Discovery of an MLLT1/3 YEATS Domain Chemical Probe

Abstract YEATS domain (YD) containing proteins are an emerging class of epigenetic targets in drug discovery. Dysregulation of these modified lysine‐binding proteins has been linked to the onset and progression of cancers. We herein report the discovery and characterisation of the first small‐molecule chemical probe, SGC‐iMLLT, for the YD of MLLT1 (ENL/YEATS1) and MLLT3 (AF9/YEATS3). SGC‐iMLLT is a potent and selective inhibitor of MLLT1/3–histone interactions. Excellent selectivity over other human YD proteins (YEATS2/4) and bromodomains was observed. Furthermore, our probe displays cellular target engagement of MLLT1 and MLLT3. The first small‐molecule X‐ray co‐crystal structures with the MLLT1 YD are also reported. This first‐in‐class probe molecule can be used to understand MLLT1/3‐associated biology and the therapeutic potential of small‐molecule YD inhibitors.


Lysineresiduesbearingacetyl(Kac)orcrotonyl(Kcr)marks
are fundamental components of the epigenetic code. [1,2] In addition to the well-studied binders of acyl lysine,s o-called bromodomains, [3,4] YEATS (YAF9, ENL, AF9, TAF14, SAS5) domain containing proteins bind acetyl and crotonyl marks on histone tails.Recent reports have suggested that the dysregulation of YEATS domain (YD) containing proteins correlates with the onset and progression of cancers. [5][6][7] There are four YD-containing genes in humans (MLLT1, YEATS2, MLLT3, and YEATS4). Despite high sequence homology (88 %YDalignment) and similar roles in complex formation, both MLLT1 and MLLT3 appear to have independent roles in cancers such as acute myeloid leukaemia (AML). [5,6] MLLT1, which associates with DOT1L, [8][9][10] has been implicated in Wilms tumour progression when mutations occur in the YD [11] and associates with the AF4 subcomponent in certain rearranged leukaemia types. [12] MLLT3 is acomponent of the super elongation complex (SEC), [7] and unlike MLLT1, it is the most common fusion partner with MLL (mixed lineage leukaemia protein) in AML (ca. 30 %o fc ases). [7] Recent reports suggested that MLLT1/3 share a" KILK" motif interaction, also present in the extra-terminal (ET) domain of BRD3, which is responsible for the recruitment of chromatin-remodelling complexes,f or example,N uRD, BAF, and INO80. [13] Despite the YD-containing proteins being correlated with other diseases, [7] no small-molecule inhibitors have been reported to further the understanding of YD-associated biology.
Building on our own and others understanding of developing acetyllysine reading domain inhibitors (bromodomains) exemplified by collaborative efforts targeting the BET bromodomains, [14] p300/CBP, [15] PCAF/GCN5, [16] and others, [17][18][19][20] we sought to apply synthetic and medicinal chemistry efforts towards the identification of the first YDcontaining protein chemical probe. [21] Owing to the high sequence homology of MLLT1 and MLLT3, particularly in the YD,achieving selective inhibitors would pose achallenge and indeed may not in fact be desired as dual inhibition may mitigate any functional redundancyinMLLT1/3.
Am edium-throughput screen of the Ontario Institute of Cancer Research (OICR) library (40 000 compounds) [22] revealed compound 1 as am icromolar inhibitor of the MLLT1 YD in an AlphaScreen (AS) assay (MLLT1:I C 50 = 2.1 mm ;F igure 1). Compound 1 posed as an attractive chemical starting point for analogue generation owing to intuitive retrosynthetic disconnections and alack of structural alerts after PA INS [23] filtering. Guided by flexible docking with ICM [24] using am odel extracted from ap reviously reported co-crystal structure of MLLT1:H3Kac27 peptide ( Figure 2A,P DB ID 5J9S), docked poses of compound 1 overlay with the amide bond present in the Kac residue in aflipped conformation ( Figure 2B). YDs demonstrate higher affinities for crotonylated lysine peptides over acetylated ones, [25,26] which is thought to be related to the presence of a p-p-p network between the crotonyl double bond and residues F28, Y78, and F59 in MLLT1. [7] Docking studies revealed ap otential interaction when compound 1 bound to MLLT1 YD between the amide bond CO and backbone NH of Y78, in addition to the amide N-H interacting with the side chain of S58. Thep iperidine ring in compound 1 was likely protonated in its bound form (predicted pK a 8.1 using ACD/ Percepta pK a ), [27] and because of an umber of polar residues close to the entrances of the YD binding channel such as E75, any additional substituents or modifications that decrease the piperidine ring basicity would likely cause ad rop off in binding affinity.I fa nalogues did indeed overlay with Kac/cr hinged on the amide bond depicted in Figure 2B,substituents capable of improving p-p-p stacking would be favoured. Although the Kac/Kcr channel in MLLT1 is linear and narrow,b oth ends contain adequate space for ligand elaboration ( Figure 2B).
Ligand development efforts focussed on the conversion of the potentially labile methyl ester of compound 1 into another substituent that would be tolerated and offer asuitable vector to identify new binding interactions.U tilising a" poised" approach, [28] compound 1 was disconnected into synthons for rapid diversification (Figure 3). Structure-activity relationship (SAR) studies were carried out on compound 1, culminating in the synthesis of > 200 analogues (selected example compounds shown Table 1, others in the Supporting Information).
Derivatives of compound 1 were synthesised from 4-nitrobenzene-1,2-diamine 2,which was treated with chloroalkyl esters under acidic conditions to form condensed chloromethyl and 2-chloroethyl benzimidazoles 3-5.B enzimidazoles 3-5 were then substituted with amines,p roviding compounds 6-36.R eduction of nitro compounds 6-36 furnished the corresponding anilines 37-67,w hich were converted into amides and sulfonamides,namely compounds 68-183.C ompounds 68-183 were screened by AS,a nd selected examples were further validated by isothermal titration calorimetry (ITC;Scheme 1).   Structural modifications of the 2-or 3-positions in the benzoyl motif of compound 1 were unfavoured (compounds 68-70,T able 1). Theintroduction of para substitutions or 3,4disubstitions was better tolerated although the binding activity still decreased relative to compound 1 (compounds 71-74). Replacement of the amide bond of ester 1 with as ulfonamide also ablated activity (compound 75). The addition of electron-poor heteroaromatic moieties at the benzoyl position to mimic the methyl ester of compound 1 (compounds 76-79)improved binding activity.With suitable methyl ester replacements identified in compounds 77-79,we focused our attention on further improving potencyb y modification of the basic amine.I ntroduction of ac hiral centre on the piperidine ring of ester 1 would potentially increase preference for ap articular conformation for as alt bridge (e.g., E75). Modifications to the basic amine involving substituted piperidine rings or homologation of the benzylic centre ablated or had no effect (compounds 80-84)onbinding affinity compared to unsubstituted compound 73.Rearrangement of the piperidyl motif to a2 -methylpyrrolidine or af used cyclopropyl-pyrrolidine in combination with the 1-methyl-1H-indazol-5-yl motif to give compounds 85 and In efforts to optimise substitution of the pyrrolidine core of compound 85,a ll stereoisomers of both 2-methyl-and 3-methylpyrrolidine derivatives were synthesised (89-92, Table 1). Interestingly,t he 2-methyl-substituted pyrrolidines 91 and 92 display al arger difference in binding activity between both enantiomers,with compound 92 displaying the most potent activity ((S)-92 MLLT1 YD IC 50 0.26 mm,(R)-91 MLLT1 YD IC 50 2.0 mm). Binding of compound 92 to MLLT1 YD was validated by ITC (MLLT1 YD K d 0.129 mm). Weaker activity was observed for the R enantiomer 91 (MLLT1 YD K d 0.83 mm), which allows it to be used as achemically similar, but less active control compound. Compound 92 is predicted to be slightly more conformationally restricted about the aliphatic pyrrolidine ring due to substitution compared with original hit piperidine 1,which may confer stabilised electrostatic interactions with charged side chain residues in MLLT1 YD.M ore potent binding observed from the introduction of heteroaromatics in place of the methyl benzoyl motif as with compound 92 may be attributed to am ore complementary p-p-p "sandwich" stack in the binding site.C ompounds 91 Profiling of compound 92 against YEATS2 and YEATS4 revealed excellent selectivity for MLLT1/3 with no activity observed (YEATS2/4 IC 50 > 10 mm). Profiling of compound 92 against as election of bromodomains showed complete selectivity:There was no inhibition of BRD4 (I), CBP,T AF1, CECR2, and FALZ (10 mm using AS). This was validated in at hermal shift assay where both the original hit compound 1 and compound 92 showed no activity against 48 bromodomains (50 mm compound concentrations;s ee the Supporting Information).
After extensive crystal soaking experiments,anX-ray cocrystal structure of compound 92 in complex with MLLT1 YD was obtained ( Figure 4A). Compound 92 occupies the Kac/ Kcr binding site of MLLT1 YD,m aking an umber of interactions with loop 1, loop 4, and loop 6( Figure 4A; cyan, magenta, and yellow,r espectively) adjacent to as tructural water molecule.I nterestingly,t he binding mode of compound 92 matches docked predictions for compound 1. Y78 adopts two conformations,n amely an "in" pose where a p stacking interaction with the amide of 92 can take place, similar to the Kcr:MLLT3 YD crystal structure (PDB ID 5HJB), along with an "out" pose in which the Y78 side chain is now displayed edge-to-face with the adjacent side chain of F28.
As demonstration of target engagement is akey criteria in chemical probe qualification, [29] we sought to demonstrate cell activity of compound 92 through multiple methods.W e demonstrated MLLT1 target engagement using ac ellular thermal shift assay (CETSA) [30] with endogenous MLLT1 in MV4;11 cells.C ompound 92 showed stabilisation of MLLT1 ( Figure 5A-B) whereas the less active control compound 91 elicited no thermal stabilisation up to 10 mm.N ext, we performed fluorescence recovery after photobleaching (FRAP) measurements using green fluorescent protein (GFP) tagged MLLT1 wild-type,M LLT1 mutant, and MLLT3 wild-type plasmids.P hotobleaching of GFP-tagged wild-type MLLT1 and MLLT3 had ah alf recovery time (t 1/2 ) of 0.46 AE 0.06 sa nd 0.6 AE 0.09 s, respectively ( Figure 5C,D). As t 1/2 was relatively short, meaning that the majority of the protein is mobile,itwas difficult to measure shorter recovery  times to study the effect of compound 92.T herefore,w e preincubated the cells with HDAC inhibitor suberoylanilide hydroxamic acid (SAHA, 2.5 mm), preserving global histone acetylation and thus increasing binding of the wild-type MLLT1 and MLLT3 but not mutant MLLT1 ( Figure 5C,D). Preincubation with SAHA increased t 1/2 to 0.7767 AE 0.09 sand 1.32 AE 0.45 sf or wild-type MLLT1 and MLLT3, respectively. Incubation of cells with compound 92 in presence of SAHA significantly decreased t 1/2 to 0.46 AE 0.05 sand 0.56 AE 0.02 sfor MLLT1 and MLLT3, respectively (MLLT1: P < 0.0001, MLLT3: P = 0.0185). On the other hand, t 1/2 of cells incubated in compound 91 was not significantly different from that of wild-type MLLT1 or MLLT3 in the presence of SAHA ( Figure 5C,D). We also developed af ull length MLLT1:Histone 3.3 (H3.3) NanoBRET assay to test compound 92. [31] Although the MLLT1 NanoBRET assay was responsive to SAHA treatment, showing as ignificant increase in BRET activity (mBU) relative to the DMSO control, there was no reduction in mBUi nr esponse to MLLT1/3 inhibitor treatment. ForMLLT3, the NanoBRET assay showed clear dosedependent displacement of full-length MLLT3-NanoLuc from histone H3.3-HaloTag (average IC 50 0.4 AE 0.08 mm)i n HEK293 cells ( Figure 5E and Figure S5).
We also investigated the metabolic stability of compound 92 in primary human hepatocytes.C ompound 92 shows moderate metabolic resistance (t 1/2 53 min, 48 %r emaining after 60 min), with ap rimary process for metabolism being Ndemethylation. An N-cyclopropyl indazole analogue,compound 94,d esigned to improve pharmacokinetics,l argely mitigates Ndealkylation observed and retains potent binding activity (MLLT1 K d 0.058 mm), which was also rationalised in ac o-crystal structure with MLLT1 YD (see the Supporting Information, PDB ID 6HT0), but the overall half-life of 94 was inferior to 92,with pyrrolidine oxidation occurring more rapidly (t 1/2 % 30 min). These analogues provide information on how to develop MLLT1/3 probes with good PK properties.
We have reported the discovery of the first small-molecule inhibitors and ac ell-potent, selective chemical probe for MLLT1 and MLLT3 YDs.Compound 1 was discovered as an initial hit in amedium-throughput biochemical screen. Simple synthesis allowed rapid generation of > 200 analogues and the chemical probe 92 (SGC-iMLLT)w ith its less active control 91 (SGC-iMLLT-N). Selectivity was demonstrated over acyllysine binding modules,Y EATS2/4, and 48 bromodomains.U sing orthogonal combinations of cell target engagement studies (NanoBRET,F RAP,a nd CETSA), submicromolar cellular activity was confirmed. SGC-iMLLT will enable researchers to design the first biological experiments exploring MLLT1/3 YD inhibition.