Spotlight pubs.acs.org/acschemicalbiology
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FLY MARKS MEET MASS SPEC
Reprinted with permission from Vianello, P. et al. J. Med. Chem., 2016, 59 (4), 1501−1517. Copyright 2016 American Chemical Society.
Reprinted with permission from Henry et al., Biochemistry 2016, DOI: 10.1021/acs.biochem.5b01070. Copyright 2016 American Chemical Society.
In eukaryotes, a repeating octamer of histone proteins packages the DNA into chromatin, the gatekeeper for DNA accessibility. Gene expression is finely regulated by post-translation modifications to histones, including methylation and acetylation of lysine residues. While many details of the histone code remain underexplored, global patterns have emerged for activation and repression. Additionally, altered acetylation patterns have been linked to cancer prognosis and other diseases. New methods continue to develop for understanding acetylation, but many have involved modification-specific antibodies and often suffer from a lack of either scalability or rigorous quantification. Now, Henry et al. (Biochemistry 2016, DOI: 10.1021/ acs.biochem.5b01070) introduce a quantitative mass spectrometry method to measure histone acetylation in fruit flies without antibodies. They first describe a procedure to acid-extract histone proteins from all the stages of Drosophila development, from ovaries through whole adults or adult tissues. They then used a mass spectrometer to simultaneously measure four different lysine acetylation events on the N-terminal tail of histone H3. While three of the lysines showed similar degrees of acetylation through development, the H3K18 mark increased as the organism progressed toward adulthood. With this method in hand, the researchers went on to test whether putative modulators of acetylation could be tested in adult flies by simply adding them to their daily diet. Their assay showed that modification of the four H3 lysines is altered to different extents when flies were exposed to a deacetylase inhibitor or the plant compound, curcumin. Finally, the study of acetylation turned to mutant fly strains with compromised DNA repair pathways. Irradiation of the flies induced DNA damage and increased the mark on H3K18 in wild type flies, but not in the repair mutants. The simplicity of the extraction combined with the quantitative nature of the data make this a valuable procedure with potential in other model organisms or in drug screens where histone modifications are the target. Jason G. Underwood © 2016 American Chemical Society
IMPROVED HISTONE DEMETHYLASE INHIBITOR INCREASES SURVIVAL IN MOUSE LEUKEMIA MODEL
Improvements to an anticancer drug undergoing preclinical development have recently been reported by a team of researchers in Italy (J. Med. Chem., 2016, 59 (4), 1501−1517). The new molecules are orally bioavailable and significantly extend survival in a mouse leukemia model relative to vehicle treated controls. High expression of histone lysine-specific demethylase 1A (KDM1A, or LSD1) has been linked to poor prognosis in several cancer types; therefore, KDM1A inhibitors have been hotly pursued as anticancer drug leads. Many drug discovery teams began by systematically modifying the monoamine oxidase (MAO) inhibitor tranylcypromine (TCPA), which is also known to inhibit KDM1A; two TCPA derivatives are currently in clinical studies. In their efforts to improve the performance of TCPA derivatives, the Italian research team focused on expanding the TCPA scaffold, seeking compounds with better selectivity for KDM1A as opposed to MAO and good in vitro activity toward leukemia cell model systems. The research team screened a panel of elongated TCPA derivates for their potency and selectivity toward isolated enzymes as well as their ability to inhibit colony formation in leukemia cell lines. The pharmacokinetics of the top candidates, initially tested as racemates, were assessed in mice, and compounds with the best oral bioavailability were selected for in vivo testing. Model mice were inoculated with leukemia cells, and after a 10-day incubation period they were orally dosed with the novel TCPA derivatives. The two enantiomers of the most effective compound, each prepared by enantioselective synthesis, were screened to determine the most active compound. The optimized derivative was well tolerated and prolonged the survival of treated mice up to 62% relative to vehicle-treated controls, prompting the authors to recommend further development of the derivative as a potential oral anticancer agent. Heidi A. Dahlmann
Published: March 18, 2016 544
DOI: 10.1021/acschembio.6b00219 ACS Chem. Biol. 2016, 11, 544−546
ACS Chemical Biology
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Spotlight
ORALLY BIOAVAILABLE SULFONAMIDE INHIBITS LYSINE METHYLTRANSFERASE SMYD3
A chemical probe designed to inhibit BRD9, a protein involved in recognizing chromatin post-translational modifications, has recently been developed by researchers at GlaxoSmithKline (J. Med. Chem. 2016, 59 (4), 1425−1439). The probe (I-BRD9), believed to be the first to selectively target BRD9 in preference to other closely related proteins, is expected to facilitate elucidation of BRD9’s cellular function and suitability as a target for anticancer therapy. The bromodomain and extra terminal domain (BET) family of bromodomains recognize acetylated lysine residues on histone tails; these modified amino acids recruit chromatin remodeling factors to regulate gene transcription. Inhibition of the BET bromodomains has been shown to have anticancer and antiinflammatory effects, making bromodomain-containing proteins (BCPs) an attractive target for drug-discovery programs. As bromodomains are highly conserved, many bromodomain inhibitors target multiple BCPs, making it difficult for researchers to home in on specific therapeutic targets. Thus, Humphreys and co-workers at GlaxoSmithKline had very specific demands for developing a molecule to probe the biological role of BRD9: the compound was required to be very potent against the BRD9 bromodomain and possess 100-fold and 30-fold selectivity over BET and non-BET bromodomains, respectively. By tailoring the size and hydrophobicity of substituents on a lead thienopyridone core structure, the research team developed I-BRD9, a probe that not only greatly exceeded the criteria for selectivity but also had favorable solubility and permeability. The research team tested the probe in a myeloid leukemia cell model, observing that I-BRD9 induced strong down-regulation of four genes previously implicated in cancer and immunology pathways but not previously known to be regulated by BRD9. Heidi A. Dahlmann
Reprinted with permission from Mitchell, L. H. et al. ACS Med. Chem. Lett., 2016, 7 (2), 134−138. Copyright 2016 American Chemical Society.
The concept that an organism’s phenotype is dictated by its DNA, which gets transcribed into RNA that is subsequently translated into proteins, has been undisputed for decades. This “central dogma” of biology could now arguably be expanded to include epigenetic regulation, or control of an organism’s gene expression by external factors that can be influenced by the organism’s environment. Epigenetic regulation occurs through a variety of mechanisms, including reversible DNA methylation and histone modification. The latter process is mediated by enzymes such as lysine methyltransferases (KMTs), which alkylate specific residues on histone proteins to turn gene expression on or off. High levels of a particular KMT known as Set and Mynd Domain containing 3 (SMYD3) are associated with poor prognosis for multiple cancer types, while genetic knockdown of SMYD3 decreases proliferation of several cancer cell lines. The attractiveness of SMYD3 as a potential therapeutic target prompted the recent exploration of small molecule inhibitors for the enzyme by researchers at Epizyme Inc. (ACS Med. Chem. Lett. 2016, 7 (2), 134−138). The research team screened the company’s library of histone methyltransferase inhibitors to identify an oxindole-based lead compound that inhibited SMYD3 at micromolar concentrations. A crystal structure of the lead compound in a ternary complex with SMYD3 and S-adenosyl-L-methionine revealed potential sites for inhibitor-enzyme interactions, enabling the research team to introduce specific substituents to their lead molecule to increase its potency. The two most potent derivatives were found to be noncompetitive inhibitors with good specificity for SMYD3 in comparison to a highly homologous SMYD protein. Furthermore, the inhibitors showed favorable metabolic stability and permeability in vitro and were orally bioavailable in mice; the authors note that these factors make their top-performing compound an ideal probe for future in vivo target validation studies. Heidi A. Dahlmann
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HOW HISTONES AND DEMETHYLATION ENZYMES FIT TOGETHER
Reprinted with permission from Burg, J. M. et al., Biochemistry, 2016, DOI: 10.1021/acs.biochem.5b01135, published online December 16, 2015. Copyright 2016 American Chemical Society.
THIENOPYRIDONE PROBE SELECTIVELY INHIBITS BRD9
Gene expression depends on the controlled spooling and unspooling of chromatin. This ultimately permits or blocks the access of transcriptional machinery to the genetic code contained in DNA. One of the elegant ways that our bodies control this “breathing” is by chemical modifications of histone proteins, one of the core building blocks of chromatin. Consequently, when the enzymes that install, erase, or read these histones’ post-translational marks are not regulated properly, they can lead to diseases such as cancer. Histones can include a plethora of chemical modifications, but only within the past few years have researchers identified enzymes that remove the methyl modification from the side chains of amino acids within histones. Now Burg and co-workers have offered clearer details of how one demethylation enzyme may recognize these histone proteins (Biochemistry, 2015, DOI: 10.1021/acs.biochem.5b01135).
Reprinted with permission from Theodoulou, N. H. et al. J. Med. Chem., 2016, 59 (4), pp 1425−1439. Copyright 2016 American Chemical Society. 545
DOI: 10.1021/acschembio.6b00219 ACS Chem. Biol. 2016, 11, 544−546
ACS Chemical Biology
Spotlight
So far, studies to better understand the substrate specificity of lysine-specific demethylase 1A (KDM1A) have led to slightly contradictory results. To efficiently catalyze demethylation, the enzyme requires a substrate of at least 21 amino acid residues representing the N-terminus of the histone H3. However, even though KDM1A has a large active site, steric limitations make it unlikely that this entire portion of histone H3 fits into the catalytic binding pocket. Therefore, the researchers speculated that histones may contact different regions of the KDM1A surface outside of the active site. To better understand how KDM1A binds to and recognizes substrates and products, Burg and co-workers designed a series of kinetic studies using peptide fragments and full-length histones. First, they looked at the catalytic activity of KDM1A and KDM1A complexes bound to constructs of CoREST1 in the presence of minimal N-terminal histone H3 peptides. These complexes showed negligible changes in catalysis as compared to wild-type KDM1A. They then looked at the kinetics of binding to full-length H3 and found that it binds tightly to KDM1A and is a competitive inhibitor of the enzyme. They independently confirmed this interaction using surface plasmon resonance experiments. The tight-binding affinity of histone H3 was in stark contrast to minimal peptides that have much more modest affinity for KDM1A. The results suggest that KDM1A may have a secondary binding site on its surface to accommodate additional residues of histone H3 that do not bind in the active site of the enzyme. This binding surface might aid in a “stick-and-catch” binding model proposed by others. In addition, this tight binding might play a role in bringing together partner proteins or other processes in the function and control of chromatin relaxation and tightening. Sarah A. Webb
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DOI: 10.1021/acschembio.6b00219 ACS Chem. Biol. 2016, 11, 544−546
