Spotlight pubs.acs.org/crt
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MODIFICATION OF A TARANTULA TOXIN TO CREATE A MICROPROTEIN SODIUM CHANNEL INHIBITOR The human voltage-gated sodium channel 1.7 (Nav1.7) plays a major role in peripheral pain pathways; loss of function mutations lead to a lack of pain perception, while gain of function mutations cause painful disorders. Small natural peptide toxins known as microproteins are animal venom components which evolved to block ion channels. Pavel Strop and his collaborators used Ceratotoxin-1 (CcoTx1), a neuronal sodium channel inhibitor derived from tarantula venom, to engineer selective and potent molecules which target Nav1.7 ((2016) JBC, doi: 10.1074/jbc.M116.725978). The authors applied directed evolution to the disulfide-linked CcoTx1 to generate microprotein clones with greater Nav1.7 potency and specificity. They determined that the variant 2670 had characteristics which improved the most upon those of the starting molecule. To refine this peptide, they used a semirational drug design approach and performed single amino acid substitution saturation mutagenesis to analyze the effects of varying amino acids on 2670. The scientists then assessed how microprotein activity was affected by post-translational modifications. They found that C-terminal amidation of 2670 increased potency and selectivity against Nav1.7, while introducing N-terminal pyroglutamate enhanced the binding strength of the variant. Solving the crystal structure of 2670 allowed the authors to identify residues that contribute significantly to the Nav1.7 binding and select point mutations that further improved potency. The final microprotein that the researchers engineered can be further developed for analgesic use. Abigail Druck Shudofsky
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CARBOXYLATE MOIETIES ALLOW NONCLASSICAL ANTIFOLATES TO DIFFUSE ACROSS BACTERIAL CELL WALLS
enzyme. Modifying the para-COOH scaffold by altering the location of the hydrogen, methyl configuration, and methoxy substitution produced different activity results depending on the DHFR species. Crystal structures of the PLA-COOH compounds 3c and 3d bound to S. aureus DHFR demonstrate differential binding modes likely due to the varying locations of the methoxy substitution, but both involve water-mediated contacts between the carboxylate and enzymatic active site residues. These PLA-COOHs have a good metabolic profile, are noncytotoxic, and demonstrate potent antibacterial activity. Abigail Druck Shudofsky
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Courtesy of Dr. Amy C. Anderson.
The metabolic enzyme dihydrofolate reductase (DHFR) is an effective drug target. Classical antifolate drugs contain a basic nitrogenous headgroup and a glutamate tail that mimic the weakly basic pterin ring and negatively charged glutamate extension present in the natural DHFR substrates that are critical for enzymatic binding. While these drugs have a high affinity for the reductase, they have limited cell membrane permeability and must enter cells through active transport, a severe limitation for use in bacteria, which lack folic acid transporters. A team led by Amy C. Anderson and Dennis L. Wright developed a class of nonclassical propargyl-linked antifolates (PLAs) that passively diffuse through cell membranes and inhibit DHFR in both Gram-positive and Gram-negative bacteria ((2016) ACS Med. Chem. Lett., DOI: 10.1021/acsmedchemlett.6b00120). To increase the bacterial cell permeability of existing PLAs such as UCP1021, the authors replaced the pyridyl function with a carboxylic acid designed to form hydrogen bonds with a basic residue in the DHFR active site. They synthesized eight PLACOOHs containing a C6-ethyl diaminopyrimidine ring that was either methyl-substituted or unsubstituted at the propargylic position and a biphenyl system with either 2′- or 3′-methoxy substituents. The researchers found that placing the carboxylic acid group in the para position resulted in the strongest affinity to Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus DHFRs, though it sacrificed selectivity over the human © 2016 American Chemical Society
INHIBITING TOPOISOMERASE 1 SUPPRESSES PATHOGEN-INDUCED INFLAMMATORY GENE EXPRESSION AND PROTECTS AGAINST LETHAL INFLAMMATION
Camptothecin. National Center for Biotechnology Information, PubChem Compound Database; CID = 24360, https://pubchem. ncbi.nlm.nih.gov/compound/24360 (accessed Jun 16, 2016).
Activation of the innate immune response occurs upon host recognition of microbial structures known as PathogenAssociated Molecular Patterns (PAMPs). The induction and expression of PAMP-response genes is critical for host defense Published: July 18, 2016 1085
DOI: 10.1021/acs.chemrestox.6b00220 Chem. Res. Toxicol. 2016, 29, 1085−1086
Chemical Research in Toxicology
Spotlight
vesicles. As such, while SiNPs are generally considered to be well-tolerated in mammalian cells, their lysosomal accumulation impairs organelle function and may cause adverse effects on autophagy-mediated protein clearance and cellular metabolic activity. Abigail Druck Shudofsky
against pathogens. Nevertheless, these genes must be regulated to prevent a hyperactive immune response. Ivan Marazzi and his team demonstrated that the Topoisomerase 1 (Top1) enzyme transcriptionally activates pro-inflammatory genes during microbial infection and that its inhibition restricts inflammatory gene overexpression ((2016) Science, DOI: 10.1126/science.aad7993). The authors discovered that the quinoline alkaloid inhibitor camptothecin, which specifically targets Top1, was effective in vitro against PAMP-mediated gene expression induced by influenza A, Ebola, and Sendai viruses. They subsequently assessed differential gene expression in influenza-infected cells with and without Top1 and discovered that 84 genes were downregulated in Top1 depleted cells; these genes were predominantly induced in response to infection and encoded inflammatory cytokines. Further work demonstrated that Top1 is necessary for viral PAMP-induced gene upregulation as it creates an environment for RNA polymerase II binding at PAMP-responsive gene promoters in infected cells, facilitating inflammatory gene expression. Inhibiting Top1 diminishes the immune response associated with microbial recognition. Similarly, Top1 is required for the activation of genes induced by bacterial PAMPs and inflammatory cytokines. The researchers hypothesized that inhibiting Top1 in vivo can protect against an overactive immune response. Indeed, they found that pretreating animals with camptothecin suppressed inflammatory cytokine expression and rescued 90% of mice from lethal endotoxic shock. Furthermore, therapeutic Top1 inhibition protected 70% of mice infected with Staphyloccocus aureus, 94% of mice coinfected with influenza and S. aureus, and 90% of mice with acute liver-failure caused by high levels of cytokine secretion, indicating its ability to treat diseases characterized by aggravated immune responses. Abigail Druck Shudofsky
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DEVELOPMENT OF A RAPID, NONINVASIVE NEWBORN SCREEN FOR NIEMANN-PICK DISEASE TYPE C
Courtesy of Dr. Daniel S. Ory.
The fatal neurodegenerative Niemann-Pick disease type C (NPC) is a cholesterol storage disorder caused by loss of function mutations in the structural lysosomal protein NPC1 or the soluble cholesterol binding protein NPC2. The accumulation of cholesterol and sphingolipids in the lysosomes of NPC patients is associated with neuronal cell loss. Delays in diagnosing this rare progressive disease average 4−5 years, denying opportunities for intervention. Diagnosis in infants would allow for early disease intervention, but current screening approaches are invasive, lengthy, and often inconclusive. A group led by Daniel S. Ory established bile acids as markers for NPC and developed a blood spot-based diagnostic assay for use in newborn screening ((2016) Sci. Transl. Med., 8, 337ra6). The researchers looked at the metabolic profile of bile acids in the plasma of NPC1 patients and discovered two bile acid species that were increased 41- and 144-fold compared to control samples. High-resolution mass spectrometry along with fragmentation pattern analysis identified the two compounds to be 5α-cholanic acid-3β,5α,6β-triol (bile acid A) and 5α-cholanic acid-3β,5α,6β-triol N-(carboxymethyl)-amide (bile acid B). Levels of both were markedly elevated in dried blood spots of NPC1 patients compared to those of healthy controls. The scientists developed and validated a two-tiered high-throughput liquid chromatography/tandem mass spectrometry assay to selectively screen blood spots for NPC subjects and carriers using bile acid B as a specific NPC1 marker. This screen had 100% specificity and sensitivity in a sample size of over 5100. Broad NPC screening of newborns would allow for intervention before the onset of neurological symptoms, potentially modifying disease course and prolonging life. Abigail Druck Shudofsky
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LYSOSOMAL ACCUMULATION OF NANOPARTICLES IMPAIRS AUTOPHAGY-MEDIATED PROTEIN DEGRADATION Nanoparticles (NPs) are promising biomedical tools for disease detection and treatment. NPs interact with cellular membranes and then are often internalized by endocytosis and delivered to the endolysosomal system, where they accumulate. As the effects of NPs on cell physiology are not completely understood, a team led by Volker Haucke investigated the biological effects of NP use ((2016) JBC, DOI: 10.1074/jbc.M115.710947). The authors used siRNA knockdown to determine that uptake of dye-labeled silica NPs (SiNPs) into human cervix carcinoma cells was primarily dependent on dynamin 2-mediated caveolar endocytosis. Once internalized, the SiNPs accumulate prominently within late endosomes and lysosomes. Lysosomal accumulation of SiNPs was associated with reduced metabolic activity but not cell death. The scientists sought to determine if the high SiNP levels disturbed lysosomal function, which includes fusing with and removing autophagosomes, and found that SiNP-treated cells displayed increased autophagosomal accumulation due to defective lysosomal degradation. Additionally, intralysosomal SiNP accumulation disrupted the ability of lysosomes to degrade internalized growth factors. The researchers found that this impaired lysosomal degradation results from an inability of SiNP-filled lysosomes to receive cargo delivered from autophagasomes and late endosomes. The authors hypothesize that lysosomes containing SiNPs are incapable of fusing with the membranes of these upstream 1086
DOI: 10.1021/acs.chemrestox.6b00220 Chem. Res. Toxicol. 2016, 29, 1085−1086
