Marcos Pires

Martelli et al. describe the design, synthesis, and characterization of novel antibiotic candidates targeting M. tuberculosis. These compounds inhibit a cell wall remodeling protein using an unconventional mechanism of action acting as effectively as carbapenem-type antibiotics. Furthermore, they kill M. tuberculosis including drug-resistant variants in vitro.

Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than outer membrane. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane. We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the cytoplasmic membrane of Pseudomonas aeruginosa, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.

A photoactivatable chemically induced dimerization (photo‐CID) system based on covalent protein‐labeling technologies was developed by rationally designing a new caged ligand. This photo‐CID system enabled light‐induced protein translocation from the cytoplasm to subcellular regions such as the nucleus, mitochondrial outer membrane, and plasma membrane. Furthermore, quick protein translocation into a laser‐illuminated subcellular microregion was achieved.

Abstract

The photoactivatable chemically induced dimerization (photo‐CID) technique for tag‐fused proteins is one of the most promising methods for regulating subcellular protein translocations and protein–protein interactions. However, light‐induced covalent protein dimerization in living cells has yet to be established, despite its various advantages. Herein, we developed a photoactivatable covalent protein‐labeling technology by applying a caged ligand to the BL‐tag system, a covalent protein labeling system that uses mutant β‐lactamase. We further developed CBHD, a caged protein dimerizer, using caged BL‐tag and HaloTag ligands, and achieved light‐induced protein translocation from the cytoplasm to subcellular regions. In addition, this covalent photo‐CID system enabled quick protein translocation to a laser‐illuminated microregion. These results indicate that the covalent photo‐CID system will expand the scope of CID applications in the optical manipulation of cellular functions.

Nature Communications, Published online: 06 April 2021; doi:10.1038/s41467-021-22308-8

Outer membrane vesicles (OMVs), non-replicative particles secreted by Gram-negative bacteria, are known for their immunostimulatory and adjuvant properties. Here, by employing a Plug-and-Display technology, the authors engineer OMVs to display tumor antigens on the surface, a platform that promotes anti-tumor immune responses in preclinical cancer models.

pH/enzyme-responsive nanoreservoirs are co-loaded with anticancer drugs and antibiotics for overcoming bacterium-induced drug resistance. They can effectively inhibit tumor growth and remarkably activate the immune system in the presence of bacteria. This work presents a safe and efficacious method to reverse bacterium-induced drug resistance and convert the intratumoral bacteria from foes to friends for cancer therapy.

Abstract

The presence of bacteria in the tumor can cause cancer resistance to chemotherapeutics. To fight against bacterium-induced drug resistance, herein we design self-traceable nanoreservoirs that are simultaneously loaded with gemcitabine (an anticancer drug) and ciprofloxacin (an antibiotic) and are decorated with hyaluronic acid for active tumor targeting. The nanoreservoirs have a pH-sensitive gate and an enzyme-responsive gate that can be opened in the acidic and hyaluronidase-abundant tumor microenvironment to control drug release rates. Moreover, the nanoreservoirs can specifically target the tumor regions without eliciting evident toxicity to normal tissues, kill the intratumoral bacteria, and inhibit the tumor growth even in the presence of the bacteria. Unexpectedly, the nanoreservoirs can activate T cell-mediated immune responses through promoting antigen-presenting dendritic cell maturation and depleting immunosuppressive myeloid-derived suppressor cells in bacterium-infected tumors.

Cell-cell communication relies on the assembly of receptor-ligand complexes at the plasma membrane. The spatiotemporal receptor organization has a pivotal role in evoking cellular responses. We studied the clustering of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) and established a photoinstructive matrix with ultrasmall lock-and-key interaction pairs to control lateral membrane organization of hormone neuropeptide Y₂ receptors in living cells by light. Within seconds, receptor clustering was modulated in size, location, and density. After in situ confinement, changes in cellular morphology, motility, and calcium signaling revealed ligand-independent receptor activation. This approach may enhance the exploration of mechanisms in cell signaling and mechanotransduction.

ABSTRACT

β-Lactams are a class of antibiotics that target the synthesis of peptidoglycan, an essential component of the cell wall. β-Lactams inhibit the function of penicillin-binding proteins (PBPs), which form the cross-links between strands of peptidoglycan. Resistance to β-lactams complicates the treatment of bacterial infections. In recent years, the spread of β-lactam resistance has increased with growing intensity. Resistance is often conferred by β-lactamases, which inactivate β-lactams, or the expression of alternative β-lactam-resistant PBPs. ^P is an extracytoplasmic function (ECF) factor that controls β-lactam resistance in the species Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis. ^P is normally held inactive by the anti- factor RsiP. ^P is activated by β-lactams that trigger the proteolytic destruction of RsiP. Here, we identify the penicillin-binding protein PbpP and demonstrate its essential role in the activation of ^P. Our data show that PbpP is required for ^P activation and RsiP degradation. Our data suggest that PbpP acts as a β-lactam sensor since the binding of a subset of β-lactams to PbpP is required for ^P activation. We find that PbpP likely directly or indirectly controls site 1 cleavage of RsiP, which results in the degradation of RsiP and, thus, ^P activation. ^P activation results in increased expression of β-lactamases and, thus, increased β-lactam resistance. This work is the first report of a PBP acting as a sensor for β-lactams and controlling the activation of an ECF factor.

IMPORTANCE The bacterial cell envelope is the target for numerous antibiotics. Many antibiotics target the synthesis of peptidoglycan, which is a central metabolic pathway essential for bacterial survival. One of the most important classes of antibiotics has been β-lactams, which inhibit the transpeptidase activity of penicillin-binding proteins to decrease the cross-linking of peptidoglycan and the strength of the cell wall. While β-lactam antibiotics have historically proven to be effective, resistance to β-lactams is a growing problem. The ECF factor ^P is required for β-lactam resistance in B. thuringiensis and close relatives, including B. anthracis. Here, we provide insight into the mechanism of activation of ^P by β-lactams.

A host–microbe chemical ecology study revealed an effective and safe immunomodulator from Alcaligenes faecalis resident in gut‐associated lymphoid tissues, Peyer's patches (see picture). The complete structures of both the lipooligosaccharide and the lipopolysaccharide from A. faecalis were characterized. Furthermore, A. faecalis lipid A molecules were synthesized with varying degrees of acylation and found to be a promising vaccine adjuvant candidate.

Abstract

Alcaligenes faecalis is the predominant Gram‐negative bacterium inhabiting gut‐associated lymphoid tissues, Peyer's patches. We previously reported that an A. faecalis lipopolysaccharide (LPS) acted as a weak agonist for Toll‐like receptor 4 (TLR4)/myeloid differentiation factor‐2 (MD‐2) receptor as well as a potent inducer of IgA without excessive inflammation, thus suggesting that A. faecalis LPS might be used as a safe adjuvant. In this study, we characterized the structure of both the lipooligosaccharide (LOS) and LPS from A. faecalis. We synthesized three lipid A molecules with different degrees of acylation by an efficient route involving the simultaneous introduction of 1‐ and 4′‐phosphates. Hexaacylated A. faecalis lipid A showed moderate agonistic activity towards TLR4‐mediated signaling and the ability to elicit a discrete interleukin‐6 release in human cell lines and mice. It was thus found to be the active principle of the LOS/LPS and a promising vaccine adjuvant candidate.

Nature, Published online: 17 March 2021; doi:10.1038/s41586-021-03368-8

HLA peptidomic analysis identifies recurrent intracellular bacteria-derived peptides presented on HLA-I and HLA-II molecules in melanoma tumours, revealing how bacteria can modulate immune functions and responses to cancer therapies.

Nature, Published online: 10 March 2021; doi:10.1038/s41586-021-03316-6

Bacteria elicit two distinct immune responses in Arabidopsis thaliana, mediated by diverse signalling receptors but working in a synergistic manner.

TP53 (tumor protein p53) is the most commonly mutated cancer driver gene, but drugs that target mutant tumor suppressor genes, such as TP53, are not yet available. Here, we describe the identification of an antibody highly specific to the most common TP53 mutation (R175H, in which arginine at position 175 is replaced with histidine) in complex with a common human leukocyte antigen–A (HLA-A) allele on the cell surface. We describe the structural basis of this specificity and its conversion into an immunotherapeutic agent: a bispecific single-chain diabody. Despite the extremely low p53 peptide-HLA complex density on the cancer cell surface, the bispecific antibody effectively activated T cells to lyse cancer cells that presented the neoantigen in vitro and in mice. This approach could in theory be used to target cancers containing mutations that are difficult to target in conventional ways.

We describe a chemical strategy to prepare sucrose derivatives with vibrational tags and the characterisation of the first optical analogue of sucrose able to image real‐time trafficking of sucrose in live plant cells.

Abstract

Sucrose is the main saccharide used for long‐distance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real‐time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small (≈50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne‐tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real‐time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy.

Nature, Published online: 24 February 2021; doi:10.1038/s41586-021-03241-8

Absolute microbial abundances delineate longitudinal dynamics of bacteria, fungi and archaea in the infant gut microbiome, uncovering drivers of microbiome development masked by relative abundances and revealing notable parallels to macroscopic ecosystem assemblies.

Although bespoke, sequence-specific proteases have the potential to advance biotechnology and medicine, generation of proteases with tailor-made cleavage specificities remains a major challenge. We developed a phage-assisted protease evolution system with simultaneous positive and negative selection and applied it to three botulinum neurotoxin (BoNT) light-chain proteases. We evolved BoNT/X protease into separate variants that preferentially cleave vesicle-associated membrane protein 4 (VAMP4) and Ykt6, evolved BoNT/F protease to selectively cleave the non-native substrate VAMP7, and evolved BoNT/E protease to cleave phosphatase and tensin homolog (PTEN) but not any natural BoNT protease substrate in neurons. The evolved proteases display large changes in specificity (218- to >11,000,000-fold) and can retain their ability to form holotoxins that self-deliver into primary neurons. These findings establish a versatile platform for reprogramming proteases to selectively cleave new targets of therapeutic interest.

ABSTRACT

Multidrug-resistant (MDR) pathogens pose a significant public health threat. A major mechanism of resistance expressed by MDR pathogens is β-lactamase-mediated degradation of β-lactam antibiotics. The diazabicyclooctane (DBO) compounds zidebactam and WCK 5153, recognized as β-lactam "enhancers" due to inhibition of Pseudomonas aeruginosa penicillin-binding protein 2 (PBP2), are also class A and C β-lactamase inhibitors. To structurally probe their mode of PBP2 inhibition as well as investigate why P. aeruginosa PBP2 is less susceptible to inhibition by β-lactam antibiotics compared to the Escherichia coli PBP2, we determined the crystal structure of P. aeruginosa PBP2 in complex with WCK 5153. WCK 5153 forms an inhibitory covalent bond with the catalytic S327 of PBP2. The structure suggests a significant role for the diacylhydrazide moiety of WCK 5153 in interacting with the aspartate in the S-X-N/D PBP motif. Modeling of zidebactam in the active site of PBP2 reveals a similar binding mode. Both DBOs increase the melting temperature of PBP2, affirming their stabilizing interactions. To aid in the design of DBOs that can inhibit multiple PBPs, the ability of three DBOs to interact with P. aeruginosa PBP3 was explored crystallographically. Even though the DBOs show covalent binding to PBP3, they destabilized PBP3. Overall, the studies provide insights into zidebactam and WCK 5153 inhibition of PBP2 compared to their inhibition of PBP3 and the evolutionarily related KPC-2 β-lactamase. These molecular insights into the dual-target DBOs advance our knowledge regarding further DBO optimization efforts to develop novel potent β-lactamase-resistant, non-β-lactam PBP inhibitors.

IMPORTANCE Antibiotic resistance is a significant clinical problem. Developing novel antibiotics that overcome known resistance mechanisms is highly desired. Diazabicyclooctane inhibitors such as zidebactam possess this potential as they readily inactivate penicillin-binding proteins, yet cannot be degraded by β-lactamases. In this study, we characterized the inhibition by diazabicyclooctanes of penicillin-binding proteins PBP2 and PBP3 from Pseudomonas aeruginosa using protein crystallography and biophysical analyses. These structures and analyses help define the antibiotic properties of these inhibitors, explain the decreased susceptibility of P. aeruginosa PBP2 to be inhibited by β-lactam antibiotics, and provide insights that could be used for further antibiotic development.

Asparaginyl ligases are catalytically efficient transpeptidases. A generalizable means of improving their transpeptidation capabilities via selectively quenching the nucleophilicity of the peptides released over the reaction course is reported. By reducing reaction reversibility, this approach facilitates efficient peptide–peptide, protein–peptide, and protein–protein conjugation reactions.

Abstract

The use of enzymes for the site‐specific modification of proteins/peptides has become a highly accessible, widespread approach to study protein/peptide functions or to generate therapeutic conjugates. Asparaginyl endopeptidases (AEPs) that preferentially catalyze transpeptidation reactions (AEP ligases) have emerged as enticing alternatives to established approaches, such as bacterial sortases, due to their catalytic efficiency and short tripeptide recognition motifs. However, under standard conditions, a substantial excess of the nucleophile to be conjugated is needed to reach desirable yields. Herein we report a versatile approach to shift the AEP‐catalyzed transpeptidation equilibrium toward product formation via selectively quenching the nucleophilicity of the competing leaving‐group peptide. Our metal‐complexation‐based strategy enables efficient peptide/protein labeling at the N‐ or C‐terminus with near‐equimolar concentrations of nucleophile label. Furthermore, we show that this approach can enhance protein–protein ligation and facilitate the formation of transpeptidation products that are otherwise unattainable.

A cooperative strategy for stapling native peptides at lysine and nearby tyrosine or arginine residues with formaldehyde is reported.

Abstract

Stapling of peptides by intramolecular crosslinking of two neighboring amino acid side chains offers an important tool to modulate the structure and properties of peptides. In comparison to the stapling of artificially engineered peptide substrates, methods for stapling native peptides are more desirable for easier accessibility and genetic encodability. However, the existing strategy for selectivity control in the stapling of native peptides is relatively limited: the site of anchoring is often dominated by Cys, and the means for achieving the position selectivity among the same type of residues at different locations is lacking. We have developed a simple and powerful strategy for stapling native peptides at lysine residues with formaldehyde by the cooperation of nearby tyrosine or arginine residues. The stapling reactions can proceed with high efficiency and residue selectivity under mild conditions, and generate linchpins with distinct physiochemical properties. The new method for peptide stapling enables unique control of position‐selectivity for substrates bearing multiple reaction sites by reactivity that can be readily built in the peptide sequence.

Nature Chemical Biology, Published online: 04 February 2021; doi:10.1038/s41589-021-00736-3

A synthetic protein quality control system (ProQC) uses RNA hybridization to enhance translation of full-length proteins in coupled transcription–translation systems to optimize production of biosynthetic enzymes for metabolic engineering efforts.

Bispecific molecules that redirect antibodies towards prostate cancer cells through affinity to the Fc region of antibody and prostate‐specific membrane antigen (PSMA) have been developed. Upon recruitment by the molecules, defucosylated antibodies showed superior cytotoxicity by ADCC than antibodies with intact N‐glycans. This report provides insight into the development of immunotherapy.

Abstract

Synthetic small molecules that redirect endogenous antibodies to target cells are promising drug candidates because they overcome the potential shortcomings of therapeutic antibodies, such as immunogenicity and the need for intravenous delivery. Previously, we reported a novel class of bispecific molecules targeting the antibody Fc region and folate receptor, named Fc‐binding antibody‐recruiting molecules (Fc‐ARMs). Fc‐ARMs can theoretically recruit most endogenous antibodies, inducing antibody‐dependent cell‐mediated cytotoxicity (ADCC) to eliminate cancer cells. Herein, we describe new Fc‐ARMs that target prostate cancer (Fc‐ARM‐Ps). Fc‐ARM‐Ps recruited antibodies to cancer cells expressing prostate‐specific membrane antigen but did so with lower efficiency compared with Fc‐ARMs targeting the folate receptor. Upon recruitment by Fc‐ARM‐P, defucosylated antibodies efficiently activated natural killer cells and induced ADCC, whereas antibodies with intact N‐glycans did not. The results suggest that the affinity between recruited antibodies and CD16a, a type of Fc receptor expressed on immune cells, could be a key factor controlling immune activation in the Fc‐ARM strategy.

A method for site‐directed conjugation to antibodies is developed, in which a first lysine both directs and activates the reagent for reaction with a neighboring lysine.

Abstract

Functionalized antibodies are an indispensable resource for diagnosis, therapy and as a research tool for chemical biology. However, simpler and better methodologies are often required to improve the labeling of antibodies in terms of selectivity and scalability. Herein, we report the development of an easily available chemical reagent that allows site‐directed labeling of native human IgG1 antibodies in good yield and mono‐labeling selectivity. The salicylaldehyde moiety of the reagent reacts with surface exposed lysine residues to transiently form an iminium ion, and this positions a semi‐reactive ester in proximity of a second lysine residue that reacts with the ester to form an amide. Interestingly, it appears that the formation of the iminium ion also has a significant activating effect of the ester. We use flow cytometry and bio‐layer interferometry to confirm that the labeled antibodies retain antigen binding.