idues that would be necessary to accommodate the bromide are energetically acceptable. To explore this possibility, we tested the ability of BrSAH to inhibit DNMT1, and determined an IC50 of 1.9 M. This suggests that binding of BrSAH to DNMT1 is accompanied by unfavorable conformational strain. Superimposing BrSAH on a cofactor molecule co-crystallized with the small molecule methyltransferase NNMT highlights moderate steric clashes with A169 that may be accommodated by the enzyme at an energetic cost. We verified that BrSAH inhibits NNMT with a moderate IC50 of 1.5 M. Finally, even though the design of BrSAH was 
derived from 5ITC, a potent inhibitor of the protein kinase haspin and adenosine kinase,14, 15 addition of the homocysteine tail is likely to preclude binding at ATP binding sites, and BrSAH is expected to be inactive against kinases. We confirmed that no binding to the haspin catalytic domain was observed with 10 M BrSAH by differential scanning fluorimetry. Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts 3. Discussion We have established that BrSAH is a novel, potent and selective DOT1L inhibitor. We note here that we were unable to find cellular activity reported for any close SAH analog against any methyltransferase, even though biochemically active compounds were reported in the literature.9, 19 We attribute this liability – an apparently general phenomenon – to the high polarity of SAH analogs, which prevents crossing cell membranes. The discovery of EPZ004777 as a potent SAM competitor teaches us an important lesson: DOT1L tolerates significant PNU-100480 web chemical modifications at the amino-acid end of the cofactor. Together, these observations outline a strategy towards the development of improved SAMcompetitive inhibitors, where addition of a bulky hydrophobic group PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19811292 such as a bromide or tri-fluoromethyl at the N7 position of the adenine ring enhances potency, increases Bioorg Med Chem. Author manuscript; available in PMC 2016 March 07. Yu et al. Page 5 selectivity, and reduces hydrophilicity of the adenosine moiety of SAH and chemical modifications at the homocysteine, such as the one observed in EPZ004777, increase hydrophobicity at the amino-acid end to further improve cell penetrance. Available structures clearly indicate the presence of a strong electrostatic potential at the amino-acid end of the native cofactor pocket that can only be satisfied with polar, and poorly bioavailable molecules. Overcoming this paradigm shared by SAM mimetics appears particularly challenging. Yet, EPZ004777, a hydrophobic SAM competitor that lacks the amino-acid moiety of the cofactor, contradicts this paradigm, suggesting that the cofactor pocket may adopt an altered conformation in the absence of the cofactor’s homocysteine moiety. The recent crystal structures of DOT1L in complex with 5-ITC and EPZ004777 confirm this hypothesis: a large loop immediately N-terminal to the conserved methyltransferase fold closes like a lid on the cofactor, but adopts dramatically altered conformations in the 5-ITC and EPZ004777 complexes 6,7. This loop motion significantly alters the size and electrostatics of the cofactor pocket, which may be sufficient to accommodate more hydrophobic and cell penetrant SAM analogs. Interestingly, this structural rearrangement observed in the DOT1L structure is reminiscent of another structural rearrangement reported for PRMTs: the alpha-X helix of CARM1, positioned at the N-terminus of the conserve