WO2017118997A2

WO2017118997A2 - Methods for determining protein targets of a ligand without ligand derivatization

Info

Publication number: WO2017118997A2
Application number: PCT/IN2017/000002
Authority: WO
Inventors: Chaitanya SAXENA
Original assignee: Saxena Chaitanya
Priority date: 2016-01-08
Filing date: 2017-01-04
Publication date: 2017-07-13
Also published as: WO2017118997A3

Abstract

The present invention concerns a method of determining whether a non -purified sample contains a cellular binding partner, the target protein, of a ligand of interest comprising the steps of (a) preparing a 'ligand specific affinity matrix' by immobilizing the ligand using its inherent intermolecuiar forces for non-covalent binding on a surface that provides possibilities of forming complementary non-covalent bonds with the ligand, (b) confirming immobilization of ligand on surface offering complementary non- covalent interactions by measuring amount of ligand present in wash solvent used for surface washing; and (c) analyzing biological samples using ligand specific affinity matrix.

Description

Title:

Method! for Determining Protein Targets of a Ligand Without Ligand Derivatization Field of Invention:

The present invention relates to a method for identification of cellular binding partners, the targets, of bioactive ligand using a unique polymer method. The invention specifically relates to use of nitrocellulose based polymer in preparing the ligand specific affinity matrix without modifying the ligand structure or derivatizing the ligand. Further, it relates to quantifying the amount of ligand on the prepared affinity matrix. Furthermore, it relates to using the ligand specific affinity matrix in determining the protein target(s) of the ligand in biological systems. In certain embodiments, the methods of the invention use a mixture of solvents of varied composition to immobilize ligand on the polymer. In other embodiments, the methods of the invention use a single solvent in varying conditions to immobilize ligand on the polymer. In certain embodiments, the methods of the invention use absorption or emission spectroscopy or mass-spectrometry to quantify the amount of ligand in polymer wash, to inversely correlate, the amount of ligand present on the polymer. In certain embodiments, the methods of the invention use western blotting or mass-spectrometry based method to determine the presence of protein targets on the polymer. In other embodiments, the methods of the invention use polymer that are not quoted with ligand to confirm the specificity of determination of protein targets in biological systems.

Background of Invention(s):

In the prior art, Patent Application no. WO2006134056A1, titled, 'Process for the identification of novel enzyme interacting compounds' describes method of characterization of protein interacting compound by using subtraction approach. In this approach, one aliquot of biological-lysate is contacted with broad spectrum enzyme- ligands immobilized on a solid support. It allows the binding of the enzyme(s) to broad- spectrum enzyme-ligand. Then all the enzymes captured on solid support are characterized by mass spectrometry. Another aliquot, having biological-lysate along with the excess concentration of unbound compound, is contacted with the broad- spectrum enzyme-ligands. Then enzymes are characterized by mass spectrometry. Enzyme characterized from both the aliquots are compared. At the end, detection of lesser amount of an enzyme, in the aliquot incubated with the excess concentration of unbound compound (second aliquot), indicates that it could be the direct protein binding partner of the compound. The primary disadvantage of this approach is that, for target capture multiple other ligands needs to be derivatized and immobilized on a solid support. Also, this application mainly concentrates on a class of enzyme and are not pan-proteome in approach.

In the prior art, US Patent number 20150133336, describes methods for determining ligand binding to a target protein using thermal shift assay. In this method, the ligand is not derivatized for determining the ligand-target interaction. In this two-step method, First, ligands incubated with non-purified biological samples are exposed to temperature that is capable of relatively enhancing precipitation of the proteins that are not bound to the ligand. In second step, said sample is analyzed for the presence of target protein using two or more affinity reagents. This method offers an approach where Ligands without modification can be used for determining the ligand-protein interactions. However, the use of the method for identifying the target of the ligand protein is limited by the fact that, it largely is successfully in identifying only soluble proteins. Also, because the method does not rely on enriching the target protein from the biological samples, low-abundant target proteins may remain undetected by this method.

In the prior art, US Patent Application Number US12621290, describes ligand binding stabilization method for drug target identification. Like thermal-shift assay, in this method also the ligand is not derivatized for determining the ligand-target interaction. The method involves contacting a sample comprising mixture of target proteins with the ligand to form a sample/ligand mixture; then contacting the mixture with a protease; and identifying a protein that is protected from protease because of ligand induced stability. This method is limited by the fundamental aspect that extent of proteolysis achieved by a specific protease is a multifactorial event and every protein may not get cleaved equally by a single protease (Saxena C. Identification of protein binding partners of small molecules using label-free methods. Expert Opin Drug Discovery 2016;11:1017- 1025.)

Another method, where the ligand is not derivatized for determining the ligand-target interaction was presented by West et al. (West GM, Tang L, Fitzgerald MC. Thermodynamic analysis of protein stability and ligand binding using a chemical modification- and mass spectrometry-based strategy. Anal Chem 2008;80:4175-85). Much like thermal shift assay, this method relies on principle that interaction of ligand with its target protein may alter thermodynamic stability of the target protein. In this method, stability of proteins from rate of oxidation was used to access the protein stability and to confirm the interaction of ligand with the target protein. One of the major limitations of this method is that it utilizes methionine oxidation as a parameter to measure the thermodynamic property and all methionine residues may not necessarily exhibit differential oxidation rates that can provide sufficient information to confirm the interaction of ligand with the target (Lomenick B, Olsen RW, Huang J. Identification of direct protein targets of small molecules. ACS Chem Biol 2011;6:34-46).

Most of the methods described above utilizes biophysical properties of the protein target in determining the ligand-target interaction. In all those cases, where said biophysical property of the protein target may not change in presence of ligand or the changes cannot be recorded due to very low concentration of the protein target in the biological system may fail to determine the protein target(s) of ligand. A method that offers advantage of underivatized ligand and yet can provide significant enrichment of the protein target(s) for its determination can circumvent most of the shortcomings of currently available method.

Object of invention:

Object of invention is to provide a method for preparing ligand specific affinity matrix, without derivatization of the ligand, and using thus prepared affinity matrix for determining the presence of protein targets of ligand in biological samples. Summary of invention:

The method for determination and identification of protein target of at least one ligand or a group of ligands, comprising the steps of, a) Immobilizing the ligand without any structural modifications on an invented nitrocellulose based polymeric matrix through non-covalent bonds,

b) Confirming immobilization of ligand through its retention pattern on the polymeric matrix and thus preparing a ligand specific affinity matrix for target capture from biological lysates,

c) Incubating biological lysate of interest with ligand immobilized polymeric matrix for a finite time,

d) Washing of polymeric matrix with appropriate solvent,

e) Determining, analyzing and identifying the target protein and protein complexes bound to polymeric matrix by mass spectrometry and appropriate biophysical and biochemical methods,

f) Deconvoluting the specific targets of ligand by comparing the protein identified from polymeric matrix without ligand immobilization and incubated with biological sample

g) Further, deconvoluting the specific targets of ligand by comparing the protein identified from polymeric matrix where ligand is immobilized but biological sample is pre-incubated with the ligand.

Brief Description of Drawing

FIG. 1- describes the design of a polymer, the key component of the method, that allows formation of complementary ηοη-covalent bonds with the ligand to prepare a 'ligand specific affinity matrix' FIG. 2- shows a picture where polymer was layered inside the plastic tube and a colored ligand, Bisindc!ylmaletmide-lll, was immobilized; in control experiment the ligand was not immobilized

FIG. 3- shows retention pattern of a ligand Bisindolylmaleimide-lll on the unique polymeric surface

FIG. 4- shows detection of target protein of Bisindolylmaleimide-lll using western- blotting method

FIG. 5- shows retention pattern of many ligands on unique polymeric surface

FIG. 6- shows retention pattern of ligands of comparable hydrophobicity

FIG. 7- shows retention pattern of a single ligand in different wash conditions

Working of Invention and Detailed Description of Drawing through Examples

The present invention is related to preparation of a ligand specific affinity matrix without derivatization of the ligand using unique polymer methodology that allows immobilization of ligand on the polymeric matrix through non-covalent interactions and then using thus prepared ligand specific affinity matrix for analyzing, determining and identifying the protein targets of ligand or ligands from biological samples.

To immobilize the ligand without any derivatization 'structural features' of the ligand are of paramount importance. Typically, structural features of any ligand allow it to make non-covalent, intermolecular bonds, that include but not limited to pi-pi, cation- pi, hydrogen, van der waals, ionic, hydrophobic, with any other neighboring matter. To immobilize the ligand without derivatization a unique polymeric surface having properties of making complementary non-covalent interactions with the ligand was invented. This polymeric surface utilizes nitrocellulose as a base and then different chemicals and solvent systems are utilized in the base matrix to prepare different matrices that provides complementary non-covalent interaction sites for immobilization of different ligand.

FIG. 1 shows the structural components of the invented polymeric matrix having chemical groups that allows non-covalent interactions with the molecule (1). The design of polymer also includes an amphophilic bottom arm (2) that makes polymer compatible for the interaction studies. Ligands are layered on the polymer and non-covalent interactions of ligand with that of polymer (3) allows the ligand to remain bound to the polymer in random orientation through multiple weak interactions and thus a 'ligand specific affinity matrix' is prepared (4).

The invention will now be described in details, in the following non-limiting examples: EXAMPLE 1

Immobilization of Ligand without Ligand Derivatization

Bisindolylmaleimide-lll (Bis-Ill) is a small-molecule ligand of Glycogen Synthase Kinase 3 - beta (GSK3-beta) protein. Bis-Ill is a colored compound and its retention on the polymer can be easily visualized with the naked eye. Invented polymer was dissolved in a volatile organic solvent and layered on a small tube. Solvent from the tubes was evaporated using vacuum drying. A 250 micromolar concentration of Bis-Ill was prepared in Tris- buffered Saline. One milliliter of this solution was placed in Tube-A. For control studies, one milliliter of 250 micromolar Bis-Ill was also placed in Tube-B were polymer was not layered. Tubes were placed on a rotary shaker for 6 hours to allow the formation of weak interaction bonds between Bis-Ill and polymeric surface. After 6 hours, the solutions from both the tubes were removed and both the tubes were washed 3 times with one milliliter of Tris-Buffered Saline. At the end of the washing, Bis-Ill was observed layered on the wall of the Tube-A where polymer was layered but not in the control tubes (FIG 2). This confirmed that polymeric surface can retain small-molecule ligand on its surface using weak intermolecu!ar interactions.

EXAMPLE 2

Quantification of Ligand Immobiiization on Polymeric Surface by Profiling Ligand's Retention Pattern

Immobilization or tightness and amount of binding of the ligand with the polymer is characterized by ligand's 'retention pattern' on the polymeric matrix. To establish retention pattern, polymeric matrix after ligand immobilization is washed several times with appropriate buffer and the amount of ligand in multiple washes is measured by appropriate Liquid Chromatography-Mass Spectrometry (LC- S) or Liquid Chromatography-Ultra Violet Absorption/Fluorescence Spectroscopy (LGUV/Flu) or equivalent method. In one of the examples, Bisindolylmaleimide-Ill (Bis-Ill), a small- molecule ligand of Glycogen Synthase Kinase 3 -beta (GSK3-beta) protein, was immobilized on the polymeric surface (Example 1). After immobilization, the polymeric surface was washed for six times over the period of two hours. Amount of Bis-Ill in the washes was measured using UV-visible absorption of Bis-Ill and subtracted from the total amount of Bis-Ill loaded on the matrix. FIG 3 shows typical retention pattern of Bis- Ill on the polymeric surface. Even after extensive washing of the polymer, > 80% of the Bis-Ill that was used for immobilization was still found to be present on the polymeric surface. This confirms that polymeric surface can retain small-molecule ligand for substantially long period and amount of ligand on the polymeric surface can be quantified. Long retention time and known amount of ligand on the polymeric surface can be used for carrying out ligand specific affinity chromatography for determining target binding.

EXAMPLE 3 Determination of Target Interaction of Ligand using the Unique Polymer Technology

Once it is confirmed that substantial amount of ligand is retained on the polymeric matrix, the matrix is considered as 'qualified ligand specific affinity matrix'. Thus prepared 'ligand specific affinity matrix' can be used for analyzing, determining and identifying the protein targets of ligand or ligands from biological samples. In one of the examples of target identification, Bisindolylrnaleimide-lll (Bis-Ill), a small-molecule ligand of Glycogen Synthase Kinase 3 -beta (GSK3-beta) protein, was used for preparing the Bis-Ill specific affinity matrix (Example 1 and 2) on an 8x8 cm² polymeric surface. Affinity matrix was washed with Tris-buffered-saline to remove the unbound Bis-Ill molecules from the surface. Retention pattern confirmed that > 2 micromoles of Bis-Ill was present on the affinity matrix, Bis-Ill matrix was incubated with He-La cell lysate (and can be incubated with any other biological lysates) having protein concentration of 1 milligram per milliliter. Based on the retention pattern of Bis-Ill (FIG 3), incubation was carried for 5 minutes. In this time, GSK3-beta, target protein of Bis-Ill, should interact with the Bis-Ill immobilized on the matrix and will be enriched on the polymeric surface. After the incubation, the cell-lysate was removed from the affinity matrix. Further, the affinity matrix, the polymeric surface, was washed one time with 1 ml of Tris-buffered Saline. The enriched cellular proteome was then eluted-off the polymeric surface through competitive elution using 1 ml of buffer that contained 1 millimolar of Bis-Ill in the elution solution. Eluted proteins were analyzed through western-blotting method, using anti-GSK-3-beta antibody, to confirm the presence of the Target protein. For control experiments, polymeric surface, where Bis-Ill was not immobilized, was treated in the same fashion. FIG 4 showing the western-blot image confirms that Target protein was specifically identified in the experiment where Bis-Ill was immobilized on the polymer surface and not on control polymer surface. This validates the hypothesis and the experiment workflow that a ligand, without any derivatization, can be immobilized on polymeric surface through non-covalent interactions and thus prepared ligand specific affinity matrix can be used for determining the protein target of the ligand.

EXAMPLE 4

Determination of Off-Target Interaction of Ligand using the Unique Polymer Technology

Efficacy and toxicity induced by a ligand is a manifestation of its interactions with different target proteins present in the biological system. A ligand's beneficial potential and its selectivity towards a specific protein target can be established by identifying its 'on and off-targets'. Following example demonstrate that ligand immobilized on polymeric surface can be effectively used in identifying 'on and off-target' of a ligand. Experiments, as described in Example 3, were carried out and towards the end, all the proteins that were eluted from the Bis-Ill polymeric surface were identified using mass- spectrometry based work flow. Proteins eluted from control experiment were also identified and compared with the proteins eluted from Bis-Ill polymeric surface. Along with GSK3-beta, the on-target of compound Bisindolylmaleimide-lll, its well-established off-targets such as Mitogen Activated Protein Kinase 1, Calcium/calmodulin-dependent protein Kinase type II were also identified. Table 1 provides the list of Protein Kinases, along with number of unique peptides, that were identified from mass-spectrometry.

Table 1- List of on and off targets (proteins kinases) of Ligand Bisindolylmaleimide-lll identified using described technology workflow

This example confirms that the method can be used for (a) characterizing selectivity of a ligand against several targets present in any biological lysates (b) profiling toxicity of a ligand by identifying off-targets, (c) establishing Mechanism of Action of Ligand by identifying On and Off-targets and (d) stratifying patients during clinical trials by identifying presence and/or absence of On/Off Targets, if the target profiling is carried out in biological lysate obtained from clinical samples.

EXAMPLE 5

Determination of Target Protein Complexes Components of Ligand using the Unique Polymer Technology

In establishing beneficial effects of a ligand, clear understanding of ligand's mechanism of action (MOA) is critical. To establish MOA of a ligand, identity of interacting primary target protein(s) and other proteins that primary target associates with, is very important. In one of the examples, experiments as described in Example 3 and 4 were carried out and S-phase kinase-associated protein was identified as a part of the protein complex. This example further confirms that the method can be used for establishing Mechanism of Action of Ligand by identifying On and Off-targets.

EXAMPLE 6

Retention pattern of Many Different Ligands on Polymeric Surface

The versatility of the unique polymeric surface in immobilizing ligands of varied type was demonstrated by following experiment. Here, many different ligands, such as Dopamine, Bis-lli, Histamine, BL11282, Curcumin, Penicillin and Probenecid, that may provide different intermolecular interactions for complementary binding to polymer, were immobilized without derivatization, through non-covalent interactions, on the polymeric surface. After immobilization, polymeric surface was washed extensively, amount of ligand in washes was determined using HPLC-UV and HPLC- S based methods and retention pattern of the ligand on the polymeric surface was characterized. FIG 5 shows the retention pattern of Dopamine, Bis-Ill, Histamine, BL11282, Curcumin, Penicillin and Probenecid. This example confirms that most of the ligands were well retained on the polymeric surface, somewhere moderately retained, and some (Probenecid) were not retained on the polymer. Considering the case of Probenecid which was not retained on the polymer, this example also demonstrates that if the ligand cannot be retained on the polymeric surface, target identification efforts using this technology can be stopped very early in the project. This decision making can save substantial time and resources in the process. This example confirms that ligands of different variety can be immobilized on the invented polymeric surface system and amount of immobilization can be quantified using retention pattern by measuring the amount of ligand in polymer washes.

EXAMPLE 7

Retention pattern of Many Different Ligands on Polymeric Surface

In determining the targets of a ligand using affinity chromatography, it is essential that surface of the ligand (the action/business site) that may interact with the target protein is accessible for mutual interaction. In case of presented method, it is possible that, to retain the ligand, polymeric surface may use that specific site/surface of ligand which is important for ligand's interaction with the target and may alter the target capture efficiency of the set-up. Following example demonstrate that polymeric surface does 'not' use a specific site/surface of a ligand for immobilization and ligand is 'randomly oriented' on the polymeric surface. Consider, for example, that it's the hydrophobic forces/surface of a ligand that is playing a critical role in immobilization, then ligands of similar hydrophobicity should be retained similarly on the polymeric surface. In the experiment, three ligands of similar hydrophobicity, in individual experiments were immobilized on the polymeric surface and their retention pattern was characterized as detailed in Example 2. Though of similar hydrophobicity, retention pattern of these ligands was significantly different (FIG 6). This observation can be explained with a rational, that it's not only hydrophobic interaction, but more than one type of interaction is retaining the ligands on the polymer. This scenario can only exist when the ligands are oriented in differential, that is, in 'random fashion' on the polymer. Rational extrapolation of the experimental evidences provided in this example confirms that ligands are randomly oriented on the polymer. The random orientation of the molecule provides a possibility that it will be able to interact with its target protein in at least a specific orientation. This example confirms that ligands are randomly oriented on the polymeric surface and method can determine the target interactions.

EXAMPLE 8

Retention pattern of Same Ligands on PoSymeric Surface in different wash conditions

To establish that ligand indeed is randomly oriented on the polymeric surface, in continuation with Example 7, the process of ligand immobilization was subjected to different immobilization and washing conditions. Consider, for example, that it's the ionic forces of a ligand that is playing a critical role in its immobilization on polymeric surface, then conditions, offering differential charges to the ligand should alter its retention pattern. In the experiment, a ligand having pKa of 7, was immobilized on the polymeric surface, in individual experiments, at pH 7 and pH 10. Later, to establish the retention of the ligand on the polymer, washing was carried either at pH 7 or at pH 10. FIG 7 shows retention profile of the ligand in different scenario and confirms that change in ionic conditions did not alter the retention profile of the same ligand. This observation can be explained with a rational, that it's not only ionic interaction, but more than one type of interaction is involved in retaining the ligands on the polymer. This scenario can only exist when the ligands are oriented i differential, that is, in 'random fashion' on the polymer. Rational extrapolation of the experimental evidences provided in this example confirms that ligands are randomly oriented on the polymer. The random orientation of the molecule provides a clear possibility that it will be able to interact with its target protein in at least a specific orientation. This example further confirms that ligands are randomly oriented on the polymeric surface and method can determine the target interactions.

In conclusion, Example 1 and 6 demonstrated that invented polymeric surface system can immobilize variety of ligand without the need of any derivatization. Example 2 and 6 demonstrated that immobilization of ligands can be confirmed and quantified using retention profile of ligands. Further, Example 2, 6, 7 and 8 together demonstrated that ligands are randomly oriented and well-retained on the polymeric surface and can be used for determining their target form biological samples. Furthermore, Example 3, 4 and 5 demonstrated that method can identify targets, profile selectivity and toxicity and clarify mechanism of action of a ligand. These examples also suggest that information of on and off targets and target associated help in understanding Mechanism of Action and stratifying patients during clinical trials.

Claims

1. A method of determining whether a non-purified sample contains a cellular binding partner, the target protein, of a ligand of interest comprising the steps of (a) preparing a 'ligand specific affinity matrix' by immobilizing the ligand, without its derivatization, using its inherent intermolecular forces for non-covalent binding on a surface that provides possibilities of forming complementary non-covalent bonds with the ligand, (b) confirming immobilization of ligand on surface offering complementary non-covalent interactions by measuring amount of ligand present in wash solvent used for surface washing; and (c) analyzing biological samples using ligand specific affinity matrix,

2. The method of claim 1 wherein surface means any chemical, physical, elemental or biological surface that is capable of forming complementary non-covalent bonds with the ligand,

3. The method of claim 1 wherein ligand means a bioactive compound of any size that interacts with one or more cellular proteins to exhibit its activity,

4. The method of claim 1 wherein amount of ligand immobilized on the surface is confirmed through measuring amount of ligand, in wash solvent used for washing the surface, by any ligand quantification method,

5. The method of claim 1 wherein biological samples means lysates or live samples prepared from biological sources,

6. The method of claim 5 where in biological samples are pre-incubated with the ligand,

7. The method of claim 1 wherein analysis is performed using specifically mass- spectrometry or western-blot analysis or equivalent biophysical, biochemical method.