Low-affinity Targets Research Articles

Abstract 1235: Impact of anti-CD3 and tumor-target binder affinities on in-vitro potency, in-vivo efficacy, and cytokine release

Abstract Background: First generation T cell bispecifics (TCBs) were developed by utilizing high CD3 and tumor target binder affinities to induce a potent response. Instrumentalizing this, TCBs have clinically proven to elicit efficacious target cell killing and anti-tumor efficacy, but they encountered hurdles related to cytokine release. Recent studies suggested that reducing CD3 binder affinity could lead to an efficient tumor killing with lower cytokine levels. However, the answer to whether cytokine release is mainly dependent on CD3 binder affinity or a multifactorial phenomenon remains ambiguous. Here, we aim to characterize the efficacy-safety relationship of different TCBs through investigating the interplay between CD3 binder affinity and the affinity of the tumor target-specific binder. For that purpose, a series of TCBs with varying CD3 affinities, and two target binders of high and low affinities were generated in the 2:1 TCB format, resulting in a bivalent binding to the tumor antigen and a monovalent one to CD3, which offers a unique opportunity to enhance the activity of TCBs. Methods: Firstly, 2:1 TCBs comprising either the high or low affinity target binder and varying CD3 binder affinities were co-cultured in-vitro with human peripheral blood mononuclear cells (PBMCs), and human tumor cell lines, in order to assess T cell mediated cytotoxicity and cytokine release at different time points. Additionally, the impact on in-vivo efficacy, cytokine release, and pharmacokinetics was assessed in humanized mice implanted with a patient-derived xenograft model. Results: Based on the dose-response in the cytotoxicity assay, the CD3 binders were classified into high, intermediate, and low levels of affinity. As expected, we observed that the in-vitro T cell cytotoxicity, in-vivo tumor growth inhibition (TGI), and cytokine release levels were maximal with the highest affine CD3 binder. Simultaneously, we have observed a decrease in cytotoxicity response with decreasing CD3 affinity compared to the high affinity binder. However, this was accompanied by an atypical decrease in cytokine release. Particularly, the TCB comprising the intermediate CD3 affinity binder illustrated a comparable in-vitro cytotoxicity and in-vivo TGI to the high affinity binder with a marked decrease in cytokine release [4x reduction in-vitro and 6x reduction in-vivo]. Upon testing the TCBs comprising the low affinity target binder with the same CD3 binders, we observed an additional decrease in cytokine release with the decreasing CD3 affinity. In conclusion, we designed and generated TCBs with varying affinities in both the CD3 and target binding arms. Reducing cytokine release while maintaining adequate efficacy is feasible through CD3 binder affinity attenuation; however, the target binder affinity should be taken into consideration when interpreting our findings and designing new molecules. Citation Format: Omar Abdelmotaleb, Anneliese Schneider, Thomas Hofer, Johannes Sam, Martin Lechmann, Inja Waldhauer, Ali Bransi, Miro Eigenmann, Anne Freimoser-Grundschober, Christian Gassner, Alex Odermatt, Peter Brünker, Sara Colombetti, Christian Klein. Impact of anti-CD3 and tumor-target binder affinities on in-vitro potency, in-vivo efficacy, and cytokine release [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1235.

Abstract 1119: Systems pharmacology approaches to study tumor drug mechanism of action

Abstract Precision cancer medicine is predicated on the ability to identify drugs whose mechanism of action (MoA) matches one or more key tumor dependencies. Although the structure of many target proteins is available and their binding affinity to specific compounds can be experimentally assessed, drug efficacy and toxicity are infrequently determined based on single target affinity. Rather, the pharmacologic property of drugs is often the result of its complex polypharmacology, as mediated by both unknown or poorly characterized off-target effects (i.e., lower-affinity binding proteins) as well as by tissue-specific secondary effector proteins (i.e., non-binding) that are undetectable by traditional assays. MoA elucidation typically relies on in vitro affinity binding assays tailored to specific protein classes, such as kinases or metabolic enzymes, which cannot reveal critical high- and low-affinity binding targets among other protein classes, as well as context-specific downstream effectors. The goal of this study is to address critical unresolved questions in cancer pharmacology by characterizing the proteome-wide MoA of oncology drugs as a critical, yet highly elusive step necessary to fully understand their clinical efficacy and toxicity. To elucidate proteome-wide, drug-mediated changes in protein activity (drug MoA), we performed network-based analyses of genome-wide RNA-seq profiles representing the response of patient-matched cell lines to a comprehensive repertoire of clinically relevant drugs. More specifically, we generated genome-wide drug perturbation profiles from 23 cancer cell representing high-fidelity models of patients in clinical cohorts representing distinct tumor subtypes, using > 700 oncology drugs. This represents the largest resources of functionally annotated, clinically-relevant, genome-wide perturbational profiles for clinically relevant drugs, named PanACEA. VIPER-based analysis of this resource effectively elucidated the effect of each individual drug on the activity of ~6,500 regulatory and signaling proteins, in each of the 23 distinct represented cancer subtypes. Analyses of these data, using a graph theory approach, helped elucidate critical functional relationship between individual drugs and group drugs into functionally-distinct modules within each cancer context, thus providing critical insight into both drug MoA and polypharmacology. These analyses also effective drug inhibitors for critical cancer dependencies represented by transcription factors and co-factors, considered undruggable, that were experimentally validated. Finally, integrative analysis of PANCEA predictions and Cancer Dependency Map data significantly improved our understanding of cancer-related drug pharmacology, thus providing a unique resource for cancer related studies, which has been used in key clinical and pre-clinical trials, both published and in review. Citation Format: Lucas Zhongming Hu, Eugene Douglass, Ron Realubit, Karan Charles, Mariano Alvarez, Andrea Califano. Systems pharmacology approaches to study tumor drug mechanism of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1119.

Facile Display of Homomultivalent Proteins for In Vitro Selections.

Low-affinity protein binders are emerging as valuable domains for therapeutic applications because of their higher specificity when presented in multivalent ligands that increase the overall strength and selectivity of receptor binding. De novo discovery of low-affinity binders would be enhanced by the large library sizes attainable with in vitro selection systems, but these platforms generally maximize recovery of high-affinity monovalent binders. Here, we present a facile technology that uses rolling circle amplification to create homomultivalent libraries. We show proof of principle of this approach in ribosome display with off-rate selections of a bivalent ligand against monovalent and bivalent targets, thereby demonstrating high enrichment (up to 166-fold) against a low-affinity target that is bivalent but not monovalent. This approach to homomultivalent library construction can be applied to any binder tolerant of N- and C-terminal fusions and provides a platform for performing in vitro display selections with controlled protein valency and orientation.

BMP9 and Fluid Shear Stress Regulate Endothelial Chromatin Accessibility and Expression of SMAD Low Affinity Target Genes

BMP9 and Fluid Shear Stress Regulate Endothelial Chromatin Accessibility and Expression of SMAD Low Affinity Target Genes

Analysis of human Tribbles 2 (TRIB2) pseudokinase.

Human Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a broad human protein interactome, including the well-studied AKT, C/EBPα and MAPK modules. Several lines of evidence indicate that human TRIB2 promotes cell survival and drug-resistance in solid tumors and blood cancers and is therefore of interest as a potential therapeutic target, although its physiological functions remain relatively poorly understood. The unique TRIB2 pseudokinase domain lacks the canonical 'DFG' motif, and subsequently possesses very low affinity for ATP in both the presence and absence of metal ions. However, TRIB2 also contains a unique cysteine-rich αC-helix, which interacts with a conserved peptide motif in its own carboxyl-terminal tail. This regulatory flanking region drives regulated interactions with distinct E3 ubiquitin ligases that serve to control the stability and turnover of TRIB2 client proteins. TRIB2 is also a low-affinity target of several known small-molecule protein kinase inhibitors, which were originally identified using purified recombinant TRIB2 proteins and a thermal shift assay. In this chapter, we discuss laboratory-based procedures for purification, stabilization and analysis of human TRIB2, including screening procedures that can be used for the identification of both reversible and covalent small molecule ligands.

The development of [18F]ACI‐12589, a high affinity and selective alpha‐synuclein radiotracer, as a biomarker for Parkinson’s disease and other synucleinopathies

AbstractBackgroundParkinson’s disease (PD) and other alpha‐synucleinopathies are characterized by the progressive accumulation of alpha‐synuclein (a‐syn) inclusions in the brain, which start in the pre‐motor stage of the disease and correlates with its severity. An imaging agent for a‐syn could allow differential diagnosis and support interventional trails. AC Immune has developed and is currently testing in a first in human (FiH) clinical trial the recently discovered compound, ACI‐12589, as a PET tracer with high affinity and selectivity for a‐syn.MethodScreening of our conformation‐specific, small‐molecular weight MorphomerTM library was followed by an iterative medicinal chemistry optimization program. The primary screening strategy included the development and use of a proprietary binding assay, that importantly involved aggregates derived from PD brain samples. Autoradiography experiments performed with human tissue samples from different alpha‐synucleinopathies provided target engagement and signal specificity data. Autoradiography techniques were also employed to assess selectivity, using tissue samples from Alzheimer’s disease or Frontotemporal lobar degeneration‐TDP donors. Selected compounds were 18F radiolabeled and evaluated for their pharmacokinetics in non‐human primates (NHPs) before proceeding to clinical studies.ResultRecent optimization efforts have led to the identification of several new, highly promising chemical scaffolds. From these, the compound ACI‐12589, has emerged with highly favorable characteristics including low nanomolar affinity and excellent target occupancy in saturation binding experiments using PD brain homogenates. Using high‐resolution autoradiography, ACI‐12589 illustrates target engagement across multiple alpha‐synucleinopathies including PD, Multiple System Atrophy, and Dementia with Lewy Bodies. In classical autoradiography experiments, ACI‐12589 demonstrates significantly higher specific signal in diseased versus non‐diseased tissue samples. Furthermore, ACI‐12589 has at least 30‐fold selectivity versus Abeta and shows no binding to Tau nor TDP‐43 aggregates. Studying [18F]ACI‐12589 in NHPs reveals rapid brain uptake and quick wash out. ACI‐12589 was considered safe in the IND‐enabling toxicology study allowing advancement into the FiH clinical study.ConclusionBased on experience with other tracers, [18F]ACI‐12589 meets the target product profile deemed necessary of a highly favorable candidate to succeed as a radiotracer for imaging a‐syn inclusions. As such, [18F]ACI‐12589 has advanced into the First in Human clinical trial in patients with Parkinson’s disease to be followed by evaluation in other alpha‐synucleinopathies.

Hydrogel-Core Microstructured Polymer Optical Fibers for Selective Fiber Enhanced Raman Spectroscopy.

A new approach of Fiber Enhanced Raman Spectroscopy (FERS) is described within this article based on the use of Hydrogel-Core microstructured Polymer Optical Fibers (HyC-mPOF). The incorporation of the hydrogel only on the core of the Hollow-Core microstructured Polymer Optical Fiber (HC-mPOF) enables to perform FERS measurements in a functionalized matrix, enabling high selectivity Raman measurements. The hydrogel formation was continuously monitored and quantified using a Principal Component Analysis verifying the coherence between the components and the Raman spectrum of the hydrogel. The performed measurements with high and low affinity target molecules prove the feasibility of the presented HyC-mPOF platform.

Structural and mechanistic insight into DNA bending by antitumour calicheamicins.

Among the class of enediyne antibiotics endowed with potent antitumour activities, the calicheamicin derivative known as ozogamicin is selectively targeted to several leukaemia cell types by means of tailor-made immunoconjugates. Binding of these drugs to the DNA minor groove in a sequence-specific fashion eventually causes double-stranded cleavage that results in cell death. Use of calicheamicin ε, an unreactive analogue of calicheamicin γ1I, has demonstrated that these structurally sophisticated molecules inflict bending on certain DNA oligonucleotides of defined sequence to the extent that they increase their circularization ratio upon ligation into multimers. By modelling and simulating several linear and circular DNA constructs containing high-affinity 5'-TCCT-3' and low-affinity 5'-TTGT-3' target sites in the presence and absence of calicheamicin ε, we have shed light into the structural distortions introduced by the drug upon binding to DNA. This new insight not only informs about the direction and magnitude of the DNA curvature but also provides a rationale for an improved understanding of the preferred structural and dynamic features associated with DNA target selection by calicheamicins.

Recognition-Enhanced Metastably Shielded Aptamer for Digital Quantification of Small Molecules.

Aptamers are recognized as competitive affinity reagents; their application, however, often suffers from their relatively low target binding affinity, especially for small molecules. We herein introduce the concept of a recognition-enhanced metastably shielded aptamer probe (RMSApt) and explore its performance for digital quantification of low-affinity small molecules. The RMSApt design employs the idea of constructing an allosteric aptamer probe conferring a minor energy gap in the recognition switch process to facilitate target binding and probe response, in turn significantly improving the recognition efficiency for low-affinity targets. The probe design strategy boosts the application of aptamers for precisely quantifying targets with a dissociation constant Kd ranging from 10-4 to 10-9 M, which would cover most of the small-molecule species that exist binding aptamers. Thus, RMSApt would facilitate the translation of aptamers for medical diagnosis, food safety, and environmental screening.

Dense Bicoid hubs accentuate binding along the morphogen gradient.

Morphogen gradients direct the spatial patterning of developing embryos; however, the mechanisms by which these gradients are interpreted remain elusive. Here we used lattice light-sheet microscopy to perform in vivo single-molecule imaging in early Drosophila melanogaster embryos of the transcription factor Bicoid that forms a gradient and initiates patterning along the anteroposterior axis. In contrast to canonical models, we observed that Bicoid binds to DNA with a rapid off rate throughout the embryo such that its average occupancy at target loci is on-rate-dependent. We further observed Bicoid forming transient "hubs" of locally high density that facilitate binding as factor levels drop, including in the posterior, where we observed Bicoid binding despite vanishingly low protein levels. We propose that localized modulation of transcription factor on rates via clustering provides a general mechanism to facilitate binding to low-affinity targets and that this may be a prevalent feature of other developmental transcription factors.

Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag.

Phenotypic screening of compound libraries is a significant trend in drug discovery, yet success can be hindered by difficulties in identifying the underlying cellular targets. Current approaches rely on tethering bioactive compounds to a capture tag or surface to allow selective enrichment of interacting proteins for subsequent identification by mass spectrometry. Such methods are often constrained by ineffective capture of low affinity and low abundance targets. In addition, these methods are often not compatible with living cells and therefore cannot be used to verify the pharmacological activity of the tethered compounds. We have developed a novel chloroalkane capture tag that minimally affects compound potency in cultured cells, allowing binding interactions with the targets to occur under conditions relevant to the desired cellular phenotype. Subsequent isolation of the interacting targets is achieved through rapid lysis and capture onto immobilized HaloTag protein. Exchanging the chloroalkane tag for a fluorophore, the putative targets identified by mass spectrometry can be verified for direct binding to the compound through resonance energy transfer. Using the interaction between histone deacetylases (HDACs) and the inhibitor, Vorinostat (SAHA), as a model system, we were able to identify and verify all the known HDAC targets of SAHA as well as two previously undescribed targets, ADO and CPPED1. The discovery of ADO as a target may provide mechanistic insight into a reported connection between SAHA and Huntington's disease.

Melatonin promotes Bax sequestration to mitochondria reducing cell susceptibility to apoptosis via the lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid

Extra-neurological functions of melatonin include control of the immune system and modulation of apoptosis. We previously showed that melatonin inhibits the intrinsic apoptotic pathway in leukocytes via stimulation of high affinity MT1/MT2 receptors, thereby promoting re-localization of the anti-apoptotic Bcl-2 protein to mitochondria. Here we show that Bcl-2 sequesters pro-apoptotic Bax into mitochondria in an inactive form after melatonin treatment, thus reducing cell propensity to apoptosis. Bax translocation and the anti-apoptotic effect of melatonin are strictly dependent on the presence of Bcl-2, and on the 5-lipoxygenase (5-LOX) metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), which we have previously shown to be produced as a consequence of melatonin binding to its low affinity target calmodulin. Therefore, the anti-apoptotic effect of melatonin requires the simultaneous, independent interaction with high (MT1/MT2) and low (calmodulin) affinity targets, eliciting two independent signal transduction pathways converging into Bax sequestration and inactivation. MT1/MT2 vs. lipoxygenase pathways are activated by 10−9 vs. 10−5M melatonin, respectively; the anti-apoptotic effect of melatonin is achieved at 10−5M, but drops to 10−9M upon addition of exogenous 5-HETE, revealing that lipoxygenase activation is the rate-limiting pathway. Therefore, in areas of inflammation with increased 5-HETE levels, physiological nanomolar concentrations of melatonin may suffice to maintain leukocyte viability.

Computational prediction and experimental validation of low-affinity target of triptolide and its analogues

ERα as a novel low affinity target for triptolide and its analogues triptonide and triptriolide.

Endogenous miRNA and Target Concentrations Determine Susceptibility to Potential ceRNA Competition

Target competition (ceRNA crosstalk) within miRNA-regulated gene networks has been proposed to influence biological systems. To assess target competition, we characterize and quantitate miRNA networks in two cell types. Argonaute iCLIP reveals that hierarchical binding of high- to low-affinity miRNA targets is a key characteristic of invivo activity. Quantification of cellular miRNA and mRNA/ncRNA target pool levels indicates that miRNA:target pool ratios and an affinity partitioned target pool accurately predict invivo Ago binding profiles and miRNA susceptibility to target competition. Using single-cell reporters, we directly test predictions and estimate that ?3,000 additional high-affinity target sites can affect active miRNA families with low endogenous miRNA:target ratios, such as miR-92/25. In contrast, the highly expressed miR-294 and let-7 families are not susceptible to increases of nearly 10,000 sites. These results show differential susceptibility based on endogenous miRNA:target pool ratios and provide a physiological context for ceRNA competition invivo.

Structural and Functional Characterization of VanG d-Ala:d-Ser Ligase Associated with Vancomycin Resistance in Enterococcus faecalis

d-Alanyl:d-lactate (d-Ala:d-Lac) and d-alanyl:d-serine ligases are key enzymes in vancomycin resistance of Gram-positive cocci. They catalyze a critical step in the synthesis of modified peptidoglycan precursors that are low binding affinity targets for vancomycin. The structure of the d-Ala:d-Lac ligase VanA led to the understanding of the molecular basis for its specificity, but that of d-Ala:d-Ser ligases had not been determined. We have investigated the enzymatic kinetics of the d-Ala:d-Ser ligase VanG from Enterococcus faecalis and solved its crystal structure in complex with ADP. The overall structure of VanG is similar to that of VanA but has significant differences mainly in the N-terminal and central domains. Based on reported mutagenesis data and comparison of the VanG and VanA structures, we show that residues Asp-243, Phe-252, and Arg-324 are molecular determinants for d-Ser selectivity. These residues are conserved in both enzymes and explain why VanA also displays d-Ala:d-Ser ligase activity, albeit with low catalytic efficiency in comparison with VanG. These observations suggest that d-Ala:d-Lac and d-Ala:d-Ser enzymes have evolved from a common ancestral d-Ala:d-X ligase. The crystal structure of VanG showed an unusual interaction between two dimers involving residues of the omega loop that are deeply anchored in the active site. We constructed an octapeptide mimicking the omega loop and found that it selectively inhibits VanG and VanA but not Staphylococcus aureus d-Ala:d-Ala ligase. This study provides additional insight into the molecular evolution of d-Ala:d-X ligases and could contribute to the development of new structure-based inhibitors of vancomycin resistance enzymes.

Accelerating three‐dimensional molecular cardiovascular MR imaging using compressed sensing

To accelerate the acquisition of three-dimensional (3D) high-resolution cardiovascular molecular MRI by using Compressed Sensing (CS) reconstruction. Molecular MRI is an emerging technique for the early assessment of cardiovascular disease. This technique provides excellent soft tissue differentiation at a molecular and cellular level using target-specific contrast agents (CAs). However, long scan times are required for 3D molecular MRI. Parallel imaging can be used to speed-up these acquisitions, but hardware considerations limit the maximum acceleration factor. This limitation is important in small-animal studies, where single-coils are commonly used. Here we exploit the sparse nature of molecular MR images, which are characterized by localized and high-contrast biological target-enhancement, to accelerate data acquisition. CS was applied to detect: (a) venous thromboembolism and (b) coronary injury and aortic vessel wall in single- and multiple-coils acquisitions, respectively. Retrospective undersampling showed good overall image quality with accelerations up to four for thrombus and aortic images, and up to three for coronary artery images. For higher acceleration factors, features with high CA uptake were still well recovered while low affinity targets were less preserved with increased CS undersampling artifacts. Prospective undersampling was performed in an aortic image with acceleration of two, showing good contrast and well-defined tissue boundaries in the contrast-enhanced regions. We demonstrate the successful application of CS to preclinical molecular MR with target specific gadolinium-based CAs using retrospective (accelerations up to four) and prospective (acceleration of two) undersampling.

Azidoblebbistatin, a photoreactive myosin inhibitor

Photoreactive compounds are important tools in life sciences that allow precisely timed covalent crosslinking of ligands and targets. Using a unique technique we have synthesized azidoblebbistatin, which is a derivative of blebbistatin, the most widely used myosin inhibitor. Without UV irradiation azidoblebbistatin exhibits identical inhibitory properties to those of blebbistatin. Using UV irradiation, azidoblebbistatin can be covalently crosslinked to myosin, which greatly enhances its in vitro and in vivo effectiveness. Photo-crosslinking also eliminates limitations associated with the relatively low myosin affinity and water solubility of blebbistatin. The wavelength used for photo-crosslinking is not toxic for cells and tissues, which confers a great advantage in in vivo tests. Because the crosslink results in an irreversible association of the inhibitor to myosin and the irradiation eliminates the residual activity of unbound inhibitor molecules, azidoblebbistatin has a great potential to become a highly effective tool in both structural studies of actomyosin contractility and the investigation of cellular and physiological functions of myosin II. We used azidoblebbistatin to identify previously unknown low-affinity targets of the inhibitor (EC(50) ≥ 50 μM) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC(50) = 5 μM). Our results demonstrate that azidoblebbistatin, and potentially other azidated drugs, can become highly useful tools for the identification of strong- and weak-binding cellular targets and the determination of the apparent binding affinities in in vivo conditions.

Antimicrobials that affect the synthesis and conformation of nucleic acids

Several antimicrobials act by inhibiting the synthesis of nucleic acids (rifamycins, sulfamides, diaminopyridines), modifying their conformation (quinolones, coumarins) or causing irreversible lesions (nitroimidazoles, nitrofurans). The resistance mechanisms are: a reduction in intracytoplasmic accumulation, modification of the target or the production of a new low-affinity target and, more rarely, enzyme inactivation. Although the mechanisms affecting the targets are specific to each family and can lead to high-level resistance, the reduced permeability of the membrane and the increased efflux are non-specific and result in low-level cross-resistance between several families. The genetic mediation is usually chromosomal for rifamycins and quinolones, although plasmid-mediated resistant genes have been observed. On the other hand, for sulfamides and trimethoprim, plasmid-borne genes are frequent. Resistance to nitroimidazoles and nitrofurans is still not widely understood.

A peptidoglycan fragment triggers β-lactam resistance in Bacillus licheniformis.

To resist to β-lactam antibiotics Eubacteria either constitutively synthesize a β-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus, a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of β-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a β-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible β-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation.

Traps and cages for universal SERS detection

In this tutorial review, we provide an overview of the recent research toward surface functionalization of plasmonic nanoparticles for the generation of advanced optical sensors that make possible the analysis of various moieties by means of surface enhanced Raman scattering (SERS). Such moieties include atomic ions, low affinity target molecules, inorganic anions, biometabolites, pathogen markers and/or other analytes of interest even under very demanding circumstances such as those related to real life samples. We expect this review to be of interest to researchers in a broad diversity of fields that can take advantage of the unprecedented sensitivity of this type of molecular spectroscopy, in a wide variety of analytical and bioanalytical problems.