Grahamin-1's effect on bacterial membrane integrity using fluorescent dyes

  • Assessment of whether Grahamin-1 enhances antibiotic uptake

  • Microscopy techniques to visualize membrane disruption and antibiotic localization

  • Flow cytometry to quantify membrane permeabilization in bacterial populations

• Biofilm Eradication Assays:

  • Crystal violet staining to quantify biofilm mass

  • Confocal laser scanning microscopy with LIVE/DEAD staining

  • Enumeration of viable cells within biofilms after treatment

  • Assessment of penetration enhancement of antibiotics into biofilms

• Resistance Development Studies:

  • Serial passage experiments with sub-MIC concentrations

  • Comparison of resistance development rates: antibiotic alone vs. combination

  • Molecular characterization of any resistance mechanisms that emerge

  • Assessment of cross-resistance profiles

• Molecular Mechanism Investigation:

  • Transcriptomics to identify altered gene expression patterns

  • Proteomics to detect changes in protein expression

  • Assessment of specific antibiotic target accessibility

  • Investigation of stress response pathways

The following table outlines a methodical experimental design for testing synergy with various antibiotic classes:

These approaches provide a comprehensive framework for characterizing potential synergistic interactions that could inform the development of novel combination therapies involving Grahamin-1 .

What genomic and transcriptomic approaches could enhance our understanding of Grahamin peptide evolution?

Advanced genomic and transcriptomic methodologies can provide deeper insights into Grahamin peptide evolution:

• Comparative Genomics:

  • Whole genome sequencing of multiple Odorrana/Rana species

  • Identification and annotation of antimicrobial peptide gene clusters

  • Analysis of genomic organization of Grahamin and related peptide genes

  • Investigation of regulatory elements controlling expression

  • Comparative analysis with other frog genera to trace evolutionary origins

• Transcriptome Profiling:

  • RNA-Seq analysis of skin tissues under various conditions:

    • Normal physiological state

    • Microbial challenge

    • Environmental stress (temperature, humidity, pH)

    • Seasonal variations

  • Quantification of expression levels of Grahamin precursor genes

  • Identification of novel antimicrobial peptide transcripts

• Single-Cell Transcriptomics:

  • Analysis of different skin cell populations

  • Identification of specific cell types producing antimicrobial peptides

  • Characterization of expression heterogeneity across the skin

  • Developmental trajectory analysis of peptide-producing cells

• Phylogenetic Analysis:

  • Construction of comprehensive evolutionary trees using:

    • Nucleotide sequences of precursor genes

    • Amino acid sequences of mature peptides

    • Whole genome comparisons

  • Dating of gene duplication and diversification events

  • Analysis of selection pressures using dN/dS ratios

  • Correlation with geographic distribution and ecological niches

• Population Genomics:

  • Sampling of multiple populations of Rana grahami

  • Assessment of genetic diversity in antimicrobial peptide genes

  • Investigation of local adaptations to different pathogen pressures

  • Analysis of copy number variations in peptide genes

• Functional Genomics:

  • CRISPR-Cas9 modification of antimicrobial peptide genes in model amphibians

  • Reporter gene assays for promoter activity analysis

  • ChIP-seq to identify transcription factors regulating expression

  • Epigenetic profiling to assess regulation mechanisms

These approaches would provide insights into how Grahamin peptides evolved from ancestral sequences, potentially revealing the adaptive significance of structural features like the Rana box . Such understanding could guide the development of novel antimicrobial therapeutics inspired by natural evolutionary processes.

How might computational modeling enhance the design of Grahamin-1 derivatives with improved properties?

Computational approaches can significantly accelerate the design of improved Grahamin-1 derivatives through the following methodological framework:

• Molecular Dynamics (MD) Simulations:

  • All-atom simulations of Grahamin-1 in different environments:

    • Aqueous solution

    • Membrane-mimetic environments

    • Bacterial versus mammalian membrane models

  • Analysis of peptide conformational dynamics

  • Identification of key residues involved in membrane interactions

  • Simulation of the disulfide bridge contribution to stability and activity

• Structure-Based Virtual Screening:

  • Development of pharmacophore models based on Grahamin-1 structure

  • High-throughput virtual screening of peptide libraries

  • Fragment-based design approaches

  • Docking studies with potential membrane or intracellular targets

• Quantitative Structure-Activity Relationship (QSAR) Modeling:

  • Collection of activity data for Grahamin-1 and related peptides

  • Development of predictive models correlating sequence/structural features with:

    • Antimicrobial potency

    • Spectrum of activity

    • Host cell toxicity

    • Stability in biological fluids

  • Validation of models with experimental testing

• De Novo Peptide Design:

  • Algorithm-based generation of novel sequences maintaining key structural features

  • Deep learning approaches trained on antimicrobial peptide databases

  • Optimization of physicochemical properties while maintaining the essential Rana box motif

  • Multi-objective optimization for balanced improvement of different properties

• Peptide Stability Enhancement Strategies:

  • In silico prediction of proteolytic cleavage sites

  • Design of modifications to increase proteolytic resistance:

    • D-amino acid substitutions

    • N-methylation of backbone

    • Terminal modifications

  • Simulation of modified peptides to ensure retention of structure and function

• Hybrid Peptide Design:

  • Computational modeling of chimeric peptides combining Grahamin-1 features with other antimicrobial peptides

  • Prediction of optimal fusion points and linker regions

  • Assessment of hybrid stability and activity profiles

  • Design of multifunctional peptides with complementary activities

The following workflow illustrates a systematic computational approach to Grahamin-1 optimization:

• Build and validate 3D model of native Grahamin-1

• Identify structural determinants of activity through MD simulations

• Generate virtual library of derivatives with targeted modifications

• Predict activity and toxicity profiles using QSAR models

• Select top candidates for experimental validation

• Refine models based on experimental feedback

• Iterate design process for continued improvement

This computational pipeline can significantly reduce the experimental burden by focusing wet-lab efforts on the most promising Grahamin-1 derivatives with enhanced antimicrobial properties and reduced toxicity .