Genetically Encoded Detection of Biosynthetic Protease Inhibitors.

Proteases are an important class of drug targets that continue to drive inhibitor discovery. These enzymes are prone to resistance mutations, yet their promise for treating viral diseases and other disorders continues to grow. This study develops a general approach for detecting microbially synthesized protease inhibitors and uses it to screen terpenoid pathways for inhibitory compounds.

Evolution-Guided Biosynthesis of Terpenoid Inhibitors.

Terpenoids, the largest and most structurally diverse group of natural products, include a striking variety of biologically active compounds, from flavors to medicines. Despite their well-documented biochemical versatility, the evolutionary processes that generate new functional terpenoids are poorly understood and difficult to recapitulate in engineered systems. This study uses a synthetic biochemical objective─a transcriptional system that links the inhibition of protein tyrosine phosphatase 1B (PTP1B), a human drug target, to the expression of a gene for antibiotic resistance in ()─to evolve a terpene synthase to produce enzyme inhibitors.

VEGFR-1/Flt-1 inhibition increases angiogenesis and improves muscle function in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy is characterized by structural degeneration of muscle, which is exacerbated by localized functional ischemia due to loss of nitric oxide synthase-induced vasodilation. Treatment strategies aimed at increasing vascular perfusion have been proposed. Toward this end, we have developed monoclonal antibodies (mAbs) that bind to the vascular endothelial growth factor (VEGF) receptor VEGFR-1 (Flt-1) and its soluble splice variant isoform (sFlt-1) leading to increased levels of free VEGF and proangiogenic signaling.

Recombinant expression and characterization of Lucilia cuprina CYP6G3: Activity and binding properties toward multiple pesticides.

The Australian sheep blowfly, Lucilia cuprina, is a primary cause of sheep flystrike and a major agricultural pest. Cytochrome P450 enzymes have been implicated in the resistance of L. cuprina to several classes of insecticides.

Soluble and membrane-bound Drosophila melanogaster CYP6G1 expressed in Escherichia coli: purification, activity, and binding properties toward multiple pesticides.

Cytochrome P450 CYP6G1 has been implicated in the resistance of Drosophila melanogaster to numerous pesticides. While in vivo and in vitro studies have provided insight to the diverse functions of this enzyme, direct studies on the isolated CYP6G1 enzyme have not been possible due to the need for a source of recombinant enzyme. In the current study, the Cyp6g1 gene was isolated from D.

Rapid and quantitative measurement of metabolic stability without chromatography or mass spectrometry.

Metabolic stability measurements are a critical component of preclinical drug development. Available measurement strategies rely on chromatography and mass spectrometry, which are expensive and labor intensive. We have developed a general method to determine the metabolic stability of virtually any compound by quantifying cofactors in the mechanism of cytochrome P450 enzymes using fluorescence intensity measurements.

Simultaneous measurement of CYP1A2 activity, regioselectivity, and coupling: Implications for environmental sensitivity of enzyme-substrate binding.

The cytochrome P450 (CYP) reaction mechanism often yields a broad array of coupled and uncoupled products from a single substrate. While it is well known that reaction conditions can drastically affect the rate of P450 catalysis, their effects on regioselectivity and coupling are not well characterized. To investigate such effects, the CYP1A2 oxidation of 7-ethoxymethoxy-3-cyanocoumarin (EOMCC) was examined as a function of buffer type, buffer concentration, pH, and temperature.