Spotlight pubs.acs.org/crt
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HYDRODYNAMICS IN POROUS HABITATS INFLUENCES BACTERIAL BIOFILM COMPETITION Most bacteria live in porous environments, surviving in the interstitial spaces in dense clusters known as biofilms. Each biofilm “patch” is made up of genetically identical bacterial cells and acquires nutrients from fluid flowing through the pores. Biofilm growth usually occurs at the top layer, where cells are exposed to nutrients; conversely, flow-induced detachment shears cells away from the biofilm surface. While environmental conditions influence the composition of microbial communities, it is unknown how changes in flow affect bacterial evolution. Using fluid dynamics and game theory, a group led by William M. Durham and Kevin R. Foster modeled the impact of porous hydrodynamics on bacterial growth and competition ((2017) PNAS, 114, E161−E170). For their analysis, the authors considered biofilm patch interactions to work in pairs. Using an Escherichia coli experimental system in which wild-type and mutant bacteria form biofilms at differing rates, they observed how flow affected biofilm growth. With low-flow, a rapidly expanding impermeable biofilm patch in a pore reduces flow through that pore and diverts it to a neighboring one, thus increasing flow access of a competing biofilm. With little flow-induced detachment, the fast-growing biofilm continued to expand, creating a positive-feedback loop which minimizes its own flow access and maximizes that of its competitor. These results suggest that rapid growth is not always a competitive advantage. The researchers developed a mathematical model to resolve how hydrodynamics affected biofilm competition dynamics. In strong flow, detachment dominated growth, favoring faster-growing biofilms due to increased cell dispersal; a weak flow permits growth over detachment, favoring slow-growing biofilms with their increased access to diverted flow. Intermediate levels of flow were found to promote the coexistence of cells that grow at different rates. This knowledge can be utilized in industrial settings to either favor a preferred bacterial species or allow diverse biofilm communities to remain metabolically active. Abigail Druck Shudofsky
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SYNTHESIS OF A FLUORESCENT CHLOROQUINE DERIVATIVE WHICH DETECTS PARASITIC STRAINS WITH REDUCED RESPONSIVENESS TO QUINOLINE ANTIMALARIAL DRUGS
is pH-dependent: the emission of 4-aminoquinoline is strongest at an alkaline pH, while that of NBD is highest at an acidic pH. The scientists determined that Fluo-CQ accumulated in the acidic digestive vacuole of CQ-sensitive P. falciparum-infected erythrocytes and emitted a strong fluorescence signal. This vacuolar fluorescence was only observed in cells infected with CQ-resistant parasites in the presence of a Pf CRT inhibitor, supporting the hypothesis that the mutated Pf CRT is responsible for Fluo-CQ efflux out of the digestive vacuole. As such, Fluo-CQ can act as a real-time reporter for Pf CRT-mediated CQ transport activity in living parasites. Abigail Druck Shudofsky
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Adapted from M. Jida et al. (2016) ACS Infect. Dis., DOI: 10.1021/acsinfecdis.5b00141. Copyright 2016 American Chemical Society.
Some strains of the Plasmodium falciparum parasite are resistant to antimalarial drugs such as 4-aminoquinoline and its derivatives. Quinoline drugs accumulate in the acidic digestive vacuole of the parasite where host hemoglobin digestion occurs. The antimalarial agents interfere with heme detoxification by preventing heme biomineralization. Quinoline-resistant parasites have polymorphisms in the gene encoding the P. falciparum chloroquine resistance transporter protein (Pf CRT) located at the digestive vacuole membrane. When mutated, the protein transports quinoline drugs away from the vacuole, rendering them ineffective. A group led by Elisabeth Davioud-Charvet and Michael Lanzer developed a novel fluorescent reporter (Fluo-CQ) by functionalizing the side chain of a short chloroquine (CQ) analogue with the fluorochrome 7-nitrobenzofurazan (NBD) ((2016) ACS Infect. Dis., DOI: 10.1021/acsinfecdis.5b00141). This fluorophore can diffuse across membranes and concentrate in the digestive vacuole, allowing for quick discrimination between Pf CRT wild-type and mutant plasmodial strains that are quinoline-sensitive and resistant, respectively. Fluo-CQ (see figure) contains two fluorescent active chromophores, 4-aminoquinoline (in blue) and NBD (in green). The researchers found that the fluorescence emission of each subunit © 2017 American Chemical Society
BRIEF TREATMENT WITH A SELECTIVE IMMUNOPROTEASOME INHIBITOR NONCYTOTOXICALLY SUPPRESSES T-CELL RESPONSES AFTER ALLOGRAFT TRANSPLANT
Structure courtesy of Dr. Gang Lin. Prolonged administration of broadly immunosuppressive drugs after organ transplant can lead to systemic cytotoxic complications. A new approach is to selectively target immune cells by inhibiting their proteasomes. While constitutive proteasomes (c-20S) are ubiquitously expressed, varying isoforms are differentially expressed in specialized cells, such as the immunoproteasome (i-20S) in T cells. Jamil Azzi, Gang Lin, Published: February 20, 2017 490
DOI: 10.1021/acs.chemrestox.7b00016 Chem. Res. Toxicol. 2017, 30, 490−491
Chemical Research in Toxicology
Spotlight
fluid and in a Caenorhabditis elegans model of Aβ42-mediated dysfunction. The researchers found that the 11 ligands inhibited both the primary and the secondary nucleation steps of Aβ42 aggregation in a molar ratio concentration-dependent manner and that 6 of them also inhibited the elongation step. After compound analysis, they found that small molecules with greater lipophilicity and steric bulk were more potent at inhibiting the primary and secondary nucleation steps of Aβ42 aggregation, while small molecules with a high relative aromaticity were more effective at inhibiting Aβ42 fibril elongation. This knowledge of how chemical features relate to inhibitory activity allows for targeted rational design for drug optimization against specific steps of Aβ42 aggregation.
Carl F. Nathan, and their collaborators investigated whether selective i-20S inhibitors could promote allograft acceptance with increased safety and efficacy compared to nonselective proteasome inhibitors which indiscriminately target c-20S and i-20S ((2016) PNAS, 113, E8425−E8432). The authors synthesized a reversible, noncovalent N,C-capped dipeptide i-20S inhibitor, DPLG3 (see figure), which inhibited i-20S over c-20S with thousands-fold selectivity, suggesting that its modulatory effect would be limited to immune cells. The scientists looked at the in vivo T-cell responses of mice treated with DPLG3 for 7 d after receiving skin grafts and found that they had decreased numbers of CD4 and CD8 effector T cells compared to that of controls. Mice which had undergone allograft heart transplants treated daily with DPLG3 for 14 d post-transplant had significantly prolonged allograft survival compared to that of the controls (13 d vs 7 d) along with decreased splenic CD4 and CD8 effector memory T cells. Heart allograft survival of murine transplant recipients treated synergistically with a single dose of the coinhibitory molecule CTLA4 Ig and 14 d of DPLG3 was >100 d, compared to 38.5 d with CTLA4 alone. The researchers discovered that i-20S expression increases in antigen-activated effector CD4 and CD8 T cells after allograft transplantation. As prolonged graft survival in transplant recipients requires impaired T cell responses, the increased expression of the drug target in these cells amplifies the selective impact of DPLG3. Taken together, these findings suggest a promising option for long-term allograft acceptance.
Abigail Druck Shudofsky
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MMV001239 structure courtesy of Dr. Sabine Ottilie.
The unicellular parasite Trypanosoma cruzi is the etiological agent of the deadly Chagas disease. The two available antitrypanosomal drugs have variable efficacy and safety, and while phenotypic screens can lead to new hit discovery, they do not identify the targets of the compounds. A group led by Elizabeth A. Winzeler successfully mapped the target of the antitrypanosomal benzamide compound MMV001239 in T. cruzi by utilizing directed evolution in Saccharomyces cerevisiae followed by whole-genome sequencing ((2016) ACS Chem. Biol., DOI: 10.1021/acschembio.6b01037). The authors found that the compound MMV001239 (see figure) was strongly cytotoxic in both T. cruzi and its surrogate species S. cerevisiae. The researchers performed directed evolution in the well-characterized haploid genome of S. cerevisiae to map the drug target. They isolated MMV001239resistant single-colonies and analyzed them using whole-genome sequencing. The scientists determined that the compound targets the product of ERG11, a gene involved in the biosynthesis of ergosterol, an essential component of the fungi and protozoan plasma membrane. ScERG11 and its T. cruzi orthologue TcCyp51 each encode the enzyme lanosterol-14-α-demethylase with a high degree of sequence similarity and functional conservation. The protein is a known azole drug target in both organisms. Further experiments with gas chromatography/mass spectrometry revealed that MMV001239 blocked the sterol biosynthesis pathway of T. cruzi in vivo by inhibiting TcCyp51. Using spectrophotometric binding assays and X-ray crystallography, the authors found the drug directly binds TcCyp51, likely through heme-iron coordination. The evolved mutated amino acids in ScERG11 which confer resistance to MMV001239 may interfere with inhibitor-heme binding by steric hindrance. This work demonstrates that resistance-conferring mutations in yeast can elucidate the targeted pathway of a drug and its binding site.
Abigail Druck Shudofsky
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MAPPING PARASITIC DRUG TARGETS USING DIRECTED EVOLUTION IN YEAST
QUANTITATIVE KINETICS DRUG DISCOVERY STRATEGY OPTIMIZES SMALL MOLECULES THAT TARGET SPECIFIC STEPS IN AMYLOID-β PEPTIDE AGGREGATION
Structures courtesy of Dr. Michele Vendruscolo.
The amyloid hypothesis states that amyloid-β peptide (Aβ) aggregation initiates molecular events which lead to the development of Alzheimer’s disease. The 42-residue form of Aβ, Aβ42, is a more hydrophobic form of the peptide and thus more prone to aggregation. A team led by Christopher M. Dobson, Tuomas P. J. Knowles, and Michele Vendruscolo developed a chemical kinetic-based drug discovery approach to systematically develop candidate compounds based on the quantitative changes they cause in each microscopic step of the Aβ42 aggregation reaction ((2017) PNAS, 114, E200−E208). As the small molecule retinoid X receptor (RXR) agonist bexarotene inhibits Aβ42 aggregation, the authors looked at other well-characterized agonists and antagonists of RXRs and their partners, retinoid A receptors (RARs). They assessed 12 small molecules in vitro for their effects on Aβ42 fibril formation and found that 11 of the compounds delayed Aβ42 aggregation, with the RAR agonist adapalene and the RAR antagonist MM11253 (see figure) the most effective molecules. Importantly, the scientists observed that adapalene was similarly effective at inhibiting Aβ42 aggregation in human cerebrospinal
Abigail Druck Shudofsky
491
DOI: 10.1021/acs.chemrestox.7b00016 Chem. Res. Toxicol. 2017, 30, 490−491
