Spotlight pubs.acs.org/acschemicalbiology
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NOVEL MULTIPLE-STAGE ANTIMALARIAL AGENT IN DEVELOPMENT
BUILDING A BIGGER PICTURE OF FAST PROTEIN FOLDING, ONE SNAPSHOT AT A TIME
Reprinted from Prigozhin, M.B., et al., Proc. Natl. Acad. Sci, U.S.A., DOI: 10.1073/pnas.1422683112. Copyright 2015 National Academy of Sciences, U.S.A. Reprinted by permission from Macmillan Publishers Ltd: Nature 522, 315, copyright 2015.
A novel antimalarial agent that is effective in multiple stages of the disease is now in advanced nonclinical development (Nature 2015, 522, 315). An international research team led by Kevin D. Read and Ian H. Gilbert reported that their 2,6disubstituted quinolone-4-carboxamide derivate, DDD107498, has broad therapeutic potential and a novel mode of action that may help combat drug resistance. The development of antimalarial agents with broad-spectrum activity is timely due to the rise in drug resistance among dangerous Plasmodium parasites that cause malaria, a disease that infects hundreds of millions and kills hundreds of thousands of people per year. The disease is difficult to target because of the complicated life cycle of Plasmodium; when the parasites are transmitted from mosquitoes to humans, they first infect liver cells and eventually infect red blood cells. The parasites can then transform to another form which can be taken up by mosquitoes during feeding and transmitted to another human host. Gilbert and co-workers discovered that DDD107498 had low-nanomolar activity in assays that modeled several stages of the Plasmodium life cycle, including the liver, blood, and mosquito-borne stages. The team also determined that the agent targets translation elongation factor 2 (eEF2), which drives GTP-dependent ribosome translocation along mRNA. Because protein synthesis is essential, this explains the multiple life-cycle stage activity of the compound. From a pharmacological perspective, the antimalarial agent performed well in metabolic stability and oral bioavailability assays and appeared to be nontoxic to human cells. The authors anticipate that DDD107498 would be reasonably affordable and that in combination therapy it could be effective in as low as a single dose.
Making predictions using computational chemistry is a valuable resource for researchers evaluating conformations and interactions in molecules ranging from small compounds to large protein complexes. Chemists continually modify modeling programs with the help of empirically derived data in a cyclic process by which experimental results are compared with computational predictions and vice versa. However, when it comes to elucidating very fast processes, such as protein folding, the ability of experimental chemists to measure local structure formation along with the rates of individual steps in a global conformational change is limited. Computational chemistry is currently powerful enough to predict the dynamics of fast protein folding, but verifying the accuracy of the predictions is still on the leading edge of protein folding experiments. A recent report by Martin Gruebele and co-workers at the University of Illinois at Urbana−Champaign illustrates how the problem of experimentally characterizing local contact formation during fast protein folding can be tackled by measuring the rates of a protein labeled with different contact probes (Proc. Nat. Acad. Sci., U.S.A., 2015, DOI: 10.1073/ pnas.1422683112). The research team investigated the fivehelix bundle protein λ6−85 by engineering tryptophan and tyrosine probes pairwise into adjacent helices, denoting the mutant proteins λij, in which i and j indicate the helices bearing a modification. In these constructs, the fluorescence of the tryptophan residue was modulated by the proximity of the quencher tyrosine residue, reporting on the formation of local contacts between different helix pairs. By monitoring the probe fluorescence during temperature-jump experiments on thermally stable mutants λ12, λ13, and λ32, the authors detected that λ13 had a slower relaxation time than did λ12 and λ32. Comparison of these results with a previously published molecular dynamics (MD) simulation allowed the research team to determine that helix 1−3 contact formation occurs slowly while helix 2 forms rapid transient contacts with helices 1 and 3. The authors comment that a higher resolution picture of multistate protein folding can be successfully developed by mapping contacts
Heidi A. Dahlmann
Published: July 17, 2015 © 2015 American Chemical Society
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DOI: 10.1021/acschembio.5b00516 ACS Chem. Biol. 2015, 10, 1577−1579
ACS Chemical Biology
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methylated histones, called H3K9me3, to control chromatin compaction and repress gene expression. Monomeric chromatin effectors are known to have relatively weak dissociation constants, so how they are able to localize with their specific biomolecular targets has been a bit of a mystery. Furthermore, studying effector binding in cells is complicated because chromatin quantity, density, and alkylation status cannot be simultaneously measured or controlled, prompting Beat Fierz and co-workers at the Swiss Federal Institute of Technology in Lausanne to attack the problem with a cell-free approach (Nat. Commun. 2015, 6, 7313, DOI: 10.1038/ncomms8313). The research team developed a chemically defined model system in which H3K9me3-modified chromatin was immobilized on a slide and exposed to fluorophore-labeled HP1α; effector-chromatin binding was detected using a technique called single-molecule total internal reflection fluorescence microscopy, which allowed the researchers to measure how long individual HP1α subunits or multimers remained bound to their target. This ultimately led to the discovery that multivalent HP1α-dimers had increased effector binding affinity relative to HP1α monomers due to an elevated association rate constant. The rate of HP1α association could also be elevated by exposing HP1α to a peptide derived from hSgoL1, a protein known to interact with the HP1α chromoshadow domain, which controls HP1α dimerization. The authors speculate that modulation of effector multivalency may be a general mechanism by which cells control dynamic protein recruitment to chromatin.
within a protein by utilizing contact probes in combination with MD modeling. Heidi A. Dahlmann
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EFFECTOR-CHROMATIN BINDING VIEWED AT MOLECULAR LEVEL
Heidi A. Dahlmann
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USING ALDEHYDE CAPTURE TO MAKE PEPTIDE BONDS
Reprinted with permission from Raj, M. et al., J. Am. Chem. Soc., 137, 6932−6940. Copyright 2015 American Chemical Society.
The ability to construct large synthetic peptides is a valuable tool both to understand the underpinnings of living systems and to develop new drugs. However, researchers have always faced obstacles in constructing large peptides from amino acids. In a new study, Raj et al. (J. Am. Chem. Soc. 2015, 137, 6932−6940) present aldehyde capture ligation (ACL), a new methodology that expands synthetic options for building novel proteins. ACL uses the same fundamental design as other peptide ligation techniques such as native chemical ligation (NCL): During the reaction, the critical amine and carboxylate groups are latched together within a critical intermediate, leading to an intramolecular reaction to form the amide bond. In ACL, the chemoselective reaction occurs between an ortho-benzaldehyde selenoester and the desired amine. To design this reaction, Raj et al. started with o-benzaldehyde esters and amines, but by using thioesters and eventually selenoesters instead, they sped up the reactions so that they occurred in less than 2 min. Their mechanistic studies support the hypothesis that these reactions primarily occur via a hemiaminal intermediate. One potential side reaction in peptide ligations is the epimerization of amino acids. Using an HPLC
Reprinted by permission from MacMillan Publishers Ltd.: Nat. Commun. Kilic, S. et al., 6, 7313, DOI: 10.1038/ncomms8313, copyright 2015. Available under the Creative Commons Attribution 4.0 International License (2015) (http://creativecommons.org/licenses/by/4.0/).
DNA molecules are extremely long; to be accommodated into cell nuclei, they are wrapped around histone proteins to form condensed chromatin fragments. The association of histone proteins with DNA is governed by electrostatic interactions between positively charged histone amino acid residues and the negatively charged DNA phosphodiester backbone. Chromatin structure is modulated by enzymes that catalyze the addition of post-translational modifications (e.g., lysine methylation) to histone residues, thereby altering interactions with DNA as well as with other proteins, such as chromatin effectors. For example, heterochromatin protein 1α (HP1α) is recruited to particularly 1578
DOI: 10.1021/acschembio.5b00516 ACS Chem. Biol. 2015, 10, 1577−1579
ACS Chemical Biology
Spotlight
the effect of m6A in the identified target mRNAs. Interestingly, m6A increased the translation efficiency of mRNAs, and RNAimediated knockdown studies with both YTHDF1 and the RNA methylase METTL3 implicatd both the reader and the writer in the enhanced efficiency. Further experiments showed that the effect resides at the step of translation initiation, and artificially tethering YTHDF1 to an mRNA can have a similar stimulatory effect. Previous studies of another family member, YTHDF2, uncovered a role for this similar protein in promoting m6Adependent RNA decay, so together, these YTH-domain proteins control a regulatory circuit pivoting around the m6A mark.
assay, the researchers confirmed that the ligation reaction is rapid compared with epimerization, and even slower ligation reactions involving valine resulted in only 2% epimerized product. Most amino acid side chains are tolerated in this reaction, except for the carboxylates of C-terminal aspartic acid and glutamic acid. The reaction even proceeds with the secondary amino acid proline. In addition, Raj et al. tested ACL methodology by selectively labeling the N-terminus of ubiquitin, a protein with several lysine residues. As a result, they provided an example of protein ligation and demonstrated that they could selectively label an N-terminal amine without labeling amines on the lysine side chains. The selenoesters can be synthesized on the solid phase, which will allow researchers to further explore this method to produce large synthetic peptides.
Jason G. Underwood
Sarah A. Webb
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RNA METHYLATION GUIDES REGULATION
Reprinted from Cell, 160, Wang, et al., 489−502, DOI: 10.1016/j.cell.2015.05.014. Copyright 2015, with permission from Elsevier, Inc.
Covalent RNA modifications have long been known to play important roles in noncoding RNA biology, most dramatically in the case of tRNAs, but also for the ribosomal, small nuclear, and nucleolar RNAs. Methylation of mRNA was discovered decades ago, but the biological function of these covalent marks has remained a mystery. Recently developed techniques combining enrichment and high throughput sequencing have generated maps of N6-methyladenosine (m6A) locations along eukaryotic coding RNAs. This modified base is more commonly found in mRNA 3′ untranslated regions or near stop codons. Additionally, the methylation is reversible, so researchers have turned to studies of the readers, writers, and erasers to better understand the m6A code. Now, Wang et al. (Cell 2015, DOI: 10.1016/j.cell.2015.05.014) uncover a role for m6A in mRNA translation efficiency by studying the human methylation reader, YTHDF1. This protein was previously confirmed to possess m6A-binding activity, but in this study, a map of YTHDF1-bound sites was generated by an in vivo cross-linking-immunoprecipitation and deep sequencing workflow. Ribosome profiling was then used to better understand 1579
DOI: 10.1021/acschembio.5b00516 ACS Chem. Biol. 2015, 10, 1577−1579
