Covalent labeling of a chromatin reader domain using proximity-reactive cyclic peptides

Chemical probes for chromatin reader proteins are valuable tools for investigating epigenetic regulatory mechanisms and evaluating whether the target of interest holds therapeutic potential. Developing potent inhibitors for the plant homeodomain (PHD) family of methylation readers remains a difficult task due to the charged, shallow and extended nature of the histone binding site that precludes effective engagement of conventional small molecules. Herein, we describe the development of novel proximity-reactive cyclopeptide inhibitors for PHD3—a trimethyllysine reader domain of histone demethylase KDM5A. Guided by the PHD3–histone co-crystal structure, we designed a sidechain-to-sidechain linking strategy to improve peptide proteolytic stability whilst maintaining binding affinity. We have developed an operationally simple solid-phase macrocyclization pathway, capitalizing on the inherent reactivity of the dimethyllysine ε-amino group to generate scaffolds bearing charged tetraalkylammonium functionalities that effectively engage the shallow aromatic ‘groove’ of PHD3. Leveraging a surface-exposed lysine residue on PHD3 adjacent to the ligand binding site, cyclic peptides were rendered covalent through installation of an arylsulfonyl fluoride warhead. The resulting lysine-reactive cyclic peptides demonstrated rapid and efficient labeling of the PHD3 domain in HEK293T lysates, showcasing the feasibility of employing proximity-induced reactivity for covalent labeling of this challenging family of reader domains.


Supplementary Figure 2
Trypsin stability assay: Peptide and trypsin (1:1 w/w) were mixed in 1x PBS buffer and the resulting mixture was incubated at RT. Proteolysis reactions at a given time point was quenched by adding HCl. The proteolysis process was monitored by subjecting the quenched mixture onto reverse-phase HPLC column monitoring at 214 nm [H2O (A):MeCN (B); 5% to 100% solvent B over 23 min].

General Experimental Procedures
Preparative HPLC was performed on a Waters Prep 150Q LC system, with a Waters 2998 Photodiode Array detector, and this system was operated using ChromScope software. All separations used linear gradients of water containing 0.1% trifluoroacetic acid and acetonitrile, at a flow rate of 10 mL/min (XSelect® Peptide CSH TM C18, OBD TM Prep Column, 130Å, 5 µM, 19 mm × 250 mm, 1/pkg). Analytical HPLC was performed on a Varian ProStar 210 Solvent Delivery Module, with a Varian ProStar 335 Photodiode Array Detector, and this system was operated using Galaxy Chromatography Data System. All separations used linear gradients of water containing 0.1% trifluoroacetic acid (solvent A) and acetonitrile (solvent B), at a flow rate of 1 mL/min (Phenomenex Luna® 10 µm C18(2) 100Å, LC column 250 × 4.6 mm, Ea).

Molecular Modelling
Design feasibility of macrocyclic peptides were assessed using MacroModel. The X-ray crystallographic structure of PHD3-H3K4me3 (PDB 3GL6) was prepared as the initial starting structure. Non-coordinating water molecules found beyond 5 Å from heteroatoms were removed. Macrocyclic peptides were prepared by mutating K4me3 and T6 with a linker which contained amide, thioether, and triazole moieties respectively. The resulting structures were minimized using MMFFs and GB/SA water solvation whilst keeping the PHD3 and amide backbone of H3K4me3 frozen. The minimized model was subsequently searched for energy conformers using the conformational search tool using the MMFFs force field and GV/SA water solvation whilst keeping the PHD3 and amide backbone of H3K4me3 frozen. The resulting conformers within 4 kJmol -1 from the lowest-energy conformer were overlayed to assess the rigidity and design feasibility.

Synthetic Procedures and Peptide Characterization Data
General Workflow for Fmoc-Solid Phase Peptide Synthesis (Fmoc-SPPS)
A solution of Ac2O/pyridine (1:9 v:v) was added to the resin. After three minutes, the liquid was discarded and the resin was washed with DMF (5 × 3 mL), DCM (5 × 3 mL) and DMF (5 × 3 mL). The resin-bound amino acid was subjected to iterative peptide assembly.
Cleavage: The resin was washed with DCM (10 × 3 mL) to remove any traces of DMF. A mixture of TFA/iPrSi3H/H2O (90:5:5 v:v:v, 3 mL) was added to the resin and agitated at room temperature for three hours, at which time the resin was washed with TFA (2 × 2 mL) and DCM (2 × 2 mL).
Workup/purification: The cleavage and wash solutions were concentrated by gently blowing over the combined solutions with air. Ice-cold diethyl ether was added to the residue, and the precipitated crude peptide was pelleted by centrifugation. The diethyl ether solution was removed, and the pellet was dried to afford a colorless-yellow powder. The crude peptide was subsequently purified by reverse phase HPLC.

Deprotection procedures
Alloc and Allyl protecting groups: The resin (1 equiv) was treated with a solution of Pd(PPh3)4 (0.5 equiv) and PhSiH3 (2 equiv) in DCM for one hour, at which time the reaction vessel was carefully vented and the dark liquid was discarded. The deprotection reaction was monitored by LCMS and repeated if necessary.
Mmt protecting group: The resin was washed with DCM (5 × 3 mL). The resin was treated with a solution of DCM/TFA/iPrSi3H (95:1:4 v:v:v) and agitated for two minutes at room temperature, at which the time the liquid is discarded. A small portion of resin was treated with TFA to monitor the deprotection. The cleavage reaction was repeated until the Mmt cation was not detected (intense orange color).
S-(tBu) protecting group: The resin was washed with DMF (5 × 3 mL) and was agitated with a solution of DTT (5 equiv) dissolved in DMF/DIPEA/H2O (95:2.5:2.5 v:v:v) for 10 minutes at RT, at which time the liquid was discarded. The reaction was repeated three more times or until the deprotection was complete, indicated by LCMS analysis.

Series C: thioether macrocyclization
Tripeptide Fmoc-Lys(me2)-Gln(Trt)-Cys(StBu)-CONH-rink amide resin (1 equiv) was prepared on Rink amide resin according to the procedures outlined in the Iterative Fmoc-SPPS workflow. The resin was washed with DMF (5 × 3 mL) and the S-(tBu) group was deprotected (see Deprotection procedures). The resin-bound tripeptide intermediate 19 was washed with DMF (5 × 3 mL) and treated with a solution of DIPEA (4 equiv) in DMF, followed by a solution of 1,2-bis(bromomethyl)benzene (2 equiv) in DMF. The resin was agitated at room temperature for one hour, whilst monitoring the reaction using LCMS analysis. Once the formation of C-18 and consumption of starting material was confirmed, the resin was washed with DMF (5 × 3 mL), DCM (5 × 3 mL) and DMF (5 × 3 mL), and the peptide was elongated following the Iterative Fmoc-SPPS workflow to afford thioether cyclopeptide C-18c.
The subsequent CuAAC reaction followed a modified literature procedure. 2 The resin was transferred to a Synthware TM cylindrical pressure vessel (15 mL O.D.) sealed with a rubber septum using 1 mL of DMF, and the reaction flask was degassed and refilled with nitrogen. 2,6-Lutidine (8 equiv) and DIPEA (8 equiv) were added to the resin followed by the addition of sodium ascorbate (3 equiv as a 1% solution in degassed DMF) and copper(I) bromide (1 equiv as a 1% solution in degassed acetonitrile) was added last. The resin mixture was stirred at room temperature under nitrogen for 2-4 hours whilst monitoring the reaction using analytical HPLC. Following consumption of starting material, the reaction was transferred to a plastic fritted syringe and washed with DMF (5 × 3 mL), DCM (5 × 3 mL) and DMF (5 × 3 mL). The cyclized tripeptide intermediates D-23 and D-24 were elongated following the procedures outlined in the Iterative Fmoc-SPPS workflow to afford triazoles D-23c and D-24c. The CuAAC reaction procedures from Condition a was used with the addition of 4-bromo-1-butyne (1.5 equiv). Following the consumption of starting material (monitored by LCMS analysis), the resin was transferred to a plastic fritted syringe and washed with DMF (5 × 3 mL), DCM (5 × 3 mL) and DMF (5 × 3 mL). The resin was treated with a solution of tetrabutylammonium iodide (2 equiv) in DMF and agitated at room temperature for 16 hours, at which time the liquid was expelled and the resin was washed with DMF (5 × 3 mL), DCM (5 × 3 mL) and DMF (5 × 3 mL). The cyclized tripeptide intermediates D-25 and D-26 were elongated following the Iterative Fmoc-SPPS workflow to afford triazoles D-25c and D-26c.
The arylfluorosulfate peptide synthesis followed a slightly modified literature procedure. 3 A solution of AISF (1.2 equiv) in THF was added to resin, followed by the addition of a solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (2.2 equiv) in THF. The resin mixture was shaken at room temperature for 10 minutes at which time the liquid was expelled and the resin was washed with DMF (5 × 3 mL), DCM (5 × 3 mL), DMF (5 × 3 mL) and DCM (5 × 3 mL). Incorporation of the fluorosulfate functionality was confirmed by LCMS analysis. The peptide was cleaved from resin and purified according to the procedures outlined in the Iterative Fmoc-SPPS workflow. LCMS MALDI Hexapeptide Boc-Ala-Arg(Pbf)-Thr(tBu)-K(me3)-Cys(StBu)-Thr(tBu)-CONH-Rink amide resin (1 equiv) was prepared on Rink amide resin according to the procedures outlined in the Iterative Fmoc-SPPS workflow. The S-(tBu) group was deprotected according to the protocols outlined in the Deprotection procedures. The resin was washed with DMF (5 × 3 mL) and treated with a solution of DIPEA (5 equiv) and 3-(bromomethyl)phenol (5 equiv) in DMF and agitated at room temperature for 30 minutes or until consumption of starting material was confirmed by LCMS analysis. The liquid was discarded, and the resin was washed with DMF (5 × 3 mL), DCM (5 × 3 mL), DMF (5 × 3 mL) and DCM (5 × 3 mL). Installation of the fluorosulfate group was achieved using the synthetic procedures outlined for arylfluorosulfate peptides 30 and 32. The peptide was cleaved from resin and purified according to the procedures outlined in the Iterative Fmoc-SPPS workflow to afford arylfluorosulfate peptide 31.

Installation of the 4-benzylsulfonyl fluoride covalent warhead
Hexapeptide Boc-Ala-Arg(Pbf)-Thr(tBu)-K(me3)-Cys(StBu)-Thr(tBu)-CONH-Rink amide resin (1 equiv) was prepared on Rink amide resin according to the procedures outlined in the Iterative Fmoc-SPPS workflow. The S-(tBu) group was deprotected according to the protocols outlined in Deprotection procedures. The resin was washed with DMF (5 × 3 mL) and treated with a solution of DIPEA (2.5 equiv) and 4-(bromomethyl)benzenesulfonyl fluoride (1.2 equiv) in DMF and agitated at room temperature for 30 minutes or until consumption of starting material was confirmed (monitored by LCMS analysis). The liquid was discarded, and the resin was washed with DMF (5 × 3 mL), DCM (5 × 3 mL), DMF (5 × 3 mL) and DCM (5 × 3 mL). The peptide was cleaved and purified according to the procedures outlined in the Iterative Fmoc-SPPS workflow to afford sulfonyl fluoride peptide 29.

Installation of the fluorosulfonylbenzamide covalent warhead
Hexapeptide Boc-Ala-Arg(Pbf)-Thr(tBu)-K(me3)-Dap(Alloc)-Thr(tBu)-CONH-Rink amide resin (1 equiv) was prepared on Rink amide resin according to the procedures outlined in the Iterative Fmoc-SPPS workflow. The Alloc group was deprotected according to the protocols outlined in Deprotection procedures. The resin was washed with DMF (5 × 3 mL), DCM (5 × 3 mL), DMF (5 × 3 mL). The Alloc-deprotected resin was treated with a solution of para, or meta-fluorosulfonylbenzoic acid (4 equiv), PyBOP (4 equiv) and DIPEA (8 equiv) in DMF and agitated at room temperature for one hour or until consumption of starting material was confirmed (monitored by LCMS analysis), at which time the liquid was discarded and the resin was washed with DMF (5 × 3 mL), DCM (5 × 3 mL), DMF (5 × 3 mL) and DCM (5 × 3 mL). The peptide was cleaved from resin and purified according to the procedures outlined in the Iterative Fmoc-SPPS workflow to afford 27 and 28.

Synthesis of covalent thioether cyclopeptide C-33
Tripeptide Fmoc-Lys(me2)-Cys(Mmt)-Cys(StBu)-CONH-Rink amide resin (1 equiv) was prepared on Rink amide resin according to the procedures outlined in the Iterative Fmoc-SPPS workflow. The tripeptide intermediate was subjected to Series C: thioether macrocyclization conditions and elongated using the procedures outlined in the Iterative Fmoc-SPPS workflow. The Mmt group was cleaved using the procedures described in Deprotection procedures, and the 4-benzylsulfonyl fluoride functionality was introduced according to the protocols outlined in Installation of the 4-benzylsulfonyl fluoride covalent warhead. The resin was washed with DMF (5 × 3 mL), DCM (5 × 3 mL), DMF (5 × 3 mL) and DCM (5 × 3 mL). The peptide was cleaved from resin and purified according to the procedures outlined in the Iterative Fmoc-SPPS workflow to afford C-33.

Synthesis of covalent thioether peptide D-34 and D-35
Tripeptide Fmoc-Lys(me2)-Cys(StBu)-Dap(N3)-CONH-Rink amide resin (1 equiv) was prepared on Rink amide resin according to the procedures outlined in the Iterative Fmoc-SPPS workflow. The tripeptide intermediate was subjected to Series D: triazole macrocyclization conditions and elongated using the procedures outlined in the Iterative Fmoc-SPPS workflow. The S-(tBu) group was cleaved using the procedures described in Deprotection procedures, and the 4-benzylsulfonyl fluoride functionality was introduced according to the protocols outlined in Installation of the 4-benzylsulfonyl fluoride covalent warhead. The resin was washed with DMF (5 × 3 mL), DCM (5 × 3 mL), DMF (5 × 3 mL) and DCM (5 × 3 mL). The peptide was cleaved from resin and purified according to the procedures outlined in the Iterative Fmoc-SPPS workflow to afford D-34 and D-35.

Synthesis of biotinylated covalent thioether cyclopeptide C-36
Tetrapeptide Fmoc-Lys(me2)-Cys(Mmt)-Cys(StBu)-Lys(Alloc)-CONH-Rink amide resin (1 equiv) was prepared on Rink amide resin according to the procedures outlined in the Iterative Fmoc-SPPS workflow. The tetrapeptide intermediate was subjected to Series C: thioether macrocyclization conditions and elongated using the procedures outlined in the Iterative Fmoc-SPPs workflow. The Alloc group was removed using the procedures outlined in Deprotection procedures. The resin was treated with a solution of biotin (4 equiv), PyBOP (4 equiv) and DIPEA (8 equiv) in DMF and agitated at room temperature for one hour. The liquid was expelled from the reaction vessel and the resin was washed with DMF (5 × 3 mL), DCM (5 × 3 mL), and DMF (5 × 3 mL). The Mmt group was cleaved using the procedures described in Deprotection procedures, and the 4benzylsulfonyl fluoride functionality was introduced according to the protocols outlined in Installation of the 4benzylsulfonyl fluoride covalent warhead. The resin was washed with DMF (5 × 3 mL), DCM (5 × 3 mL), DMF (5 × 3 mL) and DCM (5 × 3 mL). The peptide was cleaved from resin and purified according to the procedures outlined in the Iterative Fmoc-SPPS workflow to afford C-36.

Fluorescence polarization assays
The binding of His6-MBP-PHD3 to H3K4me3 WT and mutant peptides, as well as the binding of recombinant His6-MBP-PHD3 lysine mutant proteins to H3K4me3 WT peptide were measured using direct or competitive fluorescence-polarization (FP) assays. 1