1H NMR (400 MHz, MeOH-= 2

1H NMR (400 MHz, MeOH-= 2.5 Hz, 1H), 7.79 (dd, = 2.1, 1.4 Hz, 1H), 7.53 C 7.49 (m, 2H), 7.35 (dq, = 2.1, 1.1 Hz, 1H), 6.85 (d, = 8.7 Hz, 1H); 19F NMR (376 MHz, MeOH-= 477.7, 479.7 [M+Na]+; Purity (AUC) 95%. 3-((5-Bromo-2-hydroxyphenyl)sulfonamido)-2-hydroxy-5-(trifluoromethoxy)benzoic acid (6j) Step A: Methyl 2-hydroxy-5-(trifluoromethoxy)benzoate 2-Hydroxy-5- (trifluoromethoxy)benzoic acid (2.0 g, 9.00 mmol) was dissolved in DCM (30 mL) and MeOH (30 mL) and cooled to 0 C. the molecules disclosed can be used as starting points for future efforts toward compounds with improved drug-like properties. to the phenol of the sulfonyl ring (6d, Table 2); gaining additional hydrophobic contacts with WDR5 in the interface occupied by a valine side chain of Myc and RBBP5 (IDVV). Table 2. Optimization of salicylic acid derivatives, 6. carbon. Interestingly, while in the salicylic acid series, the cyclohexyl derivatives 6s, 6t exhibited high binding affinity, this pattern did not hold for the amide derivatives 7l, 7m. During this exercise we explored alternate amide groups, including 7n, 7o, but, with the exception of 7n, we observed very flat SAR when exploring different amine partners (data not shown). We hypothesize that this amide group is usually directed toward solvent and does not make positive interactions with the protein, which led to the choice of this vector for attaching the small molecule FITC-probes used for the assay (structure of probe shown in Supplemental Physique S1). In a separate salicylate replacement effort, we were able to demonstrate that this acid/amide moiety could be exchanged with a sulfone; for example, methyl sulfone made up of compounds 7p-7z were synthesized. Their design was driven in part to enhance physicochemical properties which might overcome some of the limitations observed in the salicylic acid subseries. Incorporation of the best-in-class pieces led to compounds such as 7w and 7z that bind with high potency to WDR5 and are comparable to analogs in the acid and amide series. Indeed, examples lacking the aniline-ring phenol were superior to the corresponding amide (compare 7b vs 7q, 7e vs 7t). Co-crystallization of WDR5 with 7x demonstrates that this sulfone also engages Q289 via a hydrogen bond (Physique 5F). Similar to the amides, the methyl group is usually directed toward bulk solvent offering a potential vector for future derivatization to tune the physicochemical properties. Phenol substitutions While we were able to discover small molecules that bind to WDR5 with excellent affinity, considering the shallow nature of the binding site, we recognize that these molecules retain functionality, such as phenols, that would likely occlude their development. Tables 2 and ?and33 already detail the synthesis of selected compounds with the aniline-ring phenol removed. Separately, we explored several strategies to remove or replace the (M)Papp (106/s)Papp (106/s)BL21 (DE3) cells. The overnight culture was used to start a 10 Adrafinil L fermentation (BioFlo 415, New Brunswick Scientific) produced at 37 C. For NMR samples, uniformly 15N-labeled protein was produced in minimal M9 medium, where 15NH4Cl (Cambridge Isotope Laboratories) and D-glucose were used as sole nitrogen and carbon sources. When the cell density reached OD600 = 2.5, the temperature was lowered to 30 C. The protein was expressed overnight with 1mM isopropyl–D-thiogalactoside (IPTG). Myc peptide (DEEEIDVVSVE) was ordered (Genscript) as HPLC purified synthetic polypeptide. It was dissolved in DMSO for further use. Cell pellets were dissolved in lysis buffer (1XPBS plus 300 mM NaCl, 20 mM imidazole, 5 mM BME, and10% glycerol), and broken by homogenization (APV-2000, APV). The lysate was cleared by centrifugation and filtering, and then applied to Adrafinil an affinity column (140 mL, ProBond, Invitrogen). Bound protein was eluted by an imidazole gradient. The His-SUMO-tag was Adrafinil removed by SUMO protease cleavage during dialysis and the subsequent subtractive second nickel-column. WDR5 protein was then purified by size-exclusion chromatography (HiLoad 26/60, Superdex 75, GE Healthcare) using NMR or crystallization buffer. HTS Screening. The Nedd4l previously described Myc peptide,16 was labelled with FITC and used as the probe for FPA assays. Adrafinil The probe.