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Biomedical subjects

Laura L Kiessling

Publications and source records attributed to Laura L Kiessling.

At least 19 recordsLinked to original sources
PubMed2026

The phenotypic landscape of the mycobacterial cell.

The Mycobacteriales are an order of diverse bacteria that thrive in many environmental and host-associated niches. Because the most notorious member of this clade, Mycobacterium tuberculosis, is a major human pathogen, research on Mycobacteriales has focused on pathogenesis, and, as a consequence, many fundamental aspects of Mycobacterial biology remain understudied. Here, we address this gap by performing a genome-wide CRISPRi chemical genomics screen using a diverse set of >35 antibiotics, detergents, and other anti-microbials predominantly targeting the cell envelope of Mycobacterium smegmatis, a saprophytic model Mycobacterium. We highlight new information derived from this screen, including the identification of novel functions for previously uncharacterized conserved and essential genes (in mycolic acid and arabinogalactan synthesis), the discovery of a new drug scaffold/protein target pair, and insights into the mechanism of action of two commonly used antibiotics. These data are also a valuable resource for the mycobacterial research community, as they provide thousands of novel phenotypes for uncharacterized genes and meaningful phenotypic correlations between annotated and uncharacterized genes.

Journal Article↗
PubMed2007

Bifunctional ligands that target cells displaying the alpha v beta3 integrin.

Strategies to eliminate tumor cells have long been sought. We envisioned that a small molecule could be used to decorate the offending cells with immunogenic carbohydrates and evoke an immune response. To this end, we describe the synthesis of bifunctional ligands possessing two functional motifs: one binds a cell-surface protein and the other binds a naturally occurring human antibody. Our conjugates combine an RGD-based peptidomimetic, to target cells displaying the alpha v beta3 integrin, with the carbohydrate antigen galactosyl-alpha(1-3)galactose [Galalpha(1-3)Gal or alpha-Gal]. To generate such bifunctional ligands, we designed and synthesized RGD mimetics 1 b and 2 c, which possess a free amino group for modification. These compounds were used to generate bifunctional derivatives 1 c and 2 d, with dimethyl squarate serving as the linchpin; thus, our synthetic approach is modular. To evaluate the binding of our peptidomimetics to the target alpha v beta3-displaying cells, we implemented a cell-adhesion assay. Results from this assay indicate that the designed, small-molecule ligands inhibit alpha v beta3-dependent cell adhesion. Additionally, our most effective bifunctional ligand exhibits a high degree of selectivity (4000-fold) for alpha v beta3 over the related alpha v beta5 integrin, a result that augurs its utility in specific cell targeting. Finally, we demonstrate that the bifunctional ligands can bind to alpha v beta3-positive cells and recruit human anti-Gal antibodies. These results indicate that both the integrin-binding and the anti-Gal-binding moieties can act simultaneously. Bifunctional conjugates of this type can facilitate the development of new methods for targeting cancer cells by exploiting endogenous antibodies. We anticipate that our modifiable alpha v beta3-binding ligands will be valuable in a variety of applications, including drug delivery and tumor targeting.

Biochemistry↗
PubMed2006

Phage display affords peptides that modulate beta-amyloid aggregation.

As the population ages, the need to develop methods to understand and intercept the processes responsible for protein aggregation diseases is becoming more urgent. The aggregation of the protein beta-amyloid (Abeta) has been implicated in Alzheimer's Disease (AD); however, whether the toxic species is a large, insoluble aggregate or some lower order form is not yet known. Agents that can modulate the aggregation state of Abeta could resolve this controversy by facilitating our understanding of the consequences of aggregation and its underlying mechanism. To date, however, ligands that bind to specific forms of Abeta have not been identified. To address this deficiency, we tested whether phage display could yield such ligands by screening libraries against Abeta in two different states: monomeric or highly aggregated. Intriguingly, the peptides selected had different effects on Abeta aggregation. Peptides selected for binding to monomeric Abeta did not perturb aggregation, but those selected using highly aggregated Abeta increase the rate of aggregation drastically. The latter also alter the morphology of the resulting aggregate. The ability of a peptide to promote aggregation correlated with its affinity for the N-terminal 10 residues of Abeta. This result indicates that the mechanism by which the peptides influence aggregation is related to their affinity for the Abeta N-terminus. Thus, the identification of compounds that bind to this Abeta section can afford agents that affect aggregation. Moreover, the data suggest that endogenous ligands that interact with the N-terminal region can influence the propensity of Abeta to form aggregates and the morphology of those that form. Our data highlight the utility of phage display for identifying ligands that bind to target proteins in different states, and they indicate that such agents can be used to perturb protein aggregation.

Amino Acid Sequence↗
PubMed2006

N-acylsulfonamide linker activation by Pd-catalyzed allylation.

N-Acylsulfonamide safety-catch linkers are versatile tools in solid-phase organic synthesis because of their stability. This stability necessitates linker activation prior to compound cleavage. Here, we demonstrate that the N-acylsulfonamide group can react with a pi-allyl palladium complex and that these mild and neutral conditions can be exploited for linker activation. The advantages of this process are illustrated by its use in the efficient synthesis of thioesters. We anticipate that this activation method will extend the utility of the N-acylsulfonamide group. [reaction: see text]

Catalysis↗
PubMed2006

Contrast agents for magnetic resonance imaging synthesized with ring-opening metathesis polymerization.

A monomer for ring-opening metathesis polymerization (ROMP) has been developed that also functions as a portion of a GdIII chelating moiety for a magnetic resonance imaging contrast agent. An increase in per GdIII relaxivity was shown upon transition from monomer to polymer. Additionally, extremely large molecular relaxivities were achieved through incorporation of multiple GdIII ions per polymer. The nature of ROMP-derived polymers allows for functionalization of the monomer units and termini through orthogonal chemistry. This strategy is the basis for a new class of highly sensitive, targeted imaging agents.

Contrast Media↗
PubMed2006

Synthetic multivalent ligands as probes of signal transduction.

Cell-surface receptors acquire information from the extracellular environment and coordinate intracellular responses. Many receptors do not operate as individual entities, but rather as part of dimeric or oligomeric complexes. Coupling the functions of multiple receptors may endow signaling pathways with the sensitivity and malleability required to govern cellular responses. Moreover, multireceptor signaling complexes may provide a means of spatially segregating otherwise degenerate signaling cascades. Understanding the mechanisms, extent, and consequences of receptor co-localization and interreceptor communication is critical; chemical synthesis can provide compounds to address the role of receptor assembly in signal transduction. Multivalent ligands can be generated that possess a variety of sizes, shapes, valencies, orientations, and densities of binding elements. This Review focuses on the use of synthetic multivalent ligands to characterize receptor function.

Animals↗
PubMed2006

Chemical probes of UDP-galactopyranose mutase.

Many pathogenic prokaryotes and eukaryotes possess the machinery required to assemble galactofuranose (Galf)-containing glycoconjugates; these glycoconjugates can be critical for virulence or viability. Accordingly, compounds that block Galf incorporation may serve as therapeutic leads or as probes of the function of Galf-containing glycoconjugates. The enzyme UDP-galactopyranose mutase (UGM) is the only known generator of UDP-galactofuranose, the precursor to Galf residues. We previously employed a high-throughput fluorescence polarization assay to investigate the Klebsiella pneumoniae UGM. We demonstrate the generality of this assay by extending it to UGM from Mycobacterium tuberculosis. To identify factors influencing binding, we synthesized a directed library containing a 5-arylidene-2-thioxo-4-thiazolidinone core, a structure possessing features common to ligands for both homologs. Our studies offer a blueprint for identifying inhibitors of the growing family of UGM homologs and provide insight into UGM inhibition.

Binding Sites↗
PubMed2005

Solid-phase synthesis of polymers using the ring-opening metathesis polymerization.

We report a general method for the solid-phase synthesis of polymers via the ring-opening metathesis polymerization (ROMP). The method involves polymerization in solution to form a block copolymer, immobilization of the polymer via reaction of one block with a resin-bound functional group, modification of the other block, and liberation of the polymer from the resin. We demonstrated the utility of this approach by generating a block copolymer with an N-hydroxysuccinimidyl ester-substituted block (for on-resin functionalization) and a maleimide-substituted block (for conjugation to the resin). We showed that the Diels-Alder reaction can be employed to immobilize the polymers and that amines of diverse structure can be used to modify the resin-bound polymers. The reversibility of the furan-maleimide Diels-Alder adduct was exploited to liberate the polymer from the support. Specifically, treatment of the resin with cyclopentadiene resulted in complete polymer release. The resulting polymers are functional: they were as potent in assays with the lectin concanavalin A as polymers generated by traditional solution routes. We anticipate that this method can be used for the rapid synthesis of diverse polymers via ROMP.

Esters↗
PubMed2005

Link between chemotactic response to Ni2+ and its adsorption onto the Escherichia coli cell surface.

Bacterial chemotaxis is of medical, biological, and geological significance. Despite its importance, current chemotaxis measurements fail to account for the speciation of the chemical effector and the protonation state of the bacterial surface. We hypothesize that adsorption of Ni2+ onto the surface of Escherichia coli can influence its effective concentration and therefore influence its ability to induce a repellent response. By measuring repellent response at different pH values, the influence of Ni2+ adsorption on chemotaxis was assessed. In addition, we tested the effect of different Ni2+ chelating agents. Our data indicate that adsorption reactions influence the chemotactic response to Ni2+. We use potentiometric titration and Ni2+ adsorption experiments to develop and constrain a thermodynamic model capable of quantifying the concentration of Ni2+ at the bacteria/solution interface. Results from this model predict that the concentration of adsorbed Ni2+ is linearly proportional to the magnitude of the chemotactic response in E. coli. If adsorption is linked to chemotaxis in other cases, then chemotactic responses in realistic settings depend on a number of environmental factors such as pH, competing binding agents (e.g., aqueous organic acids, natural organic matter, mineral surfaces, etc.), and ionic strength. Our modeling approach quantifies adsorbed species on bacterial surfaces and may be used to predict the responses of different species to a variety of chemoeffectors. Our data suggest that specified changes in environmental conditions can be used to tune chemotactic responses in natural biological and geological settings.

Adsorption↗
PubMed2005

Large increases in attractant concentration disrupt the polar localization of bacterial chemoreceptors.

In bacterial chemotaxis, the chemoreceptors [methyl-accepting chemotaxis proteins (MCPs)] transduce chemotactic signals through the two-component histidine kinase CheA. At low but not high attractant concentrations, chemotactic signals must be amplified. The MCPs are organized into a polar lattice, and this organization has been proposed to be critical for signal amplification. Although evidence in support of this model has emerged, an understanding of how signals are amplified and modulated is lacking. We probed the role of MCP localization under conditions wherein signal amplification must be inhibited. We tested whether a large increase in attractant concentration (a change that should alter receptor occupancy from c. 0% to > 95%) would elicit changes in the chemoreceptor localization. We treated Escherichia coli or Bacillus subtilis with a high level of attractant, exposed cells to the cross-linking agent paraformaldehyde and visualized chemoreceptor location with an anti-MCP antibody. A marked increase in the percentage of cells displaying a diffuse staining pattern was obtained. In contrast, no increase in diffuse MCP staining is observed when cells are treated with a repellent or a low concentration of attractant. For B. subtilis mutants that do not undergo chemotaxis, the addition of a high concentration of attractant has no effect on MCP localization. Our data suggest that interactions between chemoreceptors are decreased when signal amplification is unnecessary.

Bacillus subtilis↗
PubMed2004

Stereoselective N-glycosylation by Staudinger ligation.

Stereoselective methods for the chemical synthesis of beta-N-glycosyl amides are needed to generate glycopeptides and glycoproteins. Here, we report that the Staudinger ligation can be used to form glycosylated asparagine derivatives. The reaction proceeds with high stereoselectivity, and a variety of glycosyl azides can function as substrates. Our results provide precedence for the use of this powerful amide-bond-forming reaction for N-glycopeptide synthesis. [reaction: see text]

Amides↗
PubMed2004

Arrays for the combinatorial exploration of cell adhesion.

A new method for the fabrication of arrays of self-assembled monolayers (SAMs) of alkane thiols (ATs) on gold to combinatorially assay surfaces for cell adhesion is reported. A fluorous SAM, which is both cytophobic and solvophobic, was used as the background between the array features. The resulting solvophobic background permits the application of an assembly after conjugation strategy for fabrication. SAMs containing mixtures of ATs and peptide-terminated ATs were generated. Multiple cell types demonstrated differential and specific binding to these surfaces. Additionally, pluripotent human embryonic stem cells proliferated on surfaces generated by this method.

Animals↗
PubMed2004

Identification of inhibitors for UDP-galactopyranose mutase.

The flavoenzyme uridine 5'-diphosphate (UDP)-galactopyranose mutase (UGM) plays a key role in the cell wall biosynthesis of many pathogens, including Mycobacterium tuberculosis. Using a synthetic fluorescent ligand, we screened 16 000 compounds in a fluorescence polarization assay. Effective inhibitors of UGM were identified.

Chromatography, High Pressure Liquid↗
PubMed2004

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.

Catalysis↗
PubMed2004

Improved chemical syntheses of 1- and 5-deazariboflavin.

The cofactor flavin adenine dinucleotide (FAD) is required for the catalytic activity of a large class of enzymes known as flavoenzymes. Because flavin cofactors participate in catalysis via a number of different mechanisms, isoalloxazine analogues are valuable for mechanistic studies. We report improved chemical syntheses for the preparation of the two key analogues, 5-deazariboflavin and 1-deazariboflavin.

Catalysis↗
PubMed2004

A polymer scaffold for protein oligomerization.

We report the design and synthesis of well-defined polymers for the noncovalent oligomerization of proteins. The reported scaffolds, which were generated by atom-transfer radical polymerization (ATRP), take advantage of the well-characterized interaction between a Ni2+ complex and an oligohistidine sequence (His tag). Thus, our polymers are designed to facilitate the oligomerization of any protein possessing a His tag. We demonstrate that they can oligomerize fibroblast growth factor-8b (FGF-8b) and promote FGF-8b-mediated cell proliferation in the absence of heparin.

Animals↗