Difference between revisions of "20.109(F21):Module 2"

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==Module 2: Drug discovery==
 
==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 400,000 deaths with 228 million cases worldwide per year. The impact of malaria is disproportionately represented in Africa where 93% of malaria cases and 94% of malaria deaths occurred in 2018 (World Health Organization).
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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 tested to ensure safety and effectiveness often requires years or decades. Here we will focus on the front-end of this process by considering what makes a good therapeutic. The basis for making this decision is often rooted in knowledge regarding the fundamental biology of the organism / disease.  Sometimes little is known about what is occurring at the molecular level and therefore it is difficult to design a therapeutic.
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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 lengthy. 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 can be useful in drug discovery when a targeted approach is not feasible. For your project you will use a small-molecule microarray (SMM) to identify molecules that putatively bind a protein target in ''Plasmodium falciparum''. The Niles Laboratory has been working to identify small, essential proteins of unknown functions from ''P. falciparum'' for drug discovery attempts. In this, multiple genes of interest have been cloned and knocked down in the parasite and if found to be essential the targets were used for drug screening by SMM.  
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Small molecule screens are foundational to drug discovery. In this module, you will learn about 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. The Niles Laboratory has been working 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 that you will study this semester is a protein that was identified using this process. Thought the structure of the protein was solved, the literature is not rich for this gene. This is one of the troublesome features of working with uncharacterized proteins but they can be good drug targets because, most likely, these genes do not have a human orthologue. Therefore, drug selectivity can be achieved.
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The protein you will work with 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. SMM and other screens have identified several interesting binding hits, but these need to be confirmed in secondary binding assays before selecting candidates to prioritize for additional characterization and optimization. As part of this module, you will have the opportunity to perform quantitative binding assays, as well as analyze and interpret these data to help inform which molecules may be stronger candidates for further study.
  
  
<font color =  #2874a6 >'''Research goal:  Identify small molecules that bind to the PF3D7_1351100 protein in ''Plasmodium falciparum'' using small-molecule microarray'''</font color>
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<font color =  #2874a6 >'''Research goal:  Identify small molecules that bind to the PF3D7_20109-F21 protein in ''Plasmodium falciparum'' using small-molecule microarray'''</font color>
  
  
[[Image:Fa20 M2 overview schematic v3.png|center|700px|thumb|Image generated using BioRender.]]
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[[Image:Fa21 M2 overview.png|center|700px|thumb|Image generated using BioRender.]]
  
  
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==Lab links: day by day==
 
==Lab links: day by day==
M2D1: [[20.109(F21):M2D1 | Complete in silico cloning of protein expression plasmid]]<br>
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M2D1: [[20.109(F21):M2D1 |Review small molecule microarray (SMM) experiment and results ]]<br>
M2D2: [[20.109(F21):M2D2 | Perform protein purification protocol]]<br>
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M2D2: [[20.109(F21):M2D2 |Complete in silico cloning of protein expression plasmid]] <br>
M2D3: [[20.109(F21):M2D3 | Assess purity and concentration of purified protein]]<br>
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M2D3: [[20.109(F21):M2D3 |Perform protein purification protocol]] <br>
M2D4: [[20.109(F21):M2D4 | Prepare small molecule microarray (SMM) slides with purified protein]]<br>
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M2D4: [[20.109(F21):M2D4 |Assess purity and concentration of purified protein]] <br>
M2D5: [[20.109(F21):M2D5 | Scan SMM slides to identify putative small molecule binders]]<br>
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M2D5: [[20.109(F21):M2D5 |Perform secondary assay to test putative small molecule binders]] <br>
M2D6: [[20.109(F21):M2D6 | Analyze SMM data to confirm putative small molecule binders]]<br>
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M2D6: [[20.109(F21):M2D6 |Complete data analysis for secondary assay]] <br>
M2D7: [[20.109(F21):M2D7 | Examine putative small molecule binders for common features]] <br>
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M2D7: [[20.109(F21):M2D7 |Plot and interpret data from secondary assay]] <br>
  
 
==Major assignments==
 
==Major assignments==
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[[Media:Sp17 M1 reference ChemBiol.pdf| Recent discoveries and applications involving small-molecule microarrays.]] ''Chemical Biology.'' (2014) 18:21-28.
 
[[Media:Sp17 M1 reference ChemBiol.pdf| Recent discoveries and applications involving small-molecule microarrays.]] ''Chemical Biology.'' (2014) 18:21-28.
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[[Media:Schmidt Skerra 2007 Streptactin.pdf | The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins.]] ''Nature Protocols.'' (2007) Vol.2. No.6:1528-1535.
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[http://www.dropbox.com/s/492hj0ct9vsc6pg/Zhang.2018.Science.pdf?dl=0 Uncovering the essential genes of the human malaria parasite ''Plasmodium falciparum'' by saturation mutagenesis.] ''Science.'' (2018) 360:506.
 
[http://www.dropbox.com/s/492hj0ct9vsc6pg/Zhang.2018.Science.pdf?dl=0 Uncovering the essential genes of the human malaria parasite ''Plasmodium falciparum'' by saturation mutagenesis.] ''Science.'' (2018) 360:506.

Latest revision as of 03:26, 31 October 2021

20.109(F21): Laboratory Fundamentals of Biological Engineering
Drawing provided by Marissa A., 20.109 student in Sp21 term.  Schematic generated using BioRender.

Fall 2021 schedule        FYI        Assignments        Homework        Class data        Communication        Accessibility

       Module 1: Genomic instability                          Module 2: Drug discovery       


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 lengthy. 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 drug discovery. In this module, you will learn about 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. The Niles Laboratory has been working 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 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. SMM and other screens have identified several interesting binding hits, but these need to be confirmed in secondary binding assays before selecting candidates to prioritize for additional characterization and optimization. As part of this module, you will have the opportunity to perform quantitative binding assays, as well as analyze and interpret these data to help inform which molecules may be stronger candidates for further study.


Research goal: Identify small molecules that bind to the PF3D7_20109-F21 protein in Plasmodium falciparum using small-molecule microarray


Image generated using BioRender.



Lab links: day by day

M2D1: Review small molecule microarray (SMM) experiment and results
M2D2: Complete in silico cloning of protein expression plasmid
M2D3: Perform protein purification protocol
M2D4: Assess purity and concentration of purified protein
M2D5: Perform secondary assay to test putative small molecule binders
M2D6: Complete data analysis for secondary assay
M2D7: Plot and interpret data from secondary assay

Major assignments

Journal club 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.

The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nature Protocols. (2007) Vol.2. No.6:1528-1535.


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