Light-mediated discovery of surfaceome nanoscale organization and intercellular receptor interaction networks.

The molecular nanoscale organization of the surfaceome is a fundamental regulator of cellular signaling in health and disease. Technologies for mapping the spatial relationships of cell surface receptors and their extracellular signaling synapses would unlock theranostic opportunities to target protein communities and the possibility to engineer extracellular signaling. Here, we develop an optoproteomic technology termed LUX-MS that enables the targeted elucidation of acute protein interactions on and in between living cells using light-controlled singlet oxygen generators (SOG). By using SOG-coupled antibodies, small molecule drugs, biologics and intact viral particles, we demonstrate the ability of LUX-MS to decode ligand receptor interactions across organisms and to discover surfaceome receptor nanoscale organization with direct implications for drug action. Furthermore, by coupling SOG to antigens we achieved light-controlled molecular mapping of intercellular signaling within functional immune synapses between antigen-presenting cells and CD8+ T cells providing insights into T cell activation with spatiotemporal specificity. LUX-MS based decoding of surfaceome signaling architectures thereby provides a molecular framework for the rational development of theranostic strategies.

Distinct conformations of the HIV-1 V3 loop crown are targetable for broad neutralization.

The V3 loop of the HIV-1 envelope (Env) protein elicits a vigorous, but largely non-neutralizing antibody response directed to the V3-crown, whereas rare broadly neutralizing antibodies (bnAbs) target the V3-base. Challenging this view, we present V3-crown directed broadly neutralizing Designed Ankyrin Repeat Proteins (bnDs) matching the breadth of V3-base bnAbs. While most bnAbs target prefusion Env, V3-crown bnDs bind open Env conformations triggered by CD4 engagement. BnDs achieve breadth by focusing on highly conserved residues that are accessible in two distinct V3 conformations, one of which resembles CCR5-bound V3. We further show that these V3-crown conformations can, in principle, be attacked by antibodies. Supporting this conclusion, analysis of antibody binding activity in the Swiss 4.5 K HIV-1 cohort (n = 4,281) revealed a co-evolution of V3-crown reactivities and neutralization breadth. Our results indicate a role of V3-crown responses and its conformational preferences in bnAb development to be considered in preventive and therapeutic approaches.

An epitope-specific chemically defined nanoparticle vaccine for respiratory syncytial virus.

Respiratory syncytial virus (RSV) can cause severe respiratory disease in humans, particularly in infants and the elderly. However, attempts to develop a safe and effective vaccine have so far been unsuccessful. Atomic-level structures of epitopes targeted by RSV-neutralizing antibodies are now known, including that bound by Motavizumab and its clinically used progenitor Palivizumab. We developed a chemically defined approach to RSV vaccine design, that allows control of both immunogenicity and safety features of the vaccine. Structure-guided antigen design and a synthetic nanoparticle delivery platform led to a vaccine candidate that elicits high titers of palivizumab-like, epitope-specific neutralizing antibodies. The vaccine protects preclinical animal models from RSV infection and lung pathology typical of vaccine-derived disease enhancement. The results suggest that the development of a safe and effective synthetic epitope-specific RSV vaccine may be feasible by combining this conformationally stabilized peptide and synthetic nanoparticle delivery system.

Identification of Genes Required for Resistance to Peptidomimetic Antibiotics by Transposon Sequencing.

Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of nosocomial infections. Due to its high intrinsic and adaptive resistance to antibiotics, infections caused by this organism are difficult to treat and new therapeutic options are urgently needed. Novel peptidomimetic antibiotics that target outer membrane (OM) proteins have shown great promise for the treatment of P. aeruginosa infections. Here, we have performed genome-wide mutant fitness profiling using transposon sequencing (Tn-Seq) to identify resistance determinants against the recently described peptidomimetics L27-11, compounds 3 and 4, as well as polymyxin B2 (PMB) and colistin (COL). We identified a set of 13 core genes that affected resistance to all tested antibiotics, many of which encode enzymes involved in the modification of the lipopolysaccharide (LPS) or control their expression. We also identified fitness determinants that are specific for antibiotics with similar structures that may indicate differences in their modes of action. These results provide new insights into resistance mechanisms against these peptide antibiotics, which will be important for future clinical development and efforts to further improve their potency.

Author Correction: Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

There is an urgent need for new antibiotics against Gram-negative pathogens that are resistant to carbapenem and third-generation cephalosporins, against which antibiotics of last resort have lost most of their efficacy. Here we describe a class of synthetic antibiotics inspired by scaffolds derived from natural products. These chimeric antibiotics contain a ß-hairpin peptide macrocycle linked to the macrocycle found in the polymyxin and colistin family of natural products. They are bactericidal and have a mechanism of action that involves binding to both lipopolysaccharide and the main component (BamA) of the ß-barrel folding complex (BAM) that is required for the folding and insertion of ß-barrel proteins into the outer membrane of Gram-negative bacteria. Extensively optimized derivatives show potent activity against multidrug-resistant pathogens, including all of the Gram-negative members of the ESKAPE pathogens1. These derivatives also show favourable drug properties and overcome colistin resistance, both in vitro and in vivo. The lead candidate is currently in preclinical toxicology studies that-if successful-will allow progress into clinical studies that have the potential to address life-threatening infections by the Gram-negative pathogens, and thus to resolve a considerable unmet medical need.

Folded Synthetic Peptides and Other Molecules Targeting Outer Membrane Protein Complexes in Gram-Negative Bacteria.

Conformationally constrained peptidomimetics have been developed to mimic interfacial epitopes and target a wide selection of protein-protein interactions. ß-Hairpin mimetics based on constrained macrocyclic peptides have provided access to excellent structural mimics of ß-hairpin epitopes and found applications as interaction inhibitors in many areas of biology and medicinal chemistry. Recently, ß-hairpin peptidomimetics and naturally occurring ß-hairpin-shaped peptides have also been discovered with potent antimicrobial activity and novel mechanisms of action, targeting essential outer membrane protein (OMP) complexes in Gram-negative bacteria. This includes the Lpt complex, required for transporting LPS to the cell surface during OM biogenesis and the BAM complex that folds OMPs and inserts them into the OM bilayer. The Lpt complex is a macromolecular superstructure comprising seven different proteins (LptA-LptG) that spans the entire bacterial cell envelope, whereas the BAM complex is a folding machine comprising a ß-barrel OMP (BamA) and four different lipoproteins (BamB-BamE). Folded synthetic and natural ß-hairpin-shaped peptides appear well-suited for interacting with proteins within the Lpt and BAM complexes that are rich in ß-structure. Recent progress in identifying antibiotics targeting these complexes are reviewed here. Already a clinical candidate has been developed (murepavadin) that targets LptD, with potent antimicrobial activity specifically against pseudmonads. The ability of folded synthetic ß-hairpin epitope mimetics to interact with ß-barrel and ß-jellyroll domains in the Lpt and Bam complexes represent new avenues for antibiotic discovery, which may lead to the development of much needed new antimicrobials to combat the rise of drug-resistant pathogenic Gram-negative bacteria.

An ultra-high affinity ligand of HIV-1 TAR reveals the RNA structure recognized by P-TEFb.

The HIV-1 trans-activator protein Tat binds the trans-activation response element (TAR) to facilitate recruitment of the super elongation complex (SEC) to enhance transcription of the integrated pro-viral genome. The Tat-TAR interaction is critical for viral replication and the emergence of the virus from the latent state, therefore, inhibiting this interaction has long been pursued to discover new anti-viral or latency reversal agents. However, discovering active compounds that directly target RNA with high affinity and selectivity remains a significant challenge; limiting pre-clinical development. Here, we report the rational design of a macrocyclic peptide mimic of the arginine rich motif of Tat, which binds to TAR with low pM affinity and 100-fold selectivity against closely homologous RNAs. Despite these unprecedented binding properties, the new ligand (JB181) only moderately inhibits Tat-dependent reactivation in cells and recruitment of positive transcription elongation factor (P-TEFb) to TAR. The NMR structure of the JB181-TAR complex revealed that the ligand induces a structure in the TAR loop that closely mimics the P-TEFb/Tat1:57/AFF4/TAR complex. These results strongly suggest that high-affinity ligands which bind the UCU bulge are not likely to inhibit recruitment of the SEC and suggest that targeting of the TAR loop will be an essential feature of effective Tat inhibitors.

Thanatin targets the intermembrane protein complex required for lipopolysaccharide transport in Escherichia coli.

With the increasing resistance of many Gram-negative bacteria to existing classes of antibiotics, identifying new paradigms in antimicrobial discovery is an important research priority. Of special interest are the proteins required for the biogenesis of the asymmetric Gram-negative bacterial outer membrane (OM). Seven Lpt proteins (LptA to LptG) associate in most Gram-negative bacteria to form a macromolecular complex spanning the entire envelope, which transports lipopolysaccharide (LPS) molecules from their site of assembly at the inner membrane to the cell surface, powered by adenosine 5'-triphosphate hydrolysis in the cytoplasm. The periplasmic protein LptA comprises the protein bridge across the periplasm, which connects LptB2FGC at the inner membrane to LptD/E anchored in the OM. We show here that the naturally occurring, insect-derived antimicrobial peptide thanatin targets LptA and LptD in the network of periplasmic protein-protein interactions required to assemble the Lpt complex, leading to the inhibition of LPS transport and OM biogenesis in Escherichia coli.

A Peptidomimetic Antibiotic Interacts with the Periplasmic Domain of LptD from Pseudomonas aeruginosa.

The outer membrane (OM) in Gram-negative bacteria is an asymmetric bilayer with mostly lipopolysaccharide (LPS) molecules in the outer leaflet. During OM biogenesis, new LPS molecules are transported from their site of assembly on the inner membrane to the OM by seven LPS transport proteins (LptA-G). The complex formed between the integral ß-barrel OM protein LptD and the lipoprotein LptE is responsible for transporting LPS from the periplasmic side of the OM to its final location on the cell surface. Because of its essential function in many Gram-negative bacteria, the LPS transport pathway is an interesting target for the development of new antibiotics. A family of macrocyclic peptidomimetics was discovered recently that target LptD and inhibit LPS transport specifically in Pseudomonas spp. The related molecule Murepavadin is in clinical development for the treatment of life-threatening infections caused by P. aeruginosa. To characterize the interaction of these antibiotics with LptD from P. aeruginosa, we characterized the binding site by cross-linking to a photolabeling probe. We used a hypothesis-free mass spectrometry-based proteomic approach to provide evidence that the antibiotic cross-links to the periplasmic segment of LptD, containing a ß-jellyroll domain and an N-terminal insert domain characteristic of Pseudomonas spp. Binding of the antibiotic to the periplasmic segment is expected to block LPS transport, consistent with the proposed mode of action and observed specificity of these antibiotics. These insights may prove valuable for the discovery of new antibiotics targeting the LPS transport pathway in other Gram-negative bacteria.

Protein Epitope Mimetics: From New Antibiotics to Supramolecular Synthetic Vaccines.

Protein epitope mimetics provide powerful tools to study biomolecular recognition in many areas of chemical biology. They may also provide access to new biologically active molecules and potentially to new classes of drug and vaccine candidates. Here we highlight approaches for the design of folded, structurally defined epitope mimetics, by incorporating backbone and side chains of hot residues onto a stable constrained scaffold. Using robust synthetic methods, the structural, biological, and physical properties of epitope mimetics can be optimized, by variation of both side chain and backbone chemistry. To illustrate the potential of protein epitope mimetics in medicinal chemistry and biotechnology, we present studies in two areas of infectology; the discovery of new antibiotics targeting essential outer membrane (OM) proteins in Gram-negative bacteria and the design of supramolecular synthetic vaccines. The discovery of new antibiotics with novel mechanisms of action, in particular to combat infections caused by Gram-negative pathogens, represents a major challenge in medicinal chemistry. We were inspired by naturally occurring cationic antimicrobial peptides to design structurally related peptidomimetics and to optimize their antimicrobial properties through library synthesis and screening. Through these efforts, we could show that antimicrobial ß-hairpin mimetics may have structures and properties that facilitate interactions with essential bacterial ß-barrel OM proteins. One recently discovered family of antimicrobial peptidomimetics targets the ß-barrel protein LptD in Pseudomonas spp. This protein plays a key role in lipopolysaccaride (LPS) transport to the cell surface during OM biogenesis. Through a highly selective interaction with LptD, the peptidomimetic blocks LPS transport, resulting in nanomolar antimicrobial activity against the important human pathogen P. aeruginosa. Epitope mimetics may also have great potential in the field of vaccinology, where structural information on complexes between neutralizing antibodies and their cognate epitopes can be taken as a starting point for B cell epitope mimetic design. In order to generate potent immune responses, an effective method of delivering epitope mimetics to relevant cells and tissues in the immune system is also required. For this, engineered synthetic nanoparticles (synthetic virus-like particles, SVLPs) prepared using supramolecular chemistry can be designed with optimal surface properties for efficient dendritic cell-mediated delivery of folded B-cell and linear T-cell epitopes, along with ligands for pattern recognition receptors, into lymphoid tissues. In this way, multivalent display of the epitope mimetics occurs over the surface of the nanoparticle, suitable for cross-linking B cell receptors. In this highly immunogenic format, strong epitope-specific humoral immune responses can be elicited that target infections caused by pathogenic microorganisms. Other potential applications of epitope mimetics in next-generation therapeutics are also discussed.

Dendritic Cell Sensing of Hydrophobic Di- and Triacylated Lipopeptides Self-Assembled within Synthetic Virus-like Particles.

Dendritic cells (DCs) play critical roles in developing immune defenses. One important aspect is interaction with pathogen-associated molecular patterns (PAMPs)/danger-associated molecular patterns, including di- and triacylated lipopeptides. Isolated or synthetic lipopeptides are potent vaccine adjuvants, interacting with cell surface TLR2 heterodimers. In contrast, deep embedment within bacteria cell walls would impair lipopeptide interaction with cell surface TLR2, requiring degradation for PAMP recognition. Accordingly, DC processing in the absence of surface TLR2 ligation was defined using synthetic virus-like particles (SVLPs) carrying hydrophobic TLR2 PAMPs within di- and triacylated lipopeptide cores (P2Cys-SVLPs and P3Cys-SVLPs) compared with SVLPs lacking immunomodulatory lipopeptides. DCs rapidly and efficiently internalized SVLPs, which was dominated by slow endocytic processing via macropinocytosis, although some caveolar endocytosis was implicated. This delivered SVLPs primarily into macropinosomes often interacting with EEA-1+ early endosomes. Although endoplasmic reticulum association was occasionally noted, association with recycling/sorting structures was not observed. Involvement of LysoTracker+ structures slowly increased with time, with SVLPs present in such structures ultimately dominating. Only SVLPs carrying di- and triacylated lipopeptide cores induced DC activation and maturation independently of surface TLR2 ligation. Intracellular recognition of SVLP TLR2 ligands was confirmed by observing SVLPs' association with internal TLR2, which had similar kinetics to SVLP association with LysoTracker. This related to inflammatory cytokine induction by SVLP+ DCs, with adaptive immune response activation ex vivo/in vivo. Importantly, particular DCs, not monocytes, recognized intracellular exposure of the TLR2 PAMPs carried by di- and triacylated SVLP cores, which indicates subset-distinct recognition of functional internal TLR2 ligands. Thus, vaccines carrying hydrophobic TLR2 ligands would interact with particular DCs for efficient induction of specific immunity in the absence of additional adjuvant.

Synthesis and antimicrobial activity against Pseudomonas aeruginosa of macrocyclic ß-hairpin peptidomimetic antibiotics containing N-methylated amino acids.

Antimicrobial resistance among Gram-negative bacteria is a growing problem, fueled by the paucity of new antibiotics that target these microorganisms. One novel family of macrocyclic ß-hairpin-shaped peptidomimetics was recently shown to act specifically against Pseudomonas spp. by a novel mechanism of action, targeting the outer membrane protein LptD, which mediates lipopolysaccharide transport to the cell surface during outer membrane biogenesis. Here we explore the mode of binding of one of these ß-hairpin peptidomimetics to LptD in Pseudomonas aeruginosa, by examining the effects on antimicrobial activity following N-methylation of individual peptide bonds. An N-methyl scan of the cyclic peptide revealed that residues on both sides of the ß-hairpin structure at a non-hydrogen bonding position likely mediate hydrogen-bonding interactions with the target LptD. Structural analyses by NMR spectroscopy further reinforce the conclusion that the folded ß-hairpin structure of the peptidomimetic is critical for binding to the target LptD. Finally, new NMe analogues with potent activity have been identified, which opens new avenues for optimization in this family of antimicrobial peptides.

Solution Structure and Dynamics of LptE from Pseudomonas aeruginosa.

LptE is an outer membrane (OM) lipoprotein found in Gram-negative bacteria, where it forms a complex with the ß-barrel lipopolysaccharide (LPS) transporter LptD. The LptD/E complex plays a key role in OM biogenesis, by translocating newly synthesized LPS molecules from the periplasm into the external leaflet of the asymmetric OM during cell growth. The LptD/E complex in Pseudomonas aeruginosa (Pa) is a target for macrocyclic ß-hairpin-shaped peptidomimetic antibiotics, which inhibit the transport of LPS to the cell surface. So far, the three-dimensional structure of the Pa LptD/E complex and the mode of interaction with these antibiotics are unknown. Here, we report the solution structure of a Pa LptE derivative lacking the N-terminal lipid membrane anchor, determined by multidimensional solution nuclear magnetic resonance (NMR) spectroscopy. The structure reveals a central five-stranded ß-sheet against which pack a long C-terminal and a short N-terminal α-helix, as found in homologues of LptE from other Gram-negative bacteria. One unique feature is an extended C-terminal helix in Pa LptE, which in a model of the Pa LptD/E complex appears to be long enough to contact the periplasmic domain of LptD. Chemical shift mapping experiments suggest only weak interactions occur between LptE and the oligosaccharide chains of LPS. The NMR structure of Pa LptE will be valuable for more detailed structural studies of the LptD/E complex from P. aeruginosa.

A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli.

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel ß-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected ß-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many ß-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of ß-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target ß-barrel proteins and the integrity of the Gram-negative OM.

A Synthetic Virus-Like Particle Streptococcal Vaccine Candidate Using B-Cell Epitopes from the Proline-Rich Region of Pneumococcal Surface Protein A.

Alternatives to the well-established capsular polysaccharide-based vaccines against Streptococcus pneumoniae that circumvent limitations arising from limited serotype coverage and the emergence of resistance due to capsule switching (serotype replacement) are being widely pursued. Much attention is now focused on the development of recombinant subunit vaccines based on highly conserved pneumococcal surface proteins and virulence factors. A further step might involve focusing the host humoral immune response onto protective protein epitopes using as immunogens structurally optimized epitope mimetics. One approach to deliver such epitope mimetics to the immune system is through the use of synthetic virus-like particles (SVLPs). SVLPs are made from synthetic coiled-coil lipopeptides that are designed to spontaneously self-assemble into 20-30 nm diameter nanoparticles in aqueous buffer. Multivalent display of epitope mimetics on the surface of SVLPs generates highly immunogenic nanoparticles that elicit strong epitope-specific humoral immune responses without the need for external adjuvants. Here, we set out to demonstrate that this approach can yield vaccine candidates able to elicit a protective immune response, using epitopes derived from the proline-rich region of pneumococcal surface protein A (PspA). These streptococcal SVLP-based vaccine candidates are shown to elicit strong humoral immune responses in mice. Following active immunization and challenge with lethal doses of streptococcus, SVLP-based immunogens are able to elicit significant protection in mice. Furthermore, a mimetic-specific monoclonal antibody is shown to mediate partial protection upon passive immunization. The results show that SVLPs combined with synthetic epitope mimetics may have potential for the development of an effective vaccine against Streptococcus pneumoniae.

Synthesis of a polymyxin derivative for photolabeling studies in the gram-negative bacterium Escherichia coli.

The antimicrobial activity of polymyxins against Gram-negative bacteria has been known for several decades, but the mechanism of action leading to cell death has not been fully explored. A key step after binding of the antibiotic to lipopolysaccharide (LPS) exposed at the cell surface is 'self-promoted uptake' across the outer membrane (OM), in which the antibiotic traverses the asymmetric LPS-phospholipid bilayer before reaching the periplasm and finally targeting and disrupting the bacterial phospholipid inner membrane. The work described here was prompted by the hypothesis that polymyxins might interact with proteins in the OM, as part of their self-promoted uptake and permeabilizing effects. One way to test this is through photolabeling experiments. We describe the design and synthesis of a photoprobe based upon polymyxin B, containing photoleucine and an N-acyl group with a terminal alkyne suitable for coupling to a biotin tag using click chemistry. The resulting photoprobe retains potent antimicrobial activity, and in initial photolabeling experiments with Escherichia coli ATCC25922 is shown to photolabel several OM proteins. This photoprobe might be a valuable tool in more detailed studies on the mechanism of action of this family of antibiotics.

Bis-chlorination of a hexapeptide-PCP conjugate by the halogenase involved in vancomycin biosynthesis.

Vancomycin is an important nosocomial antibiotic containing a glycosylated, cross-linked and doubly chlorinated heptapeptide backbone. During the biosynthesis of the vancomycin aglycone, two ß-hydroxytyrosine (Bht) residues are inserted at positions-2 and -6 into the heptapeptide backbone by a non-ribosomal peptide synthetase. A single flavin-dependent chlorinase (VhaA) is responsible for chlorinating both Bht residues at some ill-defined point in the assembly process. We show here using in vitro assays that VhaA is able to introduce a chlorine atom into each aromatic ring of both Bht residues at positions-2 and -6 of a peptide carrier protein-bound hexapeptide. The results suggest that VhaA can recognize and chlorinate two quite different sites within a linear hexapeptide intermediate during vancomycin biosynthesis.

Structural studies of ß-hairpin peptidomimetic antibiotics that target LptD in Pseudomonas sp.

We report structural studies in aqueous solution on backbone cyclic peptides that possess potent antimicrobial activity specifically against Pseudomonas sp. The peptides target the ß-barrel outer membrane protein LptD, which plays an essential role in lipopolysaccharide transport to the outer membrane. The peptide L27-11 contains a 12-residue loop (T(1)W(2)L(3)K(4)K(5)R(6)R(7)W(8)K(9)K(10)A(11)K(12)) linked to a DPro-LPro template. Two related peptides were also studied, one with various Lys to ornithine or diaminobutyric acid substitutions as well as a DLys(6) (called LB-01), and another containing the same loop sequence, but linked to an LPro-DPro template (called LB-02). NMR studies and MD simulations show that L27-11 and LB-01 adopt ß-hairpin structures in solution. In contrast, LB-02 is more flexible and importantly, adopts a wide variety of different backbone conformations, but not ß-hairpin conformations. L27-11 and LB-01 show antimicrobial activity in the nanomolar range against Pseudomonas aeruginosa, whereas LB-02 is essentially inactive. Thus the ß-hairpin structure of the peptide is important for antimicrobial activity. An alanine scan of L27-11 revealed that tryptophan side chains (W(2)/W(8)) displayed on opposite faces of the ß-hairpin represent key groups contributing to antimicrobial activity.