Our Approach
Target Identification
Streamlined Identification of Validated Drug Targets
VLST has developed a robust and highly productive bioinformatic-proteomic platform for the prediction of viral virulence factors and the identification of interactions with host proteins. Key steps include:
- Bioinformatic mining of viral genomes for virulence factors
- Synthesis of tandem affinity tagged forms of the viral genes
- Transient expression of the tagged viral proteins
- Screening of cell lysates and conditioned media using viral proteins
- Capture of host protein targets interacting with viral proteins using tandem affinity tags
- Identification of targets by mass spectrometry
- Confirmation of identified targets by independent methods
Bioinformatic Mining for Viral Virulence Factors
VLST has taken the search for viral virulence factors to a genomic scale. Medium and large genome double-stranded DNA viruses were chosen as they contain many virulence factors and typically have fewer multiple functional genes than smaller viruses, potentially simplifying virulence gene identification. VLST has analyzed the genome sequences for over 260 pox, herpes, adeno and asfar viruses to predict protein sequences and cluster them based upon their relatedness. Virulence genes were then predicted based upon multiple criteria used in various combinations using proprietary bioinformatic tools. These criteria include homology to human proteins, non-essentiality for viral replication, protein topology and protein structural motifs.
Proteomic Identification of Host Targets of Viral Virulence Factors
Predicted viral virulence genes are synthesized and then corresponding proteins transiently expressed as tandem affinity tagged forms in mammalian cells. Viral proteins are used to probe cellular sources for interacting proteins. A wide range of cellular sources are used including cell lysates and conditioned media from cell lines and primary cells of immune origin. Targets, i.e., host proteins interacting with the viral proteins, are then recovered using proprietary tandem affinity purification tags. Targets are then identified using optimized mass spectrometry methods. False positives are minimized, e.g., by filtering out common contaminants out using a proprietary database based on thousands of mass spectrometry runs. Targets are verified by independent experiments using orthogonal methods such as flow cytometry with cell lines engineered to express the target, surface plasmon resonance with recombinant target or co-immunoprecipitation with recombinant target.