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. 2018 Feb 8;61(3):918-933.
doi: 10.1021/acs.jmedchem.7b01406. Epub 2018 Jan 5.

Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766)

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Free PMC article

Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766)

Nicola E A Chessum et al. J Med Chem. .
Free PMC article

Abstract

Demonstrating intracellular protein target engagement is an essential step in the development and progression of new chemical probes and potential small molecule therapeutics. However, this can be particularly challenging for poorly studied and noncatalytic proteins, as robust proximal biomarkers are rarely known. To confirm that our recently discovered chemical probe 1 (CCT251236) binds the putative transcription factor regulator pirin in living cells, we developed a heterobifunctional protein degradation probe. Focusing on linker design and physicochemical properties, we generated a highly active probe 16 (CCT367766) in only three iterations, validating our efficient strategy for degradation probe design against nonvalidated protein targets.

Conflict of interest statement

The authors declare the following competing financial interest(s): The Institute of Cancer Research has a potential financial interest in ligands of pirin and operates a Rewards to Discoverers scheme.

Figures

Figure 1
Figure 1
(top) Pirin (5JCT)/CRBN (5CI1)/PDP ternary complex design model. The PDP can either stabilize a PPI or simply bring the proteins in close proximity depending on the role of the linker. (bottom left) Cartoon representation of the chemical probe 1 (yellow) bound to recombinant pirin (5JCT). The cloud represents the shape of the binding pocket with key residues shown in black and the metal in orange. Red = oxygen, blue = nitrogen. Hydrogens and solvent are omitted for clarity except the water coordinated to the metal, shown as a red sphere. Both representations were generated using the PyMOL Molecular Graphics System, version 1.8; Schrödinger, LLC. (bottom right) Key residues in the binding site and the clear solvent exposed vector for the chemical probe 1 binding to pirin are shown adapted from an analysis using MOE 2014.09. The ethyl pyrrolidine solubilizing group of chemical probe 1 was not resolved in the crystal structure and therefore is not drawn in the analysis.
Scheme 1
Scheme 1. Design and Synthesis of the First Generation PDP 3 Based on the Chemical Probe 1
Reagents and conditions: (a) (i) PPh3, tbutyl 2-hydroxyacetate, DTBAD, THF, 0 °C → RT, 16 h, 75%, (ii) HCO2H, DCM, 40 °C, 16 h, 54%, (iii) HATU, DIPEA, DMF, tbutyl (2-(2-aminoethoxy)ethyl)carbamate, RT, 16 h, 81%, (iv) 4 M HCl in dioxane, 0.5 h, 100%; (b) ethyl 4-bromobutanoate, K2CO3, DMF, RT, 16 h, 37%; (c) Herrmann’s palladacycle,tBu3PHBF4, MoCO6, DBU, 2-(trimethylsilyl)ethan-1-ol, 130 °C, 62%; (d) TBAF, THF, RT, 16 h; (e) (i) HATU, DIPEA, DMF, RT, 38% (over 2 steps), (ii) LiOH·H2O, MeOH/THF/H2O, RT, 48 h, 18%; (f) HATU, DIPEA, DMF, 74%. For the synthesis of hydroxythalidomide 2, see ref (28).
Scheme 2
Scheme 2. Synthesis of Second (10) and Third (16) Generation Probes and Control Compounds
Reagents and conditions: (a) (i) X = F 12, 2-methylquinoline carboxylic acid, oxalyl chloride, DMF, DCM, RT, 3 h, then pyridine, 18 h quant, (ii) Fe(0), NH4Cl, EtOH/H2O, 90 °C, 1 h, quant; (b) 2,3-dihydrobenzo-[b][1,4]-dioxine-6-carboxylic acid, oxalyl chloride, DMF, DCM, RT, 3 h, then pyridine, RT, 2 h, 85%; (c) (i) SeO2, 1,4-dioxane/DMF, reflux, 1 h, (ii) N-Boc-piperazine, DCM, RT, 12 h then NaBH(OAc)3, DCM, RT, 2 h, 94% (over 2 steps), (iii) TFA, DCM, RT, 2 h, 69%; (d) (i) 2, PPh3, tbutyl 2-hydroxyacetate, DTBAD, THF, 0 °C → RT, 16 h, 75%, (ii) HCO2H, DCM, 40 °C, 16 h, 54%, (iii) HATU, DIPEA, DMF, tbutyl 3-(2-aminoethoxy)propanoate, RT, 16 h, 72%, (iv) HCO2H, DCM, 40 °C, 6 h, 93%; (e) HATU, DIPEA, DMF, RT, 16 h, 52%; (f) (i) X = Cl 17, 2-methylquinoline carboxylic acid, oxalyl chloride, DMF, DCM, RT, 3 h, then pyridine 2 h, 88%, (ii) Fe(0), NH4Cl, EtOH/H2O, 90 °C, 1 h, quant; (g) 2,3-dihydrobenzo-[b][1,4]-dioxine-6-carboxylic acid, oxalyl chloride, DMF, DCM, RT, 3 h, then pyridine 2 h, 61%; (h) (i) SeO2, DMF, 1,4-dioxane, 50 °C, 16 h, (ii) N-Boc-piperazine, NaBH3CN, AcOH, DMF, 0 °C → RT, 16 h, (iii) 4 M HCl in dioxane, MeOH 0 °C → RT, 16 h, 32% over 3 steps; (i) 2-[2-(2-hydroxyethoxy)ethoxy]ethyl 4-methylbenzenesulfonate, K2CO3, DMF, RT, 16 h, 48%; (j) PPh3, DTBAD, THF, RT, 2 h, 27%; (k) (i) 23, 2,3-dihydro-1,4-benzodioxine-5-carboxylic acid, oxalyl chloride, DMF, DCM, RT, 2 h, then pyridine 48 h, 84%, (ii) Pd/C, EtOH/DCM, H2 (1 atm), 77%, (iii) 2-methylquinoline carboxylic acid, oxalyl chloride, DMF, DCM, RT, 2 h, then pyridine 16 h, 58%, then same procedure as from 18, 6% yield over 5 steps. (l) (i) SeO2, 1,4-dioxane/DMF, 50 °C, 5.5 h, (ii) N-ethylpiperazine, DCM, RT, 20 h, then NaBH(OAc)3, DCM, RT, 2 h, 35% (over 2 steps).
Figure 2
Figure 2
(A) Representative SPR sensorgram of the third generation PDP 16 and recombinant pirin. (B) Representative binding curve of PDP 16 in the CRBN-DDB1 FP-assay.
Figure 3
Figure 3
(A) Immunoblot of SK-OV-3 human ovarian cancer cells demonstrating the depletion of pirin protein using the third generation PDP 16 and the time-dependent hook-effect. (B) Immunoblot demonstrating the concentration-dependent depletion of pirin protein after 2 h exposure in SK-OV-3 cells. (C) Capillary electrophoresis and immunoassay were used to quantify the pirin protein expression after 2 h exposure with PDP 16, and all values are normalized to vinculin loading control and relative to the measured basal pirin protein expression, all bars represent the arithmetic mean of n = 3 independent biological repeats, error bars are SEM. (D) Proteomics analysis of the third generation PDP 16 (50 nM) exposure (4 h) in SK-OV-3 cells compared to vehicle control, using a tandem mass tagging (TMT) MS2 protocol on the cell lysate, 8547 quantifiable proteins were identified; each blue dot represents a single quantifiable protein, pirin is marked in red (adjusted p value = 1.4 × 10–4), p values were calculated using a linear modeling based t test and corrected for multiple comparisons using the Benjamini–Hochberg method to give the p(adj) values shown, dotted lines represent 2-fold depletion of the protein and a p(adj) = 0.05.
Figure 4
Figure 4
Intracellular competition studies with PDP 16 and the chemical probes. SK-OV-3 cells were pretreated with increasing concentrations of chemical probe for 4 h before exposing to PDP 16 for 2 h at the concentrations shown. Cells were then lysed and protein expression analyzed using immunoblot. For clarity, gel images have been cropped where appropriate.

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