Sub-Cellular Location Specific Technology (SCLS™)

Sub-Cellular Location Specific (SCLSTM) technology is Shantani’s proprietary and most comprehensive methodology for target identification, validation and for determination of the mechanism of action. SCLSTM is specially advantageous as it:

    • Determines the sub-cellular location of the drug targets through corresponding functionality assays, which reducing the complexity of the proteome to be analyzed 
    • Utilizes only the functionally relevant ligand concentration to capture the target(s). 
    • Generate target information with highest confidence.

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’.

Figure: Schematic representation of SCLS Peptides


In SCLS, the ‘bait-molecule’ is coupled to three different peptide probes. These peptide probes allow enrichment of ‘bait-molecule’ in three different sub-cellular locations. The biological activity of the peptide-coupled bait molecule from these sub-cellular locations is recorded in functional activity assays. A particular sub-cellular location that provides maximum activity is considered as target-rich location and then target capture experiments are carried out specifically from target rich sub-cellular fraction. Peptides coupled bait-molecule along with the target proteins is isolated from sub-cellular 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 sub-cellular 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.


Key Advantages

  • Provides information on identity and sub-cellular location of the target
  • Can be directly linked to a specific functional assay
  • Low false positive identification rate
  • Capture of targets from live cells providing physiologically relevant target information

Points of Consideration

  • ‘Bait-Molecule’ Structure Activity Relationship (SAR) must be well defined
  • ‘Bait-Molecule’ needs to be derivatized/modified to accommodate the coupling of peptides
  • A cell-based functional activity assay should be available


Before starting each project, Shantani lays out the experiment plans and client appraisal timelines. While Shantani’s expertise in chemical proteomics helps identify the technology to use and the righful target(s), we firmly believe that the client is the one who can best use such ‘-omics’ experiments to truly understand the MoA of your molecule. Hence, we send frequent updates to our clients as targets get identified & validated.

Technical Note 1:     This note explains various steps involved in deconvoluting the right target(s) of small molecule using Shantani’s proprietary workflow.

(PDF, 232KB)

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

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

Application Note 3:  This note demonstrates the capability of sub-cellular location specific probes in identifying the location of the activity of the target protein
(PDF, 64KB)            in particular cellular compartment.
Frequently Asked Questions (FAQ’s)   



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. 

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.  

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 change 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.  

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 the minimal loss in the specific activity compared to the primary molecule is then utilized for coupling to the 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. 

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 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. 

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 conclude that a lower concentration of the probe (compare to an uncoupled molecule) may provide the same function/phenotype. 

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 at least three different cell-lines for more than 48 hours; we found <5% cell-death as compared to control cells. 

The design of target capture experiment depends on the active cellular location. If the activities are identified in the 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. 

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.