Biomedical subjects

John B Biggins

Publications and source records attributed to John B Biggins.

8 recordsLinked to original sources

PubMed2006

Chemical biology of steroid and nuclear hormone receptors.

The nuclear hormone receptors are ligand-gated transcription factors that modulate gene expression by directly acting upon genomic DNA, and have been of profound interest across all biological disciplines. Recent advancements in this area have included the expansion of transgene activation through ligand-receptor engineering, drug development from structural design and the exploitation of innate ligand-specific associations towards developing novel conditional protein-based recombinant and diagnostic tools. These advancements come on the heels of exciting new modes of hormone action that challenge and expand upon the classic paradigms of hormone receptor function.

Animals↗

PubMed2006

A continuous assay for DNA cleavage using molecular break lights.

Exploring the properties of molecules that cleave DNA (i.e., enzymatic nucleases, chemical footprinting agents, and naturally occurring DNA cleaving antibiotics) has been an ongoing process with benefits extending toward both laboratory and clinical applications. Despite the progress that has been made toward understanding the mechanics of DNA cleavage, a simple and continuous assay for detecting DNA cleavage has been lacking. Herein, we describe the molecular break light assay, wherein a single oligo-nucleotide modified by a 5'-fluorophore-3'-quencher pair adopting a stem-loop structure with an appropriate DNA recognition site, provides for the rapid assaying of DNA cleavage with high sensitivity. Furthermore, the described methodology is highly convenient in that it is readily adaptable to common laboratory fluorometers and multi-well plate/ array systems, which may provide the basis for high-throughput screening of novel DNA cleaving agents. This assay may also be further extended to natural or "unnatural" transcription factor protection assay systems.

DNA↗

PubMed2005

Ligand-receptor engineering and its application towards the complementation of genetic disease and target identification.

In some instances, small molecules can restore function to proteins that are impaired by genetic mutations. There are now many examples where non-specific molecules or specific ligands can act as chemical chaperones to fold proteins or stabilize folded proteins harboring genetic mutations. In contrast a few recent examples have shown that functionally impaired proteins that are stably folded can be "functionally rescued" by appropriate small molecules. Compounds that can rescue functionally impaired proteins may provide new strategies for the treatment of genetic diseases such as rickets and resistance to thyroid hormone (RTH). In addition mutant-complementing analogs and substrates that act exclusively on mutant proteins are providing important tools for the study of complex biological systems that are controlled by molecules that have multiple cellular targets.

Animals↗

PubMed2003

Resistance to enediyne antitumor antibiotics by CalC self-sacrifice.

Antibiotic self-resistance mechanisms, which include drug elimination, drug modification, target modification, and drug sequestration, contribute substantially to the growing problem of antibiotic resistance among pathogenic bacteria. Enediynes are among the most potent naturally occurring antibiotics, yet the mechanism of resistance to these toxins has remained a mystery. We characterize an enediyne self-resistance protein that reveals a self-sacrificing paradigm for resistance to highly reactive antibiotics, and thus another opportunity for nonpathogenic or pathogenic bacteria to evade extremely potent small molecules.

Aminoglycosides↗

PubMed2003

Studies on the substrate specificity of Escherichia coli galactokinase.

In vitro glycorandomization (IVG) technology is dependent upon the ability to rapidly synthesize sugar phosphates. Compared with chemical synthesis, enzymatic (kinase) routes to sugar phosphates would be attractive for this application. This work focuses upon the development of a high-throughput colorimetric galactokinase (GalK) assay and its application toward probing the substrate specificity and kinetic parameters of Escherichia coli GalK. The demonstrated dinitrosalicylic assay should also be generally applicable to a variety of sugar-processing enzymes. [reaction: see text]

Colorimetry↗

PubMed2003

Substrate specificity of NovM: implications for novobiocin biosynthesis and glycorandomization.

[reaction: see text] In an effort to expand the scope of natural product in vitro glycorandomization (IVG), the substrate specificity of NovM was investigated. A test of four aglycon analogues and over 40 nucleotide sugars revealed NovM has a surprisingly stringent substrate specificity and provided only three new "unnatural" natural products. On the basis of the determined substrate specificity, an alternative to the sugar nucleotide biosynthetic dogma and a cautionary note for the general applicability of IVG are introduced.

Anti-Bacterial Agents↗

PubMed2002

Expanding pyrimidine diphosphosugar libraries via structure-based nucleotidylyltransferase engineering.

In vitro "glycorandomization" is a chemoenzymatic approach for generating diverse libraries of glycosylated biomolecules based on natural product scaffolds. This technology makes use of engineered variants of specific enzymes affecting metabolite glycosylation, particularly nucleotidylyltransferases and glycosyltransferases. To expand the repertoire of UDP/dTDP sugars readily available for glycorandomization, we now report a structure-based engineering approach to increase the diversity of alpha-d-hexopyranosyl phosphates accepted by Salmonella enterica LT2 alpha-d-glucopyranosyl phosphate thymidylyltransferase (E(p)). This article highlights the design rationale, determined substrate specificity, and structural elucidation of three "designed" mutations, illustrating both the success and unexpected outcomes from this type of approach. In addition, a single amino acid substitution in the substrate-binding pocket (L89T) was found to significantly increase the set of alpha-d-hexopyranosyl phosphates accepted by E(p) to include alpha-d-allo-, alpha-d-altro-, and alpha-d-talopyranosyl phosphate. In aggregate, our results provide valuable blueprints for altering nucleotidylyltransferase specificity by design, which is the first step toward in vitro glycorandomization.

Amino Acid Substitution↗

PubMed2002

Incorporation of glucose analogs by GtfE and GtfD from the vancomycin biosynthetic pathway to generate variant glycopeptides.

Analogs of the glycopeptide antibiotics vancomycin and teicoplanin with alterations in one or both sugar moieties of the disaccharide have been prepared by tandem action of the vancomycin pathway glycosyltransferases GtfE and GtfD. All four regioisomers (2-, 3-, 4-, 6-) of TDP-deoxyglucoses and UDP/TDP-aminoglucoses were prepared, predominantly by action of D-glucopyranosyl-1-phosphate thymidylyltransferase, E(p). GtfE transferred the deoxyglucoses or aminoglucoses onto the 4-OH of 4-hydroxyphenylglycine of both the vancomycin and teicoplanin aglycone scaffolds. Kinetic analysis indicated the 2-, 3-, 4-, and 6-amino-glucoses were transferred by GtfE with only a 4- to 30-fold drop in k(cat) and no effect on K(m) compared to the native substrate, UDP/TDP-glucose, suggesting preparative utility. The next enzyme, GtfD, could utilize the variant glucosyl-peptides as substrates for transfer of L-4-epi-vancosamine. The aminosugar moieties in these variant glycopeptides introduce sites for acylation or reductive alkylation.

Anti-Bacterial Agents↗