Marcos Pires

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.

Vaccine adjuvants are required for the generation of robust and long‐lasting immune responses of antigen vaccines. Screening of a newly constructed chemical library of self‐assembling molecules led to the rapid discovery of cholicamide (6), as a potent vaccine adjuvant. Just like viruses, the nanoassembly of cholicamide enters the cells and is recognized by an endosomal Toll‐like receptor to elicit potent innate immune responses.

Abstract

Immune potentiators, termed adjuvants, trigger early innate immune responses to ensure the generation of robust and long‐lasting adaptive immune responses of vaccines. Presented here is a study that takes advantage of a self‐assembling small‐molecule library for the development of a novel vaccine adjuvant. Cell‐based screening of the library and subsequent structural optimization led to the discovery of a simple, chemically tractable deoxycholate derivative (molecule 6, also named cholicamide) whose well‐defined nanoassembly potently elicits innate immune responses in macrophages and dendritic cells. Functional and mechanistic analyses indicate that the virus‐like assembly enters the cells and stimulates the innate immune response through Toll‐like receptor 7 (TLR7), an endosomal TLR that detects single‐stranded viral RNA. As an influenza vaccine adjuvant in mice, molecule 6 was as potent as Alum, a clinically used adjuvant. The studies described here pave the way for a new approach to discovering and designing self‐assembling small‐molecule adjuvants against pathogens, including emerging viruses.

Uropathogenic Escherichia coli (UPEC) is the leading cause of human urinary tract infections (UTIs), and many patients experience recurrent infection after successful antibiotic treatment. The source of recurrent infections may be persistent bacterial reservoirs in vivo that are in a quiescent state and thus are not susceptible to antibiotics. Here, we show that multiple UPEC strains require a quorum to proliferate in vitro with glucose as the carbon source. At low cell density, the bacteria remain viable but enter a quiescent, nonproliferative state. Of the clinical UPEC isolates tested to date, 35% (51/145) enter this quiescent state, including isolates from the recently emerged, multidrug-resistant pandemic lineage ST131 (i.e., strain JJ1886) and isolates from the classic endemic lineage ST73 (i.e., strain CFT073). Moreover, quorum-dependent UPEC quiescence is prevented and reversed by small-molecule proliferants that stimulate colony formation. These proliferation cues include d-amino acid-containing peptidoglycan (PG) tetra- and pentapeptides, as well as high local concentrations of l-lysine and l-methionine. Peptidoglycan fragments originate from the peptidoglycan layer that supports the bacterial cell wall but are released as bacteria grow. These fragments are detected by a variety of organisms, including human cells, other diverse bacteria, and, as we show here for the first time, UPEC. Together, these results show that for UPEC, (i) sensing of PG stem peptide and uptake of l-lysine modulate the quorum-regulated decision to proliferate and (ii) quiescence can be prevented by both intra- and interspecies PG peptide signaling.

IMPORTANCE Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). During pathogenesis, UPEC cells adhere to and infiltrate bladder epithelial cells, where they may form intracellular bacterial communities (IBCs) or enter a nongrowing or slowly growing quiescent state. Here, we show in vitro that UPEC strains at low population density enter a reversible, quiescent state by halting division. Quiescent cells resume proliferation in response to sensing a quorum and detecting external signals, or cues, including peptidoglycan tetra- and pentapeptides.

Nature Communications, Published online: 23 September 2020; doi:10.1038/s41467-020-18139-8

The bacterial periplasmic predator Bdellovibrio bacteriovorus deacetylates the peptidoglycan of the prey bacterium early upon invasion. Here, the authors identify and characterize a Bdellovibrio lysozyme that acts specifically on deacetylated peptidoglycan and is important for periplasmic exit.

ABSTRACT

Staphylococcus aureus is a human pathogen causing life-threatening diseases. The increasing prevalence of multidrug-resistant S. aureus infections is a global health concern, requiring development of novel therapeutic options. Peptidoglycan-degrading enzymes (peptidoglycan hydrolases, PGHs) have emerged as a highly effective class of antimicrobial proteins against S. aureus and other pathogens. When applied to Gram-positive bacteria, PGHs hydrolyze bonds within the peptidoglycan layer, leading to rapid bacterial death by lysis. This activity is highly specific and independent of the metabolic activity of the cell or its antibiotic resistance patterns. However, systemic application of PGHs is limited by their often low activity in vivo and by an insufficient serum circulation half-life. To address this problem, we aimed to extend the half-life of PGHs selected for high activity against S. aureus in human serum. Half-life extension and increased serum circulation were achieved through fusion of PGHs to an albumin-binding domain (ABD), resulting in high-affinity recruitment of human serum albumin and formation of large protein complexes. Importantly, the ABD-fused PGHs maintained high killing activity against multiple drug-resistant S. aureus strains, as determined by ex vivo testing in human blood. The top candidate, termed ABD_M23, was tested in vivo to treat S. aureus-induced murine bacteremia. Our findings demonstrate a significantly higher efficacy of ABD_M23 than of the parental M23 enzyme. We conclude that fusion with ABD represents a powerful approach for half-life extension of PGHs, expanding the therapeutic potential of these enzybiotics for treatment of multidrug-resistant bacterial infections.

IMPORTANCE Life-threatening infections with Staphylococcus aureus are often difficult to treat due to the increasing prevalence of antibiotic-resistant bacteria and their ability to persist in protected niches in the body. Bacteriolytic enzymes are promising new antimicrobials because they rapidly kill bacteria, including drug-resistant and persisting cells, by destroying their cell wall. However, when injected into the bloodstream, these enzymes are not retained long enough to clear an infection. Here, we describe a modification to increase blood circulation time of the enzymes and enhance treatment efficacy against S. aureus-induced bloodstream infections. This was achieved by preselecting enzyme candidates for high activity in human blood and coupling them to serum albumin, thereby preventing their elimination by kidney filtration and blood vessel cells.

ABSTRACT

Disruption of the outer membrane (OM) barrier allows for the entry of otherwise inactive antimicrobials into Gram-negative pathogens. Numerous efforts to implement this approach have identified a large number of OM perturbants that sensitize Gram-negative bacteria to many clinically available Gram-positive active antibiotics. However, there is a dearth of investigation into the strengths and limitations of this therapeutic strategy, with an overwhelming focus on characterization of individual potentiator molecules. Herein, we look to explore the utility of exploiting OM perturbation to sensitize Gram-negative pathogens to otherwise inactive antimicrobials. We identify the ability of OM disruption to change the rules of Gram-negative entry, overcome preexisting and spontaneous resistance, and impact biofilm formation. Disruption of the OM expands the threshold of hydrophobicity compatible with Gram-negative activity to include hydrophobic molecules. We demonstrate that while resistance to Gram-positive active antibiotics is surprisingly common in Gram-negative pathogens, OM perturbation overcomes many antibiotic inactivation determinants. Further, we find that OM perturbation reduces the rate of spontaneous resistance to rifampicin and impairs biofilm formation. Together, these data suggest that OM disruption overcomes many of the traditional hurdles encountered during antibiotic treatment and is a high priority approach for further development.

IMPORTANCE The spread of antibiotic resistance is an urgent threat to global health that necessitates new therapeutics. Treatments for Gram-negative pathogens are particularly challenging to identify due to the robust outer membrane permeability barrier in these organisms. Recent discovery efforts have attempted to overcome this hurdle by disrupting the outer membrane using chemical perturbants and have yielded several new peptides and small molecules that allow the entry of otherwise inactive antimicrobials. However, a comprehensive investigation into the strengths and limitations of outer membrane perturbants as antibiotic partners is currently lacking. Herein, we interrogate the interaction between outer membrane perturbation and several common impediments to effective antibiotic use. Interestingly, we discover that outer membrane disruption is able to overcome intrinsic, spontaneous, and acquired antibiotic resistance in Gram-negative bacteria, meriting increased attention toward this approach.

Nature Communications, Published online: 18 September 2020; doi:10.1038/s41467-020-18377-w

The dTAG system is used to rapidly deplete tagged target proteins in vitro and in vivo, but there are context- and protein-specific differences in its effectiveness. Here, the authors develop a second generation dTAG molecule that can degrade previously recalcitrant target proteins in cells and mice.

Associations between chronic kidney disease (CKD) and the gut microbiota have been postulated, yet questions remain about the underlying mechanisms. In humans, dietary protein increases gut bacterial production of hydrogen sulfide (H₂S), indole, and indoxyl sulfate. The latter are uremic toxins, and H₂S has diverse physiological functions, some of which are mediated by posttranslational modification. In a mouse model of CKD, we found that a high sulfur amino acid–containing diet resulted in posttranslationally modified microbial tryptophanase activity. This reduced uremic toxin–producing activity and ameliorated progression to CKD in the mice. Thus, diet can tune microbiota function to support healthy host physiology through posttranslational modification without altering microbial community composition.

Microfluidic vesicle traps were developed to trap and reversibly deform giant unilamellar vesicles (GUVs) and in doing so manipulate membrane geometry and tension. We use these traps to show that filaments of the bacterial cytoskeletal protein FtsZ orientate themselves along the short axis of elongated GUVs and that membrane tension drives reversible FtsZ reorganization from filaments into rings, which facilitates membrane shape changes.

Abstract

The geometry of reaction compartments can affect the local outcome of interface‐restricted reactions. Giant unilamellar vesicles (GUVs) are commonly used to generate cell‐sized, membrane‐bound reaction compartments, which are, however, always spherical. Herein, we report the development of a microfluidic chip to trap and reversibly deform GUVs into cigar‐like shapes. When trapping and elongating GUVs that contain the primary protein of the bacterial Z ring, FtsZ, we find that membrane‐bound FtsZ filaments align preferentially with the short GUV axis. When GUVs are released from this confinement and membrane tension is relaxed, FtsZ reorganizes reversibly from filaments into dynamic rings that stabilize membrane protrusions; a process that allows reversible GUV deformation. We conclude that microfluidic traps are useful for manipulating both geometry and tension of GUVs, and for investigating how both affect the outcome of spatially‐sensitive reactions inside them, such as that of protein self‐organization.

Nature Chemical Biology, Published online: 14 September 2020; doi:10.1038/s41589-020-0641-7

Incorporation of the non-canonical amino acid 3-aminotyrosine into the chromophores of green fluorescent protein-based biosensors systematically red-shifts their fluorescent properties while maintaining brightness, dynamic range and responsiveness.

Zheng et al. rationally combine antibiotics using insights on bacterial metabolism to identify drug-drug combinations that sterilize bacterial cultures of both metabolically active and persister cells, while dose-sparing toxic antibiotics.

Nature, Published online: 09 September 2020; doi:10.1038/s41586-020-2699-5

Exposing Caenorhabditis elegans to non-coding small RNAs from pathogenic Pseudomonas aeruginosa induces avoidance behaviours in treated worms and their progeny, which reveals how C. elegans discriminates between bacterial species in its microbial environment.

Functionalization of peptides and proteins through the trapping of peptide radical species is described. This novel and versatile strategy exploits the process of desulfurization and deselenization of cysteine and selenocystine residues to enable the site‐selective installation of a broad range of groups appended to a persistent radical trap.

Abstract

The development of site‐selective chemistry targeting the canonical amino acids enables the controlled installation of desired functionalities into native peptides and proteins. Such techniques facilitate the development of polypeptide conjugates to advance therapeutics, diagnostics, and fundamental science. We report a versatile and selective method to functionalize peptides and proteins through free‐radical‐mediated dechalcogenation. By exploiting phosphine‐induced homolysis of the C−Se and C−S bonds of selenocysteine and cysteine, respectively, we demonstrate the site‐selective installation of groups appended to a persistent radical trap. The reaction is rapid, operationally simple, and chemoselective. The resulting aminooxy linker is stable under a variety of conditions and selectively cleavable in the presence of a low‐oxidation‐state transition metal. We have explored the full scope of this reaction using complex peptide systems and a recombinantly expressed protein.

The de novo design of proteins that bind highly functionalized small molecules represents a great challenge. To enable computational design of binders, we developed a unit of protein structure—a van der Mer (vdM)—that maps the backbone of each amino acid to statistically preferred positions of interacting chemical groups. Using vdMs, we designed six de novo proteins to bind the drug apixaban; two bound with low and submicromolar affinity. X-ray crystallography and mutagenesis confirmed a structure with a precisely designed cavity that forms favorable interactions in the drug–protein complex. vdMs may enable design of functional proteins for applications in sensing, medicine, and catalysis.

The chemical interactions of microbes shape and structure their communities and have important effects on the health and diseases of multicellular hosts. This special collection sheds light on some of these interactions and elucidates the molecules involved in them.

Abstract

The chemical interactions of microbes shape and structure their communities and have important effects on the health and diseases of multicellular hosts. This special collection on Microbial Biosynthesis and Interactions sheds light on some of these interactions and takes a closer look at the molecules involved in them.

Probiotic Escherichia coli Nissle 1917 (EcN) has been widely studied for the treatment of intestinal inflammatory diseases and infectious diarrhea, but the mechanisms by which they communicate with the host are n...

The application of proteinaceous toxins for cell ablation is limited by their high on- and off-target toxicity, severe side effects, and a narrow therapeutic window. The selectivity of targeting can be improved by intein-based toxin reconstitution from two dysfunctional fragments provided their cytoplasmic delivery via independent, selective pathways. While the...

McDonald et al., used gnotobiotic mice and in vivo imaging to show that clearance of circulating pathogens by Kupffer cells in the liver is governed by the gut microbiota and its production of D-lactate, which reaches the liver via the portal vein and programs Kupffer cells to capture and kill pathogens in the bloodstream.

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.

Nature Communications, Published online: 14 August 2020; doi:10.1038/s41467-020-17940-9

Staphylococcus aureus is thought to divide in three alternating orthogonal planes over three consecutive divisions. Here the authors dispel this idea, showing that one out of the multiple planes perpendicular to the septum can be used in daughter cells irrespective of its orientation in relation to the penultimate division plane.

Nature, Published online: 12 August 2020; doi:10.1038/s41586-020-2597-x

Structural and physiological studies show that the inner membrane protein PbgA is a crucial sensor of lipopolysaccharide (LPS) and regulates the activity of the LPS biosynthesis enzyme LpxC.

The preservation of biological samples in formaldehyde induces intra‐ and inter‐crosslinking of peptides, which hampers the mass spectrometric detection of those analytes. Chemical reversal of formaldehyde‐induced peptidyl crosslinking permits mass spectrometry imaging of intact peptides, while protecting the spatiochemical characteristics of analytes in formalin‐fixed paraffin‐embedded tissues.

Abstract

Linking molecular and chemical changes to human disease states depends on the availability of appropriate clinical samples, mostly preserved as formalin‐fixed paraffin‐embedded (FFPE) specimens stored in tissue banks. Mass spectrometry imaging (MSI) enables the visualization of the spatiotemporal distribution of molecules in biological samples. However, MSI is not effective for imaging FFPE tissues because of the chemical modifications of analytes, including complex crosslinking between nucleophilic moieties. Here we used an MS‐compatible inorganic nucleophile, hydroxylamine hydrochloride, to chemically reverse inter‐ and intra‐crosslinks from endogenous molecules. The analyte restoration appears specific for formaldehyde‐reactive amino acids. This approach enabled the MSI‐assisted localization of pancreatic peptides expressed in the alpha, beta, and gamma cells. Pancreatic islet‐like distributions of islet hormones were observed in human FFPE tissues preserved for more than five years, demonstrating that samples from biobanks can effectively be investigated with MSI.

by Jingwei Zeng, Leo C. James

Nature, Published online: 05 August 2020; doi:10.1038/s41586-020-2564-6

A mouse model of systemic versus mucosal exposure to microbial taxa reveals that the former provokes a flexible B cell response with a diverse immunoglobulin repertoire, whereas the latter generates a more-restricted response.

ABSTRACT

Mycobacterium tuberculosis, which causes tuberculosis (TB), is estimated to infect one-third of the world’s population. The overall burden and the emergence of drug-resistant strains of Mycobacterium tuberculosis underscore the need for new therapeutic options against this important human pathogen. Our recent work demonstrated the success of natural product discovery in identifying novel compounds with efficacy against Mycobacterium tuberculosis. Here, we improve on these methods by combining improved isolation and Mycobacterium tuberculosis selective screening to identify three new anti-TB compounds: streptomycobactin, kitamycobactin, and amycobactin. We were unable to obtain mutants resistant to streptomycobactin, and its target remains to be elucidated. We identify the target of kitamycobactin to be the mycobacterial ClpP1P2C1 protease and confirm that kitamycobactin is an analog of the previously identified compound lassomycin. Further, we identify the target of amycobactin to be the essential protein secretion pore SecY. We show further that amycobactin inhibits protein secretion via the SecY translocon. Importantly, this inhibition is bactericidal to nonreplicating Mycobacterium tuberculosis. This is the first compound, to our knowledge, that targets the Sec protein secretion machinery in Mycobacterium tuberculosis. This work underscores the ability of natural product discovery to deliver not only new compounds with activity against Mycobacterium tuberculosis but also compounds with novel targets.

IMPORTANCE Decreasing discovery rates and increasing resistance have underscored the need for novel therapeutic options to treat Mycobacterium tuberculosis infection. Here, we screen extracts from previously uncultured soil microbes for specific activity against Mycobacterium tuberculosis, identifying three novel compounds. We further define the mechanism of action of one compound, amycobactin, and demonstrate that it inhibits protein secretion through the Sec translocation machinery.