The study, published in Discover Chemistry, focused on an enzyme called phospholipase A1, or PlaF, a membrane-bound virulence factor first characterized in P. aeruginosa by Bleffert and colleagues in 2022. PlaF sits embedded in the cytoplasmic membrane, where it remodels glycerophospholipids through the so-called Lands cycle, generating medium-chain fatty acids that alter the biophysical properties of the bacterial membrane and regulate signaling cascades. This remodeling allows the pathogen to adapt to nutrient limitation, stress, and host-mediated attacks, and it coordinates iron acquisition through the pyoverdine pathway, a key element of virulence. Crucially, infection models have shown that bacteria lacking PlaF display dramatically reduced virulence without any change in growth kinetics, meaning the enzyme acts as a regulator of pathogenesis rather than a survival determinant.

That distinction matters enormously for drug design. Traditional antibiotics exert strong selective pressure by killing bacteria or halting their growth, which inevitably breeds resistance. Anti-virulence strategies, by contrast, aim to disarm the pathogen while leaving it metabolically viable, theoretically reducing the evolutionary incentive to develop resistance. PlaF also offers a structural advantage for selective targeting: although it shares the classical alpha/beta-hydrolase fold and the conserved Ser-His-Asp catalytic triad with human ABHD6, showing 27 percent sequence identity and 49 percent similarity, its membrane-associated substrate-access tunnels and a unique dimerization-dependent regulatory mechanism diverge substantially from the human enzyme. Recent work confirming that repurposed human phospholipase inhibitors such as GW4869 can sensitize P. aeruginosa to imipenem in a PlaF-dependent manner validated the enzyme as druggable, but those existing inhibitors are bulky, lipophilic molecules designed for mammalian systems, not optimized for bacterial membrane penetration or tunnel-specific engagement.

To fill that gap, the researchers assembled a library of 114 small molecules spanning seven chemical classes, drawn from their previous work on anticancer and anti-tuberculosis compounds, on the rationale that serine hydrolase inhibitors often exhibit cross-domain activity. The collection included thiophene-2-carboxamides, methyl 4-(4-amidoaryl)-3-methoxythiophene-2-carboxylates, urea and thiourea bearing thiophene derivatives, pyrimidine-5-carboxamides, triazinyl carboxamides, thienopyrimidine ureas, and pyrrolopyridine carboxamides. Using the crystal structure of PlaF available in the Protein Data Bank under the identifier 6I8W, the team deployed the Schrodinger Suite to prepare ligands with the OPLS4 force field, generate ionization states and tautomers with Epik, and filter candidates through Lipinski’s rule of five using the QikProp module. One hundred seven compounds survived drug-likeness screening and advanced to molecular docking.

The docking campaign used Glide’s extra precision protocol, ranking compounds by binding affinity and prioritizing those with scores of −6.0 kcal/mol or better. Subsequent MM/GBSA free energy calculations, which combine molecular mechanics energies with solvation models, flagged compounds with binding free energies of −50 kcal/mol or lower. Standouts emerged across the series: compound F11 from the thienopyrimidine class achieved an MM/GBSA score of −56.72 kcal/mol, while D17 from the pyrimidine carboxamides posted the highest binding free energy of the entire screen at −67.22 kcal/mol, attributed to robust halogen bonding. Structural interaction fingerprint analysis then converted residue-level contacts into comparable binary codes, revealing a consistent pharmacophoric pattern across high performers: roughly seven to ten hydrophobic contacts, three to four aromatic stacking interactions with residues such as PHE174 and TYR236, and limited but strategic hydrogen bonding with ARG233 or LEU232. Notably, compounds with excessive polar substitution or bulky aromatic expansion increased total contact counts without improving predicted stability, indicating that geometric complementarity within the narrow substrate-access tunnel matters more than interaction density.

The team then moved from computation to the bench, testing all 107 drug-like compounds against P. aeruginosa strain ATCC 27853 using a micro broth dilution assay with resazurin dye as a viability indicator, with ciprofloxacin as a positive control at a measured MIC of 0.25 micrograms per milliliter. Several compounds showed measurable activity, with minimum inhibitory concentrations of 100 to 200 micrograms per milliliter: B4 and B5 from the methoxythiophene series, C2 from the urea-thiourea thiophenes, D6 and D7 from the pyrimidine carboxamides, E6 and E8 from the triazines, F4 from the thienopyrimidines, and G2, G3, G11, and G16 from the pyrrolopyridines. While these MIC values are far weaker than conventional antibiotics, the researchers emphasize that PlaF inhibition is an anti-virulence strategy, so modest growth inhibition is expected and does not negate mechanistic relevance.

To probe how ligands would fare against a shifting target, the investigators engineered point mutations that replaced residues lining the T2 substrate-access tunnel—Asp74, Arg217, Ala218, Ala221, and Asn225—with bulky tryptophan, physically constricting the passage. Docking against these mutants confirmed the T2 tunnel as the principal route for ligand binding and revealed that some mutations actually enhanced affinity for certain scaffolds. Compounds D6 and E6 achieved improved docking scores of −10.55 and −9.38 kcal/mol against the TRP74 and TRP217 variants, while others, such as G16 against the TRP218 mutant, suffered sharp losses in binding, likely from steric clashes. The mutational blueprint suggests that halogenated, aromatic, and amide-bearing scaffolds capable of diverse non-covalent interactions are best positioned to remain effective even if the tunnel architecture shifts.

Molecular dynamics simulations provided the final filter. Each of fourteen protein-ligand complexes, embedded in a POPC lipid bilayer with explicit water and physiological salt, was simulated for 100 nanoseconds under semi-isotropic pressure coupling appropriate for membrane proteins. Six compounds—A3, B4, C2, D6, E6, and F4—maintained backbone RMSD values between 1.5 and 2.8 angstroms, preserved tunnel architecture, and showed restricted ligand mobility throughout the trajectory, outperforming even ciprofloxacin, which drifted beyond 4.5 angstroms in later simulation stages. Radius of gyration, solvent-accessible surface area, molecular surface area, and polar surface area analyses all confirmed that these six ligands kept the tunnel compact and the catalytic electrostatic environment stable, both in the wild-type enzyme and in the more permissive Ala218Trp and Ala221Trp mutant backgrounds.

Toxicity predictions from the ProTox 3.0 server, which applies machine-learning models across 61 toxicity endpoints, narrowed the field further. Compounds A3, D6, E6, and F4 fell into toxicity class 4, with predicted oral LD50 values between 1000 and 1502 milligrams per kilogram and flagged respiratory, immunotoxic, and neurotoxic endpoints. Compounds B4, a methyl 3-methoxythiophene derivative bearing a fluorinated benzamido group, and C2, a thioureido-substituted compound, landed in the more favorable class 5, with predicted LD50 values of 4000 and 4500 milligrams per kilogram respectively, though mild hepatotoxicity risk means neither can be considered non-toxic pending experimental validation. The authors are careful to note that computational binding stability does not guarantee whole-cell target engagement, and that efflux pumps may limit intracellular concentrations. Future work, they argue, should include biochemical PlaF inhibition assays, lipidomic confirmation that glycerophospholipid remodeling is disrupted, complementation studies with the delta-plaF mutant to verify target engagement, and in vivo infection models. If those steps succeed, B4 and C2 could seed a genuinely novel class of anti-virulence agents—one that fights the superbug by stripping away its weapons rather than threatening its life, and in doing so may sidestep the resistance cycle that has defeated so many antibiotics before.

Subject of Research: Structure-based discovery of inhibitors targeting the phospholipase A1 virulence factor of Pseudomonas aeruginosa

Article Title: Structure-based discovery of phospholipase A1 (PlaF) inhibitors in Pseudomonas aeruginosa using integrated in silico and experimental approaches

Article References: Vemula, D., Gulipalli, K. C., Bodige, S., Seelam, N., & Bhandari, V. (2026). Structure-based discovery of phospholipase A1 (PlaF) inhibitors in Pseudomonas aeruginosa using integrated in silico and experimental approaches. Discover Chemistry, 3(1), Article 457. https://doi.org/10.1007/s44371-026-00802-6

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00802-6

Keywords: Pseudomonas aeruginosa, phospholipase A1, PlaF, anti-virulence, antimicrobial resistance, molecular docking, molecular dynamics, virtual screening, drug discovery, MM/GBSA, virulence factor, computer-aided drug design

The biological target of the study is the central event in sickle cell pathology: the polymerization of hemoglobin S. The disease stems from a single point mutation in the beta-globin gene, in which glutamic acid at the sixth position is replaced by valine. When oxygen levels fall, this seemingly small change causes hemoglobin molecules to clump into rigid fibers, deforming red blood cells into their characteristic sickle shape. These rigid cells clog capillaries, a process known as vaso-occlusion, triggering painful crises, hemolysis and progressive organ damage. Repeated cycles of polymerization and depolymerization also generate oxidative stress, depleting nitric oxide in the vasculature and fueling inflammation that involves platelets, neutrophils and the endothelium.

Current therapies reflect the limits of treating a downstream process. Hydroxyurea raises fetal hemoglobin levels and reduces sickling, voxelotor binds hemoglobin reversibly to keep it in its oxygenated state, and crizanlizumab blocks platelet-mediated adhesion. L-glutamine offers antioxidant support, while regular transfusions manage severe cases. The only curative option, hematopoietic stem cell transplantation from a matched sibling donor, is limited by donor scarcity, cost and serious risks including graft-versus-host disease. Gene editing therapies are emerging but remain inaccessible to most patients globally. This therapeutic gap motivated the Indian team to search for small molecules that could directly inhibit hemoglobin S polymerization while simultaneously counteracting oxidative stress.

The researchers chose two pharmacophores with complementary biological credentials. The first is caffeic acid, a phenolic compound found in coffee, wine, tea and propolis, which has documented antioxidant, anti-inflammatory and anti-carcinogenic properties. Previous experimental work showed that caffeic acid increases the delay time for hemoglobin polymerization, a direct anti-sickling effect, and significantly lowers the concentration of ferryl hemoglobin formed during peroxide reactions. The second is phthalimide, whose derivatives, including the thalidomide family, have been shown to increase gamma-globin expression and fetal hemoglobin production while lowering tumor necrosis factor alpha, giving them both anti-sickling and anti-inflammatory potential.

Using an AI-assisted online tool called Mol-Opt, the team generated bioisosteric analogues of both scaffolds, systematically swapping functional groups to improve potency, pharmacokinetics and safety while preserving the molecular features responsible for activity. This yielded 373 candidate analogues across the two series. Each was then screened through a battery of computational filters. The ADMET Lab 3.0 platform predicted absorption, distribution, metabolism, excretion and toxicity parameters, while the Osiris Property Explorer provided drug-likeness and drug-score assessments. Encouragingly, every analogue satisfied Lipinski’s rule of five, the Pfizer rules and, with few exceptions, the Golden Triangle criteria, suggesting the designed molecules occupy drug-like chemical space with reasonable prospects for oral absorption.

The pharmacokinetic and toxicity screens narrowed the field considerably. Among the caffeic acid derivatives, compounds such as C-168, C-2, C-29, C-195 and C-27 stood out for combining high drug scores with low predicted toxicity. Analogue C-2 proved especially notable, showing the lowest hemotoxicity and genotoxicity values in its series. In the phthalimide series, P-60 and P-41 displayed excellent ADME balance with minimal tissue accumulation and very low skin sensitization, although several otherwise promising analogues, including P-155, P-171, P-202 and P-248, were rejected because of alarmingly high predicted genotoxicity, illustrating how computational toxicity screening can eliminate liabilities before any laboratory synthesis takes place.

The decisive test came from molecular docking against the crystal structure of deoxyhemoglobin S, available in the Protein Data Bank under the identifier 2HBS at 2.05 angstrom resolution. Using the Pyrex implementation of AutoDock Vina, the team docked the analogues into the hemoglobin active site and visualized the interactions with ICM Browser Pro. The phthalimide analogue P-2 achieved the study’s best binding affinity at minus 10.4 kilocalories per mole, followed by P-56 at minus 10.3. Among the caffeic acid derivatives, C-2 led with minus 9.0 kilocalories per mole, ahead of C-72 and C-68. For comparison, the approved drugs hydroxyurea, L-glutamine and voxelotor were docked under the same conditions, and the top analogues outperformed them, forming networks of hydrogen bonds and hydrophobic contacts with residues including LYS-66, ASN-102, ASP-99, VAL-98, LEU-28 and PHE-41.

Because a good docking score does not guarantee that a ligand remains bound in the dynamic environment of a real protein, the two lead compounds were subjected to 100-nanosecond molecular dynamics simulations using the Desmond package in the Schrödinger suite. The P-2 hemoglobin complex proved remarkably stable: after an initial equilibration period, the ligand’s root mean square deviation, fitted to the protein, settled within 1.2 to 2.2 angstroms and remained steady for the rest of the simulation. The C-2 complex showed somewhat greater fluctuation, with average protein RMSD values between 0.8 and 3.2 angstroms, but still maintained its binding pose within the active pocket. Contact analysis revealed that the ligands were held in place by a combination of hydrogen bonds, hydrophobic interactions and water bridges involving residues such as PHE-41, LYS-95, VAL-98, ASP-99 and GLU-43, providing a plausible structural basis for sustained anti-sickling activity.

To probe the electronic foundations of the two leads, the researchers performed density functional theory calculations using the ORCA 5.0.4 program at the B3LYP level with the def2-TZVP basis set. Compound C-2 displayed a HOMO energy of minus 8.63 electron volts localized over its catechol pi-system, identifying it as the principal electron-donating region, while its LUMO at minus 5.23 electron volts spread across the nicotinate ring and ester carbonyl groups. The resulting HOMO-LUMO gap of 3.40 electron volts indicates moderate electronic delocalization and efficient charge-transfer pathways, properties associated with reversible electron transfer and enhanced biological activity. Compound P-2 showed a smaller gap of 2.62 electron volts, with the HOMO distributed over its aromatic indole and benzoyl amide regions and the LUMO concentrated at the carbonyl nicotinate position, suggesting good conjugation and moderate chemical reactivity across the amide framework.

The authors conclude that the combined computational workflow, spanning bioisosteric design, ADMET prediction, docking, dynamics and quantum chemistry, has delivered two credible lead candidates, P-2 and C-2, that merit progression toward laboratory validation. Experimental studies to confirm their anti-sickling activity are reportedly underway. As with any purely computational study, the predictions remain hypotheses until synthesis, binding assays and animal or clinical testing are completed, and the high genotoxicity flags on several analogues underline the need for careful optimization. Nevertheless, the work demonstrates how a rational, multi-method in silico pipeline can compress the early stages of drug discovery, potentially cutting costs and timelines for a disease that has waited decades for a truly transformative small-molecule therapy.

Subject of Research: Computational screening of phthalimide and caffeic acid analogues as potential anti-sickling agents for sickle cell disease

Article Title: Computational screening of some newer potential anti-sickling compounds using docking, ADMET, DFT, and molecular dynamics approaches

Article References: Verma, D., Ahirwar, B., Vaishnav, Y., & Gahrewal, M. (2026). Computational screening of some newer potential anti-sickling compounds using docking, ADMET, DFT, and molecular dynamics approaches. Discover Chemistry, 3(1), Article 495. https://doi.org/10.1007/s44371-026-00942-9

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00942-9

Keywords: sickle cell disease, hemoglobin S polymerization, molecular docking, ADMET, molecular dynamics simulation, DFT, bioisosteres, phthalimide, caffeic acid, drug discovery, computer-aided drug design, vaso-occlusion

The therapeutic logic behind the work rests on the deep biological overlap between inflammatory and allergic disease. COX-2 catalyzes the conversion of arachidonic acid into prostaglandins when tissues become inflamed, driving pain, fever, and swelling, while histamine acting through H1 receptors triggers vasodilation, smooth muscle contraction, and itching. In conditions such as allergic rhinitis with sinus inflammation or chronic urticaria with an inflammatory component, both pathways fire at once. A compound that engages both could, in principle, deliver synergistic benefit, lower the effective dose of each mechanism, reduce drug-drug interactions, and improve patient adherence. The authors point to rupatadine, which combines H1 antagonism with platelet-activating factor inhibition, as an existing proof that multi-target antihistamines can succeed clinically.

The benzimidazole scaffold was a deliberate choice. This fused benzene-imidazole ring system structurally resembles the purine bases of DNA, which helps it form favorable contacts with a wide range of biological targets. Benzimidazole drugs already span an impressive pharmacological range, from anti-inflammatory and antimicrobial to antiviral, anticancer, and antiparasitic activity. Previous studies have shown that suitably substituted benzimidazoles can act as selective COX-2 inhibitors with improved gastrointestinal safety compared with traditional NSAIDs; one reported series of methyl sulfonyl-substituted analogues achieved sub-micromolar inhibitory potency in enzyme assays. What had not been done before, according to the authors, was a systematic dual-target evaluation of a single benzimidazole series against both COX-2 and the H1 receptor.

The design itself was methodical. All eleven compounds, labeled SP1 through SP11, share a 2-phenyl-1H-benzimidazole core connected through an acetamide linker at the N-1 position to a variable substituent. That substituent was varied deliberately across chemical space: aromatic groups such as aniline and resorcinol, aldehyde-bearing moieties including salicylaldehyde and isovanillin, carboxylic acids ranging from formic to benzoic, chloroacetic, and salicylic acid, and simple aliphatic propanol and butanol chains. Each variation tunes lipophilicity, hydrogen-bonding capacity, and steric profile in a different way, allowing the team to read out structure-activity relationships directly from the docking results.

Before any docking was attempted, the compounds had to pass basic drug-likeness filters. Using the SwissADME web platform, the researchers confirmed that every derivative satisfied Lipinski’s Rule of Five and Veber’s oral bioavailability criteria without a single violation. Molecular weights ranged from 279.29 to 385.42 daltons, comfortably below the 500-dalton ceiling, while calculated LogP values between 1.02 and 3.72 suggested a workable balance between membrane permeability and aqueous solubility. Topological polar surface area values of 46.92 to 84.22 square angstroms and rotatable bond counts of five to seven fell within Veber’s limits, predicting efficient passive diffusion across the gut wall. In short, all eleven molecules looked like plausible oral drugs on paper.

Safety screening came next, using ProTox-III, a machine-learning platform trained on curated toxicological datasets. Predicted acute oral LD50 values ranged from 500 to 2000 milligrams per kilogram, placing every compound in GHS Toxicity Class IV, a category comparable to many approved pharmaceuticals. Human intestinal absorption was predicted to exceed 80 percent for the entire series, and carcinogenicity and mutagenicity predictions were negative for nearly all derivatives. Notably, low predicted blood-brain barrier penetration for most compounds hints at a peripherally acting antihistamine with reduced sedation risk, the defining drawback of first-generation H1 antagonists. One caution did emerge: SP7 and SP8 showed moderate predicted cardiotoxicity, a signal the authors stress must be verified with hERG channel assays, since cardiac liability contributed to the market withdrawal of some earlier COX-2 inhibitors.

The docking results were where the study delivered its headline findings. Using Schrödinger’s Glide Extra Precision protocol against human COX-2, the crystal structure of which was solved bound to ibuprofen, the team found that SP2, a resorcinol derivative, scored minus 8.032 kilocalories per mole and SP8, a salicylic acid derivative, scored minus 9.566 kilocalories per mole, both far ahead of ibuprofen’s benchmark of minus 5.779. The protocol was validated by re-docking the co-crystallized ligands, yielding root-mean-square deviations of 1.83 and 1.78 angstroms for COX-2 and the H1 receptor respectively, both under the accepted 2.0-angstrom threshold. SP8’s strength appears to come from its ortho-hydroxyl and carboxylate groups working in concert: the carboxylate forms a salt bridge with Arg120 while hydrogen bonds anchor to Tyr355 and Ser530, and hydrophobic packing into the enzyme’s channel stabilizes the complex.

Against the histamine H1 receptor, solved by cryo-electron microscopy in complex with desloratadine, the top performers were SP2 at minus 8.021 and SP11, a butanol-bearing derivative, at minus 8.627 kilocalories per mole, both surpassing the reference antihistamine astemizole at minus 6.519. These compounds engage the orthosteric binding pocket through hydrogen bonds with Asp107, Tyr108, Ser111, and Asn198, complemented by aromatic stacking against Trp428, Tyr431, and Phe432. The extended butyl chain of SP11 appears to fill hydrophobic sub-pockets lined by Phe432, Phe435, and Trp428, consistent with established structure-activity rules for lipophilic H1 ligands. Intriguingly, the SAR analysis suggested that lengthening the alkyl chain preferentially boosts H1 affinity over COX-2 affinity, giving medicinal chemists a tunable dial for target balance.

When docking affinity, drug-likeness, and predicted safety were weighed together, three compounds rose to the top for different reasons. SP2 shows the most balanced dual-target engagement, with its COX-2 and H1 docking scores within 0.1 kilocalories per mole of each other. SP8 is the strongest single COX-2 binder of the series, and SP11 the strongest H1 binder. A fourth compound, SP9, showed promising H1-selective trends thanks to a methoxy group that occupies a hydrophobic sub-pocket absent from COX-2, but its overall dual-target and safety profile did not outperform the three leads, so it was not carried forward. The authors are careful to note that GlideScores are empirical rankings, not thermodynamic binding free energies, and cannot substitute for measured inhibition constants.

That caveat frames the study’s honest limitations. Static docking cannot capture protein flexibility or induced-fit effects, and machine-learning ADMET models can be unreliable for chemotypes poorly represented in their training data. The authors recommend a clear experimental roadmap: synthesize SP2, SP8, and SP11; run in vitro COX-2 inhibition and H1 receptor binding assays; establish COX-1/COX-2 selectivity to gauge gastrointestinal risk; complete full experimental ADMET profiling including hERG testing; and then move to in vivo efficacy studies in inflammation and anaphylaxis models, with molecular dynamics simulations guiding further optimization. Until those experiments are done, the dual-acting promise of these molecules remains a well-supported hypothesis rather than a demonstrated therapy. Still, as a rational, hypothesis-generating screen, the work offers a concrete starting point for a next generation of medicines aimed at the frequent clinical overlap between allergy and inflammation.

Subject of Research: Computational evaluation of novel benzimidazole derivatives as dual COX-2 inhibitors and histamine H1 receptor antagonists

Article Title: Integrated computational evaluation of novel benzimidazole scaffolds as dual COX-2 inhibitory and histamine H1 antagonistic activity

Article References: Pawar, S., & Panzade, P. (2026). Integrated computational evaluation of novel benzimidazole scaffolds as dual COX-2 inhibitory and histamine H1 antagonistic activity. Discover Chemistry, 3(1), Article 566. https://doi.org/10.1007/s44371-026-01024-6

Image Credits: AI Generated

DOI: 10.1007/s44371-026-01024-6

Keywords: benzimidazole, COX-2 inhibition, histamine H1 receptor, molecular docking, drug-likeness, ADMET prediction, SwissADME, ProTox-III, Glide XP docking, structure-activity relationship, computer-aided drug design, dual-target therapeutics

The research team designed ten new naproxen derivatives, labeled ND1 through ND10, by replacing the drug’s terminal carboxylic acid with an amide linkage attached to a variety of aromatic, heterocyclic, and aliphatic fragments. The naphthalene methoxy backbone that the cyclooxygenase enzymes recognize was left untouched, preserving the pharmacophore responsible for anti-inflammatory action. The logic is rooted in well-established medicinal chemistry: the acidic group anchors classical NSAIDs inside the COX channel through a salt bridge with the conserved Arg120 residue, but that same ionizable acid undergoes ion trapping in the acidic gastric lumen, directly irritates the epithelial barrier, and inhibits mitochondrial oxidative phosphorylation in stomach lining cells. Swapping the acid for a neutral amide could, in principle, keep the anchoring interactions while eliminating the gastric toxicity, and the amide’s terminal fragment could even reach into the less conserved side pocket that distinguishes COX-2 from COX-1, a strategy long used to convert non-selective NSAIDs into COX-2-preferring inhibitors.

To test this idea without synthesizing a single molecule, the researchers deployed a multi-layered computational pipeline. Density functional theory calculations at the B3LYP/6-31+G(d,p) level probed the electronic character of each derivative, yielding frontier molecular orbital energies, chemical hardness, softness, electronegativity, and electrophilicity. The results were striking: derivatives ND1 and ND4 showed the smallest HOMO-LUMO energy gaps, around 0.30 and 0.31 electron volts, compared with 0.87 electron volts for naproxen itself, indicating far greater electronic softness and reactivity. Soft molecules polarize more readily, which favors effective dispersion forces and electronic redistribution during non-covalent binding within the hydrophobic COX-2 pocket. ND2, meanwhile, posted the highest dipole moment at over 30 Debye, suggesting the strongest potential for polar interactions with the receptor. Molecular electrostatic potential mapping reinforced the picture, revealing that the high-polarization derivatives, including ND1, ND4, ND5, ND6, and ND9, retained sharply localized electron-rich and electron-poor regions capable of directional hydrogen bonding, effectively replacing the electrostatic role of the discarded carboxylic acid.

Before any binding predictions could be trusted, the docking protocol itself had to be validated. The researchers re-docked the co-crystallized ligands into the active sites of COX-1 and COX-2, using crystal structures 3KK6 and 3NT1 from the Protein Data Bank, and obtained root-mean-square deviations of 1.48 and 0.936 angstroms respectively, well within accepted thresholds. With the protocol confirmed, all ten derivatives were docked against both enzymes using AutoDock Vina and AutoDock4, alongside reference drugs including diclofenac, aspirin, mefenamic acid, and indomethacin. Every single derivative outperformed the parent drug. Naproxen scored roughly minus 7.5 kilocalories per mole against COX-2, while the derivatives ranged from minus 8.3 to minus 10.1 kilocalories per mole under Vina scoring. ND10, bearing an aminomethyl benzoic acid terminus, achieved the most favorable AutoDock4 score of minus 11.49 kilocalories per mole, driven largely by superior van der Waals contacts, while ND5’s bulky cycloheptylethyl group delivered a minus 10.43 kilocalories per mole result.

The docking poses revealed how the amide modification preserves potency through a clever molecular sleight of hand. Although converting the carboxylate into an amide removes the classical ionic salt bridge with Arg120, the amide carbonyl steps in as an alternative hydrogen-bond partner for the Arg120 guanidinium group in complexes ND3, ND5, and ND10. This neutral interaction, combined with deep penetration of the derivatives into the hydrophobic core near GLY526 and ALA527, provides a rational basis for COX-2 selectivity. Several complexes, including ND2, ND4, ND5, ND6, ND7, ND9, and ND10, reproduced the essential binding pattern of naproxen through pi-pi stacking with aromatic residues such as PHE381, TYR385, and TRP387, while ND4, ND6, and ND10 added hydrogen bonds and pi-sigma interactions with TYR385. By replacing the ionic anchor essential for COX-1 recognition with amide-based hydrogen bonding and expanded aromatic stacking, the derivatives exploit the larger active-site volume of COX-2 while potentially reducing the COX-1-mediated gastrointestinal harm of the parent acid.

Pharmacokinetic profiling through SwissADME and ProTox 3.0 added a crucial safety dimension. All ten derivatives fell safely below the 500 grams per mole Lipinski ceiling, satisfied hydrogen-bond donor and acceptor limits, and showed topological polar surface areas between 38 and 76 square angstroms, comfortably within the threshold for passive oral absorption. Predicted gastrointestinal absorption was high across the series, and all compounds were predicted non-cardiotoxic. The toxicity results were particularly encouraging: naproxen showed an LD50 of 248 milligrams per kilogram in toxicity class 3, whereas ND1 and ND8 exhibited LD50 values of 4000 and 5000 milligrams per kilogram in the far safer class 5. ND1, ND8, and ND10 were additionally predicted to be non-neurotoxic, and nearly all compounds showed inactive nephrotoxicity predictions, in contrast to active nephrotoxicity for naproxen itself. Caveats remain, however, as several analogues were predicted to inhibit multiple cytochrome P450 isoforms, flagging a potential risk of drug-drug interactions that will require experimental confirmation.

The three most promising candidates, ND2, ND4, and ND10, were then subjected to 100-nanosecond molecular dynamics simulations in GROMACS using the CHARMM36 force field, solvated in explicit water with physiological ion concentrations. The trajectories told a dramatic story. Naproxen repeatedly dissociated and rebounded at three distinct binding-site regions over the simulation, maintaining only intermittent hydrogen bonding of zero to two bonds and showing the highest conformational fluctuation of any system studied. ND4, by contrast, locked into its binding pose almost immediately and maintained a steady four to six hydrogen bonds, occasionally reaching seven, throughout the entire 100 nanoseconds. Its average root-mean-square deviation of 0.215 nanometers was the lowest of the series, and its solvent-accessible surface area of 255.90 square nanometers indicated the most compact, tightly bound complex. ND10 required roughly 50 nanoseconds to settle but then held its final pose with remarkable rigidity, achieving the steadiest plateau in the RMSD analysis.

Free-energy calculations using the MM/GBSA method over 501 trajectory snapshots quantified these differences with impressive precision. Naproxen’s binding free energy averaged minus 13 kilocalories per mole, while ND2 reached minus 16.5 and ND10 minus 25.4 kilocalories per mole. ND4 was in a class of its own at minus 46 kilocalories per mole, powered by an enormous van der Waals contribution of nearly minus 49 kilocalories per mole and an electrostatic term of minus 27 kilocalories per mole, reflecting its extended hydrogen-bond network with TYR324, ARG89, and ARG120. Per-residue energy decomposition confirmed that Arg120 contributed favorably to ND4 binding, demonstrating that the amide linkage had successfully recreated the anchoring interaction that the carboxylic acid once provided. Conformational analysis added a final layer of insight: 75 percent of the ND4 trajectory occupied a single conformational family, compared with only 43 percent for naproxen, whose energy landscape was fragmented across many shallow, poorly defined basins.

The authors are careful to frame these findings as exploratory rather than conclusive. Computational docking, dynamics, and ADMET predictions cannot fully capture the complexity of living systems, and predicted binding affinities do not always translate to in vitro or in vivo efficacy and safety. Experimental verification of the amide modification’s impact on cyclooxygenase selectivity, anti-inflammatory activity, metabolic stability, and gastrointestinal protection remains essential. Still, the study stands as a compelling demonstration of modern computer-aided drug design, showing how density functional theory, electrostatic mapping, validated docking, long-timescale simulation, and ensemble free-energy calculations can converge to identify ND4 and ND10 as lead candidates worthy of synthesis and biological testing. If laboratory work confirms what the simulations suggest, the humble painkiller in millions of medicine cabinets could one day be replaced by a safer, more potent descendant of itself.

Subject of Research: Computational design of naproxen amide derivatives as improved anti-inflammatory COX inhibitors

Article Title: Structure-based computational design of naproxen analogues with improved anti-inflammatory activity by DFT analysis, ADMET assessment, molecular docking, and molecular dynamics simulations

Article References: Abdullah, A., & Akhtar, M. (2026). Structure-based computational design of naproxen analogues with improved anti-inflammatory activity by DFT analysis, ADMET assessment, molecular docking, and molecular dynamics simulations. Results in Chemistry, 31, Article 103933. https://doi.org/10.1016/j.rechem.2026.103933

Image Credits: AI Generated

DOI: 10.1016/j.rechem.2026.103933

Keywords: naproxen, NSAIDs, COX-2 inhibition, computer-aided drug design, molecular docking, molecular dynamics simulation, DFT, ADMET, MM/GBSA, amide derivatives, gastrointestinal toxicity, drug discovery