Synthesis, Optimization, and Evaluation of Novel Small Molecules as Antagonists of WDR5-MLL Interaction

Abstract

The WD40-repeat protein WDR5 plays a critical role in maintaining the integrity of MLL complexes and fully activating their methyltransferase function. MLL complexes, the trithorax-like family of SET1 methyltransferases, catalyze trimethylation of lysine 4 on histone 3, and they have been widely implicated in various cancers. Antagonism of WDR5 and MLL subunit interaction by small molecules has recently been presented as a practical way to inhibit activity of the MLL1 complex, and N-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides were reported as potent and selective antagonists of such an interaction. Here, we describe the protein crystal structure guided optimization of prototypic compound 2 (K dis = 7 μM), leading to identification of more potent antagonist 47 (K dis = 0.3 μM). KEYWORDS: WDR5, MLL, SET1 methyltransferase complex, peptide binding site, small molecule antagonists T he SET1-family of human histone methyltransferases (SET1A, SET1B, MLL1, MLL2, MLL3, and MLL4) contribute to active chromatin via mono-, di-, and trimethylation of lysine 4 on histone H3. 1−3 MLLs are S-adenosyl methionine (SAM) dependent lysine methyltransferases that work optimally when in complex with other components; WDR5 (WD40 repeat protein 5), RbBP5 (retinoblastomabinding protein-5), ASH2L (absent small homeotic disks-2-like), and DPY-30 (WRAD). 4 WDR5 has been shown to be essential for complex integrity and for fully activating MLL methyltransferase function. 5−7 In the peptide-bound structure, WDR5 adopts a donutshaped WD40-repeat fold with a deep central cavity that normally accommodates an arginine side chain of interacting peptides. 8 WDR5 binds to the "WDR5 INteracting" (WIN) motif of MLL1 through the peptidyl arginine-binding cleft. 8−12 A crystal structure of WDR5 in complex with MLL1 WIN peptide revealed the critical role of arginine 3765 of MLL1 in complex formation. 9 A similar interaction has been reported for WDR5 and the WIN region of all SET1 family lysine methyltransferases, with reported K D values of 1.13, 0.16, 2.03, 0.03, 0.26, and 0.10 μM for MLL1-4, SET1A, and SET1B WIN peptides, respectively. 13 Although there is significant divergence in the amino acid sequences of WIN motifs close to the critical arginine residue, the plasticity of the WDR5 arginine binding pocket allows such an interaction with the WIN motifs of all MLLs. 13 MLLs have been reported to be essential for embryonic development and biological processes 14−17 and have been widely implicated in a variety of cancers. 18−22 As almost all MLLs show minimal or no activity in the absence of the complex components (WDR5 in particular), 13 identifying WDR5−MLL interaction antagonists has been proposed as a potentially selective alternative to active site directed inhibitors of MLL methyltransferase activity. Such antagonists could serve as a starting point for the development of potential therapeutics to treat MLL-rearranged leukemias or other related cancers. 23 Recent reports detailing the use of short arginine contain...

CCI-007, a novel small molecule with cytotoxic activity against infant leukemia with MLL rearrangements

“…The rapid response to CCI-007 treatment in the sensitive MLL-r cell lines, involving a marked reduction in HOXA9 , MEIS1, CMYC and BCL2 mRNA levels within the first hours after treatment, a concomitant decrease in protein levels and induction of caspase-dependent apoptosis within 24 hours after treatment, is in sharp contrast to the effects of newly developed MLL1-complex inhibitors that target proteins that are recruited by the aberrant MLL1 fusion proteins or germline MLL1 protein such as Dot1L, Menin or WDR5. While each of these inhibitors has been described to decrease mRNA levels of HOXA9 , MEIS1 or other MLL target genes, the reduction in expression of these genes requires several days of treatment to become apparent [53, 54, 59, 61–65]. Moreover, in contrast to the rapid induction of caspase-dependent apoptosis with minimal effects on cell cycle progression seen following CCI-007 treatment, the primary effects of these MLL1-complex inhibitors include induction of cell cycle block and an increase in differentiation which typically occur over the time course of several days [53, 54, 59, 61–65].…”

“…While each of these inhibitors has been described to decrease mRNA levels of HOXA9 , MEIS1 or other MLL target genes, the reduction in expression of these genes requires several days of treatment to become apparent [53, 54, 59, 61–65]. Moreover, in contrast to the rapid induction of caspase-dependent apoptosis with minimal effects on cell cycle progression seen following CCI-007 treatment, the primary effects of these MLL1-complex inhibitors include induction of cell cycle block and an increase in differentiation which typically occur over the time course of several days [53, 54, 59, 61–65]. The mechanism of action of CCI-007 as well as its selectivity towards a subpopulation of MLL-r leukemia cells is thus clearly distinct from that of any of these recently developed MLL1-inhibitors.…”

“…Recent developments in targeted therapy for MLL-r leukemia have been mainly focused on inhibiting the interaction between MLL1 or the MLL1-fusion protein and collaborating binding partners such as the Disruptor of Telomeric Silencing 1-like (Dot1L) (EPZ4777/EPZ-5676) [52–56], the Multiple Endocrine Neoplasia (Menin) protein (MI-2/MI-3) [57–62] or the WD repeat-containing protein 5 (WDR5) [63–65]. When studied in vitro , these inhibitors typically reduce the number of MLL -translocated cells over a period of days due to their modes of action through epigenetic modification [53, 54, 59, 63, 64].…”

WD40 repeat domain proteins: a novel target class?

“…The WDR5 and EED inhibitors occupy the central pocket and bind with nanomolar potency 24–26 , while the other inhibitors occupy side cavities and bind in the micromolar range 89,90 (Figure 4a). OICR-9429, a potent and selective chemical probe targeting WDR5, was derived from a hit from a medium-throughput screen of a diverse library of 16,000 compounds using a fluorescence polarization assay to measure the displacement of MLL1 peptide, followed by multiple rounds of structure-guided optimization 24,91–93 . OICR-9429 binds WDR5 with an affinity of 50 nM as measured by isothermal titration calorimetry, inhibits the co-immunoprecipitation of an MLL peptide with WDR5 with an IC50 of 223 nM, and elicits a clear phenotypic response in cellular assays at 5 μM 24 .…”

Functional and Structural Characterization of Bub3·BubR1 Interactions Required for Spindle Assembly Checkpoint Signaling in Human Cells

“…targeting G␤␥, WDR5, or CDC20 (53)(54)(55)(56)(57)(58), meaning that such an endeavor could be rewarding in the case of Bub3. Of note, the first small molecule inhibitor targeting the WDR5-MLL protein-protein interaction by blocking the funnel of the WD40 protein WDR5 has been recently published (54,55) indicating that this might be a generally applicable principle. Along these lines, here we have devised a cellular system to investigate the feasibility and biological effects of putative Bub3 inhibitors in human tumor cells.…”