https://pubs.rsc.org/en/content/articlepdf/2019/NP/C8NP00092A

Marine natural products.

Tetracyclines were discovered in the 1940s and exhibited activity against a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. They are inexpensive antibiotics, which have been used extensively in the prophlylaxis and therapy of human and animal infections and also at subtherapeutic levels in animal feed as growth promoters. The first tetracycline-resistant bacterium, Shigella dysenteriae, was isolated in 1953. Tetracycline resistance now occurs in an increasing number of pathogenic, opportunistic, and commensal bacteria. The presence of tetracycline-resistant pathogens limits the use of these agents in treatment of disease. Tetracycline resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines or for a protein that protects bacterial ribosomes from the action of tetracyclines. Many of these genes are associated with mobile plasmids or transposons and can be distinguished from each other using molecular methods including DNA-DNA hybridization with oligonucleotide probes and DNA sequencing. A limited number of bacteria acquire resistance by mutations, which alter the permeability of the outer membrane porins and/or lipopolysaccharides in the outer membrane, change the regulation of innate efflux systems, or alter the 16S rRNA. New tetracycline derivatives are being examined, although their role in treatment is not clear. Changing the use of tetracyclines in human and animal health as well as in food production is needed if we are to continue to use this class of broad-spectrum antimicrobials through the present century.

Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance

The Canonical Notch Signaling Pathway: Unfolding the Activation Mechanism

Differential scanning fluorimetry (DSF) is a rapid and inexpensive screening method to identify low-molecular-weight ligands that bind and stabilize purified proteins. The temperature at which a protein unfolds is measured by an increase in the fluorescence of a dye with affinity for hydrophobic parts of the protein, which are exposed as the protein unfolds. A simple fitting procedure allows quick calculation of the transition midpoint; the difference in the temperature of this midpoint in the presence and absence of ligand is related to the binding affinity of the small molecule, which can be a low-molecular-weight compound, a peptide or a nucleic acid. DSF is best performed using a conventional real-time PCR instrument. Ligand solutions from a storage plate are added to a solution of protein and dye, distributed into the wells of the PCR plate and fluorescence intensity measured as the temperature is raised gradually. Results can be obtained in a single day.

The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-alpha (TNFalpha) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and transforming growth factor-alpha (TGFalpha). The phenotype of mice lacking TACE suggests an essential role for soluble TGFalpha in normal development and emphasizes the importance of protein ectodomain shedding in vivo.

https://science.sciencemag.org/content/sci/282/5392/1281.full.pdf

An Essential Role for Ectodomain Shedding in Mammalian Development

Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery.

Calichemicins, a novel family of antitumor antibiotics. 2. Chemistry and structure of calichemicin .gamma.1I

Tumor necrosis factor-alpha (TNFalpha) is a cytokine that induces protective inflammatory reactions and kills tumor cells but also causes severe damage when produced in excess, as in rheumatoid arthritis and septic shock. Soluble TNFalpha is released from its membrane-bound precursor by a membrane-anchored proteinase, recently identified as a multidomain metalloproteinase called TNFalpha-converting enzyme or TACE. We have cocrystallized the catalytic domain of TACE with a hydroxamic acid inhibitor and have solved its 2.0 A crystal structure. This structure reveals a polypeptide fold and a catalytic zinc environment resembling that of the snake venom metalloproteinases, identifying TACE as a member of the adamalysin/ADAM family. However, a number of large insertion loops generate unique surface features. The pro-TNFalpha cleavage site fits to the active site of TACE but seems also to be determined by its position relative to the base of the compact trimeric TNFalpha cone. The active-site cleft of TACE shares properties with the matrix metalloproteinases but exhibits unique features such as a deep S3' pocket merging with the S1' specificity pocket below the surface. The structure thus opens a different approach toward the design of specific synthetic TACE inhibitors, which could act as effective therapeutic agents in vivo to modulate TNFalpha-induced pathophysiological effects, and might also help to control related shedding processes.

https://www.pnas.org/content/pnas/95/7/3408.full.pdf

Crystal structure of the catalytic domain of human tumor necrosis factor-alpha-converting enzyme.

Calicheamicins: discovery, structure, chemistry, and interaction with DNA

Staphylococcus aureus sortase (SrtA) is a thiol transpeptidase. The enzyme catalyzes a cell wall sorting reaction in which a surface protein with a sorting signal containing a LPXTG motif is cleaved between the threonine and glycine residues. The resulting threonine carboxyl end of this protein is covalently attached to a pentaglycine cross-bridge of peptidoglycan. The transpeptidase activity of sortase has been demonstrated in in vitro reactions between a LPETG-containing peptide and triglycine. When a nucleophile is not available, sortase slowly hydrolyzes the LPETG peptide at the same site. In this study, we have analyzed the steady-state kinetics of these two types of reactions catalyzed by sortase. The kinetic results fully support a ping-pong mechanism in which a common acyl-enzyme intermediate is formed in transpeptidation and hydrolysis. However, each reaction has a distinct rate-limiting step:  the formation of the acyl-enzyme in transpeptidation and the hydrolysis of the same acyl-enzyme in the ...

George A. Ellestad

Papers

Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery.

Calichemicins, a novel family of antitumor antibiotics. 2. Chemistry and structure of calichemicin .gamma.1I

Crystal structure of the catalytic domain of human tumor necrosis factor-alpha-converting enzyme.

Calicheamicins: discovery, structure, chemistry, and interaction with DNA

Kinetic mechanism of Staphylococcus aureus sortase SrtA.