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Process Diagram

krishna Mohan

Created on April 15, 2024

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Transcript

Levels

HT -SPR

Binding at 2 different concentrations

Binding and dose response behavior

1- A

HT Screen

ADP Glow assay

4-5 weeks

Inhibition at 3 differen concentrations

TR-FRET

Inhibition at 3 differen concentrations

TSA

SPR-KD determination

1-B

Binding at 10 different concentrations

IC50 /KD Determination

steady-state curve fitting

3 weeks

Biochemical IC50

Inhibition at 10 differen concentrations

Cell based assays

Cell-based assays

Binding at 10 different concentrations

control assays with IL-3 to check toxicity

Kinase specificty

Kinase specificity

NMR-binding site confirmation

Structural Charecterization

crystallography

co-crystallizing top-hits

Enzyme Kinetics

Determining Mechanism of action andKinetic Parameters

HitCharecterization

Further Cellular assays

Cellular phosphorylation assaysnano-BRET assays

CEST experiments gfor stabilizers

5-A

Develop SAR, screen analogs

Lead optimization

Next

TR-FRET

Krishna Time taken per assay: plating- planning and Echo- Half day Assay- half day Analysis- 3 hours

  • Capacity : 200 compounds in two weeks
  • Capacity needed : 200 compounds per week
  • Possible solutions : accelarating protein production, Decan T300

Developing SAR and lead optimization

Krishna- depend on only one kind of assay for screening. - Charecterize on other assays

  • Capacity :
  • Capacity needed : 3-5 lead series per month
  • Possible solutions :
  • dedicated FTE for SAR development/ Assays

CEST experiments to further charecterize the Stabilizers

Scarlett- Planning, set-up, analysis

  • Capacity :
  • Capacity needed : Unknown
  • Possible solutions :

Cell based assay- BAF3 cells

Inhibition checked at 10 concentrations to derive EC50
assays run in the presence of IL3 to check for toxicity

Jeremy- Planning and supervision Krishna- Planning, co-ordination, protocols , analysis Somu- running the assays Anastasiia- culturing the cells

  • Capacity : 20 compounds in two weeks
  • Capacity needed : 50 compounds per week
  • Possible solutions :
  • dedicated FTE for cell-based assays for ligand discovery

Crystallization

Sparse-screening for co-crystallization
assays run in the presence of IL3 to check for toxicity

Darcie Miller - Supervision/ expert inputs, synchrotron Krishna- Planning, co-ordination, data processing Prasanna- Setting up plates

  • Capacity : 10 compounds in two months
  • Capacity needed : 20 compounds per month
  • two Kinases per month
  • Possible solutions :
  • dedicated FTE for crystallization set-up.

Kinase assay-Kinetic

Inhibition checked at 10 concentrations to derive IC50

Krishna Time taken per assay: plating- planning and Echo- Half day Assay- half day Analysis- 3 hours

  • Capacity : 50 compounds in two weeks
  • Capacity needed : 50 compounds per week
  • Possible solutions : Switching to AssayQuant will make the assay much easier.
  • An additional person to help with the assay going forward for the other Kinases - Prasanna?

ADP glow assay

Inhibition checked at 3 concentrations

Krishna Time taken per assay: plating- planning and Echo- Half day Assay- half day Analysis- 3 hours

  • Capacity : 200 compounds in two weeks
  • Capacity needed : 400 compounds per week
  • Possible solutions : Switching to AssayQuant will make the assay much easier.
  • An additional person to help with the assay going forward for the other Kinases -

Kelly

Assay design, running the assays, analysis
  • capcity : 200 compounds per 2 weeks including preparation and analysis
  • Capacity needed : 200 compounds per week
  • Additional manpower required : someone to help with plating, DLS and biotinylation.

Krishna

Assay design, plating-echo, analysis

Preparations needed:

  • Protein production , biotinylation and Quality control - Done by Sashi
  • DLS of the compounds - Krishna does this now
  • Assay plate design and plating
  • Bench marking, running the controls, finding the right regenration conditions

NMR studies-protein detected

one point protein HSQCs (1:10)

Scarlett- Planning and running the assays Who will make the protein ? ILV samples?

  • Capacity : 10 compounds per month
  • Capacity needed :

Kelly

Assay design, running the assays, analysis
  • capcity : 200 compounds per 2 weeks including preparation and analysis
  • Capacity needed : 200 compounds per week
  • Additional manpower required : someone to help with plating, DLS and biotinylation.

Krishna

Assay design, plating-echo, analysis

Preparations needed:

  • Protein production , biotinylation and Quality control - Done by Sashi
  • DLS of the compounds - Krishna does this now
  • Assay plate design and plating
  • Bench marking, running the controls, finding the right regenration conditions

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Let the communication flow!

With Genially templates, you can include visual resources to wow your audience. You can also highlight a particular sentence or piece of information so that it sticks in your audience’s minds, or even embed external content to surprise them: Whatever you like! Do you need more reasons to create dynamic content? No problem! 90% of the information we assimilate is received through sight and, what’s more, we retain 42% more information when the content moves.

  • Generate experiences with your content.
  • It’s got the Wow effect. Very Wow.
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Enzyme Kinetics

Important to determine the Kinetic Parameters of the inhbitors
For eg. assays run in the presence of ATP to check Mechanism .

Krishna- Planning, set-up, analysis

  • Capacity : 5 compounds per month
  • Capacity needed : 20 compounds per month
  • two Kinases per month (0.33 FTE)
  • Possible solutions :
  • dedicated FTE for assays eventually- additional person for helping with assays for now.

Got an idea?

Let the communication flow!

With Genially templates, you can include visual resources to wow your audience. You can also highlight a particular sentence or piece of information so that it sticks in your audience’s minds, or even embed external content to surprise them: Whatever you like! Do you need more reasons to create dynamic content? No problem! 90% of the information we assimilate is received through sight and, what’s more, we retain 42% more information when the content moves.

  • Generate experiences with your content.
  • It’s got the Wow effect. Very Wow.
  • Make sure your audience remembers the message.

Got an idea?

Let the communication flow!

With Genially templates, you can include visual resources to wow your audience. You can also highlight a particular sentence or piece of information so that it sticks in your audience’s minds, or even embed external content to surprise them: Whatever you like! Do you need more reasons to create dynamic content? No problem! 90% of the information we assimilate is received through sight and, what’s more, we retain 42% more information when the content moves.

  • Generate experiences with your content.
  • It’s got the Wow effect. Very Wow.
  • Make sure your audience remembers the message.

Further Cellular Charecterization

Multiple assays_ Cellular phosphorylation assays, Western blots,Downstream pathway assays

Jeremy- Planning, set-up, analysis

  • Capacity : 5 compounds per month, 1 Kinase per month
  • Capacity needed : Unknown (0.5 FTE)
  • Possible solutions :
  • dedicated FTE for Cellular assays

Kelly

Assay design, running the assays, analysis
  • capcity : 20 compounds per 2 weeks including preparation and analysis
  • Capacity needed : 50 compounds per week. ( Decan T300 might increase the capcity)
  • Additional manpower required : someone to help with plating

Krishna

plating ,analysis

Preparations needed:

  • Protein production , biotinylation and Quality control - Done by Sashi
  • Assay plate design and plating
  • fitting the curves, running corss-conformational screens

Thermal Shift assay

  • Krishna
  • We will do this for all the hits with the respective constructs (WT/IS1/IS2)
  • Current Capacity: (100 compounds per 2 weeks )
  • Required Capacity : 700 -1000 compounds per month