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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

PRODUCT SPECIFICITY AND INHIBITION OF PROTEIN N-TERMINAL METHYLTRANSFERASE 1/2

Guangping Dong (11250960) 09 August 2021 (has links)
<div>Protein N-terminal methyltransferases (NTMTs) are a family of enzymes that methylate the α-N-terminus of a variety of protein substrates. Both NTMT1 and NTMT2 recognize a unique N-terminal X-P-K/R motif (X represents any amino acid other than D/E) to install 1-3 methyl group(s) on the substrates. NTMT1 plays important roles in mitosis regulation, chromatin interactions, and DNA damage repair. Another member NTMT2 shares ~50% sequence similarity and the same substrate recognition motif although NTMT2 was initially characterized as a mono-methyltransferase. To understand the molecular mechanism of NTMT2, we obtained the first co-crystal structure of NTMT2 in complex with its peptide substrate. After an extensive investigation of substrate recognition and methylated products of NTMT1/2, we found out that NTMT2 can fully methylate G/P-PKRIA peptides despite a predominant mono-methyltransferase. Moreover, we identified a gatekeeper N89 in NTMT2 that controls the substrate entry and the product specificity of NTMT2.</div><div>To elucidate the biological functions of NTMT1/2-catalyzed N-terminal methylation, we applied two different strategies to discover cell-potent inhibitors. Guided by the co-crystal structures of NTMT1 in complex with previously reported inhibitors, we designed and synthesized a series of new peptidomimetic inhibitors. By introducing more hydrophobic groups, the most cell-potent peptidomimetic inhibitor GD562 (IC50 = 0.93 ± 0.04 µM) exhibited over 2-fold increased inhibition on cellular N-terminal methylation levels with an IC50 value of ~50 µM compared to previously reported peptidomimetic inhibitor DC541. Meanwhile, we also discovered the first potent small molecule inhibitor Genz-682452 (IC50 = 0.5 ± 0.04 µM) after screening ~58,000 compounds. Subsequent structural modifications led to the discovery of GD433 (IC50 = 27 ± 0.5 nM) with a 20-fold increased potency compared to the initial hit Genz-682452. Inhibition mechanism indicated both inhibitors bind to peptide-binding pocket and co-crystal structures of both Genz-682452 and GD433 with NTMT1 confirmed their binding modes. Furthermore, GD433 shows over 7-fold selectivity over other major 40 protein methyltransferases and DNA methyltransferase and exhibits improved selectivity for NTMT1 over glucosylceramide synthase (GCS). GD433 significantly decreases the cellular N-terminal methylation level of NTMT1 substrates RCC1 and SET at 10 nM in both HEK293 and HCT116 cells, providing a valuable probe for cell-based studies in the future.<br></div><p><br></p>

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