Difference between revisions of "20.109(S23):M1D7"

Introduction

Protocols

Part 2: Complete by-eye analysis of hits identified in SMM using TDP43

Using the analysis workflow described in Part 1 and the results of a biological assay completed by 109ers in Sp21 (ask your Instructor if you are interested in the details!), five small molecule compounds were selected for your research this semester. In this exercise you will evaluate the results and structures of the small molecules, then you will select which hits you want to test using functional assays.

Evaluate raw data for hits

Visually validate the raw data images for each of the small molecule compounds in the table above using the guidelines provided in Part 1.

In your laboratory notebook, complete the following:

• For each small molecule, answer the questions below:
  • Is the z-score above the threshold value you define in Part 1?
  • Do the images captured on the 532 nm channel appear as expected? Why or why not?
  • Do the images captured on the 635 nm channel appear as expected? Why or why not?
  • Are the images for the replicates consistent?

Part 1: Identify common features in SMM hits

One method for assessing protein-small molecule binding is to visually inspect known small molecule binders for common features / structures. To do this you will carefully examine the hits and identify any common features / structures. As in the image below, it is possible that multiple features will be present within the same small molecule.

Review the hits that were identified in the SMM screen completed for TDP43-RRM12. To see the chemical structures, translate the SMILES strings using one of the methods described in the text below the table. It may be easier to copy / paste the small molecule images into a powerpoint file so you can readily see all of the structures. Also, it may be helpful to use a color-coding system (like the one in the image provided above) to highlight features / structures that are common to the hits.

These online resources may be helpful to learning more about the hits identified in the SMM:

• Cloud version of ChemDraw here.
  • Copy and paste the small molecule smiles into the work space to get a chemical structure
• Platform to transform the smiles information into a PubChem ID here.
  • Copy and paste the smiles into the input ID search to determine the ID number.
• PubChem database of chemical information here.
  • Includes small molecule molecular weight and other useful information.

In your laboratory notebook, complete the following:

• How many features did you identify that are present in two or more of the small molecules that putatively bind TDP43-RRM12? Are there more or less than you expected?
• Is there a feature present in all of the identified small molecules? What might this suggest about the binding site(s) and / or binding ability of TDP43-RRM12?
• Can you assign the identified small molecules to sub-groups based on the common features that are present?
• What might the different features represent? More specifically, consider whether each subgroup has a unique binding site on the target protein or if each subgroup represents different solutions for interacting with the same binding site.
• How might you make modifications to the small molecules / features to probe binding? As a hint, consider how different functional groups could be positioned at a given site without altering qualitative binding in the SMM assay to translate that into some testable ideas.

Reagents list

Navigation links

Next day: Organize Data summary figures and results