Structure-Based Optimization of a Small Molecule Antagonist of the Interaction Between WD Repeat-Containing Protein 5 (WDR5) and Mixed-Lineage Leukemia 1 (MLL1).

WD repeat-containing protein 5 (WDR5) is an important component of the multiprotein complex essential for activating mixed-lineage leukemia 1 (MLL1). Rearrangement of the MLL1 gene is associated with onset and progression of acute myeloid and lymphoblastic leukemias, and targeting the WDR5-MLL1 interaction may result in new cancer therapeutics. Our previous work showed that binding of small molecule ligands to WDR5 can modulate its interaction with MLL1, suppressing MLL1 methyltransferase activity. Initial structure-activity relationship studies identified N-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides as potent and selective antagonists of this protein-protein interaction. Guided by crystal structure data and supported by in silico library design, we optimized the scaffold by varying the C-1 benzamide and C-5 substituents. This allowed us to develop the first highly potent (Kdisp < 100 nM) small molecule antagonists of the WDR5-MLL1 interaction and demonstrate that N-(4-(4-methylpiperazin-1-yl)-3'-(morpholinomethyl)-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide 16d (OICR-9429) is a potent and selective chemical probe suitable to help dissect the biological role of WDR5.

Bromo-deaza-SAH: a potent and selective DOT1L inhibitor.

Chemical inhibition of proteins involved in chromatin-mediated signaling is an emerging strategy to control chromatin compaction with the aim to reprogram expression networks to alter disease states. Protein methyltransferases constitute one of the protein families that participate in epigenetic control of gene expression, and represent a novel therapeutic target class. Recruitment of the protein lysine methyltransferase DOT1L at aberrant loci is a frequent mechanism driving acute lymphoid and myeloid leukemias, particularly in infants, and pharmacological inhibition of DOT1L extends survival in a mouse model of mixed lineage leukemia. A better understanding of the structural chemistry of DOT1L inhibition would accelerate the development of improved compounds. Here, we report that the addition of a single halogen atom at a critical position in the cofactor product S-adenosylhomocysteine (SAH, an inhibitor of SAM-dependent methyltransferases) results in an 8-fold increase in potency against DOT1L, and reduced activities against other protein and non-protein methyltransferases. We solved the crystal structure of DOT1L in complex with Bromo-deaza-SAH and rationalized the observed effects. This discovery reveals a simple strategy to engineer selectivity and potency towards DOT1L into the adenosine scaffold of the cofactor shared by all methyltransferases, and can be exploited towards the development of clinical candidates against mixed lineage leukemia.

(R)-ß-lysine-modified elongation factor P functions in translation elongation.

Post-translational modification of bacterial elongation factor P (EF-P) with (R)-ß-lysine at a conserved lysine residue activates the protein in vivo and increases puromycin reactivity of the ribosome in vitro. The additional hydroxylation of EF-P at the same lysine residue by the YfcM protein has also recently been described. The roles of modified and unmodified EF-P during different steps in translation, and how this correlates to its physiological role in the cell, have recently been linked to the synthesis of polyproline stretches in proteins. Polysome analysis indicated that EF-P functions in translation elongation, rather than initiation as proposed previously. This was further supported by the inability of EF-P to enhance the rate of formation of fMet-Lys or fMet-Phe, indicating that the role of EF-P is not to specifically stimulate formation of the first peptide bond. Investigation of hydroxyl-(ß)-lysyl-EF-P showed 30% increased puromycin reactivity but no differences in dipeptide synthesis rates when compared with the ß-lysylated form. Unlike disruption of the other genes required for EF-P modification, deletion of yfcM had no phenotypic consequences in Salmonella. Taken together, our findings indicate that EF-P functions in translation elongation, a role critically dependent on post-translational ß-lysylation but not hydroxylation.

Small-molecule inhibition of MLL activity by disruption of its interaction with WDR5.

WDR5 (WD40 repeat protein 5) is an essential component of the human trithorax-like family of SET1 [Su(var)3-9 enhancer-of-zeste trithorax 1] methyltransferase complexes that carry out trimethylation of histone 3 Lys4 (H3K4me3), play key roles in development and are abnormally expressed in many cancers. In the present study, we show that the interaction between WDR5 and peptides from the catalytic domain of MLL (mixed-lineage leukaemia protein) (KMT2) can be antagonized with a small molecule. Structural and biophysical analysis show that this antagonist binds in the WDR5 peptide-binding pocket with a Kd of 450 nM and inhibits the catalytic activity of the MLL core complex in vitro. The degree of inhibition was enhanced at lower protein concentrations consistent with a role for WDR5 in directly stabilizing the MLL multiprotein complex. Our data demonstrate inhibition of an important protein-protein interaction and form the basis for further development of inhibitors of WDR5-dependent enzymes implicated in MLL-rearranged leukaemias or other cancers.

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

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).