Karuppiah Chockalingam

Directed evolution of enzymes and pathways for industrial biocatalysis

Directed evolution has become a powerful tool for developing enzyme and whole cell based biocatalysts. Significant recent advances include the creation of novel enzyme functions and the development of several new efficient directed evolution methods. The combination of directed evolution and rational design promises to accelerate the development of biocatalysts for applications in the pharmaceutical, chemical and food industries.

A cell protection screen reveals potent inhibitors of multiple stages of the hepatitis C virus life cycle

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 line—n4mBid. 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.

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

ABSTRACT 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 (IC50s) 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.

Directed Evolution of Orthogonal Ligand Specificity in a Single Scaffold

Gene-regulation systems that provide temporal and spatial regulation of target-gene expression in response to smallmolecule ligands (small-molecule-dependent gene switches or circuits) are powerful tools for gene therapy, tissue engineering, metabolic engineering, and functional genomics. 2] Notably, small-molecule-dependent gene switches were recently used to generate induced pluripotent stem cells (iPS) for regenerative medicine. 4] The need for orthogonal regulatory elements is evidenced by the surge in interest in the creation of gene circuits, inspired by their electrical counterparts. Numerous orthogonal small-molecule-dependent gene switches have been developed to exert control over transcription, translation, or protein function. However, most either use bacterial components that involve regulation of a specific operator sequence bound by an antibioticregulated repressor, or use nuclear-receptor mutants that respond to synthetic hormones, such as RU486, which in large doses affect human physiology. An ideal gene-switch system would be nonimmunogenic, would show promoter flexibility through different DNA-binding domains, and would be expandable to new inducers. Nuclear hormone receptors (NHRs) offer desirable characteristics as gene switches for transcriptional control. The binding of a small ligand results in NHR dimerization, translocation, and the activation of promoters that harbor specific responsive elements. With distinct domains for ligand binding, DNA binding, and activation/repression functions, NHRs offer protein engineers the flexibility to create chimeric transcriptional activators or repressors by modular design. The ability to access small-molecule hormonelike compounds by organic synthesis makes these natural allosteric transcriptional switches attractive targets for engineering. Nuclear hormone receptors enable a wide range of target-protein-expression levels, which are tunable through variation of the dose of a ligand. NHR ligand-binding domains (LBDs) have also been combined with other proteins to enable posttranslational control of protein function. Specificity-reengineering approaches involving NHRs have typically involved mutation of the LBD to a form that is not activated by the natural ligand but instead by a synthetic small molecule that is inactive against the wild-type LBD. The LBD of the human estrogen receptor a (hERa) has proven to be a particularly versatile platform for the creation of orthogonal ligand–receptor pairs. 13] Despite these efforts and studies on other NHR LBDs, the creation of unique, NHR-based independently functioning ligand–receptor pairs that are not only orthogonal to cellular elements but that do not cross-interact with one another has yet to be demonstrated. Previously, we identified residues in the ligand-binding pocket of the hERa LBD that are important for ligand specificity. After four cycles of saturation mutagenesis and one cycle of random mutagenesis we identified two hERa LBD variants, 4S and 5E, which are activated by 4,4’dihydroxybenzil (DHB) but not 17b-estradiol (E2; Scheme 1). To further increase the sensitivity of mutant 4S

Creation and characterization of a cell-death reporter cell line for hepatitis C virus infection.

The present study describes the creation and characterization of a hepatoma cell line, n4mBid, that supports all stages of the hepatitis C virus (HCV) life cycle and strongly reports HCV infection by a cell-death phenotype. The n4mBid cell line is derived from the highly HCV-permissive Huh-7.5 hepatoma cell line and contains a modified Bid protein (mBid) that is cleaved and activated by the HCV serine protease NS3-4A. N4mBid exhibited a 10-20-fold difference in cell viability between the HCV-infected and mock-infected states, while the parental Huh-7.5 cells showed <2-fold difference under the same conditions. The pronounced difference in n4mBid cell viability between the HCV- and mock-infected states in a 96-well plate format points to its usefulness in cell survival-based high-throughput screens for anti-HCV molecules. The degree of cell death was found to be proportional to the intracellular load of HCV. HCV-low n4mBid cells, expressing an anti-HCV short hairpin RNA, showed a significant growth advantage over naïve cells and could be rapidly enriched after HCV infection, suggesting the possibility of using n4mBid cells for the cell survival-based selection of genetic anti-HCV factors.

Engineering and characterization of human manganese superoxide dismutase mutants with high activity and low product inhibition

Human manganese superoxide dismutase is a mitochondrial metalloenzyme that is involved in protecting aerobic organisms against superoxide toxicity, and has been implicated in slowing tumor growth. Unfortunately, this enzyme exhibits strong product inhibition, which limits its potential biomedical applications. Previous efforts to alleviate human manganese superoxide dismutase product inhibition utilized rational protein design and site‐directed mutagenesis. These efforts led to variants of human manganese superoxide dismutase at residue 143 with dramatically reduced product inhibition, but also reduced catalytic activity and efficiency. Here, we report the use of a directed evolution approach to engineer two variants of the Q143A human manganese superoxide dismutase mutant enzyme with improved catalytic activity and efficiency. Two separate activity‐restoring mutations were found − C140S and N73S − that increase the catalytic efficiency of the parent Q143A human manganese superoxide dismutase enzyme by up to five‐fold while maintaining low product inhibition. Interestingly, C140S is a context‐dependent mutation, and the C140S–Q143A human manganese superoxide dismutase did not follow Michaelis–Menten kinetics. The re‐engineered human manganese superoxide dismutase mutants should be useful for biomedical applications, and our kinetic and structural studies also provide new insights into the structure–function relationships of human manganese superoxide dismutase.

Development of a bacteriophage-based system for the selection of structured peptides.

Short structured peptides can provide scaffolds for protease-resistant peptide therapeutics, serve as useful building blocks in biomedical and biotechnological applications, and shed light on the role of secondary structure elements in protein folding. It is well known that directed evolution is a powerful method for creating proteins and peptides with novel properties, and a system for the selection of short peptides based on structure from a randomized library would be an important advancement. In this study, phage particles monovalently displaying a short peptide and an N-terminal 6xHis tag on their P3 coat protein were bound to nickel agarose resin and were subsequently challenged with a protease that specifically cleaves at a site within the peptide. The extent to which phage is proteolytically released from the resin was found to be dependent on the structural properties of the inserted peptide sequences. As proofs-of-concept, a structured peptide has been isolated from a pool of flexible peptides using a trypsin selection, and a flexible peptide has been isolated from a pool of structured peptides using a chymotrypsin selection. This selection system will be a strong technological platform for the creation of short peptides with interesting structural properties using directed evolution.