info@shantani.com | Tel: +91-20-64103918, +91-20-64009991


 

Sub-Cellular Location-Specific (SCLS) Target Capture Technology

SCLS is Shantani's proprietary and most comprehensive method for target identification, validation and for determination of the mechanism of action.
[High confident identification of target and its active subcellular location .]


Working Principle

This method relies on a validated hypothesis that ‘bait-molecule’ can be sent to different subcellular compartments using chemical-biological tools. A particular subcellular compartment that shows maximum activity at a given concentration of ‘bait-molecule’ can be considered as ‘target-rich’ compartment. Target capture experiments are then performed from these target rich compartments for the higher likelihood of capturing the right target of the ‘bait-molecule’.



Workflow

In SCLS, the ‘bait-molecule’ is coupled to three different peptide probes. These peptide probes allow enrichment of ‘bait-molecule’ in three different subcellular locations. Biological activity of the peptide-coupled bait molecule from these subcellular locations is recorded in functional-activity assays. A particular subcellular location that provides maximum activity is considered as target-rich location and then target capture experiments are carried out specifically from target rich subcellular fraction. Peptides coupled bait-molecule along with the target proteins is isolated from subcellular fraction using antibody against the peptide. Isolated proteins are then identified using mass-spectrometry and specific target proteins are de-convoluted by comparing the protein profile obtained from bait-molecule specific target capture and multiple control experiments.




Our peptide-based probes:
  • penetrate a wide variety of mammalian cells & then remain confined to their subcellular locations (membrane, cytoplasm and nucleus).
  • are engineered to avoid interference with the molecule-target binding mechanism.
  • are 6-10 amino acid long, and do not form secondary structures.
  • contain no known peptidase sequence and are designed to remain protected from the 'xeno-degradation' machinery of the cells.
  • are not toxic to the cells.


These peptide-based probes:

  1. First determine the subcellular location of the drug targets through corresponding functionality assays, thus reducing the complexity of the proteome to be analysed for target capture experiments, and
  2. Utilize only the functionally relevant ligand (concentration of target capturing probes) concentration in capturing the targets - to capture the target(s) that are relevant for the functions of the ligand at given concentration.


Key Advantages


Points of Consideration


References
Technical Note 1
(PDF, 232KB)
This note explains various steps involved in deconvoluting the right target(s) of small molecule using Shantani's proprietary workflow.

Application Note 1
(PDF, 893KB)
This note describes the application of the technology in identifying/deconvoluting true positive targets and in understanding the action mechanism of a 'test/bait' molecule SB202190.

Application Note 2
(PDF, 82KB)
This note describes the application of the technology in capturing membrane bound targets, given the reality that more than 60% of the marketed medicines work through target proteins that are membrane bound.

Application Note 3
(PDF, 64KB)
This note demonstrates the capability of subcellular location specific probes in identifying the location of the activity of the target protein in particular cellular compartment.


FAQs
1. Does the 'bait/test' molecule need to be derivatized? Yes, the 'bait/test' molecule does need to be derivatized in such a way that it can be coupled to sub-cellular location specific probes. A functional moiety either primary amine (-NH2), alcohol (-OH) and carboxylic acid (-COOH) at propyl position is good to go.


2. Will Shantani help in derivatizing the molecule? Shantani does have a small chemistry group that can assist in derivatizing the molecule at minimal cost to client. We have worked with many small drug-like molecules and are confident of our chemistry skills. However, in case the molecule is outside our skill-set (such as natural products with multiple functional groups, multiple chiral centres, etc.) we shall inform you accordingly.


3. Is the functional activity of the molecule preserved after derivatization? The first step in Shantani's work-flow is to run the functional activity assay for the derivatized molecule and/or for the sub-cellular location specific probe coupled molecule. If derivatization does substantially changed the functional activity of the molecule, we conclude that the project cannot proceed further using our workflow. In such (unlikely) cases we will inform you and suggest alternative solutions.


4. What if the Structure-Activity-Relationship (SAR) information of the 'bait/test' molecule is not available? In cases where the SAR is not available, Shantani (or you) will prepare 3 different derivatives of the molecules where the functional group required for probe coupling is placed at 3 different locations of the molecule. These derivatized molecules are then tested (before probe coupling) for their functions. The particular derivative that shows minimal loss in the specific activity compare to the primary molecule is then utilized for coupling to the probes.


5. The assays where our molecules were screened are complicated and proprietary. Can we run the functional activity assays on Shantani's target capturing probes? Absolutely. We can couple our sub-cellular location specific probes to derivatized molecules and send them back to your labs with specific protocols. Probes can then be tested in your lab and later you can inform Shantani as to which probe shows the most activity. Eventually, Shantani can perform the pull-down experiments and identify the targets.


6. Has Shantani tested sub-cellular location specific peptides for their sub-cellular location specificity? After coupling the 'bait/test' molecule, will peptides still be able to maintain their location specificity? The picture below provides a typical scenario of sub-cellular location specificity of the probes. The probes were coupled to FITC (fluorescein) to visualize and confirm their location specificity.



The above experiment will demonstrate whether - after coupling - the 'bait/test' molecule peptide will still be location specific. FITC (fluorescein) is a small molecule and after its coupling to the peptides, the construct maintains its location specificity.


7. Are the molecules being artificially dragged to a sub-cellular location because of the peptides? Yes they are. The functionality experiments tell us which cellular location exhibit the most activity (specifically for the molecule). If more molecules enter a specific location because they get dragged along with the peptides, we can conlude that a lower concentration of the probe (compare to an uncoupled molecule) may provide the same function/phenotype.


8. Don't such sub-cellular location specific peptides get chopped-off by cellular machinery? Also, are these peptides safe for mammalian cells? Peptide sequences are designed in a way that they don't contain any known peptidase site. To confirm, peptides were allowed to interact with the cells. Peptide recovery and LC/MS based analysis confirmed that the peptides were intact.

In cell-viability experiments we have incubated these peptides with atleast three different cell-lines for more than 48 hours; we found <5% cell-death as compared to control cells.


9. What is the specificity of the peptides with their respective antibody (ies) that are used to isolate the small-molecule-protein complexes? Currently, antibody peptide recognition Kd is in nanomolar range.






10. After identifying the active cellular location, are targets are captured from live cells or are the cells lyzed and the active proteome enriched, before capturing targets? The design of target capture experiment depends on the active cellular location. If the activities are identified in cytosolic or nuclear compartment, then direct live cell capture is possible. However if we conclude that it is the membrane fraction where most activity is located, then first we prepare the membrane fractions and then perform the target capture experiments.


11. Are the proteins captured on peptide antibody directly analysed using the mass-spec, or is a secondary enrichment step, such as elution, involved? This depends on the type of cellular fractions that we are working with. If it is cytosolic or nuclear fraction we elute the target protein from the peptide antibody column using ~ 1 mM concentration of free molecule in elution buffer. If we are working with membrane fractions we analyse them directly.