Gfp-Amplification Mutagenesis Assay (Gma): Quantitative, Scalable Detection Of Chemical Mutgenesis

University of California System: University of California, Santa Cruz
posted on 10/25/2011

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Genotoxic (DNA damage-inducing) chemicals often increase the risk of cancer. Genotoxicity testing is mandatory for approval of new drugs as an indicator of potential carcingenicity and is therefore genotoxicity is generally undesired. However, genotoxic activity can also in some cases be deliberately screened for to help identify DNA-targeting compounds, as leads for the development of antimicrobial, immunosuppressive, or antitumor agents. The most popular genotoxicity assay is the Ames’ test. This test uses Salmonella and E. coli as model organisms because of the exquisite conservation of DNA damage and repair mechanisms across kingdoms. The Ames’ test detects point mutations and frameshifts based on the reversion of inactivating mutations in the biosynthesis operon of a given amino acid. Because this assay detects mutations rather than more indirect effects of genotoxicity such as changes in gene expression, it has a high degree of specificity (77% compared to ~50% for other genotoxicity tests). However, the classic Ames’ test presents a number of technical limitations, notably the large amount of test sample required. The Ames’ test is not easily amenable to high throughput screening. Some high-throughput versions of the Ames’ test have been developed, and are based on fluctuation analysis in liquid culture. In this case the readout is binary ( i.e. growth vs. no growth) in individual wells of a 96-well plate. UCSC researchers’ newly developed technology generates a quantitative signal that is proportional to the number of random mutations present in the culture. Thus, the information provided in a 96-well plate for the liquid Ames’ test could be obtained in one well, dramatically reducing the amount of test sample required.

Because of the savings in test sample and the ease with which this assay can be adapted to a 96-well format, UCSC’s proprietary mutagenesis assay is ideally suited for three types of applications:

1)     High-throughput screening of mutagens:  examples of classes of mutagens that can be screened for include:

a.     Identification of DNA-targeting compounds from chemical or natural product libraries, as potential antimicrobial or antitumor agents.

b.     Identification of compounds that decrease fidelity of replication, as potential antiviral and antitumor agents.

c.      Identification of mutagenic nucleotide analogs, for the generation of random mutant libraries for protein engineering purposes.

2)     Early detection of carcinogenic potential: to remove agents from the drug development pipeline early on, representing a cost-savings opportunity.

3)     Quality control for random mutant libraries: directed evolution (evolution of a given protein in the laboratory) is a standard approach for the optimization of enzymatic activities and for the generation of new biochemical activities. The genetic diversity required for these experiments is generated by random mutagenesis. The mutation spectrum and load is critical for success. Thus, our reporter can be used to monitor the quality of random mutant libraries to ensure adequate genetic diversity.

The provisional patent describing this technology obtained a very favorable opinion from the Patent Office, showing that UCSC’s technology is novel, non-obvious and useful. Our patent covers any reporter that is based on a reversion assay based on expression of a reversion mutagenesis reporter fused to a fluorescent or luminescent gene. While this assay was developed in E. coli, the same principle should work in mammalian cells.

Compared to genotoxicity tests that are based on indirect (changes in gene expression) readouts, this assay preserves the exquisite specificity of the Ames’ test while its quantitative readout dramatically decreases the amount of sample and of reagents necessary to carry out the test. This represents a major cost-savings opportunity, particularly for compounds in early stages of development.