The study, published in the journal Molecular Diversity, was led by Binglin Huang and Bijuan Lin of Fujian Medical University Union Hospital, together with colleagues at Fujian Medical University and Nanjing University of Chinese Medicine. Rather than relying on the well-characterized binding groove that VHL uses to recognize hydroxylated HIF-1α, the team took a different route. They used DDMut-PPI, a graph-based deep-learning tool that predicts how mutations at a protein-protein interface affect the interaction, to nominate residues that might serve as auxiliary contact points beyond the canonical binding site. Those predicted residues were then used to build an alternative pharmacophore model, a simplified three-dimensional map of the chemical features a molecule would need to wedge itself into the interface and interfere with the HIF-1α/VHL handshake.

With that model in hand, the researchers screened a library of 24,893 molecules computationally, using docking tools including AutoDock4, which allows selective receptor flexibility during the docking calculation. Candidates that docked favorably against the alternative pharmacophore were then carried into the laboratory, where a fluorescence-polarization assay measured how well each compound competed with a labeled HIF-1α peptide for binding to VHL. Four molecules emerged with half-maximal inhibitory concentrations, or IC50 values, below 10 micromolar, a respectable starting point for compounds discovered without any structure-guided medicinal chemistry.

The standout was a compound the authors labeled Cmpd16, which turned out to be ixazomib, a proteasome inhibitor already approved for use in multiple myeloma under the trade name Ninlaro. In the fluorescence-polarization assay, Cmpd16 showed the highest affinity of the four hits, with an IC50 of 0.41 micromolar. The finding that an existing drug can bind VHL and interfere with HIF-1α recognition is notable because it opens the possibility of repurposing: a molecule with known pharmacokinetics, safety data, and manufacturing routes could, in principle, be repositioned for a completely different indication, provided the new activity holds up in more demanding biological systems.

The team then asked whether the compound actually did what a VHL inhibitor should do inside cells. In their reported assays, Cmpd16 showed no detectable loss of cell viability, an important early safety signal, and it produced a VHL-dependent pattern of stabilization affecting both HIF-1α and the hydroxylated form of the protein. In other words, when VHL’s grip is loosened, even hydroxylated HIF-1α, which would normally be destroyed regardless of oxygen levels, lingers in the cell. That is precisely the biochemical signature expected from a molecule that blocks the VHL recognition step downstream of the oxygen-sensing hydroxylases, and it mirrors the mechanism of dedicated VHL inhibitors developed over the past decade by academic and industrial groups.

To test whether this molecular effect translated into protection under stress, the researchers used an oxygen-glucose deprivation and reoxygenation model, a standard laboratory mimic of ischemia and reperfusion injury in which cells are deprived of both oxygen and nutrients and then resupplied. At a concentration of 10 micromolar, Cmpd16 improved the migration of endothelial cells and their ability to form tube-like structures, processes central to the formation of new blood vessels that resupply damaged tissue. The treated cells also showed increased levels of vascular endothelial growth factor, VEGF, and the glucose transporter GLUT1, both of which are canonical HIF-1α target genes that support angiogenesis and metabolic adaptation. At the same time, the compound was associated with reduced accumulation of reactive oxygen species and reduced levels of cleaved caspase-3, a hallmark of apoptosis, suggesting that the cells were not only adapting but genuinely surviving the insult better.

Because the initial hit came from a computational model rather than from a crystal structure of a ligand bound in the interface, the authors took care to interrogate how plausible their predicted binding mode really was. They ran three independently initialized molecular dynamics simulations of 200 nanoseconds each using the Desmond engine. Across the replicas, the simulations showed recurring contacts between the ligand and two residues, Pro99 and His110, in VHL, but the overall dynamics of both the protein and the ligand varied from replica to replica. The authors are explicit about what this means: the simulations support a computationally plausible orientation for Cmpd16 rather than a unique, well-defined one. That honesty matters, because molecular dynamics can be seductive, and overstating a single binding pose from a handful of trajectories is a well-known pitfall in computational drug discovery.

The team also compared two selected ten-compound panels to probe whether their deep-learning-informed pharmacophore enriched for active molecules more effectively than a conventional approach. The comparison, analyzed with a two-sided Fisher’s exact test, yielded a p-value of 0.0867, which falls short of conventional statistical significance. The authors describe this analysis as exploratory, and they further caution that the causal role of the predicted interface residues remains unproven until experimental mutagenesis is performed. These caveats are worth emphasizing: the study demonstrates a viable discovery pipeline and a promising hit, but the mechanistic details of where and how the compound binds VHL are still hypotheses awaiting direct experimental confirmation.

The broader context makes the work interesting on several fronts. The HIF pathway has long been a drug-discovery target, though most clinical success has come from a different angle: inhibitors of the prolyl hydroxylase enzymes, such as daprodustat and enarodustat, which are approved or in late-stage development for anemia in chronic kidney disease. Those drugs prevent HIF-1α from being tagged in the first place. Directly blocking the VHL-HIF interaction is a more recent strategy, and potent chemical probes with nanomolar affinities have been reported, largely built around a conserved hydroxyproline mimetic scaffold. The structural diversity of known VHL ligands has remained narrow, which is exactly the gap the new study set out to address by looking for chemically distinct molecules through an alternative interface model. Finding an approved proteasome inhibitor within that chemically distinct set is an unexpected twist, since ixazomib was designed to inhibit the proteasome’s catalytic activity, not to bind an E3 ubiquitin ligase.

There is also a pleasing irony in the biology. VHL ligands were originally developed largely as tools for targeted protein degradation, forming the binding arm of PROTAC molecules that recruit VHL to destroy disease-causing proteins. Here, the same interaction is being blocked for the opposite purpose: to prevent VHL from destroying a protein the body needs during oxygen crisis. Whether Cmpd16, or molecules inspired by it, can eventually protect tissues in living models of ischemia remains to be seen, and the distance from an endothelial-cell dish to a patient’s heart or kidney is considerable. But the study offers a concrete example of how deep-learning predictions of interface residues can reshape the search space in virtual screening, and it adds a repurposing candidate, and a set of testable residue hypotheses, to a pathway whose pharmacological importance continues to grow. The next steps, mutagenesis of the predicted contact residues, co-crystallography or cryo-EM of the ligand bound to VHL, and validation in animal models of ischemic injury, will determine whether this computational detour leads to a genuinely new way of protecting cells when oxygen runs out.

Subject of Research: Computational discovery of small-molecule inhibitors of the HIF-1α/VHL protein-protein interaction for protection of cells under hypoxic stress

Article Title: Computational discovery of HIF-1α/VHL protein–protein interaction inhibitors for hypoxic cell protection

Article References: Computational discovery of HIF-1α/VHL protein–protein interaction inhibitors for hypoxic cell protection. (n.d.). https://doi.org/10.1007/s11030-026-11706-z

Image Credits: AI Generated

DOI: 10.1007/s11030-026-11706-z

Keywords: HIF-1α, VHL, protein-protein interaction, virtual screening, deep learning, ixazomib, hypoxia, ischemia, molecular dynamics, drug repurposing, endothelial cells, pharmacophore

The synthetic route itself is elegantly simple. Substituted aromatic aldehydes, methyl cyanoacetate and barbituric acid are combined in a 1:1 mixture of ethanol and water at 85 degrees Celsius with just 0.03 mol percent of Bi(OTf)3. The catalyst orchestrates a domino Knoevenagel-Michael sequence: it coordinates to the ester oxygen of methyl cyanoacetate, sharpening the electrophilicity of the carbonyl carbon, while the aldehyde-derived enolate attacks to form a carbon-carbon bond. The resulting alpha,beta-unsaturated intermediate is then struck by the weakly nucleophilic nitrogen of barbituric acid, and intramolecular cyclization closes the fused pyridopyrimidine ring. Eight derivatives, bearing nitro, bromo, hydroxyl, methyl, methoxy or unsubstituted phenyl groups, were isolated in yields ranging from 76 to 91 percent within two to five hours, with minimal side products and no need to purify intermediates.

What makes the protocol genuinely green is the solvent system and the catalyst. Bismuth(III) salts have gained a reputation as environmentally benign alternatives to conventional Lewis acids because they are air- and moisture-stable, functionally tolerant, commercially available and far less corrosive than many metal halides. Compared with earlier methods, the advantages are clear. A DMAP-catalyzed protocol requires ultrasonic irradiation in toxic DMF, ZrO2 nanoparticles demand nanomaterial synthesis and raise agglomeration concerns, and the sulfonated SBA-15 catalyst involves a cumbersome multistep preparation. The bismuth route runs in a drinkable solvent mixture under ordinary reflux, and the catalyst can be recovered, although reusability tests showed a gradual decline: the model reaction yielded 79 percent in the first cycle, 74 percent in the second, 65 percent in the third and only 33 percent in a fourth run that stretched to nine hours. The authors suggest that immobilization strategies could extend catalyst lifetime in future work.

On the computational side, the team optimized all eight molecules at the B3LYP/6-31G(d,p) level of density functional theory, confirming that each geometry corresponds to a true energy minimum. Frontier molecular orbital analysis revealed that the nitro-substituted compounds 4a and 4b possess the smallest HOMO-LUMO gaps, at roughly 0.135 electron volts, making them the most electronically soft and reactive members of the series, primed for donor-acceptor interactions with biological macromolecules. In contrast, the bromo, methyl and unsubstituted derivatives 4c, 4f and 4g showed the largest gaps and the greatest hardness, indicating higher kinetic stability. Mulliken charge analysis and molecular electrostatic potential maps pinpointed the carbonyl oxygens and ring nitrogens as the electron-rich hotspots most likely to engage in hydrogen bonding with protein targets, while the N-H hydrogens carried the complementary positive charge.

The biological evaluation used the broth dilution method to determine minimum inhibitory concentrations against four bacterial strains, the Gram-negative Escherichia coli and Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus and Streptococcus pyogenes, benchmarked against ampicillin, and three fungal strains, Candida albicans, Aspergillus niger and Aspergillus clavatus, benchmarked against griseofulvin. The structure-activity trends were striking. The unsubstituted compound 4g and the methoxy-bearing 4h were the standout antibacterial agents, with MIC values as low as 65 and 62 micrograms per milliliter respectively against E. coli and S. aureus, figures that actually beat ampicillin on those strains. The methyl derivative 4f was the most potent antifungal, matching griseofulvin’s 100 micrograms per milliliter against A. niger. Electron-donating groups such as methyl and methoxy generally enhanced activity, likely by improving lipophilicity and membrane penetration, while nitro groups tended to raise MIC values.

To explain these observations mechanistically, the researchers docked all eight compounds into two clinically significant enzymes: bacterial beta-lactamase, the molecular engine of antibiotic resistance, and sterol 14-alpha demethylase CYP51, the fungal enzyme that builds ergosterol membranes. Using AutoDock 4.2 with a Lamarckian genetic algorithm, they found that compounds 4b and 4h bound beta-lactamase most favorably, with estimated free energies of binding of minus 9.30 and minus 9.43 kilocalories per mole, stabilized by networks of hydrogen bonds, pi-alkyl contacts and pi-cation interactions with residues such as ARG 661, PRO 298 and TYR 611. Against CYP51, compound 4b again led with minus 7.79 kilocalories per mole, and the methyl-substituted 4f formed six hydrogen bonds within the active site, consistent with its antifungal potency. Torsional free energies were nearly uniform across the series, indicating that differences in affinity stem from noncovalent interaction networks rather than ligand flexibility.

Molecular dynamics simulations then stress-tested the most promising complexes over 100-nanosecond production runs using the Maestro-Desmond package with the OPLS3e force field. The 4f-beta-lactamase complex held a backbone root mean square deviation averaging 1.60 angstroms, comfortably below the 2.0 angstrom threshold that validates docking reliability, and stabilized after an initial 30-nanosecond equilibration. The 4d-CYP51 complex averaged 1.40 angstroms, closely tracking the reference antifungal ligand VT1 at 1.35 angstroms. Root mean square fluctuation analysis showed that flexibility was confined to loop and terminal regions, leaving the binding pockets rigid. Contact histograms revealed that water-bridged hydrogen bonds, involving residues such as Ser64, Gln120, Thr122 and Tyr132, together with hydrophobic contacts, were the main glue holding the complexes together throughout the simulations.

The pharmacoinformatic layer of the study added yet another dimension. POM analysis, which classifies bioactivity based on dipolar interactions between electron-rich and electron-deficient centers, identified three distinct antitumor pharmacophore regions built from NH donor to carbonyl oxygen acceptor pairs, plus a single antibacterial pharmacophore involving an NH-CO motif. OSIRIS toxicity screening found no major mutagenic, tumorigenic, irritant or reproductive risks across the series, with compounds 4d and 4g earning the highest drug scores at 0.70 and 0.87. Molinspiration calculations confirmed that all eight molecules respect Lipinski’s criteria almost entirely: molecular weights below 500 daltons, five or fewer rotatable bonds, moderate polarity and acceptable lipophilicity, with only the heavily nitrated pair showing a single violation.

Taken together, the work demonstrates a rare degree of coherence between experiment and computation. The compounds with the best MIC values, notably 4f, 4g and 4h, also posted strong docking energies and stable dynamic behavior, while the electrostatic potential maps independently flagged the same carbonyl and ring nitrogen atoms that docking identified as interaction points. The authors conclude that pyrido[2,3-d]pyrimidines represent a promising scaffold for further antimicrobial development, and that targeted substitution of the aromatic ring is the key lever for tuning activity. With antimicrobial resistance declared one of the top global public health threats, a synthetic method that is cheap, green and high-yielding, paired with a validated computational pipeline for prioritizing candidates, offers exactly the kind of integrated approach the field has been calling for. The next step will be translating these laboratory and in silico hits into lead compounds capable of surviving the far harsher tests of animal models and, eventually, the clinic.

Subject of Research: Green synthesis and antimicrobial evaluation of pyrido[2,3-d]pyrimidine derivatives catalyzed by bismuth(III) triflate

Article Title: Bi(OTf)₃-catalyzed one-pot synthesis and antimicrobial evaluation of bioactive pyridopyrimidines supported by DFT, molecular dynamics, POM and pharmacophore analyses

Article References: Mehta, A. W., Abdel-Megid, M., Patil, R. C., Ahmed, S., Salem, M. E., Abu-Rayyan, A., Shtaiwi, A., Hajam, Y. A., Bhat, A. R., Eissa, M. E., Agisho, H. A., Mujahid, M. H., Yamari, I., Raza, N., & Zbancioc, A. M. (2026). Bi(OTf)₃-catalyzed one-pot synthesis and antimicrobial evaluation of bioactive pyridopyrimidines supported by DFT, molecular dynamics, POM and pharmacophore analyses. Results in Chemistry, 30, Article 103849. https://doi.org/10.1016/j.rechem.2026.103849

Image Credits: AI Generated

DOI: 10.1016/j.rechem.2026.103849

Keywords: pyridopyrimidines, bismuth(III) triflate, multicomponent reaction, green chemistry, antimicrobial activity, molecular docking, molecular dynamics, DFT, beta-lactamase, CYP51, drug discovery, pharmacophore