Lecture 5: Developing anti-viral therapies Flashcards
Describe the importance of vaccines (immunotherapy) and their drawbacks?
- Anti-viral vaccines have provided very significant health benefits. Many vaccines available to day are based on very simple strategy of challenging immune system with “live” or dead” virus (Salk and Sabin polio vaccines). The immune system sorts it out!
- Many unsuccessful vaccine strategies are due to immune systems inability to mount a response; either with antibodies or T cells.
- Identifying anti-viral antibodies is a fruitful strategy to develop a vaccine, but these are rare (only a very small number in any individual).
- However, mounting an immune response is time consuming. Rarely an immediate treatment.
What are three properties of drugs (pharmacological intervention) that make them desirable?
Immediate effect
Specific
Convenient
What type of drug targets are favoured and why? What parts of the virus can be targeted?
[Part I: Drug discovery based on a known target]
Direct acting anti-virals (DDAs) are favored; target viral not cellular process, therefore low toxicity and high specificity. These are antagonists. Agonists can be anti-viral drugs but not favored.
All viruses have similar features in replication
Any of these processes can be targeted.
Drug targets are typically viral enzymes which are unique to virus and not cellular process.
There is a move in drug discovery towards targeting processes such as virion assembly as they, too, are unique to virus replication, but this field is less advanced.
[Part I: Drug discovery based on a known target]
All approaches rely on an intimate understanding of viral protein function e.g. enzymatic catalysis; this a what the discovery assay will be based on.
“…..knowledge of the three-dimensional structure of the protein can enable visualization of its active site and understanding of its mechanism of action, and can also be used to design or refine small molecules that can block the active site and the protein’s action.” – Douglas Richman (Fields Virology, 2016)
To design a discovery assay, we need to know the function of the enzyme, describe how?
[Part I: Drug discovery based on a known target]
From above statement if we know structure we can somewhat guess its function.
We take a protein out of the infected cell –> we either know or suspect its function –> use that as a discovery assay
Identify enzyme inhibitors using a biochemical assay then test against virus replication in cell culture assays.
Pol is protein that produces…
[Part I: Drug discovery based on a known target]
….viral enzymes, in the case of HIV it makes: Protease, reverse transcriptase and integrase
What are Reverse transcriptase inhibitors?
[Part I: Drug discovery based on a known target]
Reverse Transcriptase (RT) is a viral enzyme unique to HIV, not found in cells.
Activity of this enzyme one of the first indicators that AIDS was cause by HIV.
Briefly describe the process of reverse transcription
[Part I: Drug discovery based on a known target]
Reverse transcriptase creates double-stranded DNA from an RNA template.
Reverse transcriptase binds vial nucleic acid
The process of reverse transcription is extremely error-prone, and it is during this step that mutations may occur. Such mutations may cause drug resistance.
How was Reverse transcriptase inhibitors identified?
[Part I: Drug discovery based on a known target]
RT was identified using a biochemical assay from cell lysate to show incorporation of radiolabeled nucleotides into a template.
This biochemical assay was used to test available drugs for anti-RT activity. Examples, include AZT. AZT is a nucleotide, therefore, directly prevents viral genome synthesis by incorporating “wrong” nucleotides. Other examples include, efavirenz, which is not a nucleotide, but inhibits RT enzyme catalysis.
All then shown to inhibit HIV replication in tissue culture.
A discovery assays was preformed based on what the general idea of the function of RT was. However, this shifted to…
[Part I: Drug discovery based on a known target]
…investigating integrates and protease as potential targets instead, however since their function is unknown, their structure had to be guessed and then the function à after which we can design drug to target them
Describe the importance of a structure based design?
[Part I: Drug discovery based on a known target]
- Very detailed structural data (derived from x-ray crystallography, nuclear magnetic resonance, electron microscopy) allows computer aided “docking” of drug molecules into structure of viral proteins.
- Allow refinement of existing drugs to increase compound and protein interactions between the protases (viral protein) and the potential drug.
- Allows identification of binding of known drugs and unknown drugs using 3D stimulation.
How do we approach anti-viral discovery without a known target?
[Part II: Identification of new drug targets]
Find out what viral and cellular proteins are required for replication!
If virus genome sequence is known then we can predict what proteins (including viral enzymes) are expressed.
We then test drugs that inhibit similar enzymes. For example, acyclovir targets the polymerase of herpes simplex virus. A related herpesvirus, human cytomegalovirus, was found to have a similar polymerase. Acyclovir-like drugs were tested and the anti-cytomegalovirus drug ganciclovir was identified. (Both are nucleosides)
What techniques can be used for Identification of cellular factors?
[Part II: Identification of new drug targets]
siRNA screening - causes gene silencing through repression of transcription occurs as follows:
- Long dsRNA is cleaved by an endo-ribonuclease called Dicer à forms short interfering RNA or siRNA
- Once siRNA enters cell it gets incorporated into other proteins to form the RISC.
- The siRNA is unwound to form single stranded siRNA.
- The strand that is thermodynamically less stable due to its base pairing at the 5´end is chosen to remain part of the RISC-complex
- The single stranded siRNA which is part of the RISC complex now can scan and find a complementary mRNA
- Once the single stranded siRNA (part of the RISC complex) binds to its target mRNA, it induces mRNA cleavage à no translation à no proteins.
Use of genetic tools to “knock down” proteins. Assay virus replication in cells treated with siRNA; no virus replication indicates the cellular protein removed by siRNA is necessary for virus replication and a drug target.
Compare between shRNA and siRNA?
[Part II: Identification of new drug targets]
- siRNA and shRNA are different!
- shRNA requires processing for loading of RNA onto dicer.
- siRNA more commonly used for screening, though shRNA screening is possible.
- shRNA more commonly used to make libraries of cell lines deficient in certain factors (but costly and time consuming).
Give an example of siRNA screening to find out what proteins in the cell are required for virus replication (which is useful to find out which reaction we want to inhibit)
[Part II: Identification of new drug targets]
Scheme;
- Treat cells with siRNA covering entire human genome, so that a different siRNA goes into each well
- Allow is RNA to act on the cell à result is gene knockdown à therefore one different protein is removed from cells in each of the wells
- Infect the cells with HIV virus
- Detect infection (readout steps)
- See if there is a difference in the expression of viral proteins or production of virus itself
- Carried out using robot automation to manipulate 384 well plates and automated microscopy.
If there is no viral protein in a well à indictaes presence of siRNA that is targetting gene konckdown taking away a protien required for viral protein production.
We also take the supernatant and carry out readouts
Now that we have siRNA screening results (have targets in mind), what are the next steps?
Drug discovery from siRNA screening results
[Part II: Identification of new drug targets]
Identification of proteins that have known inhibitors. This includes drug “repurposing”; using a drug for one indication for another. For ex: anti-cancer drug repurposed for HIV
Identification of proteins without known inhibitors. Proteins similar in sequence, structure or function may be inhibited by drugs targeting proteins with similar characteristics. Ex: Acyclovir and ganciclovir
Identification of processes that can be inhibited indirectly. For example, nucleotides are require for virus genome replication. Can we target nucleotide production?
In each case assay drug to ensure that virus replication is inhibited.
The use of siRNA (knock-down) to screen for potential proteins that can be targeted is useful however it is rather…
[Part II: Identification of new drug targets]
…inefficient – so if you are trying to look through the entire genome to find a target we essentially need to screen 30,000 different siRNAs!
Therefore, we started the use of haploid cell screening (knock-out) instead which is much more rapid. With time we have also moved away from haploid cell screening to CRISPR/Cas9 screening (knock-out).
Describe Drug discovery from haploid cells screening results?
[Part II: Identification of new drug targets]
Same as the Drug discovery from siRNA screening (above answer)
Describe the process of Haploid cell screening?
[Part II: Identification of new drug targets]
In this process take advantage of the fact that haploid cells only have one chrm of each
- Infect haploid cells with retrovirus (gene trap virus)
- Retrovirus will randomly integrate into any open reading frame and prevents gene expression
- Infect with virus of interest
- Only cells that virus does not grown in survive
- Sequence those cells and find out what is knocked out
- Identify the protein of the virus required for its replication
This is a rapid method
We can do many different random library screens at any one time
What does CRISPR/Cas9 mean?
[Part II: Identification of new drug targets]
“Clustered Regularly Interspaced Short Palindromic Repeats” bacterial system using bacterial Cas9 protein
- Guide RNA (has a sequence for any open reading frame of a GOI)
- This is recognized by Cas9 protein and inserted into genomic DNA
- This manipulation of open reading frame prevents expression of protein from that open reading frame (knock-out)
High throughput drug screen (instead of focusing on a target like before, we just look for something that prevent viral growth)
[Part III: Screening of drug libraries]
Scheme;
- Treat cells with drugs using many plates at the same time
- Infect cells with HIV
- Detect infection: expression of viral protein OR production of virus using IF staining
- Take supernatant and put over fresh cells
- Through IF staining see whether or no there is infection of fresh cells
Carried out using robot automation to manipulate 384 well plates and automated microscopy to look at wells.
