Sinefungin Derivatives as Inhibitors and Structure Probes of Protein Lysine Methyltransferase SETD2

Zheng, Weihong; Ibáñez, Glorymar; Wu, Hong; Blum, Gil; Zeng, Hong; Dong, Aiping; Li, Fengling; Hajian, Taraneh; Allali-Hassani, Abdellah; Amaya, M.F.; Siarheyeva, Alena; Yu, Wenyu; Brown, Peter J.; Schapira, Matthieu; Vedadi, Masoud; Min, Jinrong

doi:10.1021/ja307060p

Cited by 123 publications

(176 citation statements)

References 49 publications

Supporting

Mentioning

172

Contrasting

Order By: Relevance

“…The initial velocities were then plotted against the concentrations of SAM and H4K20 to generate double-reciprocal curves (Fig. S1 B and C) (44,45). Here, the linear regression converged in the second quadrant rather than on the y axis and thus excluded a ping-pong mechanism and a rapid-equilibrium ordered sequential mechanism (46).…”

Section: Resultsmentioning

confidence: 99%

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

Linscott

Kapilashrami

Wang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)-PKMT-substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (S N 2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MSbased method to measure KIEs and binding isotope effects (BIEs) of the cofactor S-adenosyl-L-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic 13 C KIE of 1.04, an inverse intrinsic α-secondary CD 3 KIE of 0.90, and a small but statistically significant inverse CD 3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical S N 2 transition state with the C-N and C-S distances of 2.35-2.40 Å and 2.00-2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steadystate kinetics with the SAM analog Se-adenosyl-L-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.S tepwise progression of an enzyme-catalyzed chemical reaction is accompanied by changes of bond orders and vibrational modes involved with specific atoms of the reactant(s) (1, 2). Such changes can be traced experimentally by measuring the ratios of turnover rates [kinetic isotope effects (KIEs)] or binding affinities [binding isotope effects (BIEs)] of the reactant(s) when the relevant atoms are replaced by heavy isotopes (3, 4). KIEs and BIEs are thus useful parameters for elucidating transition-state (TS) structures and catalytic mechanisms, which sometimes cannot be elucidated readily through sole measurement of steady-state kinetics (5-9). A sufficient set of KIEs and BIEs at the positions involved with bond motions can afford electrostatic and geometric constraints, when combined with computational modeling, to define an enzymatic TS (10-12). This information provides not only the atomic resolution of the transient structure at the highest energy summit along the reaction path, but also structural guidance for designing tight-binding TS analog inhibitors (13...

show abstract

Section: Resultsmentioning

confidence: 99%

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

Linscott

Kapilashrami

Wang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Given this similarity, we compared the structure of the SETD2 and Set2 SET domains to identify highly conserved residues for further study. The structure of the SET domain in SETD2 has been solved by crystallography (34), whereas the SET domain of Set2 was predicted here using I-TASSER (35)(36)(37)(38). When the predicted structure of the Set2 SET domain was aligned with the crystal structure of the SETD2 SET domain, the structures were strikingly similar (Fig.…”

Section: Setd2 and Set2 Share A High Degree Of Structural And Sequencmentioning

confidence: 93%

“…The primary sequences of the SET and SRI domains of the enzyme responsible for H3K36 methylation from H. sapiens, S. cerevisiae, X. tropicalis, D. melanogaster, D. rerio, and M. musculus were aligned using ClustalOmega (56) and annotated with reported SETD2 mutations in ccRCC (1,4,11,12). The structures of the SETD2 SET domain (34), SETD2 SRI domain (44), and Set2 SRI domain (43) have been reported previously. To predict the structure of the yeast SET domain, the amino acid sequence (UniProtKB, P46995) was submitted to I-TASSER using the default parameters (35)(36)(37)(38).…”

Section: Methodsmentioning

confidence: 99%

Structure/Function Analysis of Recurrent Mutations in SETD2 Protein Reveals a Critical and Conserved Role for a SET Domain Residue in Maintaining Protein Stability and Histone H3 Lys-36 Trimethylation

Hacker

Fahey

Shinsky

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumorsuppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer.Cancer is increasingly characterized by alterations in chromatin-modifying enzymes (1). SETD2, a non-redundant histone H3 lysine 36 (H3K36) 4 methyltransferase (2), has been found to be mutated in a growing list of tumor types, most notably in clear cell renal cell carcinoma (ccRCC) (1, 3, 4), but also in high grade gliomas (5), breast cancer (6), bladder cancer (7), and acute lymphoblastic leukemia (8 -10). Recent studies exploring intratumor heterogeneity in ccRCC identified distinct mutations in SETD2 from spatially distinct subsections of an individual tumor, suggesting that mutation of SETD2 is a critical and selected event in ccRCC cancer progression (11). Mutations in SETD2 are predominantly inactivating, such as early nonsense or frameshift mutations, which lead to nonfunctional protein and global loss of H3K36 trimethylation (H3K36me3) (4,11,12). Missense mutations tend to cluster in two domains (1,4,12,13): the SET domain, which catalyzes H3K36me3 (14), and the Set2 Rpb1 interaction (SRI) domain, which mediates the interaction between SETD2 and the hyperphosphorylated form of RNA polymerase II (RNAPII) (13).SETD2 and its yeast counterpart, Set2, both associate with RNAPII in a co-transcriptional manner (13,15,16). In yeast, Set2 mediates all H3K36 methylation states (H3K36me1/me2/ me3) (17) and regulates the recruitment of chromatin-remodeling enzymes (Isw1b) and a histone deacetylase (Rpd3) (18) that functions to keep gene bodies deacetylated, thereby maintaining a more compact chromatin structure (19,20) that is more resistant to inappropr...

show abstract

“…Interestingly, the autoinhibitory loop in the crystal structure of the related NSD3 SET domain is disordered [129], further suggesting that autoinhibitory loop dynamics are characteristic of the NSD and related SET domains. In the case of SETD2, both auto-inhibited and open conformations of the SET domain were observed by crystallography [122] (Figure 3B). When bound to S-adenosyl homocysteine (SAH), SETD2 adopts an autoinhibited conformation similar to NSD1 and ASH1L, with the sidechain of Arg1670 in the autoinhibitory loop extending into the putative substrate lysine-binding channel ( Figure 3B).…”

mentioning

confidence: 95%

H3K36 Methyltransferases as Cancer Drug Targets: Rationale and Perspectives for Inhibitor Development

2016

View full text Add to dashboard Cite

Methylation at histone 3, lysine 36 (H3K36) is a conserved epigenetic mark regulating gene transcription, alternative splicing and DNA repair. Genes encoding H3K36 methyltransferases (KMTases) are commonly overexpressed, mutated or involved in chromosomal translocations in cancer. Molecular biology studies have demonstrated that H3K36 KMTases regulate oncogenic transcriptional programs. Structural studies of the catalytic SET domain of H3K36 KMTases have revealed intriguing opportunities for design of small molecule inhibitors. Nevertheless, potent inhibitors for most H3K36 KMTases have not yet been developed, underlining the challenges associated with this target class. As we now have strong evidence linking H3K36 KMTases to cancer, drug development efforts are predicted to yield novel compounds in the near future. Cellular development and differentiation are controlled by post-translational modifications on DNA and histone proteins, forming an epigenetic (above the genes) regulatory network. Disruption of epigenetic pathways is a nearly universal feature of cancer, causing aberrations in gene expression and genome integrity (reviewed in [1]). A key group of epigenetic enzymes disrupted in cancer is the lysine methyltransferases (KMTases), which install methyl groups on histone proteins. In cancer cells, methylation performed by KMTases drives proliferation and halts differentiation by modifying gene transcription and other DNA-templated processes [2][3][4]. Over the past decade, KMTases have emerged as important drug targets for both industrial and academic research groups. Some progress has been made, most notably by selective inhibitors for the EZH2 and DOT1L KMTases that have reached clinical trials for the treatment of nonHodgkin lymphoma and acute leukemia, respectively [5,6]. Nevertheless, selective and cell permeable inhibitors for many KMTases remain unavailable.Methylation at H3K36 represents a particularly important chromatin mark implicated in diverse forms of cancer. KMTases with specificity toward H3K36 are overexpressed in cancer cells and have been characterized as regulators of cell growth, differentiation, stemness and DNA repair pathways [7][8][9]. However, very few selective and cell-active small molecule inhibitors of H3K36-specfic KMTases have been reported to date. In this article, we provide an overview of cellular pathways involving H3K36 methylation and discuss the diverse functions carried out by the eight different human H3K36-specific KMTases. Then we analyze structural characteristics of the catalytic SET domain of H3K36 KMTases and evaluate prospects for their inhibition by small molecules. Review Rogawski, Grembecka & Cierpicki We conclude with a discussion of the challenges and o pportunities for targeting these proteins. SPECIAL FOCUS y Epigenetic drug discovery H3K36 methylation regulates diverse processes implicated in cancerH3K36 methylation participates in a wide variety of nuclear pathways. Many of these processes, including transcriptional regulation, alternative spli...

show abstract

Sinefungin Derivatives as Inhibitors and Structure Probes of Protein Lysine Methyltransferase SETD2

Cited by 123 publications

References 49 publications

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

Structure/Function Analysis of Recurrent Mutations in SETD2 Protein Reveals a Critical and Conserved Role for a SET Domain Residue in Maintaining Protein Stability and Histone H3 Lys-36 Trimethylation

H3K36 Methyltransferases as Cancer Drug Targets: Rationale and Perspectives for Inhibitor Development

Contact Info

Product

Resources

About