Modulator assay

Patent metadata

Field	Value
Publication number	US-2018172702-A1
Application number	US-201515736520-A
Country	US
Kind code	A1
Filing date	Oct 22, 2015
Priority date	Jun 18, 2015
Publication date	Jun 21, 2018
Grant date	—

Abstract

It has been demonstrated that certain compounds bind to TNF and stabilise a conformation of trimeric TNF that binds to the TNF receptor. Accordingly, these compounds can be used as modulators of TNF. A new assay for identifying compounds with this mechanism of action is also disclosed.

First claim

1 . A method for identifying a compound that is capable of binding to a trimeric protein that is a tumor necrosis factor (TNF) superfamily member, whereby the compound-trimer complex binds to the requisite TNF superfamily receptor and modulates the signalling of the receptor, comprising:
a) identifying the binding of the compound to the trimeric form of the TNF superfamily member in a sample; and/or b) measuring the stability of the trimeric form of the TNF superfamily member in a sample comprising the compound; and/or c) measuring the level of trimeric TNF superfamily member bound to the requisite receptor in a sample comprising the compound; and/or d) measuring the competition of the compound with a probe compound for binding to the trimeric form of the TNF superfamily member;
and comparing the binding of the compound to the trimeric form of the TNF superfamily member in (a), and/or the stability of the trimeric form of the TNF superfamily member in (b), and/or the level of trimeric TNF superfamily member bound to the requisite receptor in (c), and/or the level of competition observed in (d) to corresponding values from control samples and selecting a compound that is capable of binding to a trimeric protein that is a TNF superfamily member, whereby the compound-trimer complex binds to the requisite TNF superfamily receptor and modulates the signalling of the receptor.

2 . The method of claim 1 , which comprises:
a) conducting a mass spectrometric analysis on a sample containing the TNF superfamily member and the compound to detect the amount of the TNF superfamily member trimer; b) comparing the amount of TNF superfamily member trimer in the sample with a control sample; and c) selecting a compound that is capable of binding to a trimeric protein that is a TNF superfamily member, whereby the compound-trimer complex binds to the requisite TNF superfamily receptor and modulates the signalling of the receptor.

3 . The method of claim 1 , which comprises:
a) performing a receptor-ligand binding assay in which a sample of TNF superfamily member and the compound is applied to the requisite TNF receptor that has been bound to a surface; b) comparing the amount of TNF superfamily member trimer bound to the requisite TNF receptor with a control sample; and c) selecting a compound that is capable of binding to a trimeric protein that is a TNF superfamily member, whereby the compound-trimer complex binds to the requisite TNF superfamily receptor and modulates the signalling of the receptor.

4 . The method of claim 1 , which comprises:
a) performing an assay to determine the thermal transition midpoint (Tm) of the trimeric form of the TNF superfamily member in a sample of the TNF superfamily member and the compound; b) comparing the Tm of the trimeric form of the TNF superfamily member in the sample with a control sample; and c) selecting a compound that is capable of binding to a trimeric protein that is a TNF superfamily member, whereby the compound-trimer complex binds to the requisite TNF superfamily receptor and modulates the signalling of the receptor.

5 . The method of claim 1 , which comprises:
a) performing a fluorescence polarization assay using the compound and a probe compound; b) comparing the degree of polarization of the probe compound in the presence of the compound with the degree of polarization in a control sample; and c) selecting a compound that is capable of binding to a trimeric protein that is a TNF superfamily member, whereby the compound-trimer complex binds to the requisite TNF superfamily receptor and modulates the signalling of the receptor.

6 . The method of claim 1 , wherein the sample containing the TNF superfamily member and the compound further comprises a destabilising agent.

7 . The method of claim 6 , wherein the destabilising agent is dimethyl sulfoxide (DMSO).

8 . The method of claim 1 , which comprises:
a) performing an isothermal calorimetric analysis to measure the binding of the TNF superfamily member for the requisite receptor in the presence of the compound; b) comparing the binding of the TNF superfamily member for the requisite receptor with a control sample; and c) selecting a compound that is capable of binding to a trimeric protein that is a TNF superfamily member, whereby the compound-trimer complex binds to the requisite TNF superfamily receptor and modulates the signalling of the receptor.

9 . The method of claim 1 , wherein step (c) comprises measuring the binding affinity of trimeric TNF superfamily member to the requisite receptor in a sample comprising the compound.

10 . The method of claim 1 or 9 , which comprises:
a) performing an assay to determine the binding affinity for a superfamily receptor (KD-r) of the trimeric form of the TNF superfamily member in a sample of the TNF superfamily member and the compound;
b) comparing the KD-r of the trimeric form of the TNF superfamily member in the sample with a control sample; and
c) selecting a compound that is capable of binding to a trimeric protein that is a TNF superfamily member, whereby the compound-trimer complex binds to the requisite TNF superfamily receptor and modulates the signalling of the receptor.

11 . The method of claim 1 , wherein the compound increases the stability of the trimeric form of the TNF superfamily member compared to the stability of the trimeric form of the TNF superfamily member in the absence of the compound.

12 . The method of claim 11 , wherein the increase in stability results in an increase in the thermal transition midpoint (Tm) of the trimeric form of the TNF superfamily member of at least 1° C.

13 - 14 . (canceled)

15 . The method of claim 1 , wherein the compound increases the binding affinity of the TNF superfamily member to the requisite receptor compared to the binding affinity of the TNF superfamily member to its receptor in the absence of the compound.

16 . The method of claim 15 , wherein the compound increases the binding affinity of the TNF superfamily member to the requisite receptor by increasing the on rate (kon-r) and/or decreasing the off rate (koff-r) compared to the kon-r and koff-r values for binding of the TNF superfamily member to its receptor in the absence of the compound.

17 . The method of claim 15 , wherein the compound increases the binding affinity of the TNF superfamily member to the requisite receptor by increasing the on rate (kon-r) compared to the kon-r value for binding of the TNF superfamily member to its receptor in the absence of the compound.

18 . The method of claim 15 , wherein the compound decreases the KD-r of the TNF superfamily member to the requisite receptor compared to the KD-r of the TNF superfamily member to its receptor in the absence of the compound, wherein:
a) the compound decreases the KD-r of the TNF superfamily member to the requisite receptor by at least 10 times compared to the KD-r of the TNF superfamily member to its receptor in the absence of the compound; b) the KD-r value of the TNF superfamily member for binding to the requisite receptor in the presence of the compound is less than 10 nM.

19 . The method of claim 15 , wherein the compound decreases the KD-r of the TNF superfamily member to the requisite receptor compared to the KD-r of the TNF superfamily member to its receptor in the absence of the compound, wherein:
a) the compound decreases the KD-r of the TNF superfamily member to the requisite receptor by at least 4 times compared to the KD-r of the TNF superfamily member to its receptor in the absence

Assignees

• Ucb Biopharma Sprl

Inventors

• O'Connell James Philip
• Porter John Robert
• Lawson Alastair
• Kroeplien Boris
• Rapecki Stephen Edward
• Norman Timothy John
• Warrellow Graham John

Classifications

• C07K2317/55

  Fab or Fab' · CPC title

• G01N33/6845 Primary

  Methods of identifying protein-protein interactions in protein mixtures · CPC title

• C07K14/70575

  NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 (NGF C07K14/48, TNF C07K14/525) · CPC title

• G01N33/6863 Primary

  Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors · CPC title

• C07K2317/92

  Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value · CPC title