Spotlight pubs.acs.org/acschemicalbiology
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TRAPPING THE TRANSITION
moieties of biphenyl. In its ground state, this compound positions the two phenyl groups at 45° from one another, but to racemize a chiral version of the compound, it must go through a transition state with either a 0° or 90° juxtaposition of the phenyl rings. To hunt for a way to stabilize the coplanar 0° TS, the researchers chose protein as a suitable scaffold and used the algorithm Rosetta to find a spot in a stable thermophilic protein’s core where a biphenyl moiety could be accommodated and form π interactions with nearby aromatic residues. A threonyl-tRNA synthetase was identified as a good candidate, and the biphenylalanine residue was incorporated site-specifically by use of a nonsense codon in the mRNA and an orthogonal suppressor tRNA/synthetase pair for delivering this unnatural amino acid to the ribosome. Using computational predictions to model the new side chain’s surroundings, the nearby amino acids were mutated in an effort to coax the biphenyl group into a coplanar state. Continued computational predictions were helpful in guiding an iterative process of mutations coupled with X-ray crystallography for structure determination. After several rounds, the high resolution structure of BIF_0 exhibits the high energy coplanar biphenyl side chain, indicating that the racemization TS can indeed be trapped by effectively mutating the solvent surroundings in the form of amino acid side chains. Jason G. Underwood, Ph.D.
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LOSING PKC ACTIVITY DRIVE CANCERS
From Pearson et al., Science, 2015, 347, 863. DOI: 10.1126/ science.aaa2424. Reprinted with permission from AAAS.
In a chemical reaction, the transition state (TS) represents a high energy, short-lived species that is structurally distinct from both the reactants and the products. In biological systems, enzymes catalyze chemical reactions by binding their substrate in a manner favoring the TS, thus lowering the energy barrier required to pass from substrate to product. Stable mimics of transition states bound to enzymes have been observed by X-ray crystallography, but seeing the chemical snapshot of a real TS is far more difficult since the lifespan of this species is often just a handful of femtoseconds, requiring ultrafast spectroscopic techniques. Now, Pearson et al. (Science 2015, 347, 863−867) take on this challenge by redesigning the solvent surroundings of a chemical reaction to trap a TS. The reaction of interest was the rotation of a C−C bond that lies between the two phenyl © 2015 American Chemical Society
Reprinted from Cell, 160, Antal. et al., Cancer-Associated Protein Kinase C Mutations Reveal Kinase’s Role as Tumor Suppressor, 489−502. DOI: 10.1016/j.cell.2015.01.001. Copyright 2015, with permissions from Elsevier.
The human genome harbors nine genes encoding protein kinase C (PKC) family members, and these isozymes act on diverse signal transduction circuits. Activation of a PKC isozyme is achieved through a ligand, often partnered with Published: March 20, 2015 638
DOI: 10.1021/acschembio.5b00162 ACS Chem. Biol. 2015, 10, 638−640
ACS Chemical Biology
Spotlight
To identify new potential antibiotics, a research team led by Mayland Chang and Shahriar Mobashery at the University of Notre Dame performed docking-and-scoring analysis of the binding of a database of 1.2 million compounds to the active site of PBP2a (J. Am. Chem. Soc. 2015, 137, 1738−1741). Over 100 of the top-scoring compounds were screened for activity against a panel of bacteria; then, 80 analogs of the bestperforming compound were synthesized and subsequently screened. This resulted in the discovery of (E)-3-(3carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one as a potent antibiotic that was demonstrated to be active even against vanomycin- and linezolid-resistant MRSA strains, among others. The authors found that the antibiotic was readily bioavailable, had favorable pharmacokinetic and toxicity profiles, and had excellent efficacy in a mouse model of MRSA infection. The team also observed that the molecule inhibited both PBP2a and PBP1 in vitro; a crystal structure showed that the antibiotic bound to an allosteric site in PBP2a. The researchers argued that the antibiotic inhibited active sites as well, since PBP1 lacks allosteric regulation. Regardless of the exact mechanism by which it works, the authors emphasize that because the quinazolinone is not a β-lactam, it can at least evade the existing mechanism by which β-lactam-resistance arises. Heidi A. Dahlmann, Ph.D.
G-protein-coupled receptor signals, resulting in generation of the lipid second messenger diacylglycerol. The downstream functional effects are often cell-type specific and range from muscle contraction to neuronal excitation. Due to its activation by tumor-promoting phorbol esters, many have proposed that PKC enzymes may be involved in cancer progression, and some have even sought inhibitors as potential therapeutics. Recently, Antal et al. (Cell 2015, 160, 489−502) took a hard look at the many PKC coding variants uncovered from recent cancer sequencing projects and revealed a different side to the story. They found that the PKC loci are frequently hit with mutations in human cancers with no apparent hot spot in the primary sequence, nor a particular family member preference. To dive deeper into the effects of these mutations, 46 were chosen for further investigation, surveying the effects of single amino acid changes in seven PKC family members. Using a FRET-based cellular reporter assay, the effects of each PKC mutation were quantified. Strikingly, 61% of the mutations were loss-of-function, and none were activating. Since most PKC mutations are found to be heterozygous, the researchers went on to use the CRISPR-Cas9 system to correct or to knock out the mutant allele in a cancer line. Together, these results showed that having just one functional copy of PKC is not sufficient and that mutant proteins sometimes act in a dominant-negative fashion to promote tumor growth. Interestingly, an analysis of which other genes are mutated in human cancers harboring a PKC mutation showed that KRAS and TP53, genes well established to be involved in oncogenic transformation, are often hit in the same tumors. Thus, a dead or mutant allele for PKC may allow other oncogenic pathways to run wild in the cell. This study indicates how sequence data coupled with functional characterization can lead to powerful hypotheses, sometimes overturning the conventional thinking on a subject. Jason G. Underwood, Ph.D.
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(−)-ENGLERIN A MODE OF ACTION ELUCIDATED
A NEW WAY TO FIGHT MRSA
Reprinted with permission from Bouley, R., et al. J. Am. Chem. Soc., 137, 1738−1741. Copyright 2015 American Chemical Society
Reprinted from Angew. Chem. Int. Ed from Wiley-VCH Verla KGaA, Akbulut, Y., et al., 2015, DOI: 10.1002/anie.201411511
Renal cell carcinoma (RCC), an insidious malignancy that often goes undetected until it has metastasized, develops in the lining of tubules in the kidneys. The poor prognosis for latestage RCC patients is compounded by the lack of curative treatments; right now, the only mainline treatments are complete or partial removal of the affected kidney and administration of kinase inhibitors that target angiogenesis. Thus, when Beutler et al. discovered in 2009 that the natural product (−)-englerin A was selectively toxic to renal cancer cells (Org. Lett. 2009, 11, 57−60), especially in comparison to normal kidney cells (Angew. Chem. Int. Ed. 2011, 50, 3998− 4002), researchers jumped on the lead. Englerin A was eventually proposed to activate a protein called PKCθ, triggering a signaling cascade leading to insulin
These days, outbreaks of methicillin-resistant Staphylococcus aureus (MRSA), sickening and killing susceptible patients in hospitals, abound. Once targeted with penicillin and other firstgeneration β-lactams, which inhibit penicillin-binding proteins (PBPs) critical for bacterial cell-wall biosynthesis, S. aureus strains have evolved to circumvent the activity of even “last resort” antibiotics including vancomycin, linezolid, daptomycin, or ceftaroline. Not only are these drugs sometimes ineffective for combating infections by the most aggressive strains of MRSA but most of them have the drawback of needing to be delivered intravenously. Thus, the search is on for novel active, orally bioavailable antibiotics for treating difficult MRSA infections. 639
DOI: 10.1021/acschembio.5b00162 ACS Chem. Biol. 2015, 10, 638−640
ACS Chemical Biology
Spotlight
resistance and glucose dependence. However, in recent studies, a research team led by Mathias Christmann, David J. Beech, and Herbert Waldmann found that the A498 cell line, which is extremely sensitive to englerin A, does not even express PKCθ (Angew. Chem. Int. Ed. DOI: 10.1002/anie.201411511). A report linking another protein, transient receptor potential canonical channel 4 (TRPC4), to RCC and the fact that TRPC4 is most highly expressed in A498 cells among all NCI60 cancer panel cell lines led the research team to investigate TRPC4. TRPC4, -5, and -1 assemble into homotetrameric and heterotetrameric calcium-permeable channels in the cell membrane, leading the research team to hypothesize that calcium influx may be related to englerin A activity. In fact, application of englerin A to TRPC heterotetramers in a model cell membrane system as well as to cells with TRPC4 or TRPC5 overexpression led to a dramatic influx of calcium across the membranes, which ultimately caused cellular calcium overload and cell death. The authors commented that in the future, englerin A and its analogs could be used as tool for studying TRPC4/5 channel activity; moreover, they suggest that TRPC channel activation may serve as a new target pathway for RCC drug discovery. Heidi A. Dahlmann, Ph.D.
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GLEEVEC BINDING SPECIFICITY EXPLAINED The anticancer drug imatinib, also known as Gleevec, works by binding to a tyrosine-kinase called Abl, inhibiting phosphorylation and eventually causing cell death. Intriguingly, Gleevec binds to Abl with 3000-fold greater affinity than to the related Src kinase, even though cocrystal structures of both Gleevecbound kinases are nearly superimposable. Both kinases equilibrate between conformations in which a portion of the activation loop, known as the DFG loop, is either “in” or “out.” Gleevec is only able to bind to kinases in the DFG-out conformation. Thus, it was hypothesized that the difference in selectivity between kinases was related to their respective DFGconformation equilibration. However, efforts to pinpoint individual residues responsible for the different binding activity of Abl and Src, which differ by 146 amino acids, were unsuccessful. New research by a group led by Dorothee Kern, a Howard Hughes Medical Institute researcher at Brandeis University, greatly clarifies the mystery of Gleevec-binding selectivity (Science 2015, 347, 882−886). The research team analyzed the phylogenetic tree of modern kinases to interpolate sequences of “ancestral” kinases bridging the gap between Abl and Src. Kern and co-workers expressed four possible ancestral proteins and found that all were catalytically active, with sensitivities to Gleevec ranging between those of Abl and Src. By carefully analyzing the kinetics of Gleevec binding and dissociation in each kinase, the research team discovered that Gleevec activity was not so much controlled by association with the DFG-out conformation as much as by the rate of a second conformational change that each kinase undergoes after substrate binding. Ultimately, the team used their ancestral reconstruction to identify specific sites in the kinases’ “P-loop” domain that control the ability of kinases to undergo the critical second conformational change. Heidi A. Dahlmann, Ph.D.
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DOI: 10.1021/acschembio.5b00162 ACS Chem. Biol. 2015, 10, 638−640
