PD 404,182 Is a Virocidal Small Molecule That Disrupts Hepatitis C Virus and Human Immunodeficiency Virus

We describe a virucidal small molecule, PD 404,182, that is effective against hepatitis C virus (HCV) and human immunodeficiency virus (HIV). The median 50% inhibitory concentrations (IC(50)s) for the antiviral effect of PD 404,182 against HCV and HIV in cell culture are 11 and 1 μM, respectively. The antiviral activity of PD 404,182 is due to the physical disruption of virions that is accompanied to various degrees (depending on the virus and exposure temperature/time) by the release of viral nucleic acids into the surrounding medium. PD 404,182 does not directly lyse liposomal membranes even after extended exposure, and it shows no attenuation in antiviral activity when preincubated with liposomes of various lipid compositions, suggesting that the compound inactivates viruses through interaction with a nonlipid structural component of the virus. The virucidal activity of PD 404,182 appears to be virus specific, as little to no viral inactivation was detected with the enveloped Dengue and Sindbis viruses. PD 404,182 effectively inactivates a broad range of primary isolates of HIV-1 as well as HIV-2 and simian immunodeficiency virus (SIV), and it does not exhibit significant cytotoxicity with multiple human cell lines in vitro (50% cytotoxic concentration, >300 μM). The compound is fully active in cervical fluids, although it exhibits decreased potency in the presence of human serum, retains its full antiviral potency for 8 h when in contact with cells, and is effective against both cell-free and cell-associated HIV. These qualities make PD 404,182 an attractive candidate anti-HIV microbicide for the prevention of HIV transmission through sexual intercourse

Engineering Estrogen Receptor-Based Gene Switches and a Superoxide Dismutase for Therapeutic Applications

137 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006.In recent years, directed evolution has rapidly emerged as a powerful approach to engineer proteins with desirable properties for application in metabolic engineering, industrial biocatalysis, and therapeutics. Despite this success, the "irrational" nature of directed evolution, where knowledge of a protein's structure-function relationship is not utilized to create mutant protein variants with altered properties, often prevents directed evolution from realizing its full potential. This dissertation describes the application of a hybrid directed evolution/rational design approach for the engineering of two different proteins with desirable properties. In the first example, stepwise saturation mutagenesis of functionally important protein sites and random point mutagenesis throughout all protein sites, accompanied by a yeast two-hybrid screening system, was used to engineer variants of the human estrogen receptor alpha (hERalpha) that bind and respond to two synthetic small molecule ligands---4,4'-dihydroxybenzil (DHB) and 2,4-di(4-hydroxyphenyl)-5ethylthiazole (L9)---with high specificity and potency. The resulting highly specific DHB/7-S receptor and L9/L7-E receptor pairs exhibit nanomolar or subnanomolar response to their respectively matched synthetic ligand, while showing negligible or no activation in response to the natural small molecule ligand for the wild type hERalpha - 17beta-estradiol. Furthermore, the two created receptor-ligand pairs do not show significant cross-interaction with each other. Both created receptor-ligand pairs have the potential to be used as gene switches for the selective regulation of gene expression in applications ranging from gene therapy and tissue engineering to metabolic engineering. In the second example, an E. coli selection system based on resistance to superoxide toxicity was used in a directed evolution approach to enhance the catalytic efficiency of a rationally designed mutant manganese superoxide dismutase enzyme exhibiting greatly reduced product inhibition and low catalytic efficiency relative to the wild type enzyme. Finally, this dissertation describes the characterization and application of a method for the creation of protein variant libraries for a rational approach to directed evolution. This method---DNA shuffling with spiked oligonucleotides (DSSO)---simultaneously randomizes multiple functionally important sites in a protein.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD