20.109(F22):Module 2

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20.109(F22): Laboratory Fundamentals of Biological Engineering

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Fall 2022 schedule        FYI        Assignments        Homework        Class data        Communication        Accessibility

       M1: Genomic instability        M2: Drug discovery        M3: Project design       


Module 2: Drug discovery

Malaria is a mosquito-borne disease caused by Plasmodium parasites. This life-threatening disease is estimated to be responsible for over 409,000 deaths from 229 million cases worldwide in 2019. The impact of malaria is disproportionately seen in Africa, where 95% of malaria cases and 95% deaths occurred (World Health Organization, World Malaria Report, 2020).

In this module, you will explore drug discovery by focusing on a specific infectious disease, malaria, as an example of how small molecules can be used in therapeutics. The process by which new therapeutics are discovered, optimized, and tested to ensure safety and effectiveness is extensive. Here, we will focus on the discovery phase and thinking through what makes a good therapeutic. This is ideally grounded by molecular level understanding of the disease process, and knowledge of specific targets or pathways that can be manipulated to improve health outcomes. In some instances, however, effective small molecule therapeutics can be developed despite incomplete biological understanding.

Small molecule screens are foundational to identifying potential therapeutics in drug discovery. In this module, you will review small-molecule microarray (SMM) technology and how this can be used to identify molecules that putatively bind to a protein from Plasmodium falciparum that is essential for its survival. Research in the Niles Laboratory works to identify and validate essential proteins of unknown function(s) from P. falciparum as potential targets for drug discovery. Through these efforts, multiple proteins have been selected as candidate targets, and are being recombinantly expressed to use in SMM screens to identify small molecules binding hits.

The protein you will work with, PfFKBP35, was identified via this process. Though much remains to be learned about its function(s) in P. falciparum, available biological data suggest that it could be a valid therapeutic target. Furthermore, it is already known that the small molecule FK506 is able to bind PfFKBP35. One caveat of using this protein as a drug target is that an ortholog of this protein is expressed by humans, FKBP12, and this human protein also binds FK506. This means that using FK506 as a drug to target PfFKBP35 could potentially interfere with the activity of the human protein and cause harm to patients.

Given that it is not ideal to use a drug in patients that can disrupt the function of a human protein, why are we using this target? Because there is a known binder, researchers can test modifications to FK506 to generate a a drug candidate that is specific to PfFKBP35 and unable to bind the human version. In this module you test small molecules that were designed using FK506 as a scaffold for specificity to PfFKBP35 versus the human protein, FKBP12.

Research goal: Test small molecules for binding to the Plasmodium falciparum FKBP35 protein using a functional assay.


Image generated using BioRender.



Lab links: day by day

M2D1: Complete in-silico cloning of protein expression plasmid
M2D2: Perform protein purification protocol
M2D3: Assess purity and concentration of purified protein
M2D4: Review small molecule microarray (SMM) technology and data analysis used to identify hits
M2D5: Prepare protein for functional assay
M2D6: Complete data analysis of functional assay results
M2D7: Evaluate protein structures

Major assignments

Journal article presentation
Research article

References

A method for the covalent capture and screening of diverse small molecules in a microarray format. Nature Protocols. (2006) 1:2344-2352.

Recent discoveries and applications involving small-molecule microarrays. Chemical Biology. (2014) 18:21-28.

Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis. Science. (2018) 360:506.

  • Reference for mutagenesis index score: plot linked here
  • Plot shows ranked order of all Plasmodium falciparum genes from most essential (left) to dispensable (right) along the x-axis. A piggyBAC insertion mutagenesis screen (mobile element that inserts into genes to disrupt production of their encoded protein) was used to generate the plotted data.

Notes for teaching faculty

Prep notes for M2