Marcos Pires

Nature Communications, Published online: 04 August 2020; doi:10.1038/s41467-020-17736-x

The human antibacterial and immunomodulatory peptide LL-37 is a hCAP-18 protein cleavage product that self-assembles. Here, the authors present the human and gorilla LL-37 (17–29) crystal structures, revealing a self-assembly of amphipathic helices into a densely packed and elongated hexameric structure with a central pore and mutagenesis experiments support the role of self-assembly for antibacterial activity.

PhotoAffinity Bit (PhABit) is a photoreactive fragment‐screening platform to covalently capture fragment–protein interactions. Hits can be profiled and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is widely applicable to novel protein targets, identifying starting points in the development of therapeutics.

Abstract

Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a disease‐modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space. Presented here is a fragment‐screening platform, termed PhABits (PhotoAffinity Bits), which utilizes a library of photoreactive fragments to covalently capture fragment–protein interactions. Hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is envisioned to be widely applicable to novel protein targets, identifying starting points in the development of therapeutics.

Nature Microbiology, Published online: 03 August 2020; doi:10.1038/s41564-020-0762-5

This study describes the mechanism by which Chlamydia trachomatis reprogrammes host glutamine metabolism in a c-Myc-dependent manner. The authors show that glutamine uptake via the SLC15A transporter and glutaminolysis are crucial for peptidoglycan synthesis and Chlamydia replication.

Nature Chemical Biology, Published online: 03 August 2020; doi:10.1038/s41589-020-0594-x

Chemical profiling in hyponeddylated cells coupled with multi-omics target deconvolution led to the identification of molecular glue degraders of cyclin K that function by inducing proximity between the CRL adaptor DDB1 and a CDK12–cyclin K complex.

Nature, Published online: 29 July 2020; doi:10.1038/s41586-020-2545-9

Lysosome-targeting chimaeras—in which a small molecule or antibody is connected to a glycopeptide ligand to form a conjugate that can bind a cell-surface lysosome-shuttling receptor and a protein target—are used to achieve the targeted degradation of extracellular and membrane proteins.

Nature, Published online: 29 July 2020; doi:10.1038/s41586-020-2541-0

In two mouse models of intestinal cancer, mutant p53 has an oncogenic effect in the distal gut but a tumour-suppressive effect in the proximal gut, and these opposing properties are determined by the gut microbiome.

The mechanisms allowing pathogens to subvert colonization resistance are poorly understood. Using a high-density mutant library, Caballero-Flores et al. identify genes required for early pathogen expansion in the presence of the gut microbiota. They find that induction of pathogen amino acid biosynthesis and increased dietary amino acids promote pathogen colonization.

The opportunistic pathogen Staphylococcus aureus is protected by a cell envelope that is crucial for viability. In addition to peptidoglycan, lipoteichoic acid (LTA) is an especially important component of the S. aureus cell envelope. LTA is an anionic polymer anchored to a glycolipid in the outer leaflet of the cell membrane. It was known that deleting the gene for UgtP, the enzyme that makes this glycolipid anchor, causes cell growth and division defects. In Bacillus subtilis, growth abnormalities from the loss of ugtP have been attributed to both the absence of the encoded protein and the loss of its products. Here, we show that growth defects in S. aureus ugtP deletion mutants are due to the long, abnormal LTA polymer that is produced when the glycolipid anchor is missing from the outer leaflet of the membrane. Dysregulated cell growth leads to defective cell division, and these phenotypes are corrected by mutations in the LTA polymerase gene, ltaS, that reduce polymer length. We also show that S. aureus mutants with long LTA are sensitized to cell wall hydrolases, beta-lactam antibiotics, and compounds that target other cell envelope pathways. We conclude that control of LTA polymer length is important for S. aureus physiology and promotes survival under stressful conditions, including antibiotic stress.

IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of community- and hospital-acquired infections and is responsible for a large fraction of deaths caused by antibiotic-resistant bacteria. S. aureus is surrounded by a complex cell envelope that protects it from antimicrobial compounds and other stresses. Here, we show that controlling the length of an essential cell envelope polymer, lipoteichoic acid, is critical for controlling S. aureus cell size and cell envelope integrity. We also show that genes involved in LTA length regulation are required for resistance to beta-lactam antibiotics in MRSA. The proteins encoded by these genes may be targets for combination therapy with an appropriate beta-lactam.

Inflammatory bowel diseases (IBDs), including Crohn’s disease and ulcerative colitis, are associated with dysbiosis of the gut microbiome. Emerging evidence suggests that small-molecule metabolites derived from bacterial breakdown of a variety of dietary nutrients confer a wide array of host benefits, including amelioration of inflammation in IBDs. Yet, in many...

Nature Communications, Published online: 24 July 2020; doi:10.1038/s41467-020-17307-0

Inflammation, immune cells and the host microbiota are intimately linked in the pathophysiology of obesity and diabetes. Here the authors show mucosal-associated invariant T cells fuel inflammation in the tissues and serve a function in promoting metabolic breakdown, polarising macrophage populations and inducing dysbiosis of the intestinal microbiota.

Excipients, considered "inactive ingredients," are a major component of formulated drugs and play key roles in their pharmacokinetics. Despite their pervasiveness, whether they are active on any targets has not been systematically explored. We computed the likelihood that approved excipients would bind to molecular targets. Testing in vitro revealed 25 excipient activities, ranging from low-nanomolar to high-micromolar concentration. Another 109 activities were identified by testing against clinical safety targets. In cellular models, five excipients had fingerprints predictive of system-level toxicity. Exposures of seven excipients were investigated, and in certain populations, two of these may reach levels of in vitro target potency, including brain and gut exposure of thimerosal and its major metabolite, which had dopamine D3 receptor dissociation constant K_d values of 320 and 210 nM, respectively. Although most excipients deserve their status as inert, many approved excipients may directly modulate physiologically relevant targets.

The tumor microenvironment plays a critical regulatory role in cancer progression, especially in central nervous system metastases. Cancer cells within the cerebrospinal fluid (CSF)–filled leptomeninges face substantial microenvironmental challenges, including inflammation and sparse micronutrients. To investigate the mechanism by which cancer cells in these leptomeningeal metastases (LM) overcome these constraints, we subjected CSF from five patients with LM to single-cell RNA sequencing. We found that cancer cells, but not macrophages, within the CSF express the iron-binding protein lipocalin-2 (LCN2) and its receptor SCL22A17. These macrophages generate inflammatory cytokines that induce cancer cell LCN2 expression but do not generate LCN2 themselves. In mouse models of LM, cancer cell growth is supported by the LCN2/SLC22A17 system and is inhibited by iron chelation therapy. Thus, cancer cells appear to survive in the CSF by outcompeting macrophages for iron.

Nature Biotechnology, Published online: 13 July 2020; doi:10.1038/s41587-020-0601-5

Off-the-shelf CAR T cells avoid immune rejection in mouse models of allogeneic cancer cell therapy.

Persisters and viable but non-culturable (VBNC) cells are two phenotypic variants known to be highly tolerant to antibiotics. Although both cell types are stained as live and often appear as nongrowing during ...

Nature, Published online: 08 July 2020; doi:10.1038/s41586-020-2474-7

A combination of gnotobiotic mouse models, transcriptomics, circuit tracing and chemogenetic manipulations identifies neuronal circuits that integrate microbial signals in the gut with regulation of the sympathetic nervous system.

Santiago et al. show that extracellular granzyme A (GzmA) contributes to gut inflammation and colorectal cancer (CRC) development by promoting IL-6 production in macrophages. Therapeutic inhibition of GzmA attenuates inflammation and CRC development, suggesting that GzmA could be useful to treat gut inflammation and prevent CRC.

ABSTRACT

Bacterial biofilms are responsible for a variety of serious human infections and are notoriously difficult to treat due to their recalcitrance to antibiotics. Further work is necessary to elicit a full understanding of the mechanism of this antibiotic tolerance. The arginine deiminase (ADI) pathway is responsible for bacterial pH maintenance and is highly expressed during biofilm growth in multiple bacterial species. Using the group A Streptococcus (GAS) as a model human pathogen, the ADI pathway was demonstrated to contribute to biofilm growth. The inability of antibiotics to reduce GAS populations when in a biofilm was demonstrated by in vitro studies and a novel animal model of nasopharyngeal infection. However, disruption of the ADI pathway returned GAS biofilms to planktonic levels of antibiotic sensitivity, suggesting the ADI pathway is influential in biofilm-related antibiotic treatment failure and provides a new strategic target for the treatment of biofilm infections in GAS and potentially numerous other bacterial species.

IMPORTANCE Biofilm-mediated bacterial infections are a major threat to human health because of their recalcitrance to antibiotic treatment. Through the study of Streptococcus pyogenes, a significant human pathogen that is known to form antibiotic-tolerant biofilms, we demonstrated the role that a bacterial pathway known for responding to acid stress plays in biofilm growth and antibiotic tolerance. This not only provides some insight into antibiotic treatment failure in S. pyogenes infections but also, given the widespread nature of this pathway, provides a potentially broad target for antibiofilm therapies. This discovery has the potential to impact the treatment of many different types of recalcitrant biofilm infections.

ABSTRACT

The bacterial pathogen Staphylococcus pseudintermedius is involved in canine otitis externa and pyoderma as well as in surgical wound and urinary tract infections. Invasion of canine epithelial cells is promoted by S. pseudintermedius fibronectin (Fn)-binding proteins SpsD and SpsL through molecular interactions that are currently unknown. By means of single-molecule experiments, we discover that both adhesins have distinct molecular mechanisms for binding to Fn. We show that the SpsD-Fn interaction has a strength equivalent to that of a covalent bond (~1.5 to 1.8 nN), which is an order of magnitude stronger than the binding force of classical receptor-ligand complexes. We suggest that this extreme mechanostability originates from the β-sheet organization of a tandem β-zipper. Upon binding to FnI modules, the intrinsically disordered binding sequences of SpsD would shift into an ordered structure by forming additional β-strands along triple peptide β-sheets in the Fn molecule. Dynamic force measurements reveal an unexpected behavior, i.e., that strong bonds are activated by mechanical tension as observed with catch bonds. By contrast, the SpsL-Fn interaction involves multiple weak bonds (~0.2 nN) that rupture sequentially under force. Together with the recently described dock, lock, and latch complex, the ultrastrong interaction unraveled here is among the strongest noncovalent biological interaction measured to date. Our findings may find applications for the identification of inhibitory compounds to treat infections triggered by pathogens engaged in tandem β-zipper interactions.

IMPORTANCE Binding of Staphylococcus pseudintermedius surface proteins SpsD and SpsL to fibronectin (Fn) plays a critical role in the invasion of canine epithelial cells. Here, we discover that both adhesins have different mechanisms for binding to Fn. The force required to separate SpsD from Fn is extremely strong, consistent with the unusual β-sheet organization of a high-affinity tandem β-zipper. By contrast, unbinding of the SpsL-Fn complex involves the sequential rupture of single weak bonds. Our findings may be of biological relevance as SpsD and SpsL are likely to play complementary roles during invasion. While the SpsD β-zipper supports strong bacterial adhesion and triggers invasion, the weak SpsL interaction would favor fast detachment, enabling the pathogen to colonize new sites.

A PROTAC composed of a selective stapled peptide for transcription coactivator SRC‐1 linked to a specific ligand for UBR E3 ligase induces the selective degradation of SRC‐1. This first‐in‐class SRC‐1 degrader efficiently impairs SRC‐1‐mediated transcription and suppresses cancer cell invasion and migration in vitro and in vivo and holds promise as an invaluable tool to probe SRC‐1 functions.

Abstract

Aberrantly elevated steroid receptor coactivator‐1 (SRC‐1) expression and activity are strongly correlated with cancer progression and metastasis. Here we report, for the first time, the development of a proteolysis targeting chimera (PROTAC) that is composed of a selective SRC‐1 binder linked to a specific ligand for UBR box, a unique class of E3 ligases recognizing N‐degrons. We showed that the bifunctional molecule efficiently and selectively induced the degradation of SRC‐1 in cells through the N‐degron pathway. Importantly, given the ubiquitous expression of the UBR protein in most cells, PROTACs targeting the UBR box could degrade a protein of interest regardless of cell types. We also showed that the SRC‐1 degrader significantly suppressed cancer cell invasion and migration in vitro and in vivo. Together, these results demonstrate that the SRC‐1 degrader can be an invaluable chemical tool in the studies of SRC‐1 functions. Moreover, our findings suggest PROTACs based on the N‐degron pathway as a widely useful strategy to degrade disease‐relevant proteins.

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small-molecule approaches has proven to be a major challenge. Here we describe the development of a pan-RAS biologic inhibitor composed of the RAS-RAP1–specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered...

Pathogen specific antibody responses need to be tightly regulated to generate protective but limit self-reactive immune responses. While loss of humoral tolerance has been associated with microbial infections, the pathways involved in balancing protective versus autoreactive antibody responses in humans are incompletely understood. Studies in classical mouse model systems have provided evidence that balancing of immune responses through inhibitory receptors is an important quality control checkpoint. Genetic differences between inbred mouse models and the outbred human population and allelic receptor variants not present in mice, however, argue for caution when directly translating these findings to the human system. By studying Borrelia burgdorferi infection in humanized mice reconstituted with human hematopoietic stem cells from donors homozygous for a functional or non-functional FcgRIIb allele, we show that the human inhibitory FcgRIIb is a critical checkpoint balancing protective and autoreactive immune responses, linking infection with induction of autoimmunity in the human immune system.

Multiple structures in distinct ligand states provide detailed mechanistic insights into NMDAR pharmacology, activation, and inhibition, which are fundamental to the brain physiology.

This Minireview describes the resistance mechanism of bacteria producing β‐lactamase and further summarize the fluorogenic probes/inhibitors of β‐lactamase, and their applications in the treatment of infectious diseases. We provide guidelines for the design of fluorogenic probes with improved selectivity/sensitivity/effectiveness to identify further inhibitors for β‐lactamases and eventually overcome bacterial resistance.

Abstract

β‐Lactam antibiotics are generally perceived as one of the greatest inventions of the 20^th century, and these small molecular compounds have saved millions of lives. However, upon clinical application of antibiotics, the β‐lactamase secreted by pathogenic bacteria can lead to the gradual development of drug resistance. β‐Lactamase is a hydrolase that can efficiently hydrolyze and destroy β‐lactam antibiotics. It develops and spreads rapidly in pathogens, and the drug‐resistant bacteria pose a severe threat to human health and development. As a result, detecting and inhibiting the activities of β‐lactamase are of great value for the rational use of antibiotics and the treatment of infectious diseases. At present, many specific detection methods and inhibitors of β‐lactamase have been developed and applied in clinical practice. In this Minireview, we describe the resistance mechanism of bacteria producing β‐lactamase and further summarize the fluorogenic probes, inhibitors of β‐lactamase, and their applications in the treatment of infectious diseases. It may be valuable to design fluorogenic probes with improved selectivity, sensitivity, and effectiveness to further identify the inhibitors for β‐lactamases and eventually overcome bacterial resistance.

Nature Microbiology, Published online: 25 June 2020; doi:10.1038/s41564-020-0744-7

Some cytosolic bacteria deform the plasma membrane to spread from cell to cell. Secretion of 25-hydroxy-cholesterol by macrophages has emerged as a protective response that depletes the cholesterol pool used by these bacteria to disseminate through epithelia.