Structure of the Epigenetic Oncogene MMSET and Inhibition by N-Alkyl Sinefungin Derivatives

Abstract

The members of the NSD subfamily of lysine methyl transferases are compelling oncology targets due to the recent characterization of gain-of-function mutations and translocations in several hematological cancers. To date, these proteins have proven intractable to small molecule inhibition. Here, we present initial efforts to identify inhibitors of MMSET (aka NSD2 or WHSC1) using solution phase and crystal structural methods. On the basis of 2D NMR experiments comparing NSD1 and MMSET structural mobility, we designed an MMSET construct with five point mutations in the N-terminal helix of its SET domain for crystallization experiments and elucidated the structure of the mutant MMSET SET domain at 2.1 Å resolution. Both NSD1 and MMSET crystal systems proved resistant to soaking or cocrystallography with inhibitors. However, use of the close homologue SETD2 as a structural surrogate supported the design and characterization of N-alkyl sinefungin derivatives, which showed low micromolar inhibition against both SETD2 and MMSET.

Identification of a peptide inhibitor for the histone methyltransferase WHSC1

“…Structure-based methods have been extremely useful in the development of several inhibitors to histone methyltransferases, such as DOT1L [ 19 – 23 ], EHMT1/2 (G9a/GLP) [ 24 – 30 ], and SMYD2/3 [ 31 – 37 ] and would be valuable for the NSD family as well. Autoinhibited structures of the NSD1 [ 38 ] and WHSC1 [ 39 ] SET domains have been solved along with a structure of the WHSC1L1 SET domain in an open loop conformation (PDB code = 5UPD, Structural Genomics Consortium). All of these structures have been solved in the presence of the substrate SAM, but to date no structure of an NSD protein has been solved in the presence of an enzyme inhibitor.…”

“…All of these structures have been solved in the presence of the substrate SAM, but to date no structure of an NSD protein has been solved in the presence of an enzyme inhibitor. Inhibitors based on sinefungin, a substrate analog of the cofactor SAM, have been reported for WHSC1 but structures of these compounds were reported in a closely related enzyme, SETD2, rather than in an NSD family protein [ 39 ]. Additional studies with two known inhibitors of G9a, UNC0638 [ 39 ] and BIX-01294 [ 40 ], have not shown experimental evidence of specific binding of these compounds to the active site of the NSD family members.…”

“…Inhibitors based on sinefungin, a substrate analog of the cofactor SAM, have been reported for WHSC1 but structures of these compounds were reported in a closely related enzyme, SETD2, rather than in an NSD family protein [ 39 ]. Additional studies with two known inhibitors of G9a, UNC0638 [ 39 ] and BIX-01294 [ 40 ], have not shown experimental evidence of specific binding of these compounds to the active site of the NSD family members. A specific inhibitor for any member of the NSD family would be a valuable addition to the chemical library for protein methyltransferases.…”

High-throughput screening with nucleosome substrate identifies small-molecule inhibitors of the human histone lysine methyltransferase NSD2

“…Likewise, the nonspecific histone lysine methyltransferase inhibitor chaetocin (IC 50 ϭ 3-6 M) showed similar inhibition of NSD1-3 (39). The natural product sinefungin is a close structural analog of SAM and a modest inhibitor of NSD2 (IC 50 ϭ 26 -30 M) (41,42). Structure-activity relationships have been reported for sinefungin analogs, the most potent of which inhibited the SET domains of NSD2 (IC 50 ϭ 1.8 M) and SETD2 (IC 50 ϭ 0.29 M) (41).…”

Small Molecule Inhibitors of the Human Histone Lysine Methyltransferase NSD2 / WHSC1 / MMSET Identified from a Quantitative High-Throughput Screen with Nucleosome Substrate

“…The natural product sinefungin is a close structural analogue of SAM and a modest inhibitor of NSD2 (IC 50 = 26 -30 µM) (41,42). Structure-activity relationships have been reported for sinefungin analogues, the most potent of which inhibited the SET domains of NSD2 (IC 50 = 1.8 µM) and SETD2 (IC 50 = 0.29 µM) (41).…”