In-vitro selections

Protein engineering is now routinely used to modify protein molecules either via site-directed mutagenesis or by combinatorial methods. Factors that are Important for the stability of proteins have been studied, such as stabilization of a helices and reducing the number of conformations in the unfolded state. Combinatorial methods produce a large number of random mutants from which those with the desired properties are selected in vitro using phage display. Specific enzyme inhibitors, increased enzymatic activity and agonists of receptor molecules are examples of successful use of this method. 

Tai CY, Escarpe PA, Sidwell RW, WiUiams MA, Lew W, Wu H, Kim CU, Mendel DB (1998) Characterization of human influenza virus variants selected in vitro in the presence of the neuraminidase inhibitor GS 4071, Antimicrob Agents Chemother 42 3234-3241 Thomson MM, Najera R (2005) Molecular epidemiology of HlV-1 variants in the global AIDS pandemic an update. AIDS Rev 7 210-224... 

Paroxetine is the most potent inhibitor of 5-HT reuptake but, in terms of distinguishing one compound from another, their preferential selectivity for inhibition of 5-HT rather than noradrenaline reuptake is the key criterion. Citalopram is by far the most selective in vitro (1500-3000-fold) and fluoxetine, the most frequently prescribed SSRI in the UK, is the least selective of all these agents (see Stanford 1999). In fact, it is worth questioning whether fluoxetine is a true SSRI at all. 

Jansen RW, Schols D, Pauwels R, De Clercq E, Meijer DKF. Novel, negatively charged, human serum albumins display potent and selective in vitro anti-human immunodeficiency virus type 1 activity. Mol Pharmacol 1993 44 1003-1007. 

Balzarini J, Holy A, Jindrich J, Naesens L, Snoeck R, Schols D, De Clercq E. Differential antiherpesvirus and antiretrovirus effects of the (S) and (R) enantiomers of acyclic nucleoside phosphonates potent and selective in vitro and in vivo antiretrovirus activities of (R)-9-(2-phosphonomethoxy-propyl)-2,6-diaminopurine. Antimicrob Agents Chemother 1993 37 332-338. 

Often, demonstration of potency and selectivity in vitro is assumed to extrapolate to effects of inhibitors administered in vivo. Unfortunately, such assumptions are often invalid, and selectivity and potency must also be assessed in vivo or ex vivo. There are several reasons for such discrepancies between in vitro and in vivo data. 

Recent studies of members of the flavonoid 20GD family show overlapping substrate and product selectivities in vitro. For example, the C. unshiu FLS has been termed a bifunctional... 

While anchoring targeting molecules such as antibodies or other ligands on the liposome surface provides target selectivity in vitro and in vivo, the cost and reproducibility of these constructs with suf-hcient purity and in sufficient quantity for pharmaceutical application continue to be barriers to their development. The lipopep-tide approach (i.e., attaching an acylated... 

Turner, H., Blottner, S., Kuhla, S., Langhammer, M., Aim, H., Tuchscherer, A. 1999. Influence of chlorocholinechloride-treated wheat on selected in vitro fertility parameters in male mice. Reproductive Toxicology 13 399-404. 

M. Famulok. G. Mayer, and M. Blind. "Nucleic Add Aptamers—From Selection in Vitro to Applications in Vivo." ACC. Chem Res. 3000,33,591.]... 

R, 3S, 4R, 75 85 12R, 13 R )-12-hydroxy-7-isothiocyanatoamphi-lecta-11(20), 14-diene for 241. Compounds 239-241 show significant and selective in vitro antimalarial activity [192]. 

Aptamers are nucleic acids which exhibit a defined structure due to their nucleotide sequence and therefore, are able to specifically bind selected targets [1] (aptus [lat.] = fitting, sticking to). Aptamers and likewise, ribozymes [2] and deoxyribozymes [3] are selected in vitro by screening nucleic acid libraries. Here we describe in detail the selection of aptamers by a process called SELEX (Systematic Evolution of Ligands by Exponential enrichment) [4]. 

Famulok, M., Mayer, G. and Blind, M. (2000) Nucleic acid aptamers-from selection in vitro to applications in vivo. Ace. Chem. Res., 33, 591-599. 

HIV-1 genetic resistance to protease inhibitors occurs via specific mutations. Genotypic analysis of the HIV protease gene from isolates selected in vitro indicated that Gly48Val and Leu90Met mutants had reduced susceptibility to saquinavir (Ohta, 1997). Indinavir and ritonavir resistance maps to residue 82, whereas for amprenavir the key mutation is at residue 50 (I50V) and confers a threefold decline in viral sensitivity to amprenavir. Two additional mutations at residues 46 and 47 follow development of mutation at position 50, resulting in a 20-fold de-... 

D. Macmillan, R. M. Bill, K. A. Sage, D. Fern, and S. Flitsch, Selective in vitro glycosylation of recombinant proteins Semi-synthesis of novel homogeneous glycoforms of human erythropoietin, Chem. Biol., 8 (2001) 133-145. 

Cidofovir is a cytosine nucleotide analog with in vitro activity against CMV, HSV-1, HSV-2, VZV, EBV, HHV-6, HHV-8, adenovirus, poxviruses, polyomaviruses, and human papillomavirus. In contrast to ganciclovir, phosphorylation of cidofovir to the active diphosphate is independent of viral enzymes. After phosphorylation, cidofovir acts both as a potent inhibitor of and as an alternative substrate for viral DNA polymerase, competitively inhibiting DNA synthesis and becoming incorporated into the viral DNA chain. Isolates with resistance to cidofovir have been selected in vitro these isolates tend to be cross-resistant with ganciclovir but retain susceptibility to foscamet. Clinically significant resistance to cidofovir has not been reported to date. 

For brevity, results from selected in vitro and in vivo studies employing either near-infrared absorption spectroscopy or Raman spectroscopy, the most commonly used techniques, are documented in Tables 12.1 and 12.2. In these tables, error estimates are reported with either CV or P in parentheses, indicating cross-validated or predicted results, respectively. For an explanation of these terms, please refer to Section 12.4. 

Semlow, D. R., and Silverman, S. K. (2005). Parallel selections in vitro reveal a preference for 2 S RNA ligation by deoxyribozyme-mediated opening of a 2,3,-cyclic phosphate. J. Mol. Evol. 61, 207-215. 

Directed evolution turns out to be a potent tool not only for improvement of polymerase function, or for detecting unexpected new activity but also for collecting information on polymerase structure and function with respect to fidelity. The work summarized in this chapter shows that polymerases with altered fidelity or altered substrate tolerance can be generated in vivo by using genetic selection, in vitro by employing a variety of assay schemes, or by combinations of these approaches. More efforts are desirable including ... 

Protein-ligand interactions can not only be secreened or selected in vitro, but also can be directly characterized for particular interaction partners. Nemoto et al. (1999) applied the mRNA-peptide fusion technology to fluorescently label the displayed proteins in order to study protein-protein interactions by fluorescence polarization measurements. 

Spencer GE, Lukowiak K, Syed NI (2000) Transmitter-receptor interactions between growth cones of identified Lymnaea neurons determine target cell selection in vitro. J Neurosci 20 8077-8086. 

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