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Methods and reagents for analyzing protein-protein interfaces

A protein, C2-C9 technology, applied in analytical materials, chemical instruments and methods, biochemical equipment and methods, etc., can solve problems such as insurmountable

Pending Publication Date: 2020-02-11
REVOLUTION MEDICINES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The other 90% is currently considered insurmountable or intractable for small molecule drug discovery

Method used

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  • Methods and reagents for analyzing protein-protein interfaces
  • Methods and reagents for analyzing protein-protein interfaces
  • Methods and reagents for analyzing protein-protein interfaces

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0510] Example 1: Synthesis of certain crosslinkers

[0511] Synthesis of Cyclosporine Analogs Containing Acrylamide

[0512]

[0513] All reagents and solvents were purchased from Sinopharm Chemical Reagent Co.Ltd. Fmoc-amino acid, HATU, HOAT, H-Ala-2-Cl-(Trt) resin (0.36mmol / g), H-Leu-2-Cl-(Trt) resin (0.30mmol / g), H-Phe- 2-Cl-(Trt) resin (0.35 mmol / g) and H-Thr(tBu)-2-Cl-(Trt) resin (0.36 mmol / g) were purchased from GLBiochem (Shanghai) Ltd.

[0514] Coupling of linear peptides was performed on an automated synthesizer using the standard Fmoc SPSS program.

[0515] General Method A: By using TETRAS TM The synthesizer synthesized linear peptides on a scale of 0.025 mmol resin. The general protocol is as follows: 2x NMP, 30 s; 1x 20% (vol / vol) piperidine in NMP, 15 min; 5x NMP, 30 s; amino acid (3 equivalents) solution in NMP was added to the vessel containing the resin, then The solutions of HATU and DIEA in DMF were added respectively, and coupled for 45min; 3x NMP ...

Embodiment 2

[0673] Example 2: Synthesis of certain conjugates

[0674] General Protocol: The protocol describes a method for forming target protein-compound conjugates.

[0675] Reagents: Compound (internal) and mammalian target protein (internal) in 100% DMSO

[0676] Equipment: Micro PROTEAN TGX gel (Bio-Rad)

[0677] Experimental protocol: The target protein and compound were mixed together in a molar ratio of 1:2 in 12.5 mM HEPES pH 7.4, 75 mM NaCl buffer containing 2% DMSO. Reactions were incubated at 37°C for 30 min and then at room temperature overnight. Cross-linking efficiency was assessed by SDS-PAGE gel. The conjugate migrates more slowly than the non-crosslinked target protein. For thiol-reactive compounds, the Cys-specific attachment of the compound to the target protein can be further confirmed by SDS-PAGE after adding 100 mM DTT to the reaction mixture, which reduces the return of the conjugate to its individual components.

[0678] A. KRAS GTP / S39C Formation of the ...

Embodiment 3

[0690] Example 3: Formation of certain complexes

[0691] General protocol: The protocol describes two methods for the formation and isolation of complexes containing presented proteins, compounds, and mammalian target proteins.

[0692] Reagents: Compound (internal), presented protein (internal), and mammalian target protein (internal) in 100% DMSO

[0693] Equipment: Micro PROTEAN TGX gel (Bio-Rad), Superdex 75 (GE Healthcare, CV 120mL)

[0694] Experimental Protocol A: Preconjugated Compounds and Proteins

[0695] The conjugate and presentation protein were mixed together in a molar ratio of 1:2 in 12.5 mM HEPES pH 7.4, 75 mM NaCl buffer containing 2% DMSO. Reactions were incubated at 37°C for 30 min and then at room temperature overnight. Pure complexes were isolated by size exclusion chromatography (SEC) purification. The reaction mixture was injected directly onto a Superdex 75 column (CV 120 mL) pre-equilibrated with a buffer containing 12.5 mM HEPES pH 7.4, 75 mM N...

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Abstract

The present disclosure provides methods and reagents useful for analyzing protein-protein interfaces such as interfaces between a presenter protein (e.g., a member of the FKBP family, a member of thecyclophilin family, or PIN1) and a target protein. In some embodiments, the target and / or presenter proteins are intracellular proteins. In some embodiments, the target and / or presenter proteins are mammalian proteins.

Description

Background technique [0001] The vast majority of small molecule drugs work by binding to functionally important pockets on target proteins, thereby modulating the activity of said proteins. For example, the cholesterol-lowering drugs statins bind to the enzymatic active site of HMG-CoA reductase, preventing the enzyme from binding to its substrate. The fact that many such drug / target interaction pairs are known may lead some to believe that small molecule modulators can be discovered for most, if not all, proteins given a reasonable amount of time, effort and resources. But in fact, it's not. Current estimates suggest that only about 10% of all human proteins can be targeted by small molecules. The other 90% is currently considered insurmountable or tricky for small molecule drug discovery. Such targets are often referred to as "undruggable". These undruggable targets include a large and largely untapped repertoire of medically important human proteins. Therefore, there i...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K5/062C07K5/097C07D405/14C07D498/08C07D401/12C07D211/60G01N33/68G01N33/566C12Q1/533A61K47/62
CPCG01N33/6845C07D405/06C07D211/10C07D237/08C07D401/12C07D403/12C07K5/02C07K7/06C07K7/64C07D498/12C12Y502/01008
Inventor M.J.穆尔维希尔M.金N.珀尔
Owner REVOLUTION MEDICINES INC
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