Drug and Alcohol Rehabilitation: Cell-Permeable Probe for Identification and Imaging of Sialidases.

Cell-permeable probe for identification and imaging of sialidases.

Filed under: Drug and Alcohol Rehabilitation

Proc Natl Acad Sci U S A. 2013 Jan 28;
Tsai CS, Yen HY, Lin MI, Tsai TI, Wang SY, Huang WI, Hsu TL, Cheng YS, Fang JM, Wong CH

Alkyne-hinged 3-fluorosialyl fluoride (DFSA) containing an alkyne group was shown to be a mechanism-based target-specific irreversible inhibitor of sialidases. The ester-protected analog DFSA (PDFSA) is a membrane-permeable precursor of DFSA designed to be used in living cells, and it was shown to form covalent adducts with virus, bacteria, and human sialidases. The fluorosialyl-enzyme adduct can be ligated with an azide-annexed biotin via click reaction and detected by the streptavidin-specific reporting signals. Liquid chromatography-mass spectrometry/mass spectrometry analysis on the tryptic peptide fragments indicates that the 3-fluorosialyl moiety modifies tyrosine residues of the sialidases. DFSA was used to demonstrate influenza infection and the diagnosis of the viral susceptibility to the anti-influenza drug oseltamivir acid, whereas PDFSA was used for in situ imaging of the changes of sialidase activity in live cells.
HubMed – drug

 

Drug repurposing gets a boost as academic researchers join the search for novel uses of existing drugs.

Filed under: Drug and Alcohol Rehabilitation

Proc Natl Acad Sci U S A. 2013 Jan 28;
Nair P, Writer S

HubMed – drug

 

Injectable protease-operated depots of glucagon-like peptide-1 provide extended and tunable glucose control.

Filed under: Drug and Alcohol Rehabilitation

Proc Natl Acad Sci U S A. 2013 Jan 28;
Amiram M, Luginbuhl KM, Li X, Feinglos MN, Chilkoti A

Peptide drugs are an exciting class of pharmaceuticals increasingly used for the treatment of a variety of diseases; however, their main drawback is a short half-life, which dictates multiple and frequent injections and an undesirable “peak-and-valley” pharmacokinetic profile, which can cause undesirable side-effects. Synthetic prolonged release formulations can provide extended release of biologically active native peptide, but their synthetic nature can be an obstacle to production and utilization. Motivated by these limitations, we have developed a new and entirely genetically encoded peptide delivery system-Protease Operated Depots (PODs)-to provide sustained and tunable release of a peptide drug from an injectable s.c. depot. We demonstrate proof-of-concept of PODs, by fusion of protease cleavable oligomers of glucagon-like peptide-1, a type-2 diabetes drug, and a thermally responsive, depot-forming elastin-like-polypeptide that undergoes a thermally triggered inverse phase transition below body temperature, thereby forming an injectable depot. We constructed synthetic genes for glucagon-like peptide-1 PODs and demonstrated their high-yield expression in Escherichia coli and facile purification by a nonchromatographic scheme we had previously developed. Remarkably, a single injection of glucagon-like peptide-1 PODs was able to reduce blood glucose levels in mice for up to 5 d, 120 times longer than an injection of the native peptide drug. These findings demonstrate that PODs provide the first genetically encoded alternative to synthetic peptide encapsulation schemes for sustained delivery of peptide therapeutics.
HubMed – drug

 

Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay.

Filed under: Drug and Alcohol Rehabilitation

Proc Natl Acad Sci U S A. 2013 Jan 28;
Vosyka O, Vinothkumar KR, Wolf EV, Brouwer AJ, Liskamp RM, Verhelst SH

Rhomboid proteases are evolutionary conserved intramembrane serine proteases. Because of their emerging role in many important biological pathways, rhomboids are potential drug targets. Unfortunately, few chemical tools are available for their study. Here, we describe a mass spectrometry-based assay to measure rhomboid substrate cleavage and inhibition. We have identified isocoumarin inhibitors and developed activity-based probes for rhomboid proteases. The probes can distinguish between active and inactive rhomboids due to covalent, reversible binding of the active-site serine and stable modification of a histidine residue. Finally, the structure of an isocoumarin-based inhibitor with Escherichia coli rhomboid GlpG uncovers an unusual mode of binding at the active site and suggests that the interactions between the 3-substituent on the isocoumarin inhibitor and hydrophobic residues on the protease reflect S’ subsite binding. Overall, these probes represent valuable tools for rhomboid study, and the structural insights may facilitate future inhibitor design.
HubMed – drug

 

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