Unique Polymer Technology (UPT™)

Unique Polymer Technology (UPT™) is Shantani’s proprietary target capture technology that helps identify the protein targets of a small molecule without any derivatization. UPT is especially advantageous in cases where: 

    • SAR information is not available.
    • Bait-molecule is a macrocyclic or a natural product and is difficult to derivatize
    • Comparative Off-Target profiling of established and follow-on compounds is required
    • Rapid Target Screening is required to identify bait-molecule specific class of target proteins

Working Principle

This affinity interaction based target identification methodology relies on a validated hypothesis that weak-molecular interaction forces of a drug-like molecule can be utilized in preparing their affinity matrix, without any derivatization of the compound. Though these complementary affinity interactions are weak in nature, the sum of multiple weak interactions will allow the molecule to stay on a surface for a time that is sufficient for it to interact and bind with its affinity protein partners in a biological sample. Thus, the prepared affinity matrix can be used to enrich the target protein from biological sources.

Workflow

In UPT, the ‘bait-molecule’, without any chemical derivatization, is immobilized on proprietary polymers of Shantani. Shantani’s unique polymeric surface allows the formation of complementary weak interaction bonds with the ‘bait-molecule’ for its transient immobilization. The prepared ‘bait-molecule’ specific affinity matrix is then exposed to relevant biological source, typically cell-lysates, for affinity capture of target proteins. After appropriate incubation, the polymer matrix is washed, and matrix-bound proteins are isolated by dissolving the polymeric matrix in organic solvents. These proteins are then identified using mass-spectrometry. Specific target proteins are then de-convoluted by comparing the protein profile obtained from bait-molecule specific affinity matrix and multiple control experiments.

Key Advantages

  • Quick Target Profiling allowing a ‘Go/No Go’ development decision for phenotypically screened compounds.
  • Rapid target profiling of multiple compounds that saving time and cost
  • Low false positive rate that allows identification of most probable targets(s)

References

  Technical Note:     This note explains various steps involved in de-convoluting the right target(s) of at small molecule using Shantani’s UPT workflow.
  (PDF, 103KB)
 
  Application Note:   This note describes the application of UPT in identifying/de-convoluting true positive targets of a non-derivatized ‘test’ molecule BIS-III.

  (PDF, 193KB)

Frequently Asked Questions (FAQ’s)

In a systematic pattern, several unique chemical groups that can provide a probability of pi-pi, cation-pi, hydrogen bonding and/or ionic interactions with drug-like molecules were incorporated in the design of the proprietary polymer. Drug-like molecules in one or other orientation forms at least more than one kind of weak-interaction with a specific chemical groups on the polymer and thus get transiently immobilized.

In routine work-flow, after the molecules are immobilized on the polymer, extensive washing of polymer is carried out and small-molecules in the washes are quantified using LC-UV / LC-MS based method. The amount of molecule present in the washes in retrospect allows quantification of the molecule left bound to the polymeric surface. In typical experiment,1-4 micromoles of the molecule on an 8 X 8 cm polymeric surface stays for 30-60 minutes under constant orbital shaking at 20 RPM. 

Yes. Based on solubility of the bait-molecule upto eight different variations of polymeric surfaces are tested, with a final goal that 1-4 micromoles of the bait-molecule should remain retained on an 8 X 8 cm polymeric surface after 6 consecutive aqueous washing. 

Typically, following properties of the compound influences its immobilization on the polymer surface (a) hydrophobic/hydrophilic nature (logP), (b) ionic bond formation capabilities and (c) molecular 3-D structure. Every molecule is unique and characterization of their retention behavior on the polymer is the only way forward. Hence, before starting any target capture experiment, retention of the molecule on the polymer is characterized. So practically, any given molecule can be used for target capture experiment as long as they can be retained on the polymer for appropriate time.  

Typically, 12-15 mg of the test molecule and 15-20 mg of proteins/cell lysate is sufficient to carry out one set of the target capture experiments. 

Agilent 6540 UHD Accurate Mass Q-TOF is employed for the purpose of protein identification. In typical workflow using ProtID CHIP 150 II and Agilent CHIP cube interface, tryptic peptides are gradually eluted and ionized. Ionized peptides are then directly infused into the mass-spec for MS and MS/MS data acquisition. Peptide sequences are established through MS and MS/MS signatures and identified peptides are then searched against redundant protein databases to link it to a protein. 

Typically. experiments are carried in duplicate or triplicate along with the control experiments (using polymeric surface where molecules are not immobilized). Proteins identified from the bait-molecule experiments are compared with the protein identified from the controls. Proteins that are (a) exclusively identified in all the triplicate bait-molecule runs and/or (b) identified with significantly greater number of peptides/higher signal intensity compared to the control runs, are considered as deconvoluted targets.