Fluorescence Polarization Experiments Research Articles

Asymmetry of Dual-Display DNA-Encoded Chemical Libraries.

While dual-display DNA-encoded chemical libraries (DELs) are increasingly employed for ligand discovery, some of their fundamental properties have not yet been studied in-depth. Aided with fluorescence polarization experiments, we demonstrate that dual-display DELs are intrinsically asymmetrical entities, and we deduce practical guidelines to perform better-informed on-DNA hit validation from these libraries.

Functional Classification and Interaction Selectivity Landscape of the Human SH3 Domain Superfamily.

SRC homology 3 (SH3) domains are critical interaction modules that orchestrate the assembly of protein complexes involved in diverse biological processes. They facilitate transient protein-protein interactions by selectively interacting with proline-rich motifs (PRMs). A database search revealed 298 SH3 domains in 221 human proteins. Multiple sequence alignment of human SH3 domains is useful for phylogenetic analysis and determination of their selectivity towards PRM-containing peptides (PRPs). However, a more precise functional classification of SH3 domains is achieved by constructing a phylogenetic tree only from PRM-binding residues and using existing SH3 domain-PRP structures and biochemical data to determine the specificity within each of the 10 families for particular PRPs. In addition, the C-terminal proline-rich domain of the RAS activator SOS1 covers 13 of the 14 recognized proline-rich consensus sequence motifs, encompassing differential PRP pattern selectivity among all SH3 families. To evaluate the binding capabilities and affinities, we conducted fluorescence dot blot and polarization experiments using 25 representative SH3 domains and various PRPs derived from SOS1. Our analysis has identified 45 interacting pairs, with binding affinities ranging from 0.2 to 125 micromolar, out of 300 tested and potential new SH3 domain-SOS1 interactions. Furthermore, it establishes a framework to bridge the gap between SH3 and PRP interactions and provides predictive insights into the potential interactions of SH3 domains with PRMs based on sequence specifications. This novel framework has the potential to enhance the understanding of protein networks mediated by SH3 domain-PRM interactions and be utilized as a general approach for other domain-peptide interactions.

Inhibitors against Two PDZ Domains of MDA-9 Suppressed Migration of Breast Cancer Cells.

Melanoma differentiation-associated gene 9 (MDA-9) is a small adaptor protein with tandem PDZ domains that promotes tumor progression and metastasis in various human cancers. However, it is difficult to develop drug-like small molecules with high affinity due to the narrow groove of the PDZ domains of MDA-9. Herein, we identified four novel hits targeting the PDZ1 and PDZ2 domains of MDA-9, namely PI1A, PI1B, PI2A, and PI2B, using a protein-observed nuclear magnetic resonance (NMR) fragment screening method. We also solved the crystal structure of the MDA-9 PDZ1 domain in complex with PI1B and characterized the binding poses of PDZ1-PI1A and PDZ2-PI2A, guided by transferred paramagnetic relaxation enhancement. The protein-ligand interaction modes were then cross-validated by the mutagenesis of the MDA-9 PDZ domains. Competitive fluorescence polarization experiments demonstrated that PI1A and PI2A blocked the binding of natural substrates to the PDZ1 and PDZ2 domains, respectively. Furthermore, these inhibitors exhibited low cellular toxicity, but suppressed the migration of MDA-MB-231 breast carcinoma cells, which recapitulated the phenotype of MDA-9 knockdown. Our work has paved the way for the development of potent inhibitors using structure-guided fragment ligation in the future.

Two Binding Sites of SARS-CoV-2 Macrodomain 3 Probed by Oxaprozin and Meclomen.

Severe acute respiratory syndrome-coronavirus-1/2 (SARS-CoV-1/2) macrodomain 3 (Mac3) is critical for replication and transcription of the viral genome and is therefore a potential therapeutic target. Here, we solved the crystal structure of SARS-CoV-2 Mac3, which reveals a small-molecule binding pocket. Two low-molecular-weight drugs, oxaprozin and meclomen, induced different patterns of nuclear magnetic resonance (NMR) chemical shift perturbations (CSPs). Meclomen binds to site I of SARS-CoV-2 Mac3 with binding pose determined by NMR CSP and transferred paramagnetic relaxation enhancement, while oxaprozin binds to site II as revealed by the crystal structure. Interestingly, oxaprozin and meclomen both perturb residues in site I of SARS-CoV Mac3. Fluorescence polarization experiments further demonstrated that oxaprozin and meclomen inhibited the binding of DNA-G4s to SARS-CoV-2 Mac3. Our work identified two adjacent ligand-binding sites of SARS-CoV-2 Mac3 that shall facilitate structure-guided fragment linking of these compounds for more potent inhibitors.

Covalent Modification of the JH2 Domain of Janus Kinase 2.

Probe molecules that covalently modify the JAK2 pseudokinase domain (JH2) are reported. Selective targeting of JH2 domains over the kinase (JH1) domains is a necessary feature for ligands intended to evaluate JH2 domains as therapeutic targets. The JH2 domains of three Janus kinases (JAK1, JAK2, and TYK2) possess a cysteine residue in the catalytic loop that does not occur in their JH1 domains. Starting from a non-selective kinase binding molecule, computer-aided design directed attachment of substituents terminating in acrylamide warheads to modify Cys675 of JAK2 JH2. Successful covalent attachment was demonstrated first through observation of enhanced binding with increasing incubation time in fluorescence polarization experiments. Covalent binding also increased selectivity to as much as ca. 30-fold for binding the JAK2 JH2 domain over the JH1 domain after a 20-h incubation. Covalency was confirmed through HPLC electrospray quadrupole time-of-flight HRMS experiments, which revealed the expected mass shifts.

Cross-reactivity to glutamate carboxypeptidase III causes undesired salivary gland and kidney uptake of PSMA-targeted small-molecule radionuclide therapeutics.

Recently, Pluvicto™ ([177Lu]Lu-PSMA-617), a small-molecule prostate-specific membrane antigen (PSMA) radioligand therapeutic, has been approved by the FDA in metastatic castration-resistant prostate cancer. Pluvicto™ and other PSMA-targeting radioligand therapeutics (RLTs) have shown side effects due to accumulation in certain healthy tissues, such as salivary glands and kidney. Until now, the molecular mechanism underlying the undesired accumulation of PSMA-targeting RLTs had not been elucidated. We compared the sequence of PSMA with the entire human proteome to identify proteins closely related to the target. We have identified glutamate carboxypeptidase III (GCPIII), N-acetylated alpha-linked acidic dipeptidase like 1 (NAALADL-1), and transferrin receptor 1 (TfR1) as extracellular targets with the highest similarity to PSMA. The affinity of compound 1 for PSMA, GCPIII, NAALADL-1, and TfR1 was measured by fluorescence polarization. The expression of the putative anti-target GCPIII was assessed by immunofluorescence on human salivary glands and kidney, using commercially available antibodies. A fluorescent derivative of Pluvicto™ (compound 1) bound tightly to PSMA and to GCPIII in fluorescence polarization experiments, while no interaction was observed with NAALADL-1 and TfR1. Immunofluorescence analysis revealed abundant expression of GCPIII both in healthy human kidney and salivary glands. We conclude that the membranous expression of GCPIII in kidney and salivary gland may be the underlying cause for unwanted accumulation of Pluvicto™ and other Glu-ureido PSMA radio pharmaceuticals in patients.

A Chemosensor of 1,8-Dihydroxyanthraquinone PMOs Prepared in a Ternary Deep Eutectic Solvent for the Sensitive Detection of Cu2.

A novel type of organic–inorganic hybrid periodic mesoporous materials based on 1,8-dihydroxyanthraquinone (DHAQ-PMOs) was synthesized in a ternary deep eutectic solvent (DES). In the process of the material synthesis, an organosiloxane precursor (DHAQ-Si) and tetraethylorthosilicate (TEOS) were used as the mixed Si source, and cetyltrimethylammonium bromide (CTAB) was used as the structure directing agent. The DES formed by choline chloride (ChCl), urea (U), and ethylene glycol (EG) was used as a green solvent. The water needed for the hydrolysis of DHAQ-Si and TEOS was provided from free water in the sodium hydroxide solution. As characterized by small-angle X-ray scattering, nitrogen adsorption/desorption isotherms, and high-resolution transmission electron microscopy, the obtained mesoporous materials exhibit a two-dimensional hexagonal (P6mm) structure. DHAQ possesses double fluorescence emission peaks at ca. 520 and 580 nm due to the effect of the excited-state intramolecular proton transfer (ESIPT). For DHAQ-PMOs, the silica framework can provide a rigid environment that makes the fluorescence properties more stable and the fluorescence emission peak appears to red-shift obviously. The DHAQ-PMOs have high selectivity and sensitivity in a wide pH range that can be used to detect Cu2+, and the limit of detection (LOD) is as low as 2.39 × 10–9 M. Fluorescence polarization experiments, high-resolution mass spectrometry, and Fourier transform infrared spectroscopy were used to study the coordination interaction between DHAQ and Cu2+. The density functional theory (DFT) was used to further prove the coordination ability and interaction between DHAQ and Cu2+. XPS spectra were also done to prove the coordination of DHAQ and DHAQ-PMO-10 with Cu2+. In addition, confocal fluorescence microscope images exhibit that DHAQ-PMOs can detect Cu2+ in living cells. The results show that DHAQ-PMOs have great application potential as a sensor for the detection of Cu2+.

Identification of Peptide Inhibitors of Gαs that Block its Effector Binding Site

Heterotrimeric G proteins (Gαβγ) are molecular switches that alternate between “on” (GTP‐bound) and “off” (GDP‐bound) states to control the flow of signals from the extracellular milieu to the cytoplasm. Gαs is a prototypical Gα subunit of heterotrimeric G proteins. When bound to GTP, Gαs promotes the synthesis of cAMP in the cytosol through stimulation of the enzymatic activity of adenylyl cyclases. Dysregulation of this mechanism leads to human diseases. For example, naturally occurring mutations like R201C lock Gαs in an active conformation that leads to signaling hyperactivity in cancer. Despite the critical role of Gαs in signaling and its involvement in disease, there are currently no chemical or peptide tools to specifically inhibit its signaling activity. We reasoned that peptides that bind to active Gαs and block the effector binding site would inhibit its ability to signal through adenylyl cyclases. For this, we screened peptides from a phage display library and identified two unrelated peptides that bound to active Gαs. These peptides bound efficiently to GTP‐bound Gαs or to the constitutively active Gαs R201C mutant, but not to inactive GDP‐bound Gαs. Fluorescence polarization experiments revealed an equilibrium dissociation constant (KD) of approximately 1 μM for the binding of these peptides to Gαs‐GTP. Mutations in the adenylyl cyclase binding region of Gαs greatly reduced binding of these two peptides, indicating that they may work as competitive inhibitors of adenylyl cyclase stimulation. Consistently, both peptides blocked Gas‐mediated stimulation of adenylyl cyclase activity in vitrowith μM potency, but had no effect on Gαs‐independent stimulation of adenylyl cyclase activity by forskolin. Protein‐protein binding experiments revealed that one of the two peptides bound to Gαs but not to Gα subunits of other G protein families, whereas the second peptide bound to Gαi‐GTP in addition to Gαs‐GTP. In summary, we have identified a peptide inhibitor specific for Gαs‐GTP that acts by binding to its effector binding site and precluding adenylyl cyclase stimulation. The peptide identified here could be leveraged as a genetically‐encoded probe or derivatized as a cell‐penetrating peptide to inhibit Gαs‐mediated cell signaling.

Studying the Interaction Mechanism of Azurin-Derived Peptide P28 and Tumor Suppressor P53

p53 is a pro-apoptotic tumor suppressor. As a transcription factor, p53 directly regulates a variety of cellular processes, including apoptosis, cell cycle, cell senescence, metabolism, and antioxidation. Azurin is a copper-containing redox protein secreted by Pseudomonas aeruginosa. It consists of 128 amino acids. Azurin has attracted much attention because it preferentially enters a variety of cancer cells and produces cytotoxicity. p28 is a segment of azurin, corresponding to residues 50 to 77. So far, several different mechanisms have been proposed to explain the anticancer activity of azurin and p28, where azurin/p28 stabilizing p53 by forming a binary complex is the most important and widely studied one. However, except the predictive structures given by molecular simulation and molecular docking, no information about the structure of p28-p53 or azurin-p53 complex is available. Consequently, it is impossible to rationally modify the amino acid sequence of p28 to further enhance the interaction between p28 and p53 and to improve the anticancer activity of p28. Through the comprehensive use of biophysical and structural biology characterization techniques and computer simulation, we systematically explored the interactions between p28 and the DNA binding domain (DBD) of p53. Through alanine mutagenesis, GST pull-down and surface plasmon resonance experiments, it was found that the L68A mutation had the greatest effect on the p28/p53 DBD binding affinity. Through fluorescence polarization experiments, the p28 binding site on p53 DBD was found to be overlapped with the DNA binding site. The p28-interacting residues on p53 DBD were further identified by nuclear magnetic resonance, including Y236, S240, V274, V173, R267, S215, I255, G245, L145, K305, M133, Y103, A161, R156, C277, and T211. Finally, the best binding model consistent with available experimental information was obtained through molecular docking.

Genetic encoding of a highly photostable, long lifetime fluorescent amino acid for imaging in mammalian cells.

Acridonylalanine (Acd) is a fluorescent amino acid that is highly photostable, with a high quantum yield and long fluorescence lifetime in water. These properties make it superior to existing genetically encodable fluorescent amino acids for monitoring protein interactions and conformational changes through fluorescence polarization or lifetime experiments, including fluorescence lifetime imaging microscopy (FLIM). Here, we report the genetic incorporation of Acd using engineered pyrrolysine tRNA synthetase (RS) mutants that allow for efficient Acd incorporation in both E. coli and mammalian cells. We compare protein yields and amino acid specificity for these Acd RSs to identify an optimal construct. We also demonstrate the use of Acd in FLIM, where its long lifetime provides strong contrast compared to endogenous fluorophores and engineered fluorescent proteins, which have lifetimes less than 5 ns.

The discovery of potential phosphopantetheinyl transferase Ppt2 inhibitors against drug-resistant Candida albicans.

With the high-frequency use or abuse of antifungal drugs, the crisis of drug-resistant fungi continues to increase worldwide; in particular, the infection of drug-resistant Candida albicans brings the great challenge to the clinical treatment. Therefore, to decelerate the spread of this resistance, it is extremely urgent to facilitate the new antifungal targets with novel drugs. Phosphopantetheinyl transferases PPTases (Ppt2 in Candida albicans) had been identified in bacterium and fungi and mammals, effects as a vital enzyme in the metabolism of organisms in C. albicans. Ppt2 transfers the phosphopantetheinyl group of coenzyme A to the acyl carrier protein Acp1 in mitochondria for the synthesis of lipoic acid that is essential for fungal respiration, so making Ppt2 an ideal target for antifungal drugs. In this study, 110 FDA-approved drugs were utilized to investigate the Ppt2 inhibition against drug-resistant Candida albicans by the improved fluorescence polarization experiments, which have enough druggability and structural variety under the novel strategy of drug repurposing. Thereinto, eight agents revealed the favourable Ppt2 inhibitory activities. Further, broth microdilution assay of incubating C. albicans with these eight drugs showed that pterostilbene, procyanidine, dichlorophen and tea polyphenol had the superior MIC values. In summary, these findings provide more valuable insight into the treatment of drug-resistant C. albicans.

Biophysical characterizations of the recognition of the AAUAAA polyadenylation signal

Most eukaryotic messenger RNA precursors must undergo 3′-end cleavage and polyadenylation for maturation. We and others recently reported the structure of the AAUAAA polyadenylation signal (PAS) in complex with the protein factors CPSF-30, WDR33, and CPSF-160, revealing the molecular mechanism for this recognition. Here we have characterized in detail the interactions between the PAS RNA and the protein factors using fluorescence polarization experiments. Our studies show that AAUAAA is recognized with ∼3 nM affinity by the CPSF-160–WDR33–CPSF-30 ternary complex. Variations in the RNA sequence can greatly reduce the affinity. Similarly, mutations of CPSF-30 residues that have van der Waals interactions with the bases of AAUAAA also lead to substantial reductions in affinity. Finally, our studies confirm that both CPSF-30 and WDR33 are required for high-affinity binding of the PAS RNA, while these two proteins alone and their binary complexes with CPSF-160 have much lower affinity for the RNA.

α-Helical Motif as Inhibitors of Toxic Amyloid-β Oligomer Generation via Highly Specific Recognition of Amyloid Surface.

SummaryAmyloid fibril surfaces can convert soluble proteins into toxic oligomers and are attractive targets for intervention of protein aggregation diseases. Thus far, molecules identified with inhibitory activity are either large proteins or flat cyclic compounds lacking in specificity. The main design difficulty is flatness of amyloid surfaces and the lack of knowledge on binding interfaces. Here, we demonstrate, for the first time, a rational design of alpha-helical peptide inhibitors targeting the amyloid-beta 40 (Aβ40) fibril surfaces, based on our in silico finding that a helical fragment of Aβ40 interacts in a unique way with side-chain arrays on the fibril surface. We strengthen the fragment's binding capability through mutations and helicity enhancement with our Terminal Aspartic acid strategy. The resulting inhibitor shows micromolar affinity for the fibril surface, effectively impedes the surface-mediated oligomerization of Aβ40, and mitigates its cytotoxicity. This work opens up an avenue to designing aggregation modulators for amyloid diseases.

Synthesis of a Fluorous-Tagged Hexasaccharide and Interaction with Growth Factors Using Sugar-Coated Microplates.

Here, we report the synthesis of a sulfated, fully protected hexasaccharide as a glycosaminoglycan mimetic and the study of its interactions with different growth factors: midkine, basic fibroblast growth factor (FGF-2) and nerve growth factor (NGF). Following a fluorous-assisted approach, monosaccharide building blocks were successfully assembled and the target oligosaccharide was prepared in excellent yield. The use of more acid stable 4,6-O-silylidene protected glucosamine units was crucial for the efficiency of this strategy because harsh reaction conditions were needed in the glycosylations to avoid the formation of orthoester side products. Fluorescence polarization experiments demonstrated the strong interactions between the synthesized hexamer, and midkine and FGF-2. In addition, we have developed an alternative assay to analyse these molecular recognition events. The prepared oligosaccharide was non-covalently attached to a fluorous-functionalized microplate and the direct binding of the protein to the sugar-immobilized surface was measured, affording the corresponding KD,surf value.

The Shigella Virulence Factor IcsA Relieves N-WASP Autoinhibition by Displacing the Verprolin Homology/Cofilin/Acidic (VCA) Domain

Shigella flexneri is a bacterial pathogen that invades cells of the gastrointestinal tract, causing severe dysentery. Shigella mediates intracellular motility and spreading via actin comet tail formation. This process is dependent on the surface-exposed, membrane-embedded virulence factor IcsA, which recruits the host actin regulator N-WASP. Although it is clear that Shigella requires N-WASP for this process, the molecular details of this interaction and the mechanism of N-WASP activation remain poorly understood. Here, we show that co-expression of full-length IcsA and the Shigella membrane protease IcsP yields highly pure IcsA passenger domain (residues 53–758). We show that IcsA is monomeric and describe the solution structure of the passenger domain obtained by small-angle X-ray scattering (SAXS) analysis. The SAXS-derived models suggest that IcsA has an elongated shape but, unlike most other autotransporter proteins, possesses a central kink revealing a distinctly curved structure. Pull-down experiments show direct binding of the IcsA passenger domain to both the WASP homology 1 (WH1) domain and the GTPase binding domain (GBD) of N-WASP and no binding to the verprolin homology/cofilin/acidic (VCA) region. Using fluorescence polarization experiments, we demonstrate that IcsA binding to the GBD region displaces the VCA peptide and that this effect is synergistically enhanced upon IcsA binding to the WH1 region. Additionally, domain mapping of the IcsA interaction interface reveals that different regions of IcsA bind to the WH1 and GBD domains of N-WASP. Taken together, our data support a model where IcsA and N-WASP form a tight complex releasing the N-WASP VCA domain to recruit the host cell machinery for actin tail formation.

Essential oils from Inula japonica and Angelicae dahuricae enhance sensitivity of MCF-7/ADR breast cancer cells to doxorubicin via multiple mechanisms

Ethnopharmacological relevanceAngelicae dahurica (Hoffm.) Benth. & Hook.f.ex Franch. & Sav combined with Pueraria and Gastrodia elata Bl. combined with Inula japonica Thunb. are widely used in herb-pairs of traditional chinese medicine. Previous studies have shown that Angelicae dahuricae essential oil (ADO) enhanced puerarin internalization into ABCB1-overexpressed Caco-2 cells. These findings suggest the possibility that essential oils may enhance the absorption via certain mechanisms related to ABCB1 and reverse multidrug resistance (MDR). Aim of the studyADO and essential oils from Inula japonica (IJO) may reverse ABCB1-mediated MDR, but this ability has not been investigated in detail in the well-established cancer cell lines. In this study, the underlying molecular mechanisms were further investigated to examine how IJO and ADO reverse MDR in the resistant human breast cancer cell line of MCF-7/ADR. Also this work may help uncover the conceivable compatibility mechanisms of above herb-pairs involved in ABCB1. Materials and methodsThe MDR human breast cancer MCF-7/ADR cells were treated with IJO, its sesquiterpene component isoalantolactone (ISO) or ADOat non- cytotoxic concentrations. The MDR ability was examined by measuring the sensitivity to doxorubicin (DOX), DOX accumulation and efflux, ABCB1 ATPase activity, ABCB1 expression, membrane fluidity, and stability and localization of lipid rafts and caveolae. Finally, the molecular modeling was performed to postulate how ISO interacts with ABCB1. ResultsTreating MCF-7/ADR cells with IJ oil, ISO or AD oil reversed MDR 2- to 3-fold, without affecting the sensitivity of the non-MDR parental cell line. Mechanistic studies showed that these oils down-regulated mRNA and protein expression of ABCB1, and reduced the stability of lipid rafts in the cell membrane, which has previously been shown to reduce ABCB1-mediated transport. On the other hand, IJO, ISO and ADO did not inhibit ABCB1 ATPase activity, and fluorescence polarization experiments showed that low concentrations of the oils did not appear to alter membrane fluidity, unlike some MDR-reversing agents, ISO showed a higher docking score than verapamil but lower than dofequidar and tariquidar. ConclusionsOur results suggest that IJO, ISO and ADO could reverse MDR by down-regulating ABCB1 expression and reducing lipid raft stability. These findings may be useful for developing safer and effective MDR reversal agents and also help find out the compatibility mechanisms.

Fluorescent DNA-Protected Silver Nanoclusters for Ligand-HIV RNA Interaction Assay.

Studying ligand-biomacromolecule interactions provides opportunities for creating new compounds that can efficiently regulate specific biological processes. Ribonucleic acid (RNA) molecules have become attractive drug targets since the discovery of their roles in modulating gene expression, while only a limited number of studies have investigated interactions between ligands and functional RNA molecules, especially those based on nanotechnology. DNA-protected silver nanoclusters (AgNCs) were used to investigate ligand-RNA interactions for the first time in this study. The anthracycline anticancer drug mitoxantrone (MTX) was found to quench the fluorescence of AgNCs. After adding human immunodeficiency virus trans-activation responsive region (TAR) RNA or Rev-response element (RRE) RNA to AgNCs-MTX mixtures, the fluorescence of the AgNCs recovered due to interactions between MTX with RNAs. The binding constants and number of binding sites of MTX to TAR and RRE RNA were determined through theoretical calculations. MTX-RNA interactions were further confirmed in fluorescence polarization and mass spectrometry experiments. The mechanism of MTX-based fluorescence quenching of the AgNCs was also explored. This study provides a new strategy for ligand-RNA binding interaction assay.

Estrogen Receptor Folding Modulates cSrc Kinase SH2 Interaction via a Helical Binding Mode.

The estrogen receptors (ERs) feature, next to their transcriptional role, important nongenomic signaling actions, with emerging clinical relevance. The Src Homology 2 (SH2) domain mediated interaction between cSrc kinase and ER plays a key role in this; however the molecular determinants of this interaction have not been elucidated. Here, we used phosphorylated ER peptide and semisynthetic protein constructs in a combined biochemical and structural study to, for the first time, provide a quantitative and structural characterization of the cSrc SH2-ER interaction. Fluorescence polarization experiments delineated the SH2 binding motif in the ER sequence. Chemical shift perturbation analysis by nuclear magnetic resonance (NMR) together with molecular dynamics (MD) simulations allowed us to put forward a 3D model of the ER-SH2 interaction. The structural basis of this protein-protein interaction has been compared with that of the high affinity SH2 binding sequence GpYEEI. The ER features a different binding mode from that of the "two-pronged plug two-hole socket" model in the so-called specificity determining region. This alternative binding mode is modulated via the folding of ER helix 12, a structural element directly C-terminal of the key phosphorylated tyrosine. The present findings provide novel molecular entries for understanding nongenomic ER signaling and targeting the corresponding disease states.

Desmethyl macrolides: synthesis and evaluation of 4-desmethyl telithromycin.

Novel sources of antibiotics are needed to address the serious threat of bacterial resistance. Accordingly, we have launched a structure-based drug design program featuring a desmethylation strategy wherein methyl groups have been replaced with hydrogens. Herein we report the total synthesis, molecular modeling, and biological evaluation of 4-desmethyl telithromycin (6), a novel desmethyl analogue of the third-generation ketolide antibiotic telithromycin (2) and our final analogue in this series. While 4-desmethyl telithromycin (6) was found to be equipotent with telithromycin (2) against wild-type bacteria, it was 4-fold less potent against the A2058G mutant. These findings reveal that strategically replacing the C4-methyl group with hydrogen (i.e., desmethylation) did not address this mechanism of resistance. Throughout the desmethyl series, the sequential addition of methyls to the 14-membered macrolactone resulted in improved bioactivity. Molecular modeling methods indicate that changes in conformational flexibility dominate the increased biological activity; moreover, they reveal 6 adopts a different conformation once bound to the A2058G ribosome, thus impacting noncovalent interactions reflected in a lower MIC value. Finally, fluorescence polarization experiments of 6 with E. coli ribosomes confirmed 6 is indeed binding the ribosome.

Polarized fluorescence microscopy analysis of patterned, polymerized perfluorotetradecanoic acid–pentacosadiynoic acid thin films

Photoillumination of mixed films comprised of the photopolymerizable fatty acid 10,12 pentacosadiynoic acid and perfluorotetradecanoic acid deposited onto glass substrates gives rise to the formation of oriented polydiacetylene photopolymer fibers. The degree of polymer fiber orientation was investigated using dual-view, polarized fluorescence microscopy of the polydiacetylene, which allowed for characterization of individual fluorescent polymer fibers after photopolymerization, as well as comparison of the orientation of different fibers within the same sample. Measurements indicated that individual fibers consisted of multiple photopolymer strands with various orientations, and that there was a preferred orientation for fibers in the film as a whole. The fibers were preferentially oriented at an angle of approximately 60° to the direction of film compression during deposition from a Langmuir trough, with orientation being the result of mechanical stress exerted by the compression barriers coupled with rotation of the polymer fibers during film draining. These measurements were complemented with conventional “bulk” fluorescence polarization experiments, and compared with mixed film structures described previously for these systems at the air–water interface using Brewster angle microscopy.