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Given the rise of parasite resistance to all currently used antimalarial drugs, the identification of novel chemotypes with unique mechanisms of action is of paramount importance. Since Plasmodium expresses a number of aspartic proteases necessary for its survival, we have mined antimalarial datasets for drug-like aspartic protease inhibitors. This effort led to the identification of spiropiperidine hydantoins, bearing similarity to known inhibitors of the human aspartic protease β-secretase (BACE), as new leads for antimalarial drug discovery. Spiropiperidine hydantoins have a dynamic structure–activity relationship profile with positions identified as being tolerant of a variety of substitution patterns as well as a key piperidine N-benzyl phenol pharmacophore. Lead compounds 4e (CWHM-123) and 12k (CWHM-505) are potent antimalarials with IC50 values against Plasmodium falciparum 3D7 of 0.310 μM and 0.099 μM, respectively, and the former features equivalent potency on the chloroquine-resistant Dd2 strain. Remarkably, these compounds do not inhibit human aspartic proteases BACE, cathepsins D and E, or Plasmodium plasmepsins II and IV despite their similarity to known BACE inhibitors. Although the current leads suffer from poor metabolic stability, they do fit into a drug-like chemical property space and provide a new class of potent antimalarial agents for further study.

Background Facioscapulohumeral dystrophy (FSHD) is a progressive muscle disease caused by mutations that lead to epigenetic derepression and inappropriate transcription of the double homeobox 4 (DUX4) gene in skeletal muscle. Drugs that enhance the repression of DUX4 and prevent its expression in skeletal muscle cells therefore represent candidate therapies for FSHD. Methods We screened an aggregated chemical library enriched for compounds with epigenetic activities and the Pharmakon 1600 library composed of compounds that have reached clinical testing to identify molecules that decrease DUX4 expression as monitored by the levels of DUX4 target genes in FSHD patient-derived skeletal muscle cell cultures. Results Our screens identified several classes of molecules that include inhibitors of the bromodomain and extra-terminal (BET) family of proteins and agonists of the beta-2 adrenergic receptor. Further studies showed that compounds from these two classes suppress the expression of DUX4 messenger RNA (mRNA) by blocking the activity of bromodomain-containing protein 4 (BRD4) or by increasing cyclic adenosine monophosphate (cAMP) levels, respectively. Conclusions These data uncover pathways involved in the regulation of DUX4 expression in somatic cells, provide potential candidate classes of compounds for FSHD therapeutic development, and create an important opportunity for mechanistic studies that may uncover additional therapeutic targets. Electronic supplementary material The online version of this article (doi:10.1186/s13395-017-0134-x) contains supplementary material, which is available to authorized users.

Click chemistry technique led to novel 1,2,3-triazole-quinine conjugates 8a-g, 10a-o, 11a-h and 13 utilizing benzotriazole-mediated synthetic approach with excellent yields. Some of the synthesized analogs (11a, 11d-h) exhibited antimalarial properties against Plasmodium falciparum strain 3D7 with potency higher than that of quinine (standard reference used) through in vitro standard procedure bio-assay. Statistically significant BMLR-QSAR model describes the bio-properties, validates the observed biological observations and identifies the most important parameters governing bio-activity.

Summary Objective Statins possess anti-inflammatory properties. This study was undertaken to characterize the mechanism of action of statin drugs on collagenase expression in primary human osteoarthritic cartilage tissue. Method Human articular chondrocytes and cartilage explants from osteoarthritic donors were exposed to simvastatin in the presence or absence of interleukin-1 beta (IL-1β). Collagenase expression was determined by quantifying levels of matrix metalloproteinase 13 (MMP-13) and MMP-1 mRNA and MMP-13 protein. The mechanism of statin action was tested by addition of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) or by using inhibitors of farnesyl transferase (FT) and geranylgeranyl transferase (GGT-1). Results Treatment of osteoarthritic chondrocytes with simvastatin decreased mRNA levels of MMP-13 and MMP-1 whether under basal conditions or during stimulation with IL-1β. MMP-13 protein secreted into the culture media was also decreased. Genes involved in cartilage synthesis (type II collagen and aggrecan) were not down-regulated by simvastatin. Exogenous addition of GGPP completely reversed the statin-mediated decrease in MMP-13 mRNA and protein levels whereas FPP partially reversed the statin-mediated effect. An inhibitor of GGT-1 mimicked the simvastatin-mediated reduction in MMP-13 expression by chondrocytes. Finally, consistent with impacts on MMP-13 and MMP-1 expression, simvastatin as well as the GGT-1 inhibitor both blocked type II collagen degradation in primary human articular cartilage explants. Conclusion These results suggest that statins modulate chondrocyte metabolism by reducing prenylation of key signaling molecules that control the expression of collagen-degrading enzymes. Our results strongly support the hypothesis that protein prenyltransferases including geranylgeranyl transferase regulate chondrocyte collagenase expression in osteoarthritis.

Summary Insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) are trophic factors for cartilage and have been shown to be chondroprotective in animal models of osteoarthritis (OA). IGFBP-5 is degraded in joint fluid and inhibition of IGFBP-5 degradation has been shown to enhance the trophic effects of IGF-I. Objective To determine the identity of IGFBP-5 protease activity in human OA joint fluid. Method OA joint fluid was purified and the purified material was analyzed by IGFBP-5 zymography. Results Both crude joint fluid and purified material contained a single band of proteolytic activity that cleaved IGFBP-5. Immunoblotting of joint fluid for complement 1s (C1s) showed a band that had the same Mr estimate, e.g., 88kDa. In gel tryptic digestion and subsequent peptide analysis by LC-MS/MS showed that the band contained human C1s. A panel of protease inhibitors was tested for their ability to inhibit IGFBP-5 cleavage by the purified protease. Three serine protease inhibitors, FUT175 and CP-143217 and CB-349547 had IC50's between 1 and 6μM. Two other serine protease inhibitors had intermediate activity (e.g., IC50's 20–40μM) and MMP inhibitors had no detectible activity at concentrations up to 300μM. Conclusion Human OA fluid contains a serine protease that cleaves IGFBP-5. Zymography, immunoblotting and LC-MS/MS analysis indicate that C1s is the protease that accounts for this activity.

Insulin-like growth factor binding protein 5 (IGFBP-5) has been proposed to promote cartilage anabolism through insulin-like growth factor (IGF-1) signaling. A proteolytic activity towards IGFBP-5 has been detected in synovial fluids from human osteoarthritic (OA) joints. The purpose of this study was to determine if protease activity towards IGFBP-5 is present in the rat medial meniscal tear (MMT) model of OA and whether inhibition of this activity would alter disease progression. Sprague-Dawley rats were subject to MMT surgery. Synovial fluid lavages were assessed for the presence of IGFBP-5 proteolytic activity. Treatment animals received intra-articular injections of vehicle or protease inhibitor peptide PB-145. Cartilage lesions were monitored by India ink staining followed by macroscopic measurement of lesion width and depth. The MMT surgery induced a proteolytic activity towards IGFPB-5 that was detectable in joint fluid. This activity was stimulated by calcium and was sensitive to serine protease inhibitors as well as peptide PB-145. Significantly, intra-articular administration of PB-145 after surgery protected cartilage from lesion development. PB-145 treatment also resulted in an increase in cartilage turnover as evidenced by increases in serum levels of procollagen type II C-propeptide (CPII) as well as synovial fluid lavage levels of collagen type II neoepitope (TIINE). IGFBP-5 metabolism is disrupted in the rat MMT model of OA, potentially contributing to cartilage degradation. Inhibition of IGFBP-5 proteolysis protected cartilage from lesion development and enhanced cartilage turnover. These data are consistent with IGFBP-5 playing a positive role in anabolic IGF signaling in cartilage.

Regulatory T cells (Tregs), which are characterized by expression of the transcription factor Foxp3, are a dynamic and heterogeneous population of cells that control immune responses and prevent autoimmunity. We recently identified a subset of Tregs in murine skin with properties typical of memory cells and defined this population as memory Tregs (mTregs). Due to the importance of these cells in regulating tissue inflammation in mice, we analyzed this cell population in humans and found that almost all Tregs in normal skin had an activated memory phenotype. Compared with mTregs in peripheral blood, cutaneous mTregs had unique cell surface marker expression and cytokine production. In normal human skin, mTregs preferentially localized to hair follicles and were more abundant in skin with high hair density. Sequence comparison of TCRs from conventional memory T helper cells and mTregs isolated from skin revealed little homology between the two cell populations, suggesting that they recognize different antigens. Under steady-state conditions, mTregs were nonmigratory and relatively unresponsive; however, in inflamed skin from psoriasis patients, mTregs expanded, were highly proliferative, and produced low levels of IL-17. Taken together, these results identify a subset of Tregs that stably resides in human skin and suggest that these cells are qualitatively defective in inflammatory skin disease.

The cellular protein AMF-1 (Gps2) positively modulates gene expression by the papillomavirus E2 protein (D. E. Breiding et al., Mol. Cell. Biol. 17:7208–7219, 1997). We show here that AMF-1 also binds the transcriptional coactivator p300 in vitro and in vivo. E2 interacted weakly with p300. These observations led to a model in which AMF-1 recruits p300 into a complex with E2. Cotransfection of AMF-1 or p300 stimulated levels of E2-dependent transcription, while cotransfection of both AMF-1 and p300 showed an additive effect. The functional significance of p300 recruitment for E2 transactivation was evidenced by repression of E2-activated transcription by adenovirus E1A, which inhibits both coactivator and acetylase activities of p300. Antibodies to AMF-1 or E2 immunoprecipitated histone acetylase activity from cell lysates. Western blotting using antibody against acetyl-lysine failed to detect acetylation of AMF-1 or E2 in complex with p300. These results suggest that AMF-1 facilitates the recruitment of p300 and its histone acetylase activity into complexes with E2 and represents a novel mechanism of transcriptional activation.

Gene delivery into human cells has been facilitated by the development of viral vector systems. These vectors have shown great potential for the efficient delivery of therapeutic genes into human cells. A problem with many of the existing systems, however, is the integration of these vectors into the chromosome which affects the length of gene expression and may promote oncogenic transformation. In an effort to develop viral vectors that can replicate extrachromosomally in human cells, we have generated human papillomavirus (HPV) plasmids containing all the elements required for replication on a single DNA molecule. HPV plasmids containing the viral E1 and E2 genes (or the E1 gene alone) and an origin of replication were shown to replicate to significant levels in the transfected human cervical carcinoma C-33A cell line. Since approaches towards the possible gene therapy of cystic fibrosis (CF) are currently under intensive investigation, we have also tested short-term replication of HPV plasmids in the IB3 cell line derived from a CF patient. Our results demonstrate that HPV plasmids are capable of extrachromosomal replication in these cell lines and may potentially be important vectors for the delivery of therapeutic genes into human cells.

Human cells are generally thought to be nonpermissive for polyomavirus (Py) DNA replication. Using transient transfection, we show that Py large T-antigen (LT) was able to support replication of a Py origin-containing plasmid in two human cell lines. Replication supported by LT in human cells was specific for the Py origin and required its enhancer sequences, as well as the previously reported critical phosphorylation sites within LT. Py replication efficiency was comparable to that of papillomavirus E1- and E2-activated DNA replication in transient assays performed in human 293 and C-33A cells. Previous analysis of DNA replication in vitro has pointed to polymerase α–primase as a specificity determinant for polyomavirus. The data presented here imply that in certain cellular environments, Py LT must functionally interact with human polymerase α–primase to permit DNA replication.

Novel drugs are required for the elimination of infections caused by filarial worms, as most commonly used drugs largely target the microfilariae or first stage larvae of these infections. Previous studies, conducted in vitro, have shown that inhibition of Hsp90 kills adult Brugia pahangi. As numerous small molecule inhibitors of Hsp90 have been developed for use in cancer chemotherapy, we tested the activity of several novel Hsp90 inhibitors in a fluorescence polarization assay and against microfilariae and adult worms of Brugia in vitro. The results from all three assays correlated reasonably well and one particular compound, NVP-AUY922, was shown to be particularly active, inhibiting Mf output from female worms at concentrations as low as 5.0 nanomolar after 6 days exposure to drug. NVP-AUY922 was also active on adult worms after a short 24 h exposure to drug. Based on these in vitro data, NVP-AUY922 was tested in vivo in a mouse model and was shown to significantly reduce the recovery of both adult worms and microfilariae. These studies provide proof of principle that the repurposing of currently available Hsp90 inhibitors may have potential for the development of novel agents with macrofilaricidal properties., Author Summary Adult filarial worms are long-lived nematode parasites that have proved very difficult to kill with existing drugs. Current campaigns for the control or elimination of these parasites are largely based on treatment with drugs such as diethylcarbamazine or ivermectin, that preferentially kill the first stage larvae of the parasite, the microfilariae. As microfilariae repopulate the body from unaffected adult worms, repeated dosing with these drugs is required over the long reproductive life span of the worm. The availability of compounds with macrofilaricidal activity would help facilitate the goal of controlling filarial infections. Hsp90 is a recognized target in tumor cells: consequently many oncology programs have developed small molecule inhibitors of Hsp90, several of which are commercially available. Here we provide proof of principle that inhibition of Hsp90 is lethal to adult Brugia worms in vivo, as well as in vitro, suggesting that these compounds may have potential for further development as macrofilaricidal drugs.

The transactivation domain (AD) of bovine papillomavirus type 1 E2 stimulates gene expression and DNA replication. To identify cellular proteins that interact with this 215-amino-acid domain, we used a transactivation-defective mutant as bait in the yeast two-hybrid screen. In vitro and in vivo results demonstrate that the cDNA of one plasmid isolated in this screen encodes a 37-kDa nuclear protein that specifically binds to an 82-amino-acid segment within the E2 AD. Mutants with point mutations within this E2 domain were isolated based on their inability to interact with AMF-1 and were found to be unable to stimulate transcription. These mutants also exhibited defects in viral DNA replication yet retained binding to the viral E1 replication initiator protein. Overexpression of AMF-1 stimulated transactivation by both wild-type E2 and a LexA fusion to the E2 AD, indicating that AMF-1 is a positive effector of the AD of E2. We conclude that interaction with AMF-1 is necessary for the transcriptional activation function of the E2 AD in mammalian cells.

Given the threat of drug resistance, there is an acute need for new classes of antimalarial agents that act via a unique mechanism of action relative to currently used drugs. We have identified a set of druglike compounds within the Tres Cantos Anti-Malarial Set (TCAMS) which likely act via inhibition of a Plasmodium aspartic protease. Structure–activity relationship analysis and optimization of these aminohydantoins demonstrate that these compounds are potent nanomolar inhibitors of the Plasmodium aspartic proteases PM-II and PM-IV and likely one or more other Plasmodium aspartic proteases. Incorporation of a bulky group, such as a cyclohexyl group, on the aminohydantion N-3 position gives enhanced antimalarial potency while reducing inhibition of human aspartic proteases such as BACE. We have identified compound 8p (CWHM-117) as a promising lead for optimization as an antimalarial drug with a low molecular weight, modest lipophilicity, oral bioavailability, and in vivo antimalarial activity in mice.

Bovine papillomavirus type 1 replication was previously shown to require both the E1 initiator protein and the E2 transactivator protein. We show here that E1, in the absence of E2, is sufficient for low-level bovine papillomavirus type 1 DNA replication in C-33A cells. In addition, studies of genetically isolated E2 point mutants demonstrate that enhancement of replication by E2 does not require its transcriptional activation function. The uncoupling of the E2 functions suggests that stimulation of transcription and replication by enhancer proteins occurs via divergent mechanisms.

Malaria is a devastating and common infectious disease,responsible for more than one million deaths each year.Quinine (Figure 1), an alkaloid isolated from the bark of thecinchona tree (1), has been the most acceptable antima-larial drug for almost 400 years. The protozoal parasiteresponsible for malaria, Plasmodium falciparum, hasgained resistance to most forms of monotherapy especiallyin South-East Asia, South America, and East Africa (2).However, quinine has an impressive track record consider-ing that most antimalarial drugs encounter parasite resis-tance within a few years of introduction. Quinine is treatedas a privileged natural structure because of its low parasiteresistance, which is difficult for the parasite to circumvent.Quinoline-containing antimalarial drugs, such as quinine,chloroquine, and mefloquine, are mainstays of chemother-apy against malaria. While the search for novel drug tar-gets and new lead structures for the treatment of malariais a critical objective, a complimentary strategy is to utilizeand develop lead structures from nature with proven effi-cacious properties like quinine or artemisinin. The synthe-sis of quinine derivatives, a strategy that led to thediscovery of chloroquine, has been largely abandoned (3)because of toxicity and reaction complexity. Artemisinin isused against chloroquine-resistant malarial parasites. Thepoor solubility of artemisinin coupled with its short plasmahalf-life led to a high rate of parasite recrudescence. Modi-fication of the cinchona alkaloids is one of the most suc-cessful strategies for antimalarial drug development (3,4).Peptide derivatives of primaquine evidenced a comparableor greater activity of conjugates with respect of the parentdrug (5).Efficient cellular drug delivery is a severe problem for manytherapeutic agents due to charge, hydrophilic character,and/or size. High drug doses, necessary to compensatefor the reduced bioavailability, often cause strong adverseeffects. Synthetic drug delivery vectors can sometimessolve this problem, if limitations like low-cellular uptake effi-ciency or cytotoxicity are overcome. Among such syntheticvectors, so-called cell-penetrating peptides have provenapplicability because of their ability as drug carriers tocross cell membranes (6). Drug–peptide conjugates arealso known to overcome multidrug resistance in chemo-therapy and bind to specific receptors expressed on tumorcells (7–9).Recently, Islahudin et al. reported that quinine can blocka cell’s ability to take up essential amino acids like trypto-phan and tyrosine; they suggested that dietary amino acidsupplements could improve the performance of quinine(10,11). Amino acids like

Replication of papillomaviruses requires an origin of replication and two virus-encoded proteins, E1 and E2. Using a transient replication assay for human papillomavirus type 18 (HPV-18) DNA, we have found that two adjacent sequences present within the origin of replication can independently support replication. The first, a 77-bp region, contains one E2 binding site (E2BS) and a 16-bp inverted repeat element that probably corresponds to the E1 binding site (E1BS). The other, an 81-bp region, includes two E2BS but lacks the putative E1BS. A synthetic 33-bp oligonucleotide containing two high-affinity E2BS was also found to function as an origin of replication. Replication of all these plasmids was absolutely dependent on the presence of the HPV-18 E1 and E2 proteins. The HPV-1a E1 and E2 proteins were also found to support replication of a plasmid containing the complete HPV-18 origin but failed to replicate a plasmid containing two E2BS alone. Our results suggest that the E2 protein can target E1 to the origin through the formation of an E1-E2 complex which is likely to be involved the initiation of replication.

Foxp3+ CD4+ T helper cells called regulatory T (T reg) cells play a key role in controlling reactivity to self-antigens and onset of autoimmunity. T reg cells either arise in thymus and are called natural T reg (nT reg) cells or are generated in the periphery through induction of Foxp3 and are called inducible T reg (iT reg) cells. The relative contributions of iT reg cells and nT reg cells in peripheral tolerance remain unclear as a result of an inability to separate these two subsets of T reg cells. Using a combination of novel TCR transgenic mice with a defined self-antigen specificity and conventional mouse models, we demonstrate that a cell surface molecule, neuropilin-1 (Nrp-1), is expressed at high levels on nT reg cells and can be used to separate nT reg versus iT reg cells in certain physiological settings. In addition, iT reg cells generated through antigen delivery or converted under homeostatic conditions lack Nrp-1 expression. Nrp-1lo iT reg cells show similar suppressive activity to nT reg cells in controlling ongoing autoimmune responses under homeostatic conditions. In contrast, their activity might be compromised in certain lymphopenic settings. Collectively, our data show that Nrp-1 provides an excellent marker to distinguish distinct T reg subsets and will be useful in studying the role of nT reg versus iT reg cells in different disease settings.© 2012 Yadav et al.

Introduction Glucocorticoids are known to attenuate bone formation in vivo leading to decreased bone volume and increased risk of fractures, whereas effects on the joint tissue are less characterized. However, glucocorticoids appear to have a reducing effect on inflammation and pain in osteoarthritis. This study aimed at characterizing the effect of glucocorticoids on chondrocytes, osteoclasts, and osteoblasts. Experimental We used four model systems to investigate how glucocorticoids affect the cells of the joint; two intact tissues (femoral head- and cartilage-explants), and two separate cell cultures of osteoblasts (2T3-pre-osteoblasts) and osteoclasts (CD14+-monocytes). The model systems were cultured in the presence of two glucocorticoids; prednisolone or dexamethasone. To induce anabolic and catabolic conditions, cultures were activated by insulin-like growth factor I/bone morphogenetic protein 2 and oncostatin M/tumor necrosis factor-α, respectively. Histology and markers of bone- and cartilage-turnover were used to evaluate effects of glucocorticoid treatment. Results Prednisolone treatment decreased collagen type-II degradation in immature cartilage, whereas glucocorticoids did not affect collagen type-II in mature cartilage. Glucocorticoids had an anti-catabolic effect on catabolic-activated cartilage from a bovine stifle joint and murine femoral heads. Glucocorticoids decreased viability of all bone cells, leading to a reduction in osteoclastogenesis and bone resorption; however, bone morphogenetic protein 2-stimulated osteoblasts increased bone formation, as opposed to non-stimulated osteoblasts. Conclusions Using highly robust in vitro models of bone and cartilage turnover, we suggest that effects of glucocorticoids highly depend on the activation and differential stage of the cell targeted in the joint. Present data indicated that glucocorticoid treatment may be beneficial for articular cartilage, although detrimental effects on bone should be taken into account.

The papillomavirus E2 proteins regulate viral replication, gene transcription, and genome maintenance by interacting with other viral and host proteins. From a yeast two-hybrid screen, we identified the cellular protein Tax1BP1 as a novel binding partner of human papillomavirus type 18 (HPV18) E2. Tax1BP1 also interacts with the HPV16 and bovine papillomavirus type 1 (BPV1) E2 proteins, with the C-terminal region of Tax1BP1 interacting with the N-terminal transactivation domain of BPV1 E2. Tax1BP1 complexes with p300 and acts synergistically as a coactivator with p300 to enhance E2-dependent transcription. Using chromatin immunoprecipitation assays, we show that Tax1BP1 and E2 localize to the long control region on the BPV1 genome. Tax1BP1 was recently reported to bind ubiquitin and to function as an essential component of an A20 ubiquitin-editing complex. We demonstrate that Tax1BP1 plays a role in the regulation of the steady-state level of E2 by preventing its proteasomal degradation. These studies provide new insights into the regulation of E2 functions.

A number of vector systems have been developed for the delivery of therapeutic genes into cells (1). Many viral vectors suffer from the disadvantage of random integration into the chromosome, making the expression of the cloned genes dependent on the chromosomal context of the inserted DNA. Papillomaviruses (PVs) are potentially important vector systems because of their extrachromosomal replication in target cells. The PVs are small DNA viruses that infect humans and a wide range of animals. Human papillomaviruses (HPVs) induce benign proliferative squamous eplthelial and fibro-epithelial lesions (warts and papillomas) in their natural hosts (2). Some HPVs are also involved in the pathogenesis of anogenital cancer and, in particular, cancer of the cervix (2). Papillomaviruses contain circular, double-stranded DNA of approx 8 kb, and usually replicate extrachromosomally at a copy number estimated to be between 10 and 100 (3). The potential advantages of PV vectors include expression of cloned genes from an extrachromosomal state that may be more amenable to uniform expression and possible elimination of problems associated with integration of DNA into transcriptionally inactive regions of the cellular chromosomes. Since PV DNA is not encapsidated, it may be possible to insert larger DNA sequences into such vectors, provided the DNA can still replicate in a stable manner. Bovine papillomavirus type 1 (BPV-1) vectors have been used to produce stable cell lines expressing foreign proteins (for reviews, see refs. 4-7). The BPV-1 vectors used in these studies contained both replication and transforming genes, and in most cases, extrachromosomal replication of these vectors was accompanied by transformation of the target cells (8-12). Recent advances in our understanding of the replication and transforming genes of PVs have resulted in renewed efforts to develop vectors that can be established as stable extrachromosomal plasmids and express foreign proteins without oncogenic transformation of the host cell. The current article deals mostly with the potential of HPVs as vectors for gene therapy.

Papillomavirus E1 and E2 proteins co-operation in viral DNA replication is mediated by protein-protein interactions that lead to formation of an E1-E2 complex. To identify the domains involved, portions of the two proteins were expressed as fusions to the DNA-binding protein LexA or the transactivation domain of VP16 and analyzed by the yeast two-hybrid system. The C-terminal 266 amino acids of BPV1 E1 (E1C266) interacted strongly with E2 in the yeast system and in a mammalian two-hybrid assay. VP16-E1C266 interacted with a region encompassing amino acids 1-200 of the transactivation domain of E2 that was fused to LexA. The interaction between E1 full length and E2 was clearly observed only when E1 was expressed as LexA-E1 chimera. In addition, we found that in the LexA context also the N-terminal region encompassing the first 340 amino acids of E1 (E1N340) interacted with E2 full length. The interactions of E1N340 and E1C266 with E2 were confirmed also by in vitro binding studies. These observations demonstrate that two distinct regions of E1 mediate the interaction with E2 in vivo.

Antisense technology provides a high-throughput and systematic approach to drug target validation and gene function discovery. In combination with other emerging technologies (such as microarrays), this technology will enable efficient evaluation of the sequence data generated by the Human Genome Project. The authors review recent advances in the antisense field and discuss the potential use of antisense technology for functional genomics.

Publisher Summary Human papillomaviruses (HPVs) contain a circular, double-stranded DNA genome of approximately 8 kb that is usually maintained in an extrachromosomal form at a relatively fixed copy number between 10 and 50. HPVs are responsible for benign epithelial proliferations (warts), and many HPVs are associated with human anogenital cancers, particularly cancer of the cervix. Replication of HPVs requires the viral E1 and E2 proteins and an origin of replication (ori). The ori contains an El-binding site and multiple E2-binding sites. The E1 and E2 proteins encoded by various HPVs are quite homologous. E1 is the replication initiator protein that is involved in the regulation of viral gene expression through its interaction with the E2 protein. This chapter describes the expression of the E1 proteins of HPV types 1a and 18 in mammalian and insect cells and the purification of the HPV-1a E1 protein from insect ceils using a baculovirus vector.

Benzotriazole-mediated syntheses led to novel bis-conjugates of quinine with quinolone antibiotics and amino acid linkers which were successfully prepared by two alternative routes with excellent yields and retention of chirality. These bis conjugates retain in vitro antimalarial activity with IC(50) values ranging from 12 to 207 nM, similar to quinine itself.

Transient replication of human papillomavirus (HPV) type 18 DNA was shown to require the viral E1 and E2 proteins. A 108-bp sequence within the long control region (nucleotides 12 to 119) was sufficient to function as the origin, but maximal replication required a region of 177 bp from positions 7800 to 7857 and 1 to 119 of HPV-18. The E1 and E2 proteins of HPV-18 also supported transient replication of plasmids containing the origins of HPV-1a and bovine papillomavirus type 1 to low levels. Interestingly, the level of replication observed with the HPV-6b origin was higher than that obtained with the homologous HPV-18 origin.