Novel coumarin aminophosphonates as potential multitargeting antibacterial agents against Staphylococcus aureus.

Unique coumarin aminophosphonates as new antibacterial agents were designed and synthesized to combat severely bacterial resistance. Bioactivity assessment identified that 3-hydroxylphenyl aminophosphonate 6f with low hemolytic activity not only exhibited excellent inhibition potency against Staphylococcus aureus at low concentration (0.5 µg/mL) in vitro but also showed considerable antibacterial potency in vivo. Meanwhile, the active compound 6f was capable of eradicating the S. aureus biofilm, thus alleviating the development of S. aureus resistance. Furthermore, the drug combination of compound 6f with norfloxacin could enhance the antibacterial efficacy. Mechanistic explorations manifested that molecule 6f was able to destroy the integrity of cell membrane, which resulted in the leakage of protein and metabolism inhibition. The cellular redox homeostasis was interfered through inducing the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), leading to the reduction of glutathione (GSH) activity and lipid peroxidation. Furthermore, compound 6f could intercalate into DNA base pair to hinder normal biological function. The above results provided powerful information for the further development of coumarin aminophosphonates as antibacterial agents.

Synthesis of 3-aryl-4-(N-aryl)aminocoumarins via photoredox arylation and the evaluation of their biological activity.

A new series of 3-aryl-4-(N-aryl)aminocoumarins was synthesized in two steps starting from the natural product 4-hydroxycoumarin using the photoredox catalysis for the key step. These conditions reactions allowed to make CC bonds is up to 95% yields in mild conditions, easy operation, in an environmentally benign way, and are compatible with several patterns of substitution. The biological activity of the new compounds was tested in vitro against MCF-7, MDA-MB-231, and CCD-1072Sk cancer cell lines, as soon as to promastigotes and intracellular amastigotes of Leishmania amazonensis. Compounds 17d, 17s and 17x showed activity against promastigote forms (IC50 = 5.96 ± 3.210, 9.05 ± 2.855 and 5.65 ± 2.078 µM respectively), and compound 17x presented the best activity against L. amazonensis amastigote intracellular form (IC50 = 9.6 ± 1.148 µM), no BALB/c peritoneal macrophage cytotoxicity at assayed concentrations (CC50 > 600 µM), and high selectivity to parasites over the mammalian cells (Selectivity Index > 62.2). There was no expressive activity for the cancer cell lines. Single crystal X-ray diffraction analysis was employed for structural elucidation of compounds 17a and 17s. In silico analyses of physicochemical, pharmacokinetic, and toxicological properties suggest that compound 17x is a potential candidate for anti-leishmaniasis drugs.

A novel heat shock protein inhibitor KU757 with efficacy in lenvatinib-resistant follicular thyroid cancer cells overcomes up-regulated glycolysis in drug-resistant cells in vitro.

BACKGROUND: Patients with advanced differentiated thyroid cancer develop resistance to lenvatinib treatment from metabolic dysregulation. Heat shock protein 90 is a molecular chaperone that plays an important role in glycolysis and metabolic pathway regulation. We hypothesize that lenvatinib-resistant differentiated thyroid cancer cells will have an increased dependency on glycolysis and that a novel C-terminal heat shock protein 90 inhibitor (KU757) can effectively treat lenvatinib-resistant cells by targeting glycolysis. METHODS: Inhibitory concentration 50 values of thyroid cancer cells were determined by CellTiter-Glo assay (Promega Corp, Madison, WI). Glycolysis was measured through Seahorse experiments. Reverse transcription-polymerase chain reaction and Western blot evaluated glycolytic pathway genes/proteins. Exosomes were isolated/validated by nanoparticle tracking analysis and Western blot. Differentially expressed long non-coding ribonucleic acids in exosomes and cells were evaluated using quantitative polymerase chain reaction. RESULTS: Extracellular acidification rate demonstrated >2-fold upregulation of glycolysis in lenvatinib-resistant cells versus parent cells and was downregulated after KU757 treatment. Lenvatinib-resistant cells showed increased expression of the glycolytic genes lactic acid dehydrogenase, pyruvate kinase M1/2, and hexokinase 2. KU757 treatment resulted in downregulation of these genes and proteins. Several long non-coding ribonucleic acids associated with glycolysis were significantly upregulated in WRO-lenvatinib-resistant cells and exosomes and downregulated after KU757 treatment. CONCLUSION: Lenvatinib resistance leads to increased glycolysis, and KU757 effectively treats lenvatinib-resistant cells and overcomes this increased glycolysis by targeting key glycolytic genes, proteins, and long non-coding ribonucleic acids.

Redox cycling of copper by coumarin-di(2-picolyl)amine hybrid molecule leads to ROS-mediated modulation of redox scavengers, DNA damage and cell death in diethylnitrosamine induced hepatocellular carcinoma.

Targeted therapy is a new strategy for cancer treatment that targets chemical entities specific to cancer cells than normal ones. One of the features associated with malignancy is the elevated copper which plays an integral role in angiogenesis. Work is in progress in our lab to identify new copper chelators to target elevated copper under targeted therapy for the killing of cancer cells. Recently, a coumarin-based copper chelator, di(2-picolyl)amine-3(bromoacetyl)coumarin hybrid molecule (ligand-L) has been synthesized by us, and also studied its copper-dependent macromolecular damage response in copper overloaded lymphocytes. The present study investigates the anticancer activity of ligand-L and its mode of action in rat model of diethylnitrosamine (DEN) induced hepatocellular carcinoma. It has been found that liver tissue has a marked increase in copper levels in DEN induced hepatocellular carcinoma. Ex vivo results showed that ligand-L inhibited cell viability, induced reactive oxygen species (ROS) generation, DNA damage, loss of mitochondrial membrane potential and caspase-3 activation in isolated hepatocellular carcinoma cells (HCC). All these effects induced by ligand-L were abrogated by neocuproine and N-acetylcysteine (ROS scavenger). Further, ligand-L treatment of animals bearing hepatocellular carcinoma results in an increment in the cellular redox scavengers, lipid peroxidation and DNA breakage in malignant hepatocytes. In vivo studies using ligand-L also showed that ligand-L possesses anticancer properties as evidenced by improvement in liver marker enzymes and liver surface morphology, and reduced alpha-fetoprotein in the treated group compared to untreated cancer-induced group. Overall, this study suggests that copper-ligand-L interaction leads to ROS generation which caused DNA damage and apoptosis in malignant cells. This study provides enough support to establish ligand-L as a clinically relevant lead molecule for the treatment of different malignancies.

4-Aminocoumarin based Aroylthioureas as Potential Jack Bean Urease Inhibitors; Synthesis, Enzyme Inhibitory Kinetics and Docking Studies.

BACKGROUND: Urease enzyme catalyzes the hydrolysis of urea into ammonia and CO2, excess ammonia causes global warming and crop reduction. Ureases are also responsible for certain human diseases such as stomach cancer, peptic ulceration, pyelonephritis, and kidney stones. New urease inhibitors are developed to get rid of such problems. OBJECTIVE: This article describes the synthesis of a series of novel 1-aroyl-3-(2-oxo-2H-chromen-4- yl)thiourea derivatives (5a-j) as Jack bean urease inhibitors. METHODS: Freshly prepared aryl isothiocyanates were reacted with 4-aminocoumarin in the same pot in an anhydrous medium of acetone. The structures of the title thioureas (5a-j) were ascertained by their spectroscopic data. The inhibitory effects against jack bean urease were determined. RESULTS: It was found that compounds 5i and 5j showed excellent activity with IC50 values 0.0065 and 0.0293, µM respectively. Compound 5i bearing 4-methyl substituted phenyl ring plays a vital role in enzyme inhibitory activity. The kinetic mechanism analyzed by Lineweavere-Burk plots revealed that compound 5i inhibits the enzyme non-competitively. The Michaelis-Menten constant Km and inhibition constants Ki calculated from Lineweavere-Burk plots for compound 5i are 4.155mM and 0.00032µM, respectively. The antioxidant activity results displayed that compound 5j showed excellent radical scavenging activity. The cytotoxic effects determined against brine shrimp assay showed that all of the synthesized compounds are non-toxic to shrimp larvae. Molecular docking studies were performed against target protein (PDBID 4H9M) and it was determined that most of the synthesized compounds exhibited good binding affinity with the target protein. Molecular dynamics simulation (MDS) results revealed that compound 5i forms a stable complex with target protein showing little fluctuation. CONCLUSIONS: Based upon our investigations, it is proposed that 5i derivative may serve as a lead structure for devising more potent urease inhibitors.

Growth Phase-Dependent Chromosome Condensation and Heat-Stable Nucleoid-Structuring Protein Redistribution in Escherichia coli under Osmotic Stress.

The heat-stable nucleoid-structuring (H-NS) protein is a global transcriptional regulator implicated in coordinating the expression of over 200 genes in Escherichia coli, including many involved in adaptation to osmotic stress. We have applied superresolved microscopy to quantify the intracellular and spatial reorganization of H-NS in response to a rapid osmotic shift. We found that H-NS showed growth phase-dependent relocalization in response to hyperosmotic shock. In stationary phase, H-NS detached from a tightly compacted bacterial chromosome and was excluded from the nucleoid volume over an extended period of time. This behavior was absent during rapid growth but was induced by exposing the osmotically stressed culture to a DNA gyrase inhibitor, coumermycin. This chromosomal compaction/H-NS exclusion phenomenon occurred in the presence of either potassium or sodium ions and was independent of the presence of stress-responsive sigma factor σS and of the H-NS paralog StpA.IMPORTANCE The heat-stable nucleoid-structuring (H-NS) protein coordinates the expression of over 200 genes in E. coli, with a large number involved in both bacterial virulence and drug resistance. We report on the novel observation of a dynamic compaction of the bacterial chromosome in response to exposure to high levels of salt. This stress response results in the detachment of H-NS proteins and their subsequent expulsion to the periphery of the cells. We found that this behavior is related to mechanical properties of the bacterial chromosome, in particular, to how tightly twisted and coiled is the chromosomal DNA. This behavior might act as a biomechanical response to stress that coordinates the expression of genes involved in adapting bacteria to a salty environment.

In silico identification and analysis of the binding site for aminocoumarin type inhibitors in the C-terminal domain of Hsp90.

Hsp90 contains two Nucleotide Binding Sites (NBS): one each in its N-terminal domain (NTD) and C-terminal domain (CTD), respectively. Previously we used computational techniques to locate a nucleotide-binding site in the CTD. Nucleotide binding at this site stabilized the structurally labile region within this domain, thus providing a rationale for increased resistance to thermal denaturation and proteolysis. A scan for ligand-binding sites in CTD revealed four potential sites with the requisite volume to accommodate aminocoumarins and -derived inhibitors. Only one of these reproducibly formed docked complexes with inhibitors and showed excellent interactions with residues lining the site. Fortuitously, it was identical to the aforementioned nucleotide-binding site thus providing an explanation for the reported direct competition between inhibitors and nucleotides. Further studies with carefully chosen inhibitors and some inactive analogues provided an explanation for the known Structure-Activity Relationships (SAR) of aminocoumarin and -derived inhibitors. We also performed similar studies of the NTD to discern the reason(s) for its inability to bind aminocoumarins, given the family resemblance to prokaryotic Top-IV and Gyr-B. Our studies permitted the identification of the putative inhibitor binding site in the CTD, an explanation for increased resistance to thermal denaturation and proteolysis upon inhibitor binding as well as direct competition with ATP.

Design and synthesis of aminocoumarin derivatives as DPP-IV inhibitors and anticancer agents.

DPP-IV "a moonlighting protein" has immerged as promising pathway to control Type 2 diabetes as well as found to play key role in earlier stages of cancer. Here we have reported design, synthesis and applications of aminocoumarin derivatives as DPP-IV inhibitors. Compounds have been synthesized and studied for their DPP-IV inhibition activity. Three compounds have shown moderate inhibition at 100 µM concentration. All compounds were also screened for their anticancer activity against A549 (Lung cancer cell line), MCF-7 (Breast cancer cell line) using MTT assay. One of the compounds has shown very good anticancer activity with IC50 value 24 ±â€¯0.1 nM against A549 cell line.

Antioxidant activity and protective role on protein glycation of synthetic aminocoumarins

Background: Synthesized aminocoumarins are heterocyclic compounds possessing potential for the treatment of insulin-dependent diabetes mellitus with unexplored anti-glycative action. Results: In this study 4-aminocoumarin derivatives (4-ACDs) were evaluated in vitro for antiglycation (AG) activities by using the human serum albumin (HSA)/glucose system, for 8 weeks of incubation. The glycation and conformational alteration of HSA in the presence of the tested compounds were evaluated by Congo red assay, fluorescence and circular dichroism spectroscopy. The antioxidant (AO) capacity were also tested by four different assays including: DPPH (2,2'-diphenyl-1-picrylhydrazyl radical), ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulphonate) diammonium salt), FRAP (ferric reducing antioxidant power) and β-carotene-linoleic acid assay. The tested compounds showed AG and AO effects. The intensity of the accomplished AO potential is related to the type of the used assay. Significant alterations in the secondary (monitored by CD spectropolarimetry) and tertiary structure (assessed by spectrofluorimetry) of HSA upon glycation were mitigated by the 4-ACDs, suggesting their suppressive role in the late stage (post-Amadori) of the HSA glycation. Conclusions: By the analogues, in vitro ascertained AO and AG properties of 4-ACD may be recognized as rationale for their protective role against oxidative changes of proteins, thereby precluding diabetic complications in humans.

Functional characterization and inhibition of the type II DNA topoisomerase coded by African swine fever virus.

DNA topoisomerases are essential for DNA metabolism and while their role is well studied in prokaryotes and eukaryotes, it is less known for virally-encoded topoisomerases. African swine fever virus (ASFV) is a nucleo-cytoplasmic large DNA virus that infects Ornithodoros ticks and all members of the family Suidae, representing a global threat for pig husbandry with no effective vaccine nor treatment. It was recently demonstrated that ASFV codes for a type II topoisomerase, highlighting a possible target for control of the virus. In this work, the ASFV DNA topoisomerase II was expressed in Saccharomyces cerevisiae and found to efficiently decatenate kDNA and to processively relax supercoiled DNA. Optimal conditions for its activity were determined and its sensitivity to a panel of topoisomerase poisons and inhibitors was evaluated. Overall, our results provide new knowledge on viral topoisomerases and on ASFV, as well as a possible target for the control of this virus.

Triggering HIV polyprotein processing by light using rapid photodegradation of a tight-binding protease inhibitor.

HIV protease (PR) is required for proteolytic maturation in the late phase of HIV replication and represents a prime therapeutic target. The regulation and kinetics of viral polyprotein processing and maturation are currently not understood in detail. Here we design, synthesize, validate and apply a potent, photodegradable HIV PR inhibitor to achieve synchronized induction of proteolysis. The compound exhibits subnanomolar inhibition in vitro. Its photolabile moiety is released on light irradiation, reducing the inhibitory potential by 4 orders of magnitude. We determine the structure of the PR-inhibitor complex, analyze its photolytic products, and show that the enzymatic activity of inhibited PR can be fully restored on inhibitor photolysis. We also demonstrate that proteolysis of immature HIV particles produced in the presence of the inhibitor can be rapidly triggered by light enabling thus to analyze the timing, regulation and spatial requirements of viral processing in real time.

Altering gene expression by aminocoumarins: the role of DNA supercoiling in Staphylococcus aureus.

BACKGROUND: It has been shown previously that aminocoumarin antibiotics such as novobiocin lead to immediate downregulation of recA expression and thereby inhibit the SOS response, mutation frequency and recombination capacity in Staphylococcus aureus. Aminocoumarins function by inhibiting the ATPase activity of DNA gyrase subunit B with a severe impact on DNA supercoiling. RESULTS: Here, we have analysed the global impact of the DNA relaxing agent novobiocin on gene expression in S. aureus. Using a novobiocin-resistant mutant, it became evident that the change in recA expression is due to gyrase inhibition. Microarray analysis and northern blot hybridisation revealed that the expression levels of a distinct set of genes were increased (e.g., recF-gyrB-gyrA, the rib operon and the ure operon) or decreased (e.g., arlRS, recA, lukA, hlgC and fnbA) by novobiocin. The two-component ArlRS system was previously found to decrease the level of supercoiling in S. aureus. Thus, downregulation of arlRS might partially compensate for the relaxing effect of novobiocin. Global analysis and gene mapping of supercoiling-sensitive genes did not provide any indication that they are clustered in the genome. Promoter fusion assays confirmed that the responsiveness of a given gene is intrinsic to the promoter region but independent of the chromosomal location. CONCLUSIONS: The results indicate that the molecular properties of a given promoter, rather than the chromosomal topology, dictate the responsiveness to changes in supercoiling in the pathogen Staphylococcus aureus.

Necroptosis induced by RIPK3 requires MLKL but not Drp1.

Necroptosis is a mechanism by which cells can kill themselves that does not require caspase activity or the presence of the pro-apoptotic Bcl-2 family members Bax or Bak. It has been reported that RIPK3 (receptor interacting protein kinase 3) activates MLKL (mixed lineage kinase domain-like) to cause cell death that requires dynamin-related protein 1 (Drp1), because survival was increased in cells depleted of Drp1 or treated with the Drp1 inhibitor mdivi-1. To analyze necroptosis in a system that does not require addition of tumor necrosis factor (TNF), we used a construct that allows RIPK3 to be induced in cells, and then dimerized via an E. coli gyrase domain fused to its carboxyl-terminus, using the dimeric gyrase binding antibiotic coumermycin. We have previously shown elsewhere that RIPK3 dimerized in this manner not only induces necroptosis but also apoptosis, which can be inhibited by the broad-spectrum caspase inhibitor Q-VD-OPh (QVD). In response to RIPK3 dimerization, wild-type mouse embryonic fibroblasts (MEFs) underwent cell death that was reduced but not completely blocked by QVD. In contrast, death upon dimerization of RIPK3 in Mlkl(-/-) MEFs was completely inhibited with QVD, confirming that MLKL is required for necroptosis. Similar to wild-type MEFs, most Drp1(-/-) MEFs died when RIPK3 was activated, even in the presence of QVD. Furthermore, overexpression of wild-type MLKL or dominant active mutants of MLKL (Q343A or S345E/S347E) caused death of wild-type and Drp1(-/-) MEFs that was not inhibited with QVD. These results indicate that necroptosis caused by RIPK3 requires MLKL but not Drp1.

New aminocoumarins from the rare actinomycete Catenulispora acidiphila DSM 44928: identification, structure elucidation, and heterologous production.

Genome mining led to the discovery of a novel aminocoumarin gene cluster in the rare actinomycete Catenulispora acidiphila DSM 44928. Sequence analysis revealed the presence of genes putatively involved in export/resistance, regulation, and biosynthesis of the aminocoumarin moiety and its halogenation, as well as several genes with so far unknown function. Two new aminocoumarins, cacibiocin A and B, were identified in the culture broth of C. acidiphila. Heterologous expression of the putative gene cluster in Streptomyces coelicolor M1152 confirmed that this cluster is responsible for cacibiocin biosynthesis. Furthermore, total production levels of cacibiocins could be increased by heterologous expression and screening of different culture media from an initial yield of 4.9 mg L(-1) in C. acidiphila to 60 mg L(-1) in S. coelicolor M1152. By HR-MS and NMR analysis, cacibiocin A was found to contain a 3-amino-4,7-dihydroxycoumarin moiety linked by an amide bond to a pyrrole-2,5-dicarboxylic acid. The latter structural motif has not been identified previously in any natural compound. Additionally, cacibiocin B contains two chlorine atoms at positions 6' and 8' of the aminocoumarin moiety.

New aminocoumarin antibiotics as gyrase inhibitors.

The aminocoumarins novobiocin, clorobiocin and coumermycin A1 are structurally related antibiotics produced by different Streptomyces strains. They are potent inhibitors of bacterial gyrase. Their binding sites and their mode of action differ from those of fluoroquinolones such as ciprofloxacin. Novobiocin has been introduced into clinical use against Staphylococcus aureus infections, and S. aureus gyrase is particularly sensitive to inhibition by aminocoumarins, while topoisomerase IV is much less sensitive. Modern genetic techniques have allowed the engineering of the producer strains, resulting in a diverse range of new aminocoumarins, including compounds which are more active than the natural antibiotics as well as a compound which is actively imported across the cell envelope of Gram-negative bacteria. A further group of aminocoumarins are the simocyclinones which bind simultaneously to two different sites of gyrase and show a completely new mode of inhibition. Both the simocyclinones and the "classical" aminocoumarins strongly inhibit the fluoroquinolone-induced activation of RecA and thereby the SOS response in S. aureus. Therefore, a combination of aminocoumarins and fluoroquinolones strongly reduced the risk of resistance development and may offer new prospects in anti-infective therapy.

Aminocoumarins inhibit osteoclast differentiation and bone resorption via downregulation of nuclear factor of activated T cells c1.

Aminocoumarins, such as coumermycin A1 and novobiocin, are natural products of streptomycetes. They are potent inhibitors of bacterial DNA gyrase and are used to suppress the growth of bacteria in inflammatory diseases. However, their effect in osteoclastogenesis has not been investigated. In this study, using mouse bone-marrow-derived macrophages (BMMs), we showed that coumermycin A1 and novobiocin suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. The inhibitory effect of coumermycin A1 was associated with impaired activation of multiple signaling events downstream of RANK, including extracellular signal-regulated kinase, p38, and c-Jun terminal kinase phosphorylation, followed by decreased c-Fos and nuclear factor of activated T cells (NFAT)c1 expression. Ectopic overexpression of a constitutively active form of NFATc1 completely rescued the anti-osteoclastogenic effect of coumermycin A1, suggesting that the anti-osteoclastogenic effect of coumermycin A1 was mainly attributable to reduction in NFATc1 expression. Coumermycin A1 also abrogated RANKL-induced expression of interleukin-1ß, tumor necrosis factor-α, and inducible nitric oxide synthase in mouse BMMs. Consistent with the in vitro anti-osteoclastogenic effect, the aminocoumarin suppressed lipopolysaccharide-induced osteoclast formation and bone loss in in vivo mouse experiments. Taken together, our data demonstrate that aminocoumarins inhibit osteoclast formation and bone resorption, and comprise a potential therapeutic strategy for treating bone destructive diseases.

Opposing effects of aminocoumarins and fluoroquinolones on the SOS response and adaptability in Staphylococcus aureus.

OBJECTIVES: RecA is the key enzyme involved in DNA repair, recombination and induction of the SOS response and is central to the development of antibiotic resistance. Here we assessed the interaction of two different gyrase inhibitors, ciprofloxacin (a fluoroquinolone) and novobiocin (an aminocoumarin), on RecA activity and the SOS response in Staphylococcus aureus. METHODS: The influence of different gyrase inhibitors on the SOS response of S. aureus (including recA and lexA mutants) was analysed by northern blot analysis, real-time RT-PCR, western blot analysis and promoter activity assays. Recombination as well as mutation frequencies were determined for the different antibiotic combinations. RESULTS: We verified that ciprofloxacin leads to RecA activation and therefore induction of the SOS response. In contrast, novobiocin treatment resulted in an inhibition of recA transcription independent of LexA. When novobiocin and ciprofloxacin were added simultaneously, recA was reduced to the same level as with novobiocin alone. In combination, novobiocin also partially reduces the ciprofloxacin-mediated induction of the LexA target gene umuC (error-prone polymerase). Apart from reducing recA and umuC expression, novobiocin also inhibited the frequency of recombination, mutation and the formation of non-haemolytic variants. CONCLUSION: In summary, aminocoumarins inhibit recA expression in S. aureus and probably delay the process of developing antibiotic resistance and gene transfer. A clinical re-evaluation of these compounds as well as designing more applicable derivatives should be considered.

Functional self-assembling polypeptide bionanomaterials.

Bionanotechnology seeks to modify and design new biopolymers and their applications and uses biological systems as cell factories for the production of nanomaterials. Molecular self-assembly as the main organizing principle of biological systems is also the driving force for the assembly of artificial bionanomaterials. Protein domains and peptides are particularly attractive as building blocks because of their ability to form complex three-dimensional assemblies from a combination of at least two oligomerization domains that have the oligomerization state of at least two and three respectively. In the present paper, we review the application of polypeptide-based material for the formation of material with nanometre-scale pores that can be used for the separation. Use of antiparallel coiled-coil dimerization domains introduces the possibility of modulation of pore size and chemical properties. Assembly or disassembly of bionanomaterials can be regulated by an external signal as demonstrated by the coumermycin-induced dimerization of the gyrase B domain which triggers the formation of polypeptide assembly.

Multiplexing interactions to control antibiotic release from cyclodextrin hydrogels.

A new strategy for affinity-based drug delivery by modification of the drug rather than modification of the device is presented. Rifampin is modified to contain either one or two PEG-adamantane arms, and the drug release properties of dimeric coumermycin are compared to novobiocin with only one biding domain. The drugs are loaded into affinity-based and diffusion-only delivery platforms, the loading efficiency is calculated, and the release kinetics is determined in vitro. The presence of additional binding domains prolongs the release of antibiotics. Release rates differ little between modified and unmodified drug from the diffusion-only system. The results demonstrate the feasibility of custom-tuning drug delivery by multiplexing interactions with an affinity-based polymer platform.

Potentiation of Helicobacter pylori CagA protein virulence through homodimerization.

Chronic infection with Helicobacter pylori cagA-positive strains is associated with atrophic gastritis, peptic ulceration, and gastric carcinoma. The cagA gene product, CagA, is delivered into gastric epithelial cells via type IV secretion, where it undergoes tyrosine phosphorylation at the EPIYA motifs. Tyrosine-phosphorylated CagA binds and aberrantly activates the oncogenic tyrosine phosphatase SHP2, which mediates induction of elongated cell morphology (hummingbird phenotype) that reflects CagA virulence. CagA also binds and inhibits the polarity-regulating kinase partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) via the CagA multimerization (CM) sequence independently of tyrosine phosphorylation. Because PAR1 exists as a homodimer, two CagA proteins appear to be passively dimerized through complex formation with a PAR1 dimer in cells. Interestingly, a CagA mutant that lacks the CM sequence displays a reduced SHP2 binding activity and exhibits an attenuated ability to induce the hummingbird phenotype, indicating that the CagA-PAR1 interaction also influences the morphological transformation. Here we investigated the role of CagA dimerization in induction of the hummingbird phenotype with the use of a chemical dimerizer, coumermycin. We found that CagA dimerization markedly stabilizes the CagA-SHP2 complex and thereby potentiates SHP2 deregulation, causing an increase in the number of hummingbird cells. Protrusions of hummingbird cells induced by chemical dimerization of CagA are further elongated by simultaneous inhibition of PAR1. This study revealed a role of the CM sequence in amplifying the magnitude of SHP2 deregulation by CagA, which, in conjunction with the CM sequence-mediated inhibition of PAR1, evokes morphological transformation that reflects in vivo CagA virulence.