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Tunable endogenous protein degradation with heterobifunctional compounds

A functional and compound technology, applied in the direction of peptide/protein composition, genetic material composition, fusion with degradation motifs, etc., can solve problems such as infeasible regulation strategies in vivo

Pending Publication Date: 2019-12-27
DANA FARBER CANCER INST INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this system is also not feasible as an in vivo regulatory strategy due to the requirement for the presence of Sield-1 in the cytoplasm to avoid degradation

Method used

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  • Tunable endogenous protein degradation with heterobifunctional compounds
  • Tunable endogenous protein degradation with heterobifunctional compounds
  • Tunable endogenous protein degradation with heterobifunctional compounds

Examples

Experimental program
Comparison scheme
Effect test

Synthetic example 1

[1639] Synthesis Example 1: Synthesis of dBET1

[1640]

[1641] (1) Synthesis of JQ-acid

[1642] JQ1 (1.0 g, 2.19 mmol, 1 equiv) was dissolved in formic acid (11 mL, 0.2M) at room temperature and stirred for 75 hours. The mixture was concentrated under reduced pressure to give a yellow solid (0.99 g, quantitative yield), which was used without purification. 1 H NMR (400MHz, methanol-d 4 )δ7.50 –7.36(m,4H),4.59(t,J=7.1Hz,1H),3.51(d,J=7.1Hz,2H),2.70(s,3H),2.45(s,3H), 1.71 (s,3H). LCMS 401.33 (M+H).

[1643] N-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-oxoisoindoline-4 was synthesized according to a previously published method -yl)oxy)acetamide trifluoroacetate (Fischer et al., Nature 512(2014):49).

[1644] (2) Synthesis of dBET1

[1645] At room temperature, JQ-acid (11.3 mg, 0.0281 mmol, 1 eq) and N-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)- 1,3-Oxoisoindolin-4-yl)oxy)acetamide trifluoroacetate (14.5 mg, 0.0281 mmol, 1 equiv) was dissolved in DMF (0.28...

Synthetic example 2

[1646] Synthesis Example 2: Synthesis of dBET4

[1647]

[1648]At room temperature, 0.1M N-(4-aminobutyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-oxoisoindoline-4 A solution of -yl)oxy)acetamide trifluoroacetate in DMF (0.438 mL) was added to (R)-JQ-acid (prepared from (R)-JQ1 in a similar manner to JQ-acid) (14.63 mg, 0.0365mmol, 1 equiv) 0.0438mmol 1.2 equiv). DIPEA (19.1 μL, 0.1095 mmol, 3 eq) and HATU (15.3 mg, 0.0402 mmol, 1.1 eq) were added and the mixture was stirred for 24 h, then diluted with MeOH and concentrated under reduced pressure. The crude material was purified by preparative HPLC to give a yellow solid (20.64 mg, 0.0263 mmol, 72%). 1 H NMR (400MHz, methanol-d 4 )δ 7.79(dd, J=8.4,7.4Hz,1H),7.51(d,J=7.3Hz,1H),7.47–7.39 (m,5H),5.11–5.06(m,1H),4.75(s, 2H),4.68(dd,J=8.8,5.5Hz,1H),3.47–3.31(m,5H),2.83–2.65(m,7H),2.44(s,3H),2.13–2.06(m,1H) ,1.68(s,3H),1.67–1.60(m,4H). 13 C NMR (100MHz, cd 3 od)δ174.43,172.40,171.29,169.92,168.24,167.82,166.71, 156.31,153.14,1...

Synthetic example 3

[1649] Synthesis Example 3: Synthesis of dBET3

[1650]

[1651] At room temperature, 0.1M N-(2-aminoethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-oxoisoindoline-4 A solution of -yl)oxy)acetamide trifluoroacetate in DMF (0.475 mL, 0.0475 mmol, 1.2 equiv) was added to Q-acid (15.86 mg, 0.0396 mmol, 1 equiv). Then DIPEA (20.7 μL, 0.1188 mmol, 3 equiv) and HATU (16.5 mg, 0.0435 mmol, 1.1 equiv) were added and the mixture was stirred for 24 h before purification by preparative HPLC to give a yellow solid (22.14 mg, 0.0292 mmol, 74%). 1 H NMR (400MHz, methanol-d 4 )δ7.82–7.75(m,1H),7.52–7.32(m,6H),5.04(dd,J=11.6,5.5Hz,1H),4.76(d,J=3.2Hz,2H),4.66(d ,J=6.6Hz,1H),3.58–3.35(m,6H),2.78–2.58(m,6H),2.48–2.41(m,3H),2.11–2.02(m,1H),1.70(d,J = 11.8 Hz, 3H). 13 C NMR (100MHz, cd 3 od)δ174.38,171.26,171.19, 170.26,168.86,168.21,167.76,166.72,156.27,153.14,138.44, 138.36,138.19,134.87,133.71,132.31,131.57,131.51,129.90, 129.86,121.81,119.36,117.95,69.48, 54.83, 50.52, 40.09, 39.76, 38.30...

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Abstract

The present invention provides a means to modulate gene expression in vivo in a manner that avoids problems associated with CRISPR endogenous protein knock-out or knock-in strategies and strategies that provide for correction, or alteration, of single nucleotides. The invention includes inserting into the genome a nucleotide encoding a heterobifunctional compound targeting protein (dTAG) in-framewith the nucleotide sequence of a gene encoding an endogenously expressed protein of interest which, upon expression, produces an endogenous protein-dTAG hybrid protein. This allows for targeted protein degradation of the dTAG and the fused endogenous protein using a heterobifunctional compound.

Description

[0001] Related applications [0002] This application claims the benefit of Provisional U.S. Patent Application No. 62 / 456,654, filed February 8, 2017, and Provisional U.S. Patent Application No. 62 / 457,127, filed February 9, 2017. The entire contents of these applications are hereby incorporated by reference for all purposes. [0003] incorporated by reference [0004] The content of the text file named "16010-025WO1_sequencelisting_ST25.txt" was created on January 26, 2018, is 287 kilobytes in size, and is hereby incorporated by reference in its entirety. technical field [0005] The present invention describes methods, compounds and compositions for modulating endogenously expressed proteins using targeted protein degradation. Background technique [0006] Many tools have been developed to manipulate gene expression to interrogate the function of a gene or protein of interest. For example, techniques such as RNA interference and antisense deoxyoligonucleotides are comm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/17A61K35/15A61K35/12A61K38/16C12N9/64C12N5/10C12N5/071A61P35/00A61P35/04
CPCA61P35/04A61P35/00C12Y502/01008C12N9/104C12N9/22C12N9/90C07K2319/20C07K2319/95A61K38/465A61K48/005A61K2300/00A61K35/407A61K48/0058A61K48/0025A61K48/0083
Inventor D·巴克利G·温特A·J·菲利普斯T·P·赫弗南J·布拉德纳J·罗伯茨B·纳贝特
Owner DANA FARBER CANCER INST INC
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