Erin E. Carlson

Natural Products as Chemical Probes

Natural products have evolved to encompass a broad spectrum of chemical and functional diversity. It is this diversity, along with their structural complexity, that enables natures small molecules to target a nearly limitless number of biological macromolecules and to often do so in a highly selective fashion. Because of these characteristics, natural products have seen great success as therapeutic agents. However, this vast pool of compounds holds much promise beyond the development of future drugs. These features also make them ideal tools for the study of biological systems. Recent examples of the use of natural products and their derivatives as chemical probes to explore biological phenomena and assemble biochemical pathways are presented here.

A unique catalytic mechanism for UDP-galactopyranose mutase.

The flavoenzyme uridine 5′-diphosphate (UDP)-galactopyranose mutase (UGM) catalyzes the interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). The latter is an essential precursor to the cell wall arabinogalactan of Mycobacterium tuberculosis. The catalytic mechanism for this enzyme had not been elucidated. Here, we provide evidence for a mechanism in which the flavin cofactor assumes a new role. Specifically, the N5 of the reduced anionic flavin cofactor captures the anomeric position of the galactose residue with release of UDP. Interconversion of the isomers occurs via a flavin-derived iminium ion. To trap this putative intermediate, we treated UGM with radiolabeled UDP-Galp and sodium cyanoborohydride; a radiolabeled flavin-galactose adduct was obtained. Ultraviolet-visible spectroscopy and mass spectrometry indicate that this product is an N5-alkyl flavin. We anticipate that the clarification of the catalytic mechanism for UGM will facilitate the development of anti-mycobacterial agents.

Chemoselective probes for metabolite enrichment and profiling.

Chemical probes that target classes of proteins based on shared functional properties have emerged as powerful tools for proteomics. The metabolome rivals, if not surpasses, the proteome in terms of size and complexity, suggesting that efforts to profile metabolites would also benefit from targeted technologies. Here we apply the principle of chemoselective probes to the metabolome, creating a general strategy to tag, enrich and profile large classes of small molecules from biological systems. Key to success was incorporation of a protease-cleavage step to release captured metabolites in a format compatible with liquid chromatography–mass spectrometry (LC-MS) analysis. This technology, termed metabolite enrichment by tagging and proteolytic release (METPR), is applicable to small molecules of any physicochemical class, including polar, labile and low-mass (<100 Da) compounds. We applied METPR to profile changes in the thiol metabolome of human cancer cells treated with the antioxidant N-acetyl-L-cysteine.

Requirement of essential Pbp2x and GpsB for septal ring closure in Streptococcus pneumoniae D39

Bacterial cell shapes are manifestations of programs carried out by multi‐protein machines that synthesize and remodel the resilient peptidoglycan (PG) mesh and other polymers surrounding cells. GpsB protein is conserved in low‐GC Gram‐positive bacteria and is not essential in rod‐shaped Bacillus subtilis, where it plays a role in shuttling penicillin‐binding proteins (PBPs) between septal and side‐wall sites of PG synthesis. In contrast, we report here that GpsB is essential in ellipsoid‐shaped, ovococcal Streptococcus pneumoniae (pneumococcus), and depletion of GpsB leads to formation of elongated, enlarged cells containing unsegregated nucleoids and multiple, unconstricted rings of fluorescent‐vancomycin staining, and eventual lysis. These phenotypes are similar to those caused by selective inhibition of Pbp2x by methicillin that prevents septal PG synthesis. Dual‐protein 2D and 3D‐SIM (structured illumination) immunofluorescence microscopy (IFM) showed that GpsB and FtsZ have overlapping, but not identical, patterns of localization during cell division and that multiple, unconstricted rings of division proteins FtsZ, Pbp2x, Pbp1a and MreC are in elongated cells depleted of GpsB. These patterns suggest that GpsB, like Pbp2x, mediates septal ring closure. This first dual‐protein 3D‐SIM IFM analysis also revealed separate positioning of Pbp2x and Pbp1a in constricting septa, consistent with two separable PG synthesis machines.

Pbp2x localizes separately from Pbp2b and other peptidoglycan synthesis proteins during later stages of cell division of Streptococcus pneumoniae D39

The relative localization patterns of class B penicillin‐binding proteins Pbp2x and Pbp2b were used as positional indicators of septal and peripheral (side‐wall‐like) peptidoglycan (PG) synthesis, respectively, in the mid‐cell regions of Streptococcus pneumoniae cells at different stages of division. We confirm that Pbp2x and Pbp2b are essential in the strain D39 genetic background, which differs from that of laboratory strains. We show that Pbp2b, like Pbp2x and class A Pbp1a, follows a different localization pattern than FtsZ and remains at division septa after FtsZ reappears at the equators of daughter cells. Pulse‐experiments with fluorescent D‐amino acids (FDAAs) were performed in wild‐type cells and in cells in which Pbp2x activity was preferentially inhibited by methicillin or Pbp2x amount was depleted. These experiments show that Pbp2x activity separates from that of other PBPs to the centres of constricting septa in mid‐to‐late divisional cells resolved by high‐resolution 3D‐SIM microscopy. Dual‐protein and protein‐fluorescent vancomycin 2D and 3D‐SIM immunofluorescence microscopy (IFM) of cells at different division stages corroborate that Pbp2x separates to the centres of septa surrounded by an adjacent constricting ring containing Pbp2b, Pbp1a and regulators, StkP and MreC. The separate localization of Pbp2x suggests distinctive roles in completing septal PG synthesis and remodelling.

A general glycomimetic strategy yields non-carbohydrate inhibitors of DC-SIGN

Shikimic acid can be transformed into monovalent and multivalent glycomimetics that target different members of the C-type lectin class, including DC-SIGN, a dendritic cell lectin that facilitates HIV transmission.

Selective Penicillin-Binding Protein Imaging Probes Reveal Substructure in Bacterial Cell Division

The peptidoglycan cell wall is a common target for antibiotic therapy, but its structure and assembly are only partially understood. Peptidoglycan synthesis requires a suite of penicillin-binding proteins (PBPs), the individual roles of which are difficult to determine because each enzyme is often dispensable for growth perhaps due to functional redundancy. To address this challenge, we sought to generate tools that would enable selective examination of a subset of PBPs. We designed and synthesized fluorescent and biotin derivatives of the β-lactam-containing antibiotic cephalosporin C. These probes facilitated specific in vivo labeling of active PBPs in both Bacillus subtilis PY79 and an unencapsulated derivative of D39 Streptococcus pneumoniae. Microscopy and gel-based analysis indicated that the cephalosporin C-based probes are more selective than BOCILLIN-FL, a commercially available penicillin V analogue, which labels all PBPs. Dual labeling of live cells performed by saturation of cephalosporin C-susceptible PBPs followed by tagging of the remaining PBP population with BOCILLIN-FL demonstrated that the two sets of PBPs are not co-localized. This suggests that even PBPs that are located at a particular site (e.g., septum) are not all intermixed, but rather that PBP subpopulations are discretely localized. Accordingly, the Ceph C probes represent new tools to explore a subset of PBPs and have the potential to facilitate a deeper understand of the roles of this critical class of proteins.

Integrated metabolomics approach facilitates discovery of an unpredicted natural product suite from Streptomyces coelicolor M145.

Natural products exhibit a broad range of biological properties and have been a crucial source of therapeutic agents and novel scaffolds. Although bacterial secondary metabolomes are widely explored, they remain incompletely cataloged by current isolation and characterization strategies. To identify metabolites residing in unexplored chemical space, we have developed an integrated discovery approach that combines bacterial growth perturbation, accurate mass spectrometry, comparative mass spectra data analysis, and fragmentation spectra clustering for the identification of low-abundant, novel compounds from complex biological matrices. In this investigation, we analyzed the secreted metabolome of the extensively studied Actinomycete, Streptomyces coelicolor M145, and discovered a low-abundant suite of 15 trihydroxamate, amphiphilic siderophores. Compounds in this class have primarily been observed in marine microorganisms making their detection in the soil-dwelling S. coelicolor M145 significant. At least 10 of these ferrioxamine-based molecules are not known to be produced by any organism, and none have previously been detected from S. coelicolor M145. In addition, we confirmed the production of ferrioxamine D1, a relatively hydrophilic family member that has not been shown to be biosynthesized by this organism. The identified molecules are part of only a small list of secondary metabolites that have been discovered since sequencing of S. coelicolor M145 revealed that it possessed numerous putative secondary metabolite-producing gene clusters with no known metabolites. Thus, the identified siderophores represent the unexplored metabolic potential of both well-studied and new organisms that could be uncovered with our sensitive and robust approach.

Biological Responses to Engineered Nanomaterials: Needs for the Next Decade.

The interaction of nanomaterials with biomolecules, cells, and organisms is an enormously vital area of current research, with applications in nanoenabled diagnostics, imaging agents, therapeutics, and contaminant removal technologies. Yet the potential for adverse biological and environmental impacts of nanomaterial exposure is considerable and needs to be addressed to ensure sustainable development of nanomaterials. In this Outlook four research needs for the next decade are outlined: (i) measurement of the chemical nature of nanomaterials in dynamic, complex aqueous environments; (ii) real-time measurements of nanomaterial–biological interactions with chemical specificity; (iii) delineation of molecular modes of action for nanomaterial effects on living systems as functions of nanomaterial properties; and (iv) an integrated systems approach that includes computation and simulation across orders of magnitude in time and space.

Activity-based probe for histidine kinase signaling.

Bacterial two-component systems (TCSs) are signaling pathways composed of two proteins: a histidine kinase (HK) and a response regulator (RR). Upon stimulation, the HK autophosphorylates at a conserved histidine. The phosphoryl group is subsequently transferred to an aspartate on an RR, eliciting an adaptive response, often up- or downregulation of gene expression. TCS signaling controls many functions in bacteria, including development, virulence, and antibiotic resistance, making the proteins involved in these systems potential therapeutic targets. Efficient methods for the profiling of HKs are currently lacking. For direct readout of HK activity, we sought to design a probe that enables detection of the phosphotransfer event; however, analysis of the phosphohistidine species is made difficult by the instability of the P-N bond. We anticipated that use of a γ-thiophosphorylated ATP analogue, which would yield a thiophosphorylated histidine intermediate, could overcome this challenge. We determined that the fluorophore-conjugated probe, BODIPY-FL-ATPγS, labels active HK proteins and is competitive for the ATP binding site. This activity-based probe provides a new strategy for analysis of TCSs and other HK-mediated processes and will facilitate both functional studies and inhibitor identification.