UMass Medical School researcher Dr. Robert Finberg & colleagues have developed a stable cell line expressing TLR2

UMass Medical School researcher Dr. Robert Finberg & colleagues have developed a stable cell line expressing TLR2, CD14 and NF-κB -driven luciferase reporter gene. This unique cell line can be used as a screening tool to rapidly identify new compounds which could potentially modulate TLR-2 mediated cytokine signaling (Antiviral Res. 2010 87: 295). Viruses such Herpes Simplex virus the causative agent for most common sexually transmitted infections, exploit this vital signaling pathway. Inhibitors for this vital signalling pathway can be novel therapeutics candidates for viral and other inflammatory disease processes

Background

Traditional methods used to determine the involvement of a specific amino acid or group of

Background

Traditional methods used to determine the involvement of a specific amino acid or group of amino acids to a protein’s structure, stability or function, are alanine scanning or random mutagenesis.  However, these approaches are significantly limited in accessing a residues contribution.  With alanine scanning, the technique only measures the function of an amino acid change to alanine, ignoring 18 other possible amino acids in the same context. Moreover, random mutagenesis, such as PCR and gene shuffling, are recognized as being error-prone. 

Technology

Dr. Daniel Bolon’s laboratory at UMass Medical School has developed a novel method to analyze the function of all possible amino acid replacements at multiple positions of a protein.  This method includes a platform to systematically generate and introduce random codons in a gene, mutational selection based on biochemical function or property, measure abundance of randomized sequence, and classify each codon replacement.  For enhanced accuracy, Dr. Bolon has also designed an accompanying software program for the automated design of oligonucleotides that can be used to mutagenize the DNA and facilitate deep-sequencing readout and abundance of each mutation in the library.    

Applications

•  Enhancement of protein function, biological or chemical or properties.
•  Identification of region(s) in a protein sensitive or resistant to mutation(s).
• Determine which amino acid substitutions lead to drug or antibody resistance or sensitivity.
• Detection of amino acid(s) involved as active or receptor binding sites.

Competitive Advantages 

• Provides improved coverage of the mutational space over existing methods by systematically substituting all possible amino acids at each position(s) of interest.
•  Cost-effective and greater efficiency- reduced experimental time and labor- with maximum results.

This technology is based, in part, on the discovery that IL6 and TNFa can serve

This technology is based, in part, on the discovery that IL6 and TNFa can serve as biomarkers for JNK inhibition as levels of IL6 and TNFa can decrease upon treatment with a JNK inhibitor. 

Background

The nucleus of a cell needs to fit a large amount of DNA into a small area

Background

The nucleus of a cell needs to fit a large amount of DNA into a small area efficiently.  While local chromatin structure has been studied, less is known about the higher order packing of DNA.  The way chromatin is packaged is a major determinant of a cell’s gene expression patterns. Regions of DNA can interact with each other despite a large distance from one site to another on a chromosome.  These interactions can either enhance or silence transcription, and classifying these sites on a genomic scale would be an important tool for understanding transcription systems in differentiation or disease states.  Identifying long-range interactions between chromatin segments can also provide an overview of the 3-dimensional structural conformation of chromosomes in the nucleus.

Technology

High-throughput chromosome capture (Hi-C) technology is a method of identifying DNA elements that interact on a genome-wide scale.  Developed by the Dekker lab at University of Massachusetts Medical School, Hi-C blends chromosome capture methods used to identify enhancers or silencers of specific genes with deep sequencing, allowing for the identification of all interacting regions in a cell.  By using Solexa sequencing methods, Hi-C improves upon the previous 3C and 5C methods by avoiding multiple PCR steps with large primer sets.  This technology also allows for the unbiased, complete identification of interacting genomic elements in any cell type.

Protocol

1.     Regions of chromatin are crosslinked.

2.     The crosslinked DNA is digested using a restriction enzyme.

3.     Interacting regions of DNA are then ligated together in the presence of a biotinylated linker.

4.     The crosslinks are then reversed and the DNA is purified.

5.     The DNA is then sonicated into smaller fragments.

6.     Interacting DNA fragments are isolated by streptavidin capture of biotinylated fragments.

7.     Solexa adaptors are added to these fragments, and the library is amplified by PCR.

8.     Solexa deep sequencing is performed to identify interacting elements.

 Applications

·      Understanding the spatial organization of the genome

·      Determination of genome architecture specific to different cell types

·      Characterization of disease states based on interactions of genetic elements

Advantages

·      High throughput method of identifying enhancer or repressor elements

·      Unbiased approach to identification

·      Can be adapted to kit format

References

Lieberman-Aiden, E., van Berkum, N.L., Williams, L., Imakaev, M., Ragoczy, T., Telling, A., Amit, I., Lajoie, B.R., Sabo, P.J., Dorschner, M.O., Sandstrom, R., Bernstein, B., Bender, M.A., Groudine, M., Gnirke, A., Stamatoyannopoulos, J.A., Mirny, L., Lander, E.S. and Dekker, J.(2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science, 326(5950): 289-293.

Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J. (2010) Hi-C: a method to study the three-dimensional architecture of genomes.  J. Vis. Exp., 39: 1869.

Background

HIV protease has proven to be a powerful drug target in the treatment of HIV

Background

HIV protease has proven to be a powerful drug target in the treatment of HIV/AIDS.  While the first generation of HIV-1 protease inhibitors (PIs) have been effective in slowing the spread of the virus, multiple drug resistant (MDR) forms of HIV have evolved that are not responsive to these inhibitors.  While current PIs were designed to inhibit wild-type HIV-1 protease, our new structure-based drug design focuses on developing PIs that bind and inhibit multiple MDR protease variants by understanding how the enzyme recognizes substrates.

Technology

UMass Medical school investigator Dr. Celia Schiffer and colleagues have designed a new class of PIs that are more potent and less likely to induce drug resistance than inhibitors that are currently available.  Dr. Schiffer and colleagues have designed competitive inhibitors based on a detailed understanding of the structures of the protease when complexed with its viral substrates (Ali et al (2006) J Med Chem 49:7342, Reddy et al (2007) J Med Chem 50:4316, Chellappan et al (2007) Chem Biol Drug Des 69:298, and Altman et al (2008) J Am Chem Soc 130:6099).  These novel inhibitors bind within the consensus substrate binding region or “substrate envelope”. Thus, with these PIs, resistance is less likely to occur while protease maintains substrate recognition. These next generation PIs are robust against resistance, demonstrating higher potency than Darunavir against patient derived MDR viruses with known protease resistance.

Competitive Advantages

l    Novel HIV-1 protease inhibitors: Designed based on a novel approach using the constraints of the substrate envelope combined with structure based drug design.

l    Potent activity: Novel inhibitors are potent against drug resistant HIV-1 variants with subnanomolar IC₅₀s

l    Resilience to mutations: Containment of these PI’s within the substrate envelope allows these PIs to retain activity as new mutations appear, increasing the time for therapeutic efficacy

l    High affinity binding: These novel PIs, although small, bind with high affinity picomolar affinity to a variety of HIV-1 protease variants that are resistant to most of the FDA approved drugs

l    Ideal drug-like property:  Calculated ClogP values compare favorably with FDA-approved HIV-1 PIs.