Scientists Discover Potent Biphenyl-Macolacin Derivatives Through Iterative Scanning and Chemical Ligation

A New Molecular Strategy Produces Biphenyl-Macolacin Antibiotic Candidates Active Against Resistant Bacteria

The search for antibiotics that can outmaneuver drug-resistant bacteria has yielded a new set of laboratory-made candidates derived from biphenyl-macolacin, a peptide antibiotic with activity against Gram-negative pathogens, including some bacteria resistant to colistin. In a study published in Molecular Diversity, researchers from China describe a chemical strategy that rapidly maps which parts of the molecule can be altered without destroying its antibacterial power. The approach generated four classes of biphenyl-macolacin derivatives, several of which performed as well as or better than the parent compound in antibacterial tests. Three representative molecules—identified as compounds 5, 18 and 46—also showed encouraging results in experiments examining the emergence of resistance and damage to red blood cells. The findings do not yet represent a new medicine, but they offer medicinal chemists a structured route for turning a promising natural-product scaffold into a broader family of potential antibiotics.

Biphenyl-macolacin belongs to a group of peptide-based molecules that are attractive in the fight against Gram-negative bacteria because their chemistry can be tuned to interact with the unusual envelope surrounding these organisms. Gram-negative cells are protected by an outer membrane rich in lipopolysaccharide, a large molecule that contributes to the barrier’s negative charge and helps restrict the entry of many conventional antibiotics. Peptide antibiotics can exploit electrostatic and hydrophobic interactions with this membrane, potentially disrupting its organization or enabling the compound to reach vulnerable targets. The clinical importance of this area is underscored by the spread of resistance to colistin, itself a last-resort drug often used against infections caused by multidrug-resistant Gram-negative bacteria. Biphenyl-macolacin has attracted interest because its reported activity includes several colistin-resistant pathogens, suggesting that its molecular architecture may provide a useful starting point for antibiotic design even when existing drugs have failed.

The challenge is that natural antibiotics are often chemically intricate. Their activity can depend on a precise three-dimensional arrangement of amino acids, unusual linkages and hydrophobic groups, meaning that changing one position may improve potency, reduce it or make no difference at all. The researchers addressed this problem with a method they call lysine-T/CDHA iterative scanning, or LTIS. In a conventional scanning experiment, individual residues in a peptide are systematically replaced to determine which positions are essential and which can tolerate modification. The LTIS strategy extends that logic by using lysine-containing substitutions and a T/CDHA design framework to interrogate the molecule’s structure in a stepwise way. Rather than relying on isolated trial-and-error modifications, the researchers used the resulting activity patterns to establish a map of modifiable sites across the biphenyl-macolacin scaffold.

That map is important because it separates the chemical “load-bearing walls” of the antibiotic from regions that can serve as attachment points for new features. A residue that is indispensable for folding, membrane recognition or biological activity is unlikely to tolerate a bulky chemical replacement. By contrast, a permissive site may accept a polar group, a hydrophobic substituent or another peptide fragment that changes solubility, stability, distribution or interaction with bacterial membranes. The researchers synthesized the scanned analogues and compared their antibacterial performance, using the results to build a systematic structure–activity relationship. In medicinal chemistry, a structure–activity relationship is more than a catalogue of compounds: it links a particular molecular change to a measurable biological consequence. Such information helps researchers prioritize the next generation of molecules instead of repeatedly rebuilding inactive candidates.

After the scanning phase, the team used chemical ligation to expand the scaffold. Chemical ligation is a form of molecular assembly in which separately prepared fragments are joined through a chemoselective reaction that favors the intended functional groups. The study used ligation chemistry based on serine or threonine-related peptide junctions, a strategy that can form natural-looking peptide bonds at positions that are difficult to access through ordinary stepwise synthesis. In practical terms, this allows chemists to construct the core and modified segments independently, purify them and then connect them under controlled conditions. For complex peptide antibiotics, that modularity can make it easier to introduce structural diversity while preserving the parts of the molecule already known to support activity. The researchers describe the ligation-based stage as a convenient way to create additional biphenyl-macolacin analogues after LTIS had identified promising sites for derivatization.

The resulting library comprised four classes of derivatives. Although the study’s abstract does not report a single numerical potency value for every compound, it states that several analogues exhibited antibacterial activities comparable to, or greater than, biphenyl-macolacin. This comparison is significant because improving a natural product is not simply a matter of making it more chemically elaborate. Each modification must preserve the balance among bacterial activity, chemical stability and compatibility with host tissues. An added group may strengthen contact with the bacterial envelope but also increase nonspecific binding to mammalian membranes. Alternatively, it may improve water solubility while weakening the hydrophobic interactions needed for antibacterial action. By combining systematic scanning with ligation, the researchers created a way to explore these trade-offs across multiple chemical families rather than focusing on a single derivative.

Compounds 5, 18 and 46 were selected for additional biological evaluation. One test examined the potential for resistance development, a crucial step for any antibiotic candidate because a molecule that works in the first experiment may quickly lose effectiveness if bacteria can adapt to it. Resistance studies typically expose bacterial populations to repeated or sustained drug pressure and monitor whether susceptibility declines over successive passages. The source study reports that these representative analogues underwent resistance-development evaluation, but the available article information does not provide the full numerical profiles or identify a universal resistance-free result. That distinction matters: a favorable laboratory trend is not proof that resistance cannot arise in patients. Nevertheless, testing resistance potential at this early stage can reveal whether a scaffold deserves deeper investigation and may help researchers compare derivatives with different structural features.

The same three compounds were also assessed in hemolysis assays, which measure whether a candidate damages red blood cells by disrupting their membranes. Hemolysis is a particularly relevant safety signal for membrane-active peptides because the same physicochemical properties that destabilize bacterial membranes can, in some circumstances, affect mammalian cells. A low hemolytic effect relative to antibacterial potency suggests a potentially useful therapeutic window, although it does not establish safety in the body. The researchers included hemolysis testing alongside antibacterial and resistance experiments, allowing activity and an important form of preliminary toxicity to be considered together. The abstract describes the efficacy of compounds 5, 18 and 46 as demonstrated through resistance-development evaluation and hemolysis assay, but it does not present enough detail to conclude that any of the candidates is ready for animal testing or clinical development.

The study’s broader contribution is therefore methodological as much as pharmacological. Antibiotic discovery increasingly depends on finding compounds that can attack resistant organisms while avoiding rapid resistance and unacceptable toxicity. Natural peptide scaffolds offer chemical possibilities, but their optimization can be slow when every analogue must be designed and synthesized independently. LTIS provides a systematic way to identify editable positions, while chemical ligation supplies a modular route for attaching new structures at those positions. Together, the methods generate a feedback loop: scanning reveals how the scaffold works, synthesis tests the proposed rules, and biological assays refine the map for the next round of design. The authors present their structure–activity relationship as a reference for future biphenyl-macolacin-based antibiotics and suggest that the strategy could also be useful for other peptide therapeutics. Further work will need to establish precise mechanisms of action, activity across clinically relevant bacterial panels, pharmacological behavior, toxicity in animals and effectiveness in infection models. For now, the molecules are promising chemical leads—not approved treatments—but they illustrate how detailed molecular engineering could help reopen the antibiotic pipeline against pathogens that have learned to resist some of medicine’s most powerful drugs.