Malcolm G. P. Page

Siderophore cephalosporins are designed to exploit bacterial nutrient uptake systems to gain accelerated uptake across the outer membrane of Gram-negative bacteria. They contain iron (III) binding motifs that allow them to form complexes that will be recognized as potential substrates by iron-siderophore transport systems. Research during the last five decades has culminated in the approval for clinical use of the siderophore cephalosporin cefiderocol, which incorporates accumulated learning from investigations of structural features that enhance resistance toward hydrolysis by β-lactamases, that promote bacterial membrane permeability, and that confer long pharmacokinetic half-life in the human host.

A standardized scheme for numbering and annotation of secondary structure features has been proposed for class D β-lactamases (F. Attana, S. Kim, J. Spencer, B. I. Iorga, et al., Antimicrob Agents Chemother 69:e00150-25, 2025, https://doi.org/10.1128/aac.00150-25 ). This will significantly facilitate the comparison of biochemical and biophysical studies performed on the disparate members of this class of enzymes and improve communication in the field.

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-β-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential β-lactamase stable β-lactam mimics. Subsequent structure–activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL–carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.

Objective: To biochemically, structurally, and microbiologically determine the ability and mechanism of 3‐(4‐phenylamino‐phenylamino)‐2‐(1H‐tetrazol‐5‐yl)‐acrylonitrile (FTA) ( Figure 1A ) as an inhibitor of PDC. Hypothesis: Given the mechanistic and structural similarities in Class A and Class C β‐lactamases, we hypothesized FTA would bind a modeled hydrophobic allosteric site in PDC analogous to the site it binds in Class A β‐lactamases. Methods: Steady state kinetic constants were determined spectrophotometrically using a nitrocefin reporter assay. β‐Lactamase variants were cloned in pET24a+ expression vectors and purified using cation exchange and size exclusion chromatography. Growth curves were determined by measuring OD ₆₀₀ readings with a microplate reader. Circular dichroism (CD) melting curves and X‐ray crystal structures of both apo and FTA‐bound PDC‐3 WT were determined. Results: FTA demonstrates competitive inhibition behavior and does so without time dependence. Apparent inhibition constant ( K _{I app} ) values ( Figure 2B ) for both PDC‐3 WT and PDC‐3 E219K (a variant notable for increased resistance to ceftolozane and ceftazidime, two key anti‐Pseudomonal β‐lactams) were both roughly an order of magnitude lower than in class A β‐lactamases. FTA also impacts the growth kinetics of E. coli expressing PDC‐3 WT in sublethal concentrations of antibiotic ( Figure 2A ), dramatically reducing the slope of growth curves through log phase in the presence of 1000 µM FTA and reducing the lowest concentration of antibiotic preventing pellet formation by five (WT) to six (E219K) dilutions. Importantly, FTA alone does not appear to be toxic to E. coli expressing PDC variants in the absence of an antibiotic (e.g. does not impact growth rates) and CD reveals negligible change in the melting curve of PDC‐3 E219K on the addition of 100 µM FTA and a melting point decrease of less than 1°C for PDC‐3 WT, substantially less than the difference between WT and E219K alone. Consistent with steady state kinetics, X‐ray crystallography ( Figure 1B ) reveals active site density after FTA soaking experiments that could fit an FTA molecule. The nitrile moiety of FTA forms a hydrogen bond with serine 64, the key residue involved in formation of the acyl‐enzyme complex, suggesting a novel inhibition mechanism. Conclusions: FTA is a better inhibitor of PDC than class A β‐lactamases and serves as a competitive, active site inhibitor in PDC in contrast to a non‐competitive, allosteric inhibitor in class A. Given that it does not resemble a β‐lactam or a currently available β‐lactamase inhibitor, FTA may serve as a starting point for the development of future β‐lactamase inhibitors. Additionally, these vastly different mechanisms in different classes will serve as the basis of further investigations into the similarities and differences in structure‐activity relationships in class A and class C β‐lactamases.

Resistance to β-lactam antibacterials, importantly via production of β-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both serine- (SBL) and metallo- (MBL) β-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C β-lactamase from Escherichia coli, by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied β-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D β-lactamases, our data support the proposal that bicyclic boronates are broad-spectrum β-lactamase inhibitors that work by mimicking a high energy ‘tetrahedral’ intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful β-lactamase inhibition.

A standard numbering scheme has been proposed for class C β-lactamases. This will significantly enhance comparison of biochemical and biophysical studies performed on different members of this class of enzymes and facilitate communication in the field.

Iron is an essential nutrient for bacterial growth, replication, and metabolism. Humans store iron bound to various proteins such as hemoglobin, haptoglobin, transferrin, ferritin, and lactoferrin, limiting the availability of free iron for pathogenic bacteria. However, bacteria have developed various mechanisms to sequester or scavenge iron from the host environment. Iron can be taken up by means of active transport systems that consist of bacterial small molecule siderophores, outer membrane siderophore receptors, the TonB-ExbBD energy-transducing proteins coupling the outer and the inner membranes, and inner membrane transporters. Some bacteria also express outer membrane receptors for iron-binding proteins of the host and extract iron directly from these for uptake. Ultimately, iron is acquired and transported into the bacterial cytoplasm. The siderophores are small molecules produced and released by nearly all bacterial species and are classified according to the chemical nature of their iron-chelating group (ie, catechol, hydroxamate, α-hydroxyl-carboxylate, or mixed types). Siderophore-conjugated antibiotics that exploit such iron-transport systems are under development for the treatment of infections caused by gram-negative bacteria. Despite demonstrating high in vitro potency against pathogenic multidrug-resistant bacteria, further development of several candidates had stopped due to apparent adaptive resistance during exposure, lack of consistent in vivo efficacy, or emergence of side effects in the host. However, cefiderocol, with an optimized structure, has advanced and has been investigated in phase 1 to 3 clinical trials. This article discusses the mechanisms implicated in iron uptake and the challenges associated with the design and utilization of siderophore-mimicking antibiotics.

Bacteria use small molecules called siderophores to scavenge iron. Siderophore-Fe³⁺complexes are recognised by outer-membrane transporters and imported into the periplasm in a process dependent on the inner-membrane protein TonB. The siderophore enterobactin is secreted by members of the family Enterobacteriaceae, but many other bacteria includingPseudomonasspecies can use it. Here, we show that thePseudomonastransporter PfeA recognises enterobactin using extracellular loops distant from the pore. The relevance of this site is supported by in vivo and in vitro analyses. We suggest there is a second binding site deeper inside the structure and propose that correlated changes in hydrogen bonds link binding-induced structural re-arrangements to the structural adjustment of the periplasmic TonB-binding motif.

Nuclear magnetic resonance and infrared spectroscopy have been used to investigate the formation of complexes of BAL30072 with Fe³⁺ and Ga³⁺ in solution and to collect geometrical parameters supporting reliable 3D structure models. Structural models for the ligand–metal complexes with different stoichiometries have been characterized using density functional theory calculations. Blind ensemble docking to the PiuA receptor from P. aeruginosa was performed for the different complexes to compare binding affinities and statistics of the residues most frequently contacted. When compared to analogues, BAL30072 was found to have an intrinsic propensity to form complexes with low ligand-to-metal stoichiometry. By using one of the sulfate oxygen atoms as a third donor in addition to the bidentate pyridinone moiety, BAL30072 can form a L₂M complex, which was predicted to be the one with the best binding affinity to PiuA. The example of BAL30072 strongly suggests that a lower stoichiometry might be the one recognized by the receptor, so that to focus only on the highest stoichiometry might be misleading for siderophores with less than six donors.

New strategies are urgently required to develop antibiotics. The siderophore uptake system has attracted considerable attention, but rational design of siderophore antibiotic conjugates requires knowledge of recognition by the cognate outer-membrane transporter. Acinetobacter baumannii is a serious pathogen, which utilizes (pre)acinetobactin to scavenge iron from the host. We report the structure of the (pre)acinetobactin transporter BauA bound to the siderophore, identifying the structural determinants of recognition. Detailed biophysical analysis confirms that BauA recognises preacinetobactin. We show that acinetobactin is not recognised by the protein, thus preacinetobactin is essential for iron uptake. The structure shows and NMR confirms that under physiological conditions, a molecule of acinetobactin will bind to two free coordination sites on the iron preacinetobactin complex. The ability to recognise a heterotrimeric iron-preacinetobactin-acinetobactin complex may rationalize contradictory reports in the literature. These results open new avenues for the design of novel antibiotic conjugates (trojan horse) antibiotics.