The Genetic Insights blog series provides perspectives on the use and impact of genetic analysis in drug development. Genetic research is fueling new discoveries that link diseases, therapies, and patient responses. To learn more, please browse our genetic analysis solutions.
Containing Viral Spread is Critical to Global Health
Emerging viruses pose a great challenge to global health care and economic systems. Given their pandemic potential, as witnessed in the case of the SARS-CoV-2 virus, rapid development and screening of drugs is needed to combat and contain the infection spread.
Rapid Development & Screening of Drugs is the Challenge
Screening Drug Candidates & Deciphering Mode of Action
In Vitro Cell-Based Assays
Screening drug candidates and deciphering their mode of action can be a laborious process. Several in vitro cell-based or biochemical/cell-free assays are routinely used for testing antiviral drugs for efficacy. Cell-based assays involve the use of 2D or 3D cell culture models to determine phenotypic and/or genomic changes in the cell after drug treatment. These assays not only provide a physiologically relevant environment but can also allow multiple targets in a single screen. One of the main challenges with cell-based assays is that variable assay conditions such as passage number of the cells, the time of cell infection, media conditions, etc., can have a profound impact on the apparent drug potency .
In contrast, biochemical assays utilize proteins purified from cell lysates. While they do not recapitulate a cellular environment, they are more consistent in terms of experimental reproducibility and are adaptable to high-throughput workflows as well as analytical readouts. Often, biochemical assays precede cell culture-based experimentation, as they are simple to control for and design.
Enter Capillary Electrophoresis
Can CE Help in Drug Discovery and Development?
CE is more sensitive, requires low sample input, and couples analyte separation with detection.
When used in tandem with biochemical assays, CE can expand their utility.
Capillary electrophoresis (CE) is an analytical technique that separates biomolecules based on their differential migration in an electric field. The migration is predominantly governed by the size and charge of the analytes, and is influenced by several factors, such as the separation matrix and current applied to the matrix. CE has recently begun to be recognized for its numerous advantages over other commonly used technologies for drug screening. In comparison to fluorescent plate readers, a popular drug screening instrument, CE is more sensitive, requires low sample input, and couples analyte separation with detection . When used in tandem with biochemical assays such as enzymatic assays, it can expand their utility by providing additional information on the reaction products, their size, and their relative concentration through fragment analysis (FA). In addition to FA, Sanger sequencing by CE has been utilized in cell-based assays such as guide RNA screening for CRISPR-based therapeutic approaches [3,4]. Given its versatility as an analytical platform, CE holds great potential in enabling drug discovery and development.
CE in Action: Screening Anti-Viral Drugs & Understanding Their Cellular Mechanism
RNA Viruses & Rapid Drug Screening Platforms
RNA viruses pose the greatest risk for zoonotic transmission , and their pandemic potential surpasses all potential pathogens . Therefore, it is imperative to develop and utilize rapid drug screening platforms to minimize the time from discovery to deployment.
RNA Polymerase (RdRp)
One of the key targets for developing drugs against these viruses is the RNA-dependent RNA polymerase (RdRp), an enzyme required to support viral replication in the infected cell. The viral RdRp has no mammalian homolog, making it an ideal target since it reduces any potential off-target effects of the drug as well as enhances potency.
SARS-CoV-2 Genomic Sequencing & Development of Remdesivir Triphosphate (RTP)
A recent article by Dangerfield and colleagues  demonstrated that remdesivir triphosphate (RTP), a nucleoside analog that inhibits RdRp , competes with ATP and serves as a better substrate for the enzyme during RNA polymerization. Using untagged, purified SARS-CoV-2 RdRp complex, the group performed in vitro RNA replication experiments. A 20-nucleotide 5’-[6-FAM] labeled primer was annealed to a 40-nucleotide SARS-CoV-2 genomic sequence and the kinetics of ATP or RTP incorporation was determined by quenching the replication reaction at various time points. The formation of the extended products was analyzed by fragment analysis through capillary electrophoresis. FA enabled kinetic modeling of RNA extension by determining the concentration of the various-sized products over time when ATP or RTP were used as substrates. The data showed that remdesivir was incorporated into the replication product faster than ATP by the viral RdRp and suppressed product extension.
A follow-up study showed that mechanistically, remdesivir modulates virus replication by stalling the polymerase after several molecules of RTP are incorporated into the replication complex . Using a similar experimental approach as described above, fragment analysis revealed that over a prolonged reaction period, several smaller-sized products were formed when RTP was used as a substrate, of which only 70% were extended to a full-sized product upon addition of all four nucleotides to the reaction.
CE in Conjunction With In Vitro Assays
Rapidly Screen Drugs and Determine Their Mode of Action
As demonstrated by these studies, CE can be used to rapidly screen drugs and determine their mode of action when used in conjunction with in vitro assays. High throughputs can be achieved by simultaneous separation of samples by 8- to 24- channel capillaries in a single run and single instrument. Additional multiplexing capabilities can be added by labeling different samples with distinct dyes in a single well and further enhance the platform’s ability to analyze potentially hundreds of samples in one plate within a few hours.
- Postnikova, E., et al., Testing therapeutics in cell-based assays: Factors that influence the apparent potency of drugs. PLoS One, 2018. 13(3): p. e0194880.
- Ouimet, C.M., I. D’Amico C, and R.T. Kennedy, Advances in capillary electrophoresis and the implications for drug discovery. Expert Opin Drug Discov, 2017. 12(2): p. 213-224.
- Ophinni, Y., et al., CRISPR/Cas9 system targeting regulatory genes of HIV-1 inhibits viral replication in infected T-cell cultures. Sci Rep, 2018. 8(1): p. 7784.
- Yin, C., et al., Functional screening of guide RNAs targeting the regulatory and structural HIV-1 viral genome for a cure of AIDS. AIDS, 2016. 30(8): p. 1163-74.
- Woolhouse, M.E., Population biology of emerging and re-emerging pathogens. Trends Microbiol, 2002. 10(10 Suppl): p. S3-7.
- Bukasov, R., D. Dossym, and O. Filchakova, Detection of RNA viruses from influenza and HIV to Ebola and SARS-CoV-2: a review. Anal Methods, 2021. 13(1): p. 34-55.
- Dangerfield, T.L., N.Z. Huang, and K.A. Johnson, Remdesivir Is Effective in Combating COVID-19 because It Is a Better Substrate than ATP for the Viral RNA-Dependent RNA Polymerase. iScience, 2020. 23(12): p. 101849.
- Gordon, C.J., et al., The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus. J Biol Chem, 2020. 295(15): p. 4773-4779.
- Bravo, J.P.K., et al., Remdesivir is a delayed translocation inhibitor of SARS-CoV-2 replication. Mol Cell, 2021. 81(7): p. 1548-1552 e4.