Herpes simplex virus internalization into epithelial cells requires Na+/H+ exchangers and p21-activated kinases but neither clathrin- nor caveolin-mediated endocytosis.

UNLABELLED: Herpes simplex virus 1 (HSV-1) is an alphaherpesvirus that has been reported to infect some epithelial cell types by fusion at the plasma membrane but others by endocytosis. To determine the molecular mechanisms of productive HSV-1 cell entry, we perturbed key endocytosis host factors using specific inhibitors, RNA interference (RNAi), or overexpression of dominant negative proteins and investigated their effects on HSV-1 infection in the permissive epithelial cell lines Vero, HeLa, HEp-2, and PtK2. HSV-1 internalization required neither endosomal acidification nor clathrin- or caveolin-mediated endocytosis. In contrast, HSV-1 gene expression and internalization were significantly reduced after treatment with 5-(N-ethyl-N-isopropyl)amiloride (EIPA). EIPA blocks the activity of Na(+)/H(+) exchangers, which are plasma membrane proteins implicated in all forms of macropinocytosis. HSV-1 internalization furthermore required the function of p21-activated kinases that contribute to macropinosome formation. However, in contrast to some forms of macropinocytosis, HSV-1 did not enlist the activities of protein kinase C (PKC), tyrosine kinases, C-terminal binding protein 1, or dynamin to activate its internalization. These data suggest that HSV-1 depends on Na(+)/H(+) exchangers and p21-activated kinases either for macropinocytosis or for local actin rearrangements required for fusion at the plasma membrane or subsequent passage through the actin cortex underneath the plasma membrane. IMPORTANCE: After initial replication in epithelial cells, herpes simplex viruses (HSVs) establish latent infections in neurons innervating these regions. Upon primary infection and reactivation from latency, HSVs cause many human skin and neurological diseases, particularly in immunocompromised hosts, despite the availability of effective antiviral drugs. Many viruses use macropinocytosis for virus internalization, and many host factors mediating this entry route have been identified, although the specific perturbation profiles vary for different host and viral cargo. In addition to an established entry pathway via acidic endosomes, we show here that HSV-1 internalization depended on sodium-proton exchangers at the plasma membrane and p21-activated kinases. These results suggest that HSV-1 requires a reorganization of the cortical actin cytoskeleton, either for productive cell entry via pH-independent fusion from macropinosomes or for fusion at the plasma membrane, and subsequent cytosolic passage to microtubules that mediate capsid transport to the nucleus for genome uncoating and replication.

Screening of small molecules affecting mammalian P-body assembly uncovers links with diverse intracellular processes and organelle physiology.

Processing bodies (P-bodies) are cytoplasmatic mRNP granules containing non-translating mRNAs and proteins from the mRNA decay and silencing machineries. The mechanism of P-body assembly has been typically addressed by depleting P-body components. Here we apply a complementary approach and establish an automated cell-based assay platform to screen for molecules affecting P-body assembly. From a unique library of compounds derived from myxobacteria, 30 specifically inhibited P-body assembly. Gephyronic acid A (GA), a eukaryotic protein synthesis inhibitor, showed the strongest effect. GA also inhibited, under stress conditions, phosphorylation of eIF2α and stress granule formation. Other hits uncovered interesting novel links between P-body assembly, lipid metabolism, and internal organelle physiology. The obtained results provide a chemical toolbox to manipulate P-body assembly and function.

Novel peptidomimetic compounds containing redox active chalcogens and quinones as potential anticancer agents.

Many types of cancer cells are associated with a disturbed intracellular redox balance and oxidative stress (OS). Among the various agents employed to modulate the intracellular redox state of cells, certain redox catalysts containing quinone and chalcogen moieties have shown considerable promise. Passerini multicomponent reaction has been developed for the synthesis of agents combining two, three or even four redox centers in one molecule in a good yield. When incubated with cancer cells these agents inhibited cell proliferation and induced apoptotic cell death. Interestingly, some of these redox active compounds exhibited quite low toxicity with normal cells. The cause was obviously OS, which was reflected by significant decrease in reduced glutathione, subsequently cell cycle arrest and induction of apoptosis.

The C terminus of the large tegument protein pUL36 contains multiple capsid binding sites that function differently during assembly and cell entry of herpes simplex virus.

The largest tegument protein of herpes simplex virus type 1 (HSV1), pUL36, is a multivalent cross-linker between the viral capsids and the tegument and associated membrane proteins during assembly that upon subsequent cell entry releases the incoming capsids from the outer tegument and viral envelope. Here we show that pUL36 was recruited to cytosolic progeny capsids that later colocalized with membrane proteins of herpes simplex virus type 1 (HSV1) and the trans-Golgi network. During cell entry, pUL36 dissociated from viral membrane proteins but remained associated with cytosolic capsids until arrival at the nucleus. HSV1 UL36 mutants lacking C-terminal portions of increasing size expressed truncated pUL36 but could not form plaques. Cytosolic capsids of mutants lacking the C-terminal 735 of the 3,164 amino acid residues accumulated in the cytosol but did not recruit pUL36 or associate with membranes. In contrast, pUL36 lacking only the 167 C-terminal residues bound to cytosolic capsids and subsequently colocalized with viral and host membrane proteins. Progeny virions fused with neighboring cells, but incoming capsids did not retain pUL36, nor could they target the nucleus or initiate HSV1 gene expression. Our data suggest that residues 2430 to 2893 of HSV1 pUL36, containing one binding site for the capsid protein pUL25, are sufficient to recruit pUL36 onto cytosolic capsids during assembly for secondary envelopment, whereas the 167 residues of the very C terminus with the second pUL25 binding site are crucial to maintain pUL36 on incoming capsids during cell entry. Capsids lacking pUL36 are targeted neither to membranes for virus assembly nor to nuclear pores for genome uncoating.

Cellular localisation of antitumoral 6-alkyl thymoquinones revealed by an alkyne-azide click reaction and the streptavidin-biotin system.

The subcellular distribution and accumulation of thymoquinone 1, a natural anticancer agent, has hitherto been unknown. We prepared 6-(dec-9-ynyl)thymoquinone 3, an alkyne-labelled derivative with anticancer activity similar to that of its parent compound 1. Alkyne 3 was seen, after a Huisgen-type click reaction with 3-azido-7-hydroxycoumarin, to accumulate in distinct compartments of the nuclei of PtK(2) potoroo kidney cells, and in adjoining regions that were stained with an antibody specific for the Golgi apparatus. In contrast, a biotinlabelled thymoquinone 4 seemed to accumulate across the entire cell nucleus upon visualisation with streptavidin; but this was less easily traceable because of co-staining of other structures such as mitochondria. In conclusion, for small drug-like molecules, visualisation by alkyne-azide cycloaddition seems to be superior to conventional visualisation by the biotin-streptavidin system.

Structure-activity relationships of precursors and analogs of natural 3-enoyl-tetramic acids.

Fragments and synthetic precursors prepared en route to the macrocyclic 3-acyltetramic acids (=3-acyl-1,5-dihydro-4-hydroxy-2H-pyrrol-2-ones) aburatubolactam and macrocidin A, as well as other analogs with variance in the ring heteroatom (N, O, S), and the residues at N(1), C(3), and C(5) were tested for cytotoxic and antimicrobial effects. Anticancer activity against various tumor cell lines in vitro did not necessarily require an intact pyrrolidin-2,4-dione ring. An acyclic ß-hydroxy-octatrienoyl amide precursor to aburatubolactam also exhibited distinct activity with an IC50 (120 h) value of <2.5 µM. The length of 3-oligoenoyl residues had little influence on the anticancer activity, but 3-alka-oligoenoyl tetramic acids were far more efficacious than their 3-(4-methoxycinnamoyl) congeners. N-H-3-acyltetramic acids were generally more active than their N-Me or N-Boc analogs, unless further polar groups necessitated an increased lipophilicity for sufficient uptake. Tetronic and thiotetronic acids were far less antiproliferative in cancer cells when compared with identically substituted tetramic acids.

A selective 3-acylation of tetramic acids and the first synthesis of ravenic acid.

3-Acyltetramic acids, including delicate 3-oligoenoyl derivatives, such as the Penicillium metabolite ravenic acid, were prepared in two high-yielding steps. Reaction of tetramic acids with the ylide Ph(3)PCCO afforded exclusively the corresponding 3-acylylidenetetramic acids. These were amenable to Wittig olefinations with aliphatic, aromatic, saturated and unsaturated aldehydes after deprotonation with KOtBu. Due to its simplicity, selectivity and tolerance of pH-sensitive groups this method is superior to the established acylation protocols by Jones and Yoshii. It is also applicable to the synthesis of 3-acyltetronic acids. The new 3-oligoenoyl tetramic acids exhibited structure-dependent antimicrobial and cytotoxic activity.

Biological activity of volatiles from marine and terrestrial bacteria.

The antiproliferative activity of 52 volatile compounds released from bacteria was investigated in agar diffusion assays against medically important microorganisms and mouse fibroblasts. Furthermore, the activity of these compounds to interfere with the quorum-sensing-systems was tested with two different reporter strains. While some of the compounds specific to certain bacteria showed some activity in the antiproliferative assay, the compounds common to many bacteria were mostly inactive. In contrast, some of these compounds were active in the quorum-sensing-tests. γ-Lactones showed a broad reactivity, while pyrazines seem to have only low intrinsic activity. A general discussion on the ecological importance of these findings is given.

Chivosazoles A and F, cytostatic macrolides from myxobacteria, interfere with actin.

Chivosazoles A and F, isolated from Sorangium cellulosum, showed high antiproliferative activity with different mammalian cell lines including human cancer cells. The chivosazoles caused a delay in G2/M phase of the cell cycle, and treated cells often contained two nuclei. By labeling F-actin it was shown that the actin cytoskeleton of the cells starts to break down after a few minutes of treatment. In vitro polymerization assays with purified G-actin revealed that the chivosazoles inhibit actin polymerization and also cause a depolymerization of pyrene-labeled F-actin microfilaments prepared in vitro. Chivosazoles are new tools for the investigation of issues concerning the actin cytoskeleton and they have a different mode of action from the known microfilament-disrupting compounds like rhizopodin and cytochalasin D.

Metal complexes of natural melophlins and their cytotoxic and antibiotic activities.

Complexes of the natural melophlins A and C with Mg, Zn, Ga, La and Ru were prepared, characterized and tested for antimicrobial and cytotoxic effects. The lanthanum complex La(melophlinato C)(3) and the ruthenium complex chlorido(eta(6)-p-cymene)(melophlinato C)ruthenium(II) inhibited cells of human A-498 kidney cancer at IC(50)=0.54 microM and 1.0 microM, respectively, and so distinctly better than free melophlin C. Another synergistic effect of coordinating melophlins to bioactive metals was found in the growth inhibition of the melophlin C-resistant bacterium Micrococcus luteus by Ga, La and Ru complexes of melophlin C.

First syntheses of melophlins P, Q, and R, and effects of melophlins on the growth of microorganisms and tumor cells.

The marine tetramic acid (=1,5-dihydro-4-hydroxy-2H-pyrrol-2-ones) derivatives melophlin P, Q, and R (1p-1r, resp.) were synthesized for the first time in only four steps. Together with the congenerous melophlins A-C and G, they were also tested for antimicrobial and cytotoxic effects. Melophlins B, C, P, Q, and R, which share a 5-Me residue, showed some antibacterial activity, mainly in Gram-positive bacteria. Melophlins B, C, and R, which have Me-branched 3-acyl side chains in common, inhibited the growth of cells of human KB-3-1 cervix carcinoma, A-498 kidney carcinoma, and U-937 leukemia with IC(50) values <10 muM. They were similar in activity to cisplatin. Melophlin Q, also Me-branched, was astoundingly specific in inhibiting A-498 kidney cancer cells, while melophlin P inhibited U-937 leukemia cells particularly well. The position of the Me branch is decisive for the magnitude of the antiproliferative effect of the melophlin couples B/C and R/Q.

Synthesis and biological properties of cylindramide derivatives: evidence for calcium-dependent cytotoxicity of tetramic acid lactams.

To gain insight into the biological properties of tetramic acid lactam cylindramide 1, the analogues 4 a-d bearing a cyclopentane ring instead of the pentalene unit were prepared by tandem conjugate addition/enolate trapping of cyclopentenone 10; a Sonogashira or Stille coupling, followed by a Julia-Kocienski olefination, macrolactamisation and Lacey-Dieckmann cyclisation were the key steps. The previous NMR structure of cylindramide 1, which was based on NOE and J coupling restraints, could be refined by including residual dipolar coupling data measured for a sample of cylindramide that was aligned in polyacrylonitrile (18 %). Biological screening of cylindramide 1 and its analogues 2-epi-1, 20 and 4 revealed promising antiproliferative activity against several tumour cell lines. It turned out that the activity is strongly correlated to the functionalised pentalene system. The configuration of the cyclopentane ring and an intact tetramic acid lactam with the correct configuration seem to play an equal role in the cytotoxicity. The antiproliferative activity was found to be calcium dependent. Phenotypic characterisation of the mode of action showed vacuolisation and vesicle formation in the endoplasmic reticulum.