Nicolò Santi

The rise of antimicrobial resistance (AMR) poses a critical threat to global health, mostly due to the proliferation of β‐lactamases, a class of enzymes responsible for the inactivation of β‐lactam antibiotics. Among the most promising strategies to restore β‐lactam efficacy is the use of β‐lactamase inhibitors (BLIs), with boronic acid derivatives emerging as a pivotal and chemically versatile class. These compounds act as transition‐state analogs, forming reversible covalent bonds with the catalytic serine of serine β‐lactamases (SBLs), effectively mimicking the tetrahedral intermediate of β‐lactam hydrolysis. Their electron‐deficient boron atom, combined with a tunable scaffold, allows fine modulation of potency, selectivity, and pharmacokinetic properties. This review traces the evolution of boronic acid‐based BLIs from early phenylboronic acids to next‐generation acyclic and cyclic derivatives, highlighting key structure‐activity relationships, binding mechanisms, and microbiological profiles. Clinically approved agents such as vaborbactam, as well as investigational compounds including taniborbactam, xeruborbactam, and benzoxaboroles, are discussed in the context of their therapeutic relevance and spectrum of activity. Particular attention is given to their ability to inhibit class A and C enzymes, with ongoing efforts aimed at extending coverage to class D and metallo‐β‐lactamases. Additionally, the review explores innovative approaches such as kinetic target‐guided synthesis and fragment‐based design to expand the chemical space of boronic acid pharmacophores. Together, these advances underscore the potential of boronic acid‐based BLIs as powerful tools in overcoming β‐lactamase‐mediated resistance and developing next‐generation antimicrobial therapies.

Penicillin-binding proteins (PBPs) are essential enzymes involved in bacterial cell wall biosynthesis and represent the primary targets of β-lactam antibiotics. However, the efficacy of these agents is threatened by β-lactamase production and PBP alterations, prompting the search for alternative strategies. In this context, boronic acids, long established as potent inhibitors of serine β-lactamases (SBLs), have been proposed as scaffolds for PBP inhibition based on the shared structural and mechanistic features of these enzyme families. This perspective provides a literature-based survey with structural analysis to evaluate emerging evidence on the potential role of boronic acids as PBP-targeting agents, with a particular focus on peptidomimetic boronic acids, repurposed β-lactamase inhibitors, and novel scaffold architectures. While early work showed limited activity against low-molecular-mass PBPs, more recent compounds, particularly certain bicyclic boronates, have demonstrated potent binding and, in some cases, antibacterial activity. Structural analyses reveal diverse binding modes and underscore the role of conformational dynamics in modulating affinity. Despite these advances, significant challenges remain, including target selectivity, membrane permeability, and species-specific differences. Nevertheless, the direct inhibition of PBPs by boronic acids, while still in early development, may offer a viable complement or alternative to β-lactam therapy, warranting further exploration through structure-guided design and comprehensive biological evaluation. Here, we analyze the potential of boronic acid inhibitors (BAIs) to target PBP enzymes, considering their promise as non-β-lactam antimicrobial agents with possible clinical relevance.

In this study, we evaluated in situ click chemistry as a platform for discovering boronic acid-based β-lactamase inhibitors (BLIs). Unlike conventional drug discovery approaches requiring multi-step synthesis, protection strategies, and extensive screening, the in situ method can allow for the generation and identification of potent β-lactamase inhibitors in a rapid, economic, and efficient way. Using KPC-2 (class A carbapenemase) and AmpC (class C cephalosporinase) as templates, we demonstrated their ability to catalyse azide-alkyne cycloaddition, facilitating the formation of triazole-based β-lactamase inhibitors. Initial screening of various β-lactamases and boronic warheads identified compound 3 (3-azidomethylphenyl boronic acid) as the most effective scaffold for kinetic target-guided synthesis (KTGS). KTGS experiments with AmpC and KPC-2 yielded triazole inhibitors with K_i values as low as 140 nM (compound 10a, AmpC) and 730 nM (compound 5, KPC-2). Competitive inhibition studies confirmed triazole formation within the active site, while an LC–MS analysis verified that the reversible covalent interaction of boronic acids did not affect detection of the in situ-synthesised product. While KTGS successfully identified potent inhibitors, limitations in amplification coefficients and spatial constraints highlight the need for optimised warhead designs. This study validates KTGS as a promising strategy for BLI discovery and provides insights for further refinement in fighting β-lactamase-mediated antibiotic resistance.

In this study we evaluated in situ click chemistry as platform for discovering boronic acid-based β-lactamase inhibitors (BLIs). Unlike conventional drug discovery approaches requiring multi-step synthesis, protection strategies, and extensive screening, the in situ method can allow the generation and identification of potent β-lactamase inhibitors in a rapid, economic and efficient way. Using KPC-2 (class A carbapenemase) and AmpC (class C cephalosporinase) as templates, we demonstrated their ability to catalyse azide-alkyne cycloaddition, facilitating the formation of triazole-based β-lactamase inhibitors. Initial screening of various β-lactamases and boronic warheads identified compound 3 (3-azidomethylphenyl boronic acid) as the most effective scaffold for Kinetic Target-Guided Synthesis (KTGS). KTGS experiments with AmpC and KPC-2 yielded triazole inhibitors with K_i values as low as 140 nM (compound 10a, AmpC) and 730 nM (compound 5, KPC-2). Competitive inhibition studies confirmed triazole formation within the active site, while LC-MS analysis verified that the reversible covalent interaction of boronic acids did not affect detection of the in situ synthesised product. While KTGS successfully identified potent inhibitors, limitations in amplification coefficients and spatial constraints highlight the need for optimised warhead designs. This study validates KTGS as a promising strategy for BLI discovery and provides insights for further refinement in fighting β-lactamase-mediated antibiotic resistance.

Class C Acinetobacter-derived cephalosporinases (ADCs) represent an important target for inhibition in the multidrug-resistant pathogen Acinetobacter baumannii. Many ADC variants have emerged, and characterization of their structural and functional differences is essential. Equally as important is the development of compounds that inhibit all prevalent ADCs despite these differences. The boronic acid transition state inhibitor, MB076, a novel heterocyclic triazole with improved plasma stability, was synthesized and inhibits seven different ADC β-lactamase variants with K_i values <1 μM. MB076 acted synergistically in combination with multiple cephalosporins to restore susceptibility. ADC variants containing an alanine duplication in the Ω-loop, specifically ADC-33, exhibited increased activity for larger cephalosporins, such as ceftazidime, cefiderocol, and ceftolozane. X-ray crystal structures of ADC variants in this study provide a structural context for substrate profile differences and show that the inhibitor adopts a similar conformation in all ADC variants, despite small changes near their active sites.

Here, we combine the use of host screening, protein crystallography and QM/MM molecular dynamics simulations to investigate how protein enviroment affects iminium catalysis by biotinylated secondary amines in a model 1,4 conjugate addition reaction.

Schistosomiasis is a neglected tropical disease that currently affects over 250 million individuals worldwide. In the absence of an immunoprophylactic vaccine and the recognition that mono-chemotherapeutic control of schistosomiasis by praziquantel has limitations, new strategies for managing disease burden are urgently needed. A better understanding of schistosome biology could identify previously undocumented areas suitable for the development of novel interventions. Here, for the first time, we detail the presence of G-quadruplexes (G4) and putative quadruplex forming sequences (PQS) within theSchistosoma mansonigenome. We find that G4 are present in both intragenic and intergenic regions of the seven autosomes as well as the sex-defining allosome pair. Amongst intragenic regions, G4 are particularly enriched in 3´ UTR regions. Gene Ontology (GO) term analysis evidenced significant G4 enrichment in thewntsignalling pathway (pwnt-related genes resolve into parallel and anti-parallel G4 motifs as elucidated by circular dichroism (CD) spectroscopy. Finally, utilising a single chain anti-G4 antibody called BG4, we confirm thein situpresence of G4 within both adult female and male worm nuclei. These results collectively suggest that G4-targeted compounds could be tested as novel anthelmintic agents and highlights the possibility that G4-stabilizing molecules could be progressed as candidates for the treatment of schistosomiasis.

Here, the streptavidin–biotin technology was applied to enable organocatalytic transfer hydrogenation. By introducing a biotin-tethered pyrrolidine (1) to the tetrameric streptavidin (T-Sav), the resulting hybrid catalyst was able to mediate hydride transfer from dihydro-benzylnicotinamide (BNAH) to α,β-unsaturated aldehydes. Hydrogenation of cinnamaldehyde and some of its aryl-substituted analogues was found to be nearly quantitative. Kinetic measurements revealed that the T-Sav:1 assembly possesses enzyme-like behavior, whereas isotope effect analysis, performed by QM/MM simulations, illustrated that the step of hydride transfer is at least partially rate-limiting. These results have proven the concept that T-Sav can be used to host secondary amine-catalyzed transfer hydrogenations.

Background: Schistosomiasis is a neglected tropical disease that currently affects over 250 million individuals worldwide. In the absence of an immunoprophylactic vaccine and the recognition that mono-chemotherapeutic control of schistosomiasis by praziquantel has limitations, new strategies for managing disease burden are urgently needed. A better understanding of schistosome biology could identify previously undocumented areas suitable for the development of novel interventions. Methodology/Principal findings: Here, for the first time, we detail the presence of G-quadruplexes (G4) and putative quadruplex forming sequences (PQS) within the Schistosoma mansoni genome. We find that G4 are present in both intragenic and intergenic regions of the seven autosomes as well as the sex- defining allosome pair. Amongst intragenic regions, G4 are particularly enriched in 3’ UTR regions. Gene Ontology (GO) term analysis evidenced significant G4 enrichment in the wnt signalling pathway ( p wnt -related genes resolve into parallel and hybrid G4 motifs as elucidated by circular dichroism (CD) spectroscopy. Finally, utilising a single chain anti-G4 antibody called BG4, we confirm the in situ presence of G4 within both adult female and male worm nuclei. Conclusion/Significance: These results collectively suggest that G4-targeted compounds could be tested as novel anthelmintic agents and highlights the possibility that G4-stabilizing molecules could be progressed as candidates for the treatment of schistosomiasis. Author Summary: Schistosoma mansoni causes schistosomiasis, a parasitic disease that affects millions of people living in resource-deprived areas of developing countries. No vaccine exists and the current drug treatment has limitations, notably inefficacy against the larval stages of the parasite. New drugs are, therefore, needed to sustainably control schistosomiasis. A further understanding of parasite biology will uncover new targets and lead to the development of novel therapies. Here, we identify the presence of G-Quadruplexes (G4s) in S. mansoni . G4s are four-stranded DNA structures that can affect gene function and, to date, have not been previously found in any parasitic helminth. Computational analysis predicted potential G4 folding sequences within the S. mansoni genome, several of which were confirmed to fold by circular dichroism spectroscopy. Analysis of G4-containing protein coding genes found an enrichment within the wnt signalling pathway, a developmental pathway crucial for axial development in the parasite. Additionally, G4s could be detected within adult worms using a fluorescent antibody that selectively recognises quadruplex structures in nucleic acids. This research describes the presence of a previously unknown structure within the parasite, which could present a new target for developing novel treatments.

In this report, the streptavidin-biotin technology was applied to enable organocatalytic aldol addition. By attaching pyrrolidine to the valeric motif of biotin and introducing it to streptavidin (Sav), a protein-based organocatalytic system was created, and the aldol addition of acetone with p-nitrobenzaldehyde was tested. The conversion of substrate to product can be as high as 93%. Although the observed enantioselectivity was only moderate (33:67 er), further protein engineering efforts can be included to improve the selectivity. These results have proven the concept that Sav can be used to host stereoselective aldol addition.