Discovery of a PCAF Bromodomain Chemical Probe

Abstract The p300/CBP‐associated factor (PCAF) and related GCN5 bromodomain‐containing lysine acetyl transferases are members of subfamily I of the bromodomain phylogenetic tree. Iterative cycles of rational inhibitor design and biophysical characterization led to the discovery of the triazolopthalazine‐based L‐45 (dubbed L‐Moses) as the first potent, selective, and cell‐active PCAF bromodomain (Brd) inhibitor. Synthesis from readily available (1R,2S)‐(−)‐norephedrine furnished L‐45 in enantiopure form. L‐45 was shown to disrupt PCAF‐Brd histone H3.3 interaction in cells using a nanoBRET assay, and a co‐crystal structure of L‐45 with the homologous Brd PfGCN5 from Plasmodium falciparum rationalizes the high selectivity for PCAF and GCN5 bromodomains. Compound L‐45 shows no observable cytotoxicity in peripheral blood mononuclear cells (PBMC), good cell‐permeability, and metabolic stability in human and mouse liver microsomes, supporting its potential for in vivo use.

Bromodomains proteins (Brds) bind to acetylated lysines (KAc) through the Brd acetyllysine-binding site.M isregulation of these proteins is linked to the onset and progression of multiple disease states,s uch as cancer. [1] Significant efforts have been made recently to interrogate the role of these targets through the development of chemical probes and inhibitors. [2] Considerable work has focused on the BET family (Brd sub-family II), [3] however non-BET [4] Brds are increasingly receiving the attention of small molecule intervention efforts,w ith the disclosure of more than 10 new chemical probes/inhibitors in 2016. [5] Thep 300/CBP-associated factor,P CAF( KAT2B), is amulti-domain protein containing asingle Brd, an N-terminal domain, and ah istone acetyltransferase (HAT) domain. Known to associate with CBP [6] and p300 [6b] during transcription, misregulation of PCAF has been linked to cancer, [7] HIV infection, [7a, 8] and neuroinflammation. [7a, 9] Despite predictions of high druggability [10] and links with inflammatory disease, [7a, 11] the role of PCAF and, more specifically,c ontributions of the Brd in such disease states are poorly understood. Thedevelopment of asmall molecule modulator of PCAF Brd would provide au seful tool for interrogating this potential therapeutic target and allow for dissociation of the roles of the Brd and enzymatic domains in disease.Initial reports of PCAF Brd inhibitors were focused on disrupting interactions between the HIV-1 peptide TAT-1a nd PCAF Brd. [8a,d] Wang et al. reported the first PCAF Brd inhibitor, compound 1 (PCAF IC 50 1.6 mm,F igure 1), which was effective at disrupting HIV-1 replication (EC 50 2.8 mm). [8c] Further efforts made by Hu et al. [12] towards more potent compounds such as 2 were described without significant increases in potencyo ri ndication of selectivity (PCAFI C 50 0.93 mm,EC 50 11.5 mm, Figure 1). Additional chemotypes have been disclosed from fragment-based screening by Chaikuad et al. [5l] Concurrent to this work, Constellation/Genentech reported compound 3 [13] and others,w hich are potent PCAF inhibitors (AlphaLISA IC 50 13 nm)b ut lack reported selectivity over other Brds (Figure 1). [7b,c] Despite recent developments of PCAF Brd inhibitors,apotent, selective,a nd cellactive chemical probe has not been reported. Thework herein describes the discovery of such ap robe.
Our first line of inquiry towards the first PCAF Brd chemical probe was focused on the core of non-selective Brd inhibitors,b romosporine [14] (PCAF isothermal titration calorimetry (ITC) K D :5 mm)a nd [1,2,4]triazolo[4,3-a]phthalazine [15] derivatives as starting points.S mall amine substituents,asincompounds 7-9 (Table 1), were designed to extend out of the narrow PCAF pocket and target glutamic acid residues E750 and E756 at the edge of the KAc-binding pocket through amine-acid salt bridge interactions (PDB: 5FE0). [5l] Commercially available 1,4-dichlorophthalazine 4 underwent ascalable (up to 20 g) tandem S N Ar/condensation reaction to furnish corresponding triazole intermediate 5 in good yields (Scheme 1). Significant efforts were employed to screen conditions using Pd-catalyzed couplings of 5 with various amine nucleophiles;d isappointing yields or lack of reactivity were observed in all of these cases.Itwas found that aK I/HCl-catalyzed S N Ar reaction allowed for at ractable divergent synthesis of various N-linked derivatives (Scheme 1).
After the synthesis of af ocused set of 20 compounds, screening conducted using adifferential scanning fluorimetry (DSF) assay revealed two hits,d imethylamino compounds 7 and 10 (Table 1). It was found that compounds 8 and 9 featuring al onger amine chain were less potent. With the 2-(dimethylamino)ethyl group of compounds 7 and 10 identified as optimal substituents,avirtual library of~12k compounds was constructed by in silico reaction of compound 5 with commercial compounds containing the 2-(dimethylamino)ethyl motif. [16] Over 60 compounds bearing at ethered 1,2-diamine motif were chosen for synthesis based on docking score,d iversity,a nd potential for new interactions with the PCAF Brd (Table 1, compounds 11-16 and Tables S1 and S2).
Derivatives were screened for PCAF Brd affinity by ITC, leading to the discovery of compound 11 (Table 1). By ITC, the stoichiometry of binding showed that all of the activity of the racemate lay in as ingle enantiomer,l ater found to have (S)-configuration after synthesis using enantiopure building blocks (11 ITC K D 0.30 mm,B rd/11 2:1; (S)-11 K D 0.28 mm, Brd/(S)-11 1:1). Groups larger than amethyl substituent at R 2 were detrimental to activity (compounds 12, 13)a sw as ab ulkier N,N-diethyl substituent (compound 14). Although aphenyl substituent at R 3 conferred potencytocompound 10, compounds 15 and 16 with smaller methyl and ethyl groups were less potent. Compound (S)-17 featuring at rifluoromethyl group at position R 1 caused aloss in activity consistent with previously reported Brd SAR of the [1,2,4]triazolo[4,3a]phthalazines. [15] In aD SF panel of 48 human Brds,c ompound (S)-11 showed binding to PCAF and GCN5 with no observable activity against other Brds ( Figure S1). To improve the potencyo f( S)-11,i tw as rationalized that ac ombination of appropriate substituents at R 2 /R 3 might improve the avidity of binding interactions and addition of an aryl group at R 3 would

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Communications 828 www.angewandte.org serve as ac hemical handle for introduction of new functionality.T he R 2 /R 3 -substituted compounds would be ahybrid of the most potent analogues 10 and (S)-11. Synthesis of aryl substituted compounds was achieved through an on-selective aza-Henry reaction with p-substituted benzaldehydes (Scheme 2). p-Substituted benzaldehydes were chosen as provisional in silico scoring of potential inhibitors suggested that o-o rm-substitutions would be less tolerated for binding.H ighly unstable olefins 18-24 were telescoped through ad iastereoselective (d.r. 4.6:1-33:1) nitro-olefin conjugate addition furnishing racemic (S*,S*)configured [17] compounds 25-31,then reduced to corresponding amines, 32-38,u sing either Pd/C-or Raney/Ni-catalyzed hydrogenation. Compounds 32-38 were isolated as single diastereomers and submitted to the aforementioned KIcatalyzed S N Ar reaction (Scheme 2) to produce compounds 39-45 in low to good yields (16-79 %). Racemic compounds were screened by ITC for PCAF-binding affinity ( Table 2). All of the compounds showed an increase in potency compared to compound (S)-11,with the simple unsubstituted derivative 45 having highest potency.
Pleasingly,i tw as found following resolution by preparative chiral stationary phase HPLC,that active enantiomer L-45,which was dubbed L-Moses,showed good binding affinity for PCAF Brd (PCAFK D 126 nm,I TC). Theo ther enantiomer D-45 showed no observable binding,i mplying its utility as an inactive control compound. Having achieved good potencya gainst PCAF Brd, L-45 was then screened for selectivity against the panel of 48 human bromodomains using DSF ( Figure 2B). Homologous Brd of GCN5 was the only other Brd that showed any affinity for L-45,confirmed by ITC (DT m + 3.6 8 8C, K D 0.55 mm). L-45 competitively displaced ab iotinylated tool derivative,c ompound 46 (Supporting Information) in ah omogeneous time-resolved resonance fluorescence (HTRF) assay (PCAFK i 47 nm), corresponding to exquisite selectivity over BRD4 (> 4500-fold selective).
In ac ellular context, L-45 was shown to displace nano-Luciferase-tagged PCAF-Brd from halo-tagged-H3.3 in an anoBRET target engagement assay at as ingle digit mm concentration ( Figure 2C). [20] Inactive enantiomer D-45 had no effect in the same assay.
Compounds DL-45 and p-fluoro derivative 39 were then tested for liver microsomal stability in vitro. DL-45 showed good metabolic stability in both human (t 1/2 40 min) and mouse (t 1/2 38 min) liver microsome preparations. para-F derivative 39 showed aslightly increased metabolic stability in both human (t 1/2 48 min) and mouse (t 1/2 65 min) liver enzymes,l ikely due to metabolic protection of the parasubstituted aryl ring. DL-45 showed good kinetic solubility (> 200 mm)and permeability in MDCK-MDR1 cells with low efflux (Figure 2A). L-45 was also tested in peripheral blood mononuclear cells and showed no observable cytotoxicity after treatment at 10 mm for 24 hours.
Although attempts to obtain ac o-crystal structure of recombinant PCAF with L-45 were unsuccessful, which was surprising given that numerous structures of less potent PCAF fragments have been reported recently. [5l] As tructure using highly homologous (64 %i dentity) Brd from Plasmodium falciparum, PfGCN5, of which L-45 is also ap otent   [ a] clogD was calculated using ChemAxon. [18] [b] Ligand efficiency. [19] ligand (ITC K D 280 nm), was successfully obtained (PDB: 5TPX, Figure 3). L-45 bound as expected in the KAc-binding site of PfGCN5 with key interactions that include asalt bridge between E1389 (conserved in PCAF as E756) and the dimethylamino motif of L-45 ( Figure 3A). Additional contacts are also observed in the form of an edge-to-face p-p stacking interaction between W1379 (conserved in PCAF as W746) and the phenyl substituent of L-45 (average distance 4.5 ); a p-p stacking interaction between Y1442 (conserved in PCAF as Y809) and pyridazo ring of the triazolophthalazine motif (average distance 3.7 ); and characteristic Hbonds from the triazolophthalazine group and N1436 residue (conserved in PCAF as N803) and aw ater molecule.I ntolerance of substitution of L-45 in R 2 and R 3 positions (compounds 12-16, Table 1) was rationalized by the tight fit of the alkyl amine chain of L-45 ( Figure 3B). Interestingly, K1383 in PfGCN5 is substituted with E750 in human PCAF, and as such the Plasmodium homologue features as lightly open KAc-binding site ( Figure 3B). Targeting this difference may allow for design of Plasmodium-selective Brd inhibitors. As previously supported by SAR, the absolute configuration of L-45 was confirmed to be (1S,2S). Fort he asymmetric synthesis of L-45,c ommercially available (1R,2S)-(À)-norephedrine was Boc-protected and cyclized to as ulfamidite and then directly oxidized using sodium periodate to boc-protected sulfamidate 46 in reasonable yields (Scheme 3). Subsequent treatment with dimethyl-amine facilitated regio-selective ring opening of sulfamidate 46, [21] extruding SO 3 and furnishing protected diamine 47 as asingle diastereoisomer with inversion of configuration at the benzylic centre.F ollowing ad eprotection of 47 to the free amine and S N Ar with aryl chloride 5, L-45 was furnished in six steps as asingle stereoisomer.
In conclusion, we report the discovery of L-45,t he first nanomolar,s elective,a nd cell-active chemical probe of the PCAF bromodomain. Iterative cycles of rational inhibitor design, in silico docking studies,and synthesis furnished L-45 after generation of af ocused PCAF inhibitor library. L-45 shows aclean toxicity profile in primary PBMCs,and disrupts interactions between PCAF Brd and H3.3 in HEK293 cells, indicating cellular target engagement.
Good cell permeability in aM DCK-MDR1 assay and stability to metabolism in both human and mouse liver microsomes indicate that L-45, dubbed L-Moses,m ay also have utility in vivo. L-Moses will allow for robust interrogation of PCAF Brd inhibition and pharmacological effects in relevant diseases models.F uture work will investigate the use of L-Moses in functional assays pertaining to PCAFassociated diseases.

Conflict of interest
Theauthors declare no conflict of interest.