Evaluation of the Stereochemistry of Staphyloferrin A for Developing Staphylococcus-specific Targeting Conjugates.

Bacteria in the genus Staphylococcus are pathogenic and harmful to humans. Alarmingly, some Staphylococcus, such as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) have spread worldwide and become notoriously resistant to antibiotics, threatening and concerning public health. Hence, the development of new Staphylococcus-targeting diagnostic and therapeutic agents is urgent. Here, we chose the S. aureus-secreted siderophore staphyloferrin A (SA) as a guiding unit. We developed a series of Staphyloferrin A conjugates (SA conjugates) and showed the specific targeting ability to Staphylococcus bacteria. Furthermore, among the structural factors we evaluated, the stereo-chemistry of the amino acid backbone of SA conjugates is essential to efficiently target Staphylococci. Finally, we demonstrated that fluorescent Staphyloferrin A probes (SA-FL probes) could specifically target Staphylococci in complex bacterial mixtures.

Spatiotemporal Investigation of Intercellular Heterogeneity via Multiple Photocaged Probes.

Intercellular heterogeneity occurs widely under both normal physiological environments and abnormal disease-causing conditions. Several attempts to couple spatiotemporal information to cell states in a microenvironment were performed to decipher the cause and effect of heterogeneity. Furthermore, spatiotemporal manipulation can be achieved with the use of photocaged/photoactivatable molecules. Here, we provide a platform to spatiotemporally analyze differential protein expression in neighboring cells by multiple photocaged probes coupled with homemade photomasks. We successfully established intercellular heterogeneity (photoactivable ROS trigger) and mapped the targets (directly ROS-affected cells) and bystanders (surrounding cells), which were further characterized by total proteomic and cysteinomic analysis. Different protein profiles were shown between bystanders and target cells in both total proteome and cysteinome. Our strategy should expand the toolkit of spatiotemporal mapping for elucidating intercellular heterogeneity.

A Versatile Strategy to Manipulate and Probe Native Carbonic Anhydrases In Cellulo Utilizing Two-Step Ligand-Directed Functionalization.

Elucidating the biological logistics and functional interplay of proteins in their natural context has long been a great challenge in biological research. Chemical modification of proteins allows understanding of their roles and their interactions. Over decades, numerous strategies have been developed to modify target proteins with desired probes in test tubes and even biological systems. Nevertheless, these approaches require the design and synthesis of different probes for different applications, even for the same target protein, which is very time- and labor-consuming. Herein, we developed a general two-step protein functionalization strategy that utilizes ligand-directed chemistry to modify a clickable tag on the intact protein in the first step. Then, the desired functional moiety can be conjugated onto the target protein via a simple bioorthogonal click reaction in the second step, thus achieving probing and activity regulation of the target protein. In this work, carbonic anhydrase (CA) was chosen as our model protein for functionalization. We successfully labeled endogenous CAs with fluorophores to allow cellular imaging. In addition, a photoswitchable ligand was conjugated to CAs such that its activity could be manipulated in a light-responsive manner.

Constructing pH-Responsive and Tunable Azo-Phenol-Based o-Nitrobenzyl Photocages.

Multiple triggered-release strategies are widely utilized to control the release of caged target molecules. Among them, photocages with conditional triggers provide extra layers of control in photorelease. In this work, a series of pH-responsive photocages was designed that could be triggered under irradiation and specific intracellular pH values. pH-sensitive phenolic groups were conjugated with o-nitrobenzyl (oNB) to form azo-phenolic NPX photocages with tunable pKa. These azo-phenol-based oNB photocages showed differentiable photoreleasing profiles at pH 5.0, 7.2 and 9.0. By attaching fluorogenic cargos, it was shown that one of the photocages, NPdiCl, could be used to differentiate between acidic pH 5.0 and neutral pH 7.2 in cells under artificial pH conditions. Finally, NPdiCl was identified as a promising pH-responsive photocage for photoreleasing cargo inside acidic tumor cells.

A Double Photocage Strategy to Construct Light-Controllable and Spatiotemporally Trackable Cathepsin B Activity-Based Probes.

Utilizing multiple cages to selectively modulate the activity of biomolecules is indispensable to achieving controllable and trackable activity manipulation. However, trackable cages that can be used to monitor the activation of biomolecules are rare. In this work, we utilized a double photocage strategy to achieve light-controllable and spatiotemporally trackable activation. To demonstrate biological applicability, we used the well-known cancer cell biomarker cathepsin B as the target and constructed double photocaged cathepsin B activity-based probe 2PPG-FK-AcRha that performed well in cancer cell cultures. Using our probe, we could monitor the light-activation by the blue fluorescence of 7-diethylamino-4-hydroxymethyl-coumarin (DEACM) and simultaneously probe the activity of cathepsin B through the green fluorescence of acetyl rhodamine (AcRha). Additionally, by partially irradiating the cell cultures, the regional photoactivation experiments also demonstrated great spatial controllability and trackability of our probe.

Selectively Targeting and Differentiating Vancomycin-Resistant Staphylococcus aureus via Dual Synthetic Fluorescent Probes.

Many Staphylococcus bacteria are pathogenic and harmful to humans. Noticeably, some Staphylococcus, including vancomycin-resistant S. aureus (VRSA), have become notoriously resistant to antibiotics and have spread rapidly, becoming threats to public health. Here, we designed a dual fluorescent probe scheme combining siderophores and antibiotics as the guiding units to selectively target VRSA and vancomycin-sensitive S. aureus (VSSA) in complex bacterial samples. Siderophore-mediated iron uptake is the key pathway by which S. aureus acquires iron in limited environments. Therefore, the siderophore-derivative probe could differentiate between S. aureus and other bacteria. Moreover, by fine-tuning the vancomycin-derivative probes, we could selectively target only VSSA, further differentiating VRSA and VSSA. Finally, by combining the siderophore-derivative probe and the vancomycin-derivative probe, we successfully targeted and differentiated between VRSA and VSSA in complicated bacterial mixtures.

Sonogenetic-Based Neuromodulation for the Amelioration of Parkinson's Disease.

Sonogenetics is a promising strategy allowing the noninvasive and selective activation of targeted neurons in deep brain regions; nevertheless, its therapeutic outcome for neurodegeneration diseases that need long-term treatment remains to be verified. We previously enhanced the ultrasound (US) sensitivity of targeted cells by genetic modification with an engineered auditory-sensing protein, mPrestin (N7T, N308S). In this study, we expressed mPrestin in the dopaminergic neurons of the substantia nigra in Parkinson's disease (PD) mice and used 0.5 MHz US for repeated and localized brain stimulation. The mPrestin expression in dopaminergic neurons persisted for at least 56 days after a single shot of adeno-associated virus, suggesting that the period of expression was long enough for US treatment in mice. Compared to untreated mice, US stimulation ameliorated the dopaminergic neurodegeneration 10-fold and mitigated the PD symptoms of the mice 4-fold, suggesting that this sonogenetic strategy has the clinical potential to treat neurodegenerative diseases.

Labeling and Characterization of Phenol-Containing Glycopeptides Using Chemoselective Probes with Isotope Tags.

Secondary metabolites are structurally diverse natural products (NPs) and have been widely used for medical applications. Developing new tools to enrich NPs can be a promising solution to isolate novel NPs from the native and complex samples. Here, we developed native and deuterated chemoselective labeling probes to target phenol-containing glycopeptides by the ene-type labeling used in proteomic research. The clickable azido-linker was included for further biotin functionalization to facilitate the enrichment of labeled substrates. Afterward, our chemoselective method, in conjunction with LC-MS and MSn analysis, was demonstrated in bacterial cultures. A vancomycin-related phenol-containing glycopeptide was labeled and characterized by our labeling strategy, showing its potential in glycopeptide discovery in complex environments.

Reaction Tracking and High-Throughput Screening of Active Compounds in Combinatorial Chemistry by Tandem Mass Spectrometry Molecular Networking.

Combinatorial synthesis has been widely used as an efficient strategy to screen for active compounds. Mass spectrometry is the method of choice in the identification of hits resulting from high-throughput screenings due to its high sensitivity, specificity, and speed. However, manual data processing of mass spectrometry data, especially for structurally diverse products in combinatorial chemistry, is extremely time-consuming and one of the bottlenecks in this process. In this study, we demonstrated the effectiveness of a tandem mass spectrometry molecular networking-based strategy for product identification, reaction dynamics monitoring, and active compound targeting in combinatorial synthesis. Molecular networking connects compounds with similar tandem mass spectra into a cluster and has been widely used in natural products analysis. We show that both the expected and side products can be readily characterized using molecular networking based on their mass spectrometry fragmentation patterns. Additionally, time-dependent molecular networking was integrated to track reaction dynamics to determine the optimal reaction time to maximize target product yields. We also present a proof-of-concept experiment that successfully identified and isolated active molecules from a dynamic combinatorial library. These results demonstrated the potential of using molecular networking for identifying, tracking, and high-throughput screening of active compounds in combinatorial synthesis.

Selective Targeting of Vibrios by Fluorescent Siderophore-Based Probes.

Siderophores are small molecules used to specifically transport iron into bacteria via related receptors. By adapting siderophores and hijacking their pathways, we may discover an efficient and selective way to target microbes. Herein, we report the synthesis of a siderophore-fluorophore conjugate VF-FL derived from vibrioferrin (VF). Using flow cytometry and fluorescence microscopy, the probe selectively labeled vibrios, including V. parahaemolyticus, V. cholerae, and V. vulnificus, even in the presence of other species such as S. aureus and E. coli. The labeling is siderophore-related and both iron-limited conditions and the siderophore moiety are required. The competitive relationship between VF-FL and VF in vibrios implies an unreported VF-related transport mechanism in V. cholerae and V. vulnificus. These studies demonstrate that the siderophore scaffold provides a method to selectively target microbes expressing cognate receptors under iron-limited conditions.

Utilizing an iron(iii)-chelation masking strategy to prepare mono- and bis-functionalized aerobactin analogues for targeting pathogenic bacteria.

A direct and selective method for the functionalization of aerobactin has been described. Selectivity was achieved by masking the functioning carboxylate via iron-chelation, leaving the two remaining carboxylates for direct modification. Both mono- and bis-functionalized aerobactin effectively targeted pathogenic bacteria, showing a facile method with prospective applications.

Stabilizing Protein Motifs with a Genetically Encoded Metal-Ion Chelator.

The N-peptide of HIV gp41 forms a trimeric coiled-coil intermediate during host cell-viral fusion. Stable mimics of this coiled coil could potentially serve as HIV vaccine candidates or inhibitors of viral entry. Therefore, a variety of approaches have been investigated to maintain the N-peptide in its trimeric helical conformation. Here, we utilize a genetic method to incorporate the metal chelating noncanonical amino acid (2,2'-bipyridin-5-yl)alanine (BpyAla) into IZN17, an established trimeric coiled-coil gp41 model. We demonstrate that BpyAla-IZN17 acquires Fe(II) during expression in Escherichia coli, resulting in the formation of a highly stable IZN17 helical trimer with a Tm > 95°C. Removal of Fe(II) results in a 27°C decrease in thermal stability. Replacement of Fe(II) with Zn(II) also affords a Tm > 90°C. Thus, the genetic introduction of BpyAla into polypeptide provides a straightforward method for stabilizing coiled-coil structural motifs in recombinantly engineered proteins.

Facile and Versatile Chemoenzymatic Synthesis of Enterobactin Analogues and Applications in Bacterial Detection.

Siderophores, such as enterobactin (Ent), are small molecules that can be selectively imported into bacteria along with iron by cognate transporters. Siderophore conjugates are thus a promising strategy for delivering functional reagents into bacteria. In this work, we present an easy-to-perform, one-pot chemoenzymatic synthesis of functionalized monoglucosylated enterobactin (MGE). When functionalized MGE is conjugated to a rhodamine fluorophore, which affords RhB-Glc-Ent, it can selectively label Gram-negative bacteria that utilize Ent, including some E. coli strains and P. aeruginosa. V. cholerae, a bacterium that utilizes linearized Ent, can also be weakly targeted. Moreover, the targeting is effective under iron-limiting but not iron-rich conditions. Our results suggest that the RhB-Glc-Ent probe is sensitive not only to the bacterial strain but also to the iron condition in the environment.

An antibody with a variable-region coiled-coil "knob" domain.

The X-ray crystal structure of a bovine antibody (BLV1H12) revealed a unique structure in its ultralong heavy chain complementarity determining region 3 (CDR3H) that folds into a solvent-exposed ß-strand "stalk" fused to a disulfide crosslinked "knob" domain. We have substituted an antiparallel heterodimeric coiled-coil motif for the ß-strand stalk in this antibody. The resulting antibody (Ab-coil) expresses in mammalian cells and has a stability similar to that of the parent bovine antibody. MS analysis of H-D exchange supports the coiled-coil structure of the substituted peptides. Substitution of the knob-domain of Ab-coil with bovine granulocyte colony-stimulating factor (bGCSF) results in a stably expressed chimeric antibody, which proliferates mouse NFS-60 cells with a potency comparable to that of bGCSF. This work demonstrates the utility of this novel coiled-coil CDR3 motif as a means for generating stable, potent antibody fusion proteins with useful pharmacological properties.

Haloduracin α binds the peptidoglycan precursor lipid II with 2:1 stoichiometry.

The two-peptide lantibiotic haloduracin is composed of two post-translationally modified polycyclic peptides that synergistically act on gram-positive bacteria. We show here that Halα inhibits the transglycosylation reaction catalyzed by PBP1b by binding in a 2:1 stoichiometry to its substrate lipid II. Halß and the mutant Halα-E22Q were not able to inhibit this step in peptidoglycan biosynthesis, but Halα with its leader peptide still attached was a potent inhibitor. Combined with previous findings, the data support a model in which a 1:2:2 lipid II:Halα:Halß complex inhibits cell wall biosynthesis and mediates pore formation, resulting in loss of membrane potential and potassium efflux.

Transpeptidase-mediated incorporation of D-amino acids into bacterial peptidoglycan.

The ß-lactams are the most important class of antibiotics in clinical use. Their lethal targets are the transpeptidase domains of penicillin binding proteins (PBPs), which catalyze the cross-linking of bacterial peptidoglycan (PG) during cell wall synthesis. The transpeptidation reaction occurs in two steps, the first being formation of a covalent enzyme intermediate and the second involving attack of an amine on this intermediate. Here we use defined PG substrates to dissect the individual steps catalyzed by a purified E. coli transpeptidase. We demonstrate that this transpeptidase accepts a set of structurally diverse D-amino acid substrates and incorporates them into PG fragments. These results provide new information on donor and acceptor requirements as well as a mechanistic basis for previous observations that noncanonical D-amino acids can be introduced into the bacterial cell wall.

Primer preactivation of peptidoglycan polymerases.

Peptidoglycan glycosyltransferases are highly conserved bacterial enzymes that catalyze glycan strand polymerization to build the cell wall. Because the cell wall is essential for bacterial cell survival, these glycosyltransferases are potential antibiotic targets, but a detailed understanding of their mechanisms is lacking. Here we show that a synthetic peptidoglycan fragment that mimics the elongating polymer chain activates peptidoglycan glycosyltransferases by bypassing the rate-limiting initiation step.

Modular synthesis of diphospholipid oligosaccharide fragments of the bacterial cell wall and their use to study the mechanism of moenomycin and other antibiotics.

We present a flexible, modular route to GlcNAc-MurNAc-oligosaccharides that can be readily converted into peptidoglycan (PG) fragments to serve as reagents for the study of bacterial enzymes that are targets for antibiotics. Demonstrating the utility of these synthetic PG substrates, we show that the tetrasaccharide substrate lipid IV (3), but not the disaccharide substrate lipid II (2), significantly increases the concentration of moenomycin A required to inhibit a prototypical PG-glycosyltransferase (PGT). These results imply that lipid IV and moenomycin A bind to the same site on the enzyme. We also show the moenomycin A inhibits the formation of elongated polysaccharide product but does not affect length distribution. We conclude that moenomycin A blocks PG-strand initiation rather than elongation or chain termination. Synthetic access to diphospholipid oligosaccharides will enable further studies of bacterial cell wall synthesis with the long-term goal of identifying novel antibiotics.

Functional and structural analysis of a key region of the cell wall inhibitor moenomycin.

Moenomycin A (MmA) belongs to a family of natural products that inhibit peptidoglycan biosynthesis by binding to the peptidoglycan glycosyltransferases, the enzymes that make the glycan chains of peptidoglycan. MmA is remarkably potent, but its clinical utility has been hampered by poor physicochemical properties. Moenomycin contains three structurally distinct regions: a pentasaccharide, a phosphoglycerate, and a C25 isoprenyl (moenocinyl) lipid tail that gives the molecule its name. The phosphoglycerate moiety links the pentasaccharide to the moenocinyl chain. This moiety contains two negatively charged groups, a phosphoryl group and a carboxylate. Both the phosphoryl group and the carboxylate have previously been implicated in target binding but the role of the carboxylate has not been explored in detail. Here we report the synthesis of six MmA analogues designed to probe the importance of the phosphoglycerate. These analogues were evaluated for antibacterial and enzyme inhibitory activity; the specific contacts between the phosphoglycerate and the protein target were assessed by X-ray crystallography in conjunction with molecular modeling. Both the phosphoryl group and the carboxylate of the phosphoglycerate chain play roles in target binding. The negative charge of the carboxylate, and not its specific structure, appears to be the critical feature in binding since replacing it with a negatively charged acylsulfonamide group produces a more active compound than replacing it with the isosteric amide. Analysis of the ligand-protein contacts suggests that the carboxylate makes a critical contact with an invariant lysine in the active site. The reported work provides information and validated computational methods critical for the design of analogues based on moenomycin scaffolds.

The role of the substrate lipid in processive glycan polymerization by the peptidoglycan glycosyltransferases.

The peptidoglycan glycosyltransferases (PGTs) catalyze the processive polymerization of a C55 lipid-linked disaccharide (Lipid II) to form peptidoglycan, the main component of the bacterial cell wall. Our ability to understand this reaction has been limited due to challenges identifying the appropriate substrate analogues to selectively interrogate the donor (the elongating strand) and acceptor (Lipid II) sites. To address this problem, we have developed an assay using synthetic substrates that can discriminate between the donor and acceptor sites of the PGTs. We have shown that each site has a distinct lipid length preference. We have also established that processive polymerization depends on the length of the lipid attached to the donor.