Display enzyme

Elvitegravir (18, GS-9137, JTK-303, EVG) is the second IN strand transfer inhibitor to advance into phase III clinical trials (Figure 7). EVG was derived from the quinolone antibiotics which do not show IN activity [39-41]. Through careful optimization, this work resulted in EVG displaying enzyme and antiviral activity of 7.2 and 0.9 nM, respectively. EVG has moderate bioavailability in preclinical species (29 and 34%), low clearance (Qp 0.5 and l.OL/h/kg) and a moderate half-life of 2.3 and 5.2h in rats and dogs, respectively [42]. It is primarily metabolized via CYP450 oxidation and shows a marked increase in human exposure with RTV boosting. 

A most significant advance in the alkyne hydration area during the past decade has been the development of Ru(n) catalyst systems that have enabled the anti-Markovnikov hydration of terminal alkynes (entries 6 and 7). These reactions involve the addition of water to the a-carbon of a ruthenium vinylidene complex, followed by reductive elimination of the resulting hydridoruthenium acyl intermediate (path C).392-395 While the use of GpRuGl(dppm) in aqueous dioxane (entry 6)393-396 and an indenylruthenium catalyst in an aqueous medium including surfactants has proved to be effective (entry 7),397 an Ru(n)/P,N-ligand system (entry 8) has recently been reported that displays enzyme-like rate acceleration (>2.4 x 1011) (dppm = bis(diphenylphosphino)methane).398... 

Fig. 6.1. Summary of methods for selecting phage-displayed enzymes on die basis of catalytic activity (S substrate P product SS suicide substrate Bt biotin SV streptavidin TSA transition state analogue).

A solution of phage-enzyme can be used like an enzyme solution for measuring kinetic parameters such as kcat and KM. We usually observed that phage-displayed enzymes behave essentially like free enzymes in solution, although interference is always possible, especially with multiple display. Note that the kcat is the turnover rate of the phage and not of the enzyme, because the level of display is generally just an evaluation (see below). 

Add 10-7M trypsin for removing the displayed enzymes (Section 6.3.1.5) and for preventing infection bias. Incubate 30 min at room temperature. Remove the trypsin by precipitating the phages with PEG. 

As illustrated in Scheme 6.1, once the covalent intermediate is formed, the complex can either follow a normal catalytic cycle or go through a suicide event leading to the irreversible labeling that is necessary for selection. The suicide inhibition efficiency depends on the ratio k /k. This ratio depends on the nature of the suicide substrate and of the enzyme. Therefore, a large excess of suicide substrate as compared to the displayed enzyme is recommended for selection experiments. 

The Evans Cu(II)- and Sn(II)-catalyzed processes are unique in their ability to mediate aldol additions to pyruvate. Thus, the process provides convenient access to tertiary a-hydroxy esters, a class of chiral compounds not otherwise readily accessed with known methods in asymmetric catalysis. The process has been extended further to include a-dike-tone 101 (Eqs. 8B2.22 and 8B2.23). It is remarkable that the Cu(II) and Sn(II) complexes display enzyme-like group selectivity, as the complexes can differentiate between ethyl and methyl groups in the addition of thiopropionate-derived Z-silyl ketene acetal to 101. As discussed above, either syn or anti diastereomers may be prepared by selection of the Cu(II) or Sn(II) catalyst, respectively. 

Love KR, Swoboda JG, Noren CJ et al (2006) Enabling glycosyltransferase evolution a facile substrate-attachment strategy for phage-display enzyme evolution. ChemBioChem 7 753-756... 

The concentration of displayed enzyme is evaluated by measuring the activity of the phage-enzyme solution with the assumption that the displayed enzyme has the same activity as the free enzyme in solution. In our experience, this is generally the case. 

Displayed enzyme Size (kD) Fusion Display Display level References... 

A product-capture strategy was also used to select improved subtiligases. Subtiligase is a double mutant of subtilisine that catalyses the ligation of peptides. A library of >109 variants involving 25 residues of the active site was constructed on the phage-displayed enzyme. Variants that ligated a biotinylated peptide on their own ex-... 

In terms of achievements, selection from libraries of phage-displayed enzymes has afforded several enzymes with modified specificities and several abzymes. A combination of in vivo selection and in vitro selection for binding to ligands unrelated to the substrates has also opened the way to the engineering of the regulation of enzymatic activity. 

Based on these observations, Wang and Caruso [237] have described an effective method for the fabrication of robust zeolitic membranes with three-dimensional interconnected macroporous (1.2 pm in diameter) stmctures from mesoporous silica spheres previously seeded with silicalite-1 nanoparticles subjected to a conventional hydrothermal treatment. Subsequently, the zeolite membrane modification via the layer-by-layer electrostatic assembly of polyelectrolytes and catalase on the 3D macroporous stmcture results in a biomacromolecule-functionalized macroporous zeolitic membrane bioreactor suitable for biocatalysts investigations. The enzyme-modified membranes exhibit enhanced reaction stability and also display enzyme activities (for H2O2 decomposition) three orders of magnitude higher than their nonporous planar film counterparts assembled on silica substrates. Therefore, the potential of such structures as bioreactors is enormous. 

Environmental issues as well as process optimization requires the replacement of liquid acids by more friendly and safe solid-acid catalysts. The use in organic syntheses of clays or clay-modified catalysts or reagents recommends itself by its simple implementation, the small amount of catalyst required (in comparison to standard Lewis acids, such as AICI3, or Brpnsted acids, such as concentrated sulfuric or nitric acids), the presence of the acidity internally (making them safe to handle), the use of more environmentally compatible conditions and/or substances, the simple recovery and/or reuse and, finally, the harmless disposal. Furthermore, clay microreactors may display enzyme-like characteristics. 

Yeast cell-surface engineering has been established to display enzymes, functional proteins, antibodies, and combinatorial protein libraries (Kondo and Ueda, 2004). The cell surface is a functional interface between the inside and outside of the cell allowing some surface proteins to extend across the plasma membrane, while others are bound by non-covalent or covalent interactions to the cell surface components. For anchoring surface-specific proteins, yeast cells have molecular systems to confine... 

Analysis by DLS, cryo-TEM, and TEM of the resulting assemblies indicate 30-nm diameter, spherical, core—shell micelles that display enzyme substrates at the surface-solution interface (Figure 6.24). 

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