Despite their versatility and power in controlling gene regulation in nature, nuclear hormone receptors (NHRs) have largely eluded utility in heterologous gene regulation applications such as gene therapy and metabolic engineering. The main reason for this void is the pleiotropic interference of the receptor-ligand combination with regulatory networks in the host organism. In recent years, numerous strategies have been developed to engineer ligandreceptor pairs that do not cross-interact with host regulatory pathways. However, these strategies have either met with limited success or cannot be readily extended to other ligand-receptor pairs. Here, we present a simple, effective, and readily generalizable strategy for reengineering NHRs to respond specifically to a selected synthetic ligand. The method involves generation of genetic diversity by stepwise individual site saturation mutagenesis of a fixed set of ligand-contacting residues and random point mutagenesis, followed by phenotypic screening based on a yeast two-hybrid system. As a test case, this method was used to alter the specificity of the NHR human estrogen receptor ␣ in favor of the synthetic ligand 4,4 -dihydroxybenzil, relative to the natural ligand 17␤-estradiol, by >10 7 -fold. The resulting ligand-receptor pair is highly sensitive to the synthetic ligand in human endometrial cancer cells and is essentially fully orthogonal to the wild-type receptor-natural ligand pair. This method should provide a powerful, broadly applicable tool for engineering receptors͞enzymes with improved or novel ligand͞substrate specificity.gene therapy ͉ nuclear hormone receptor ͉ protein engineering ͉ orthogonal ligand-receptor pairs T he ability to manipulate naturally occurring proteins to bind and respond to synthetic ligands in a manner independent, or orthogonal, from the influence of natural proteins and ligands, constitutes a significant challenge in protein engineering (1). Such a tool has important utility in the creation of gene switches for the control of heterologous gene expression in applications such as gene therapy and metabolic engineering (2, 3), as well as in the selective regulation of cellular processes such as apoptosis, genetic recombination, signal transduction, and motor protein function (4).To date, numerous synthetic ligand-mutant receptor pairs have been created that are orthogonal to the analogous natural interaction to varying degrees. Among the proteins used for this work, nuclear hormone receptors (5), naturally occurring transcription factors, have served as a prime target, owing largely to their ''gene switch-like'' attributes: rapid induction kinetics (6-8), dose-dependent ligand response, and readily interchangeable functional modules (9, 10). Despite the extensive work carried out to engineer new specific ligand-receptor pairs from nuclear hormone receptors, the absence of a conceptually simple, generally applicable engineering approach remains a concern. Rational design of ligands to rematch interaction with a given mutant of human...

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The hepatitis C virus (HCV) life cycle involves multiple steps, but most current drug candidates target only viral replication. The inability to systematically discover inhibitors targeting multiple steps of the HCV life cycle has hampered antiviral development. We present a simple screen for HCV antivirals based on the alleviation of HCV-mediated cytopathic effect in an engineered cell linen4mBid. This approach obviates the need for a secondary screen to avoid cytotoxic false-positive hits. Application of our screen to 1280 compounds, many in clinical trials or approved for therapeutic use, yielded >200 hits. Of the 55 leading hits, 47 inhibited one or more aspects of the HCV life cycle by >40%. Six compounds blocked HCV entry to levels similar to an antibody (JS-81) targeting the HCV entry receptor CD81. Seven hits inhibited HCV replication and/or infectious virus production by >100-fold, with one (quinidine) inhibiting infectious virus production by 450-fold relative to HCV replication levels. This approach is simple and inexpensive and should enable the rapid discovery of new classes of HCV life cycle inhibitors.cell death | drug | high throughput | rescue | virus assembly H epatitis C virus (HCV) infection is a major public health problem, putting~180 million infected individuals worldwide (3% of the world's population) at risk for developing cirrhosis, hepatocellular carcinoma, and liver failure. Chronic hepatitis C is a leading cause for liver transplantation in the Western world. The current standard IFN-based therapy is costly, time-consuming, riddled with serious side effects, and cures only ∼50% of patients infected with the most common genotype. Although it is anticipated that small molecules that effectively inhibit the HCV life cycle can complement or even replace IFN therapy, no anti-HCV drugs have yet been approved for human use. Efforts to develop HCV antivirals mostly target only viral replication due to widespread dependence on the HCV replicon model (1-4), which effectively reproduces the RNA replication aspects of the HCV life cycle but does not allow screens to identify inhibitors of entry or infectious virus assembly and release.The identification of a genotype 2a isolate of HCV that reproduces the entire viral life cycle in cell culture (5-7) promised to accelerate the discovery of antivirals targeting all aspects of HCV growth in a more true-to-life model system. Several recent reports describe systems that can in principle screen for inhibitors targeting different steps of the HCV life cycle via the prevention of the spread of cell culture-derived hepatitis C virus (HCVcc) infection in a cell monolayer (8-11). Very recently, one such screen based on a colorimetric readout following immunostaining with an anti-E2 antibody identified inhibitors of multiple stages of the HCV life cycle (12). Although effective, this approach is labor-intensive and not readily amenable to high-throughput application, requiring antibodies and 12 washing steps in addition to a secondary screen to assess drug ...

The ability of peptides and proteins to change conformations in response to external stimuli such as temperature, pH and the presence of specific small molecules is ubiquitous in nature. Exploiting this phenomenon, numerous natural and designed peptides have been used to engineer stimulus-responsive systems with potential applications in important research areas such as biomaterials, nanodevices, biosensors, bioseparations, tissue engineering and drug delivery. This review describes prominent examples of both natural and designed synthetic stimulus-responsive peptide systems. While the future looks bright for stimulus-responsive systems based on natural and rationally engineered peptides, it is expected that the range of stimulants used to manipulate such systems will be significantly broadened through the use of combinatorial protein engineering approaches such as directed evolution. These new proteins and peptides will continue to be employed in exciting and high-impact research areas including bionanotechnology and synthetic biology.

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