Role of microsomal metabolism in bromfenac-induced cytotoxicity.

This study delves into the intricate mechanisms underlying drug-induced liver injury (DILI) with a specific focus on bromfenac, the withdrawn nonsteroidal anti-inflammatory drug. DILI is a pervasive concern in drug development, prompting market withdrawals and posing significant challenges to healthcare. Despite the withdrawal of bromfenac due to DILI, the exact role of its microsomal metabolism in inducing hepatotoxicity remains unclear. Herein, employing HepG2 cells with human liver microsomes and UDP-glucuronic acid (UDPGA), our investigation revealed a substantial increase in bromfenac-induced cytotoxicity in the presence of UDPGA, pointing to the significance of UDP-glucuronosyltransferase (UGT)-dependent metabolism in augmenting toxicity. Notably, among the recombinant UGTs examined, UGT2B7 emerged as a pivotal enzyme in the metabolic activation of bromfenac. Metabolite identification studies disclosed the formation of reactive intermediates, with bromfenac indolinone (lactam) identified as a potential mediator of hepatotoxic effects. Moreover, in cytotoxicity experiments, the toxicity of bromfenac lactam exhibited a 34-fold increase, relative to bromfenac. The toxicity of bromfenac lactam was mitigated by nicotinamide adenine dinucleotide phosphate-dependent metabolism. This finding underscores the role of UGT-dependent metabolism in generating reactive metabolites that contribute to the observed hepatotoxicity associated with bromfenac. Understanding these metabolic pathways and the involvement of specific enzymes, such as UGT2B7, provides crucial insights into the mechanisms of bromfenac-induced liver injury. In conclusion, this research sheds light on the metabolic intricacies leading to cytotoxicity induced by bromfenac, especially emphasizing the role of UGT-dependent metabolism and the formation of reactive intermediates like bromfenac lactam. These findings offer insight into the mechanistic basis of DILI and emphasize the importance of understanding metabolism-mediated toxicity.

The morphogenic protein CopD controls the spatio-temporal dynamics of PBP1a and PBP2b in Streptococcus pneumoniae.

IMPORTANCE: Penicillin-binding proteins (PBPs) are essential for proper bacterial cell division and morphogenesis. The genome of Streptococcus pneumoniae encodes for two class B PBPs (PBP2x and 2b), which are required for the assembly of the peptidoglycan framework and three class A PBPs (PBP1a, 1b and 2a), which remodel the peptidoglycan mesh during cell division. Therefore, their activities should be finely regulated in space and time to generate the pneumococcal ovoid cell shape. To date, two proteins, CozE and MacP, are known to regulate the function of PBP1a and PBP2a, respectively. In this study, we describe a novel regulator (CopD) that acts on both PBP1a and PBP2b. These findings provide valuable information for understanding bacterial cell division. Furthermore, knowing that ß-lactam antibiotic resistance often arises from PBP mutations, the characterization of such a regulator represents a promising opportunity to develop new strategies to resensitize resistant strains.

Metabolic engineering of Corynebacterium glutamicum for the high-level production of valerolactam, a nylon-5 monomer.

Valerolactam (VL) is an important precursor chemical for nylon-5 and nylon 6,5. It has been produced by petroleum-based route involving harsh reaction conditions and generating toxic wastes. Here, we report the complete biosynthesis of VL by metabolically engineered Corynebacterium glutamicum overproducing L-lysine. The pathway comprising L-lysine monooxygenase (davB) and 5-aminovaleramide amidohydrolase (davA) from Pseudomonas putida, and ß-alanine CoA transferase (act) from Clostridium propionicum was introduced into the C. glutamicum GA16 strain. To increase the VL flux, competitive pathways predicted from sRNA knockdown target screening were deleted. This engineered C. glutamicum strain produced VL as a major product, but still secreted significant amount of its precursor, 5-aminovaleric acid (5AVA). To circumvent this problem, putative 5AVA transporter genes were screened and engineered in the genome, thereby reuptaking 5AVA excreted. Also, multiple copies of the act gene were integrated into the genome to strengthen the conversion of 5AVA to VL. The final VL10 (pVL1) strain was constructed by enhancing glucose uptake system, which produced 9.68 g/L of VL in flask culture. Fed-batch fermentation of the VL10 (pVL1) strain produced 76.1 g/L of VL from glucose with the yield and productivity of 0.28 g/g and 0.99 g/L/h, respectively, showcasing a high potential for bio-based production of VL from renewable resources.

Reconstituting Spore Cortex Peptidoglycan Biosynthesis Reveals a Deacetylase That Catalyzes Transamidation.

Some bacteria survive in nutrient-poor environments and resist killing by antimicrobials by forming spores. The cortex layer of the peptidoglycan cell wall that surrounds mature spores contains a unique modification, muramic-δ-lactam, that is essential for spore germination and outgrowth. Two proteins, the amidase CwlD and the deacetylase PdaA, are required for muramic-δ-lactam synthesis in cells, but their combined ability to generate muramic-δ-lactam has not been directly demonstrated. Here we report an in vitro reconstitution of cortex peptidoglycan biosynthesis, and we show that CwlD and PdaA together are sufficient for muramic-δ-lactam formation. Our method enables characterization of the individual reaction steps, and we show for the first time that PdaA has transamidase activity, catalyzing both the deacetylation of N-acetylmuramic acid and cyclization of the product to form muramic-δ-lactam. This activity is unique among peptidoglycan deacetylases and is notable because it may involve the direct ligation of a carboxylic acid with a primary amine. Our reconstitution products are nearly identical to the cortex peptidoglycan found in spores, and we expect that they will be useful substrates for future studies of enzymes that act on the spore cortex.

Synthesis of Muramyl-δ-Lactam in Spore Peptidoglycan of Clostridioides difficile.

Clostridioides difficile is a spore-forming human pathogen responsible for significant morbidity and mortality. Infections by this pathogen ensue dysbiosis of the intestinal tract, which leads to germination of the spores. The process of spore formation requires a transition for the cell-wall peptidoglycan of the vegetative C. difficile to that of spores, which entails the formation of muramyl-δ-lactam. We describe a set of reactions for three recombinant C. difficile proteins, GerS, CwlD, and PdaA1, with the use of four synthetic peptidoglycan analogs. CwlD and PdaA1 excise the peptidoglycan stem peptide and the acetyl moiety of N-acetyl muramate, respectively. The reaction of CwlD is accelerated in the presence of GerS. With the use of a suitable substrate, we document that PdaA1 catalyzes a novel zinc-dependent transamidation/transpeptidation reaction, an unusual reaction that requires excision of the stem peptide as a pre-requisite.

Host lysolipid differentially modulates virulence factor expression and antimicrobial susceptibility in Pseudomonas aeruginosa.

Lysophosphatidic acid (LPA) occurs naturally in inflammatory exudates and has previously been shown to increase the susceptibility of Pseudomonas aeruginosa to ß-lactam antibiotics whilst concomitantly reducing accumulation of the virulence factors pyoverdine and elastase. Here it is demonstrated that LPA can also exert inhibitory effects upon pyocyanin production in P. aeruginosa, as well as influencing susceptibility to a wide range of chemically diverse non ß-lactam antimicrobials. Most strikingly, LPA markedly antagonizes the effect of the polycationic antibiotics colistin and tobramycin at a concentration of 250 µg ml-1 whilst conversely enhancing their efficacy at the lower concentration of 8.65 µg ml-1, approximating the maximal physiological concentrations found in inflammatory exudates. Transcriptomic responses of the virulent strain UCBPP-PA14 to LPA were analysed using RNA-sequencing along with BioLog phenoarrays and whole cell assays in attempts to delineate possible mechanisms underlying these effects. The results strongly suggest involvement of LPA-induced carbon catabolite repression together with outer-membrane (OM) stress responses whilst raising questions about the effect of LPA upon other P. aeruginosa virulence factors including type III secretion. This could have clinical relevance as it suggests that endogenous LPA may, at concentrations found in vivo, differentially modulate antibiotic susceptibility of P. aeruginosa whilst simultaneously regulating expression of virulence factors, thereby influencing host-pathogen interactions during infection. The possibility of applying exogenous LPA locally as an enhancer of select antibiotics merits further investigation.

Structural Basis of the Main Proteases of Coronavirus Bound to Drug Candidate PF-07321332.

The high mutation rate of COVID-19 and the prevalence of multiple variants strongly support the need for pharmacological options to complement vaccine strategies. One region that appears highly conserved among different genera of coronaviruses is the substrate-binding site of the main protease (Mpro or 3CLpro), making it an attractive target for the development of broad-spectrum drugs for multiple coronaviruses. PF-07321332, developed by Pfizer, is the first orally administered inhibitor targeting the main protease of SARS-CoV-2, which also has shown potency against other coronaviruses. Here, we report three crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome (MERS)-CoV bound to the inhibitor PF-07321332. The structures reveal a ligand-binding site that is conserved among SARS-CoV-2, SARS-CoV, and MERS-CoV, providing insights into the mechanism of inhibition of viral replication. The long and narrow cavity in the cleft between domains I and II of the main protease harbors multiple inhibitor-binding sites, where PF-07321332 occupies subsites S1, S2, and S4 and appears more restricted than other inhibitors. A detailed analysis of these structures illuminated key structural determinants essential for inhibition and elucidated the binding mode of action of the main proteases from different coronaviruses. Given the importance of the main protease for the treatment of SARS-CoV-2 infection, insights derived from this study should accelerate the design of safer and more effective antivirals. IMPORTANCE The current pandemic of multiple variants has created an urgent need for effective inhibitors of SARS-CoV-2 to complement vaccine strategies. PF-07321332, developed by Pfizer, is the first orally administered coronavirus-specific main protease inhibitor approved by the FDA. We solved the crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and MERS-CoV that bound to the PF-07321332, suggesting PF-07321332 is a broad-spectrum inhibitor for coronaviruses. Structures of the main protease inhibitor complexes present an opportunity to discover safer and more effective inhibitors for COVID-19.

Expanded profiling of ß-lactam selectivity for penicillin-binding proteins in Streptococcus pneumoniae D39.

Penicillin-binding proteins (PBPs) are integral to bacterial cell division as they mediate the final steps of cell wall maturation. Selective fluorescent probes are useful for understanding the role of individual PBPs, including their localization and activity during growth and division of bacteria. For the development of new selective probes for PBP imaging, several ß-lactam antibiotics were screened, as they are known to covalently bind PBP in vivo. The PBP inhibition profiles of 16 commercially available ß-lactam antibiotics were evaluated in an unencapsulated derivative of the D39 strain of Streptococcus pneumoniae, IU1945. These ß-lactams have not previously been characterized for their PBP inhibition profiles in S. pneumoniae and these data augment those obtained from a library of 20 compounds that we previously reported. We investigated seven penicillins, three carbapenems, and six cephalosporins. Most of these ß-lactams were found to be co-selective for PBP2x and PBP3, as was noted in our previous studies. Six out of 16 antibiotics were selective for PBP3 and one molecule was co-selective for PBP1a and PBP3. Overall, this work expands the chemical space available for development of future ß-lactam-based probes for specific pneumococcal PBP labeling and these methods can be used for the development of probes for PBP labelling in other bacterial species.

Exploring the Molecular Basis of Substrate and Product Selectivities of Nocardicin Bifunctional Thioesterase.

D-amino acid introduction in peptides can enrich their biological activities and pharmacological properties as potential drugs. This achievement of stereochemical inversion usually owes to an epimerase or racemase. Interestingly, a unique bifunctional thioesterase (NocTE), which is incorporated in nonribosomal peptide synthetase (NRPS) NocA-NocB assembly line for the biosynthesis of monocyclic ß-lactam antibiotic nocardicin A, can control the generation of D-products with high stereochemical purity. However, the molecular basis of NocTE selectivity on substrates and products is still unclear. Herein, we constructed a series of systems with different peptides varying in stereochemistry, length, and composition to investigate the substrate selectivity. The studies on binding affinities and loading conformations elucidated the important roles of peptide length and ß-lactam ring in substrate selectivity. Through energy decomposition and interaction analyses, some key residues involved in substrate selectivity were captured. On the other hand, natural product undergoing epimerization was found to be liberated from the active pocket more easily in comparison with its diastereomer (epi-nocardicin G), explaining the superiority of nocardicin G. These results provide detailed molecular insights into the exquisite control of substrate and product scopes for NocTE, and encourage to diversification of substrates and final products for NRPS assembly line. The molecular insights into substrate and product selectivities of unique bifunctional thioesterase NocTE were illustrated via several molecular simulations and free energy calculations, contributing to expanding substrate and product scopes of nonribosomal peptide synthetases.

Combinatorial Biosynthesis of Oxidized Combamides Using Cytochrome P450 Enzymes from Different Polycyclic Tetramate Macrolactam Pathways.

Polycyclic tetramate macrolactams (PoTeMs) are a family of natural products containing a tetramic acid moiety and a polycyclic system. Due to the valuable biological activities of different PoTeMs and the genetic simplicity of their biosynthetic genes, it is highly desirable to manipulate the biosynthesis of PoTeMs by swapping modification genes between different pathways. Herein, by combining the cytochrome P450 (CYP) enzymes from different PoTeM pathways with the combamides' biosynthetic genes, the new combamides G (3), I (5), and J (6) along with the known combamides B (1), D (2), and H (4) were identified from the recombinant strains. Combamides G (3), H (4), and J (6) displayed cytotoxic activity against human cancer cell lines. Furthermore, our results demonstrated for the first time the substrate specificity of the PoTeM-related CYPs in vivo, which will facilitate the engineered biosynthesis of other PoTeMs in the future.

Thiocysteine lyases as polyketide synthase domains installing hydropersulfide into natural products and a hydropersulfide methyltransferase.

Nature forms S-S bonds by oxidizing two sulfhydryl groups, and no enzyme installing an intact hydropersulfide (-SSH) group into a natural product has been identified to date. The leinamycin (LNM) family of natural products features intact S-S bonds, and previously we reported an SH domain (LnmJ-SH) within the LNM hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line as a cysteine lyase that plays a role in sulfur incorporation. Here we report the characterization of an S-adenosyl methionine (SAM)-dependent hydropersulfide methyltransferase (GnmP) for guangnanmycin (GNM) biosynthesis, discovery of hydropersulfides as the nascent products of the GNM and LNM hybrid NRPS-PKS assembly lines, and revelation of three SH domains (GnmT-SH, LnmJ-SH, and WsmR-SH) within the GNM, LNM, and weishanmycin (WSM) hybrid NRPS-PKS assembly lines as thiocysteine lyases. Based on these findings, we propose a biosynthetic model for the LNM family of natural products, featuring thiocysteine lyases as PKS domains that directly install a -SSH group into the GNM, LNM, or WSM polyketide scaffold. Genome mining reveals that SH domains are widespread in Nature, extending beyond the LNM family of natural products. The SH domains could also be leveraged as biocatalysts to install an -SSH group into other biologically relevant scaffolds.

A lipoprotein allosterically activates the CwlD amidase during Clostridioides difficile spore formation.

Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. During gemination, spores must degrade their cortex layer, which is a thick, protective layer of modified peptidoglycan. Cortex degradation depends on the presence of the spore-specific peptidoglycan modification, muramic-∂-lactam (MAL), which is specifically recognized by cortex lytic enzymes. In C. difficile, MAL production depends on the CwlD amidase and its binding partner, the GerS lipoprotein. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind Zn2+ stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to Zn2+, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of Zn2+ co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.

Computational simulations on the binding and reactivity of a nitrile inhibitor of the SARS-CoV-2 main protease.

We present a detailed computational analysis of the binding mode and reactivity of the novel oral inhibitor PF-07321332 developed against the SARS-CoV-2 3CL protease. Alchemical free energy calculations suggest that positions P3 and P4 could be susceptible to improvement in order to get a larger binding strength. QM/MM simulations unveil the reaction mechanism for covalent inhibition, showing that the nitrile warhead facilitates the recruitment of a water molecule for the proton transfer step.

Challenges of short substrate analogues as SARS-CoV-2 main protease inhibitors.

Specific anti-coronaviral drugs complementing available vaccines are urgently needed to fight the COVID-19 pandemic. Given its high conservation across the betacoronavirus genus and dissimilarity to human proteases, the SARS-CoV-2 main protease (Mpro) is an attractive drug target. SARS-CoV-2 Mpro inhibitors have been developed at unprecedented speed, most of them being substrate-derived peptidomimetics with cysteine-modifying warheads. In this study, Mpro has proven resistant towards the identification of high-affinity short substrate-derived peptides and peptidomimetics without warheads. 20 cyclic and linear substrate analogues bearing natural and unnatural residues, which were predicted by computational modelling to bind with high affinity and designed to establish structure-activity relationships, displayed no inhibitory activity at concentrations as high as 100 µM. Only a long linear peptide covering residues P6 to P5' displayed moderate inhibition (Ki = 57 µM). Our detailed findings will inform current and future drug discovery campaigns targeting Mpro.

Escherichia coli-Derived γ-Lactams and Structurally Related Metabolites Are Produced at the Intersection of Colibactin and Fatty Acid Biosynthesis.

Colibactin is a genotoxic hybrid polyketide-nonribosomal peptide that drives colorectal cancer initiation. While clinical data suggest colibactin genotoxicity in vivo is largely caused by the major DNA-cross-linking metabolite, the colibactin locus produces a diverse collection of metabolites with mostly unknown biological activities. Here, we describe 10 new colibactin pathway metabolites (1-10) that are dependent on its α-aminomalonyl-carrier protein. The most abundant metabolites, 1 and 2, were isolated and structurally characterized mainly by nuclear magnetic resonance spectroscopy to be γ-lactam derivatives, and the remaining related structures were inferred via shared biosynthetic logic. Our proposed formation of 1-10, which is supported by stereochemical analysis, invokes cross-talk between colibactin and fatty acid biosynthesis, illuminating further the complexity of this diversity-oriented pathway.

Evolutionary and functional analysis of an NRPS condensation domain integrates ß-lactam, á´ -amino acid, and dehydroamino acid synthesis.

Nonribosomal peptide synthetases (NRPSs) are large, multidomain biosynthetic enzymes involved in the assembly-line-like synthesis of numerous peptide natural products. Among these are clinically useful antibiotics including three classes of ß-lactams: the penicillins/cephalosporins, the monobactams, and the monocyclic nocardicins, as well as the vancomycin family of glycopeptides and the depsipeptide daptomycin. During NRPS synthesis, peptide bond formation is catalyzed by condensation (C) domains, which couple the nascent peptide with the next programmed amino acid of the sequence. A growing number of additional functions are linked to the activity of C domains. In the biosynthesis of the nocardicins, a specialized C domain prepares the embedded ß-lactam ring from a serine residue. Here, we examine the evolutionary descent of this unique ß-lactam-synthesizing C domain. Guided by its ancestry, we predict and demonstrate in vitro that this C domain alternatively performs peptide bond formation when a single stereochemical change is introduced into its peptide starting material. Remarkably, the function of the downstream thioesterase (TE) domain also changes. Natively, the TE directs C terminus epimerization prior to hydrolysis when the ß-lactam is made but catalyzes immediate release of the alternative peptide. In addition, we investigate the roles of C-domain histidine residues in light of clade-specific sequence motifs, refining earlier mechanistic proposals of both ß-lactam formation and canonical peptide synthesis. Finally, expanded phylogenetic analysis reveals unifying connections between ß-lactam synthesis and allied C domains associated with the appearance of ᴅ-amino acid and dehydroamino acid residues in other NRPS-derived natural products.

Holomycin, an Antibiotic Secondary Metabolite, Is Required for Biofilm Formation by the Native Producer Photobacterium galatheae S2753.

While the effects of antibiotics on microorganisms are widely studied, it remains less well understood how antibiotics affect the physiology of the native producing organisms. Here, using a marine bacterium, Photobacterium galatheae S2753, that produces the antibiotic holomycin, we generated a holomycin-deficient strain by in-frame deletion of hlmE, the core gene responsible for holomycin production. Mass spectrometry analysis of cell extracts confirmed that the ΔhlmE strain did not produce holomycin and that the mutant was devoid of antibacterial activity. Biofilm formation of the ΔhlmE strain was significantly reduced compared to that of wild-type S2753 and was restored in an hlmE complementary mutant. Consistent with this, exogenous holomycin, but not its dimethylated and less antibacterial derivative, S,S'-dimethyl holomycin, restored the biofilm formation of the ΔhlmE strain. Furthermore, zinc starvation was found to be essential for both holomycin production and biofilm formation of S2753, although the molecular mechanism remains elusive. Collectively, these data suggest that holomycin promotes biofilm formation of S2753 via its ene-disulfide group. Lastly, the addition of holomycin at subinhibitory concentrations also enhanced the biofilms of four other Vibrionaceae strains. P. galatheae likely gains an ecological advantage from producing holomycin as both an antibiotic and a biofilm stimulator, which facilitates nutrition acquisition and protects P. galatheae from environmental stresses. Studying the function of antibiotic compounds in the native producer will shed light on their roles in nature and could point to novel bioprospecting strategies.IMPORTANCE Despite the societal impact of antibiotics, their ecological functions remain elusive and have mostly been studied by exposing nonproducing bacteria to subinhibitory concentrations. Here, we studied the effects of the antibiotic holomycin on its native producer, Photobacterium galatheae S2753, a Vibrionaceae bacterium. Holomycin provides a distinct advantage to S2753 both as an antibiotic and by enhancing biofilm formation in the producer. Vibrionaceae species successfully thrive in global marine ecosystems, where they play critical ecological roles as free-living, symbiotic, or pathogenic bacteria. Genome mining has demonstrated that many have the potential to produce several bioactive compounds, including P. galatheae To unravel the contribution of the microbial metabolites to the development of marine microbial ecosystems, better insight into the function of these compounds in the producing organisms is needed. Our finding provides a model to pursue this and highlights the ecological importance of antibiotics to the fitness of the producing organisms.

Enzymatic Formation of Indolactam Scaffold by C-N Bond-Forming Cytochrome P450 Oxidases in Teleocidin Biosynthesis.

Teleocidins are potent protein kinase C activators, and possess a unique indole-fused nine-membered lactam structure. Teleocidin biosynthesis starts from the formation of a dipeptide by non-ribosomal peptide synthetase (NRPS), followed by oxidative C-N bond formation by a cytochrome P450 oxidase, reverse-prenylation by a prenyltransferase, and methylation-initiated terpene cyclization by a C-methyltransferase. This minireview focuses on recent research progress toward the elucidation of the molecular basis for the remarkable P450-catalyzed intramolecular C-N bond-forming reaction, which is challenging in synthetic chemistry, to generate the indolactam scaffold. In addition, precursor-directed biosynthesis with the promiscuous P450 enzymes led to the formation of a series of unnatural and novel molecular scaffolds, including a sulfur-substituted indolactam with a different conformation from that of indolactam V.

Heterologous Expression of the Biosynthetic Gene Cluster for Verticilactam and Identification of Analogues.

Verticilactam and the new geometric isomers, verticilactams B and C, were produced by heterologous expression of the biosynthetic gene cluster for verticilactam using the Streptomyces avermitilis SUKA17 strain. Only verticilactam, a compound with a characteristic ß-ketoamide unit within a 16-membered polyketide macrolactam conjugated with an octalin skeleton, had been previously reported having been isolated from Streptomyces spiroverticillatus JC-8444. In this report, minor verticilactam derivatives were isolated from the transformed strain, and their structures elucidated by spectral analysis. Verticilactam B was a geometric isomer at Δ17 and Δ19, and verticilactam C was the Δ19 and Δ21 isomer. In addition, the absolute configuration of verticilactam was confirmed by ECD analysis and NMR chemical shifts. The stereochemistry assignments of the hydroxy groups at C-10 and C-12 were supported by the domain organization of the polyketide synthase identified in the verticilactam gene cluster. Verticilactam showed moderate activity against the malaria parasite Plasmodium falciparum 3D7 strain with no significant cytotoxicity or antimicrobial effects.

Boosting the sensitivity of in vitroß-lactam allergy diagnostic tests.

The synthesis of structurally new haptens and the development of suitable antigens are essential for boosting the sensitivity of drug allergy diagnostic testing. Unprecedented structural antigens for benzylpenicillin and amoxicillin are characterised and evaluated in a cohort of 70 subjects with a turnkey solution based on consumer electronics.