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Preventing transmission is an important element of malaria control. However, most of the current available methods to assay for malaria transmission blocking are relatively low throughput and cannot be applied to large chemical libraries. We have developed a high-throughput and cost-effective assay, the Saponin-lysis Sexual Stage Assay (SaLSSA), for identifying small molecules with transmission-blocking capacity. SaLSSA analysis of 13,983 unique compounds uncovered that >90% of well-characterized antimalarials, including endoperoxides and 4-aminoquinolines, as well as compounds active against asexual blood stages, lost most of their killing activity when parasites developed into metabolically quiescent stage V gametocytes. On the other hand, we identified compounds with consistent low nanomolar transmission-blocking activity, some of which showed cross-reactivity against asexual blood and liver stages. The data clearly emphasize substantial physiological differences between sexual and asexual parasites and provide a tool and starting points for the discovery and development of transmission-blocking drugs., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The development of artemisinin resistance in the Greater Mekong Subregion poses a significant threat to malaria elimination. Artemisinin-based combination therapies including artemether-lumefantrine (AL) are recommended by WHO as first-line treatment for uncomplicated Plasmodium falciparum malaria. This article provides a comprehensive review of the existing and latest data as a basis for interpretation of observed variability in parasite sensitivity to AL over the last 5 years. Clinical efficacy and preclinical data from a range of endemic countries are summarized, including potential molecular markers of resistance. Overall, AL remains effective in the treatment of uncomplicated P. falciparum malaria in most regions. Establishing validated molecular markers for resistance and strict efficacy monitoring will reinforce timely updates of treatment policies.

Aminopyrazoles are a new class of antimalarial compounds identified in a cellular antiparasitic screen with potent activity against Plasmodium falciparum asexual and sexual stage parasites. To investigate their unknown mechanism of action and thus identify their target, we cultured parasites in the presence of a representative member of the aminopyrazole series, GNF-Pf4492, to select for resistance. Whole genome sequencing of three resistant lines showed that each had acquired independent mutations in a P-type cation-transporter ATPase, PfATP4 (PF3D7_1211900), a protein implicated as the novel Plasmodium spp. target of another, structurally unrelated, class of antimalarials called the spiroindolones and characterized as an important sodium transporter of the cell. Similarly to the spiroindolones, GNF-Pf4492 blocks parasite transmission to mosquitoes and disrupts intracellular sodium homeostasis. Our data demonstrate that PfATP4 plays a critical role in cellular processes, can be inhibited by two distinct antimalarial pharmacophores, and supports the recent observations that PfATP4 is a critical antimalarial target.

An ultrahigh-throughput screen was performed to identify novel small molecule inhibitors of influenza virus replication. The screen employed a recombinant influenza A/WSN/33 virus expressing Renilla luciferase and yielded a hit rate of 0.5%, of which the vast majority showed little cytotoxicity at the inhibitory concentration. One of the top hits from this screen, designated S20, inhibits HA-mediated membrane fusion. S20 shows potent antiviral activity (IC 50 = 80 nM) and low toxicity (CC 50 = 40 μM), yielding a selectivity index of 500 and functionality against all of the group 1 influenza A viruses tested in this study, including the pandemic H1N1 and avian H5N1 viruses. Mechanism of action studies proved a direct S20-HA interaction and showed that S20 inhibits fusion by stabilizing the prefusion conformation of HA. In silico docking studies were performed, and the predicted binding site in HA2 corresponds with the area where resistance mutations occurred and correlates with the known role of this region in fusion. This high-throughput screen has yielded many promising new lead compounds, including S20, which will potentially shed light on the molecular mechanisms of viral infection and serve as research tools or be developed for clinical use as antivirals.

New chemotherapeutic agents are urgently required to combat the global spread of multidrug-resistant tuberculosis (MDR-TB). The mycobacterial enoyl reductase InhA is one of the few clinically validated targets in tuberculosis drug discovery. We report the identification of a new class of direct InhA inhibitors, the 4-hydroxy-2-pyridones, using phenotypic high-throughput whole-cell screening. This class of orally active compounds showed potent bactericidal activity against common isoniazid-resistant TB clinical isolates. Biophysical studies revealed that 4-hydroxy-2-pyridones bound specifically to InhA in an NADH (reduced form of nicotinamide adenine dinucleotide)-dependent manner and blocked the enoyl substrate-binding pocket. The lead compound NITD-916 directly blocked InhA in a dose-dependent manner and showed in vivo efficacy in acute and established mouse models of Mycobacterium tuberculosis infection. Collectively, our structural and biochemical data open up new avenues for rational structure-guided optimization of the 4-hydroxy-2-pyridone class of compounds for the treatment of MDR-TB., (Copyright © 2015, American Association for the Advancement of Science.)

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 μM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 μM, and PQ, 0.84 μM; for developing liver stages, KAI407, 0.64 μM, and PQ, 0.37 μM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure.

New chemotherapeutic compounds against multidrug-resistant Mycobacterium tuberculosis (Mtb) are urgently needed to combat drug resistance in tuberculosis (TB). We have identified and characterized the indolcarboxamides as a new class of antitubercular bactericidal agent. Genetic and lipid profiling studies identified the likely molecular target of indolcarboxamides as MmpL3, a transporter of trehalose monomycolate that is essential for mycobacterial cell wall biosynthesis. Two lead candidates, NITD-304 and NITD-349, showed potent activity against both drug-sensitive and multidrug-resistant clinical isolates of Mtb. Promising pharmacokinetic profiles of both compounds after oral dosing in several species enabled further evaluation for efficacy and safety. NITD-304 and NITD-349 were efficacious in treating both acute and chronic Mtb infections in mouse efficacy models. Furthermore, dosing of NITD-304 and NITD-349 for 2 weeks in exploratory rat toxicology studies revealed a promising safety margin. Finally, neither compound inhibited the activity of major cytochrome P-450 enzymes or the hERG (human ether-a-go-go related gene) channel. These results suggest that NITD-304 and NITD-349 should undergo further development as a potential treatment for multidrug-resistant TB.

Indole-2-carboxamides have been identified as a promising class of antituberculosis agents from phenotypic screening against mycobacteria. One of the hits, indole-2-carboxamide analog (1), had low micromolar potency against Mycobacterium tuberculosis (Mtb), high mouse liver microsomal clearance, and low aqueous solubility. Structure-activity relationship studies revealed that attaching alkyl groups to the cyclohexyl ring significantly improved Mtb activity but reduced solubility. Furthermore, chloro, fluoro, or cyano substitutions on the 4- and 6-positions of the indole ring as well as methyl substitution on the cyclohexyl ring significantly improved metabolic stability. 39 and 41, the lead candidates, displayed improved in vitro activity compared to most of the current standard TB drugs. The low aqueous solubility could not be mitigated because of the positive correlation of lipophilicity with Mtb potency. However, both compounds displayed favorable oral pharmacokinetic properties in rodents and demonstrated in vivo efficacy. Thus, indole-2-carboxamides represent a promising new class of antituberculosis agents.

Tetrahydropyrazolo[1,5-a]pyrimidine scaffold was identified as a hit series from a Mycobacterium tuberculosis (Mtb) whole cell high through-put screening (HTS) campaign. A series of derivatives of this class were synthesized to evaluate their structure-activity relationship (SAR) and structure-property relationship (SPR). Compound 9 had a promising in vivo DMPK profile in mouse and exhibited potent in vivo activity in a mouse efficacy model, achieving a reduction of 3.5 log CFU of Mtb after oral administration to infected mice once a day at 100 mg/kg for 28 days. Thus, compound 9 is a potential candidate for inclusion in combination therapies for both drug-sensitive and drug-resistant TB.

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.

On the basis of the initial success of optimization of a novel series of imidazolopiperazines, a second generation of compounds involving changes in the core piperazine ring was synthesized to improve antimalarial properties. These changes were carried out to further improve the potency and metabolic stability of the compounds by leveraging the outcome of a set of in vitro metabolic identification studies. The optimized 8,8-dimethyl imidazolopiperazine analogues exhibited improved potency, in vitro metabolic stability profile and, as a result, enhanced oral exposure in vivo in mice. The optimized compounds were found to be more efficacious than the current antimalarials in a malaria mouse model. They exhibit moderate oral exposure in rat pharmacokinetic studies to achieve sufficient multiples of the oral exposure at the efficacious dose in toxicology studies.

Starting from a hit series from a GNF compound library collection and based on a cell-based proliferation assay of Plasmodium falciparum, a novel imidazolopiperazine scaffold was optimized. SAR for this series of compounds is discussed, focusing on optimization of cellular potency against wild-type and drug resistant parasites and improvement of physiochemical and pharmacokinetic properties. The lead compounds in this series showed good potencies in vitro and decent oral exposure levels in vivo. In a Plasmodium berghei mouse infection model, one lead compound lowered the parasitemia level by 99.4% after administration of 100 mg/kg single oral dose and prolonged mice survival by an average of 17.0 days. The lead compounds were also well-tolerated in the preliminary in vitro toxicity studies and represents an interesting lead for drug development.

The search for novel therapeutic interventions for viral disease is a challenging pursuit, hallmarked by the paucity of antiviral agents currently prescribed. Targeting of viral proteins has the inextricable challenge of rise of resistance. Safe and effective vaccines are not possible for many viral pathogens. New approaches are required to address the unmet medical need in this area. We undertook a cell-based high-throughput screen to identify leads for development of drugs to treat respiratory syncytial virus (RSV), a serious pediatric pathogen. We identified compounds that are potent (nanomolar) inhibitors of RSV in vitro in HEp-2 cells and in primary human bronchial epithelial cells and were shown to act postentry. Interestingly, two scaffolds exhibited broad-spectrum activity among multiple RNA viruses. Using the chemical matter as a probe, we identified the targets and identified a common cellular pathway: the de novo pyrimidine biosynthesis pathway. Both targets were validated in vitro and showed no significant cell cytotoxicity except for activity against proliferative B- and T-type lymphoid cells. Corollary to this finding was to understand the consequences of inhibition of the target to the host. An in vivo assessment for antiviral efficacy failed to demonstrate reduced viral load, but revealed microscopic changes and a trend toward reduced pyrimidine pools and findings in histopathology. We present here a discovery program that includes screen, target identification, validation, and druggability that can be broadly applied to identify and interrogate other host factors for antiviral effect starting from chemical matter of unknown target/mechanism of action.

Plasmodium vivax causes 25-40% of malaria cases worldwide, yet research on this human malaria parasite has been neglected. Nevertheless, the recent publication of the P. vivax reference genome now allows genomics and systems biology approaches to be applied to this pathogen. We show here that whole-genome analysis of the parasite can be achieved directly from ex vivo-isolated parasites, without the need for in vitro propagation. A single isolate of P. vivax obtained from a febrile patient with clinical malaria from Peru was subjected to whole-genome sequencing (30× coverage). This analysis revealed over 18,261 single-nucleotide polymorphisms (SNPs), 6,257 of which were further validated using a tiling microarray. Within core chromosomal genes we find that one SNP per every 985 bases of coding sequence distinguishes this recent Peruvian isolate, designated IQ07, from the reference Salvador I strain obtained in 1972. This full-genome sequence of an uncultured P. vivax isolate shows that the same regions with low numbers of aligned sequencing reads are also highly variable by genomic microarray analysis. Finally, we show that the genes containing the largest ratio of nonsynonymous-to-synonymous SNPs include two AP2 transcription factors and the P. vivax multidrug resistance-associated protein (PvMRP1), an ABC transporter shown to be associated with quinoline and antifolate tolerance in Plasmodium falciparum. This analysis provides a data set for comparative analysis with important potential for identifying markers for global parasite diversity and drug resistance mapping studies.

Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine-imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.

The antiplasmodial activity of a series of spirotetrahydro beta-carbolines is described. Racemic spiroazepineindole (1) was identified from a phenotypic screen on wild type Plasmodium falciparum with an in vitro IC(50) of 90 nM. Structure-activity relationships for the optimization of 1 to compound 20a (IC(50) = 0.2 nM) including the identification of the active 1R,3S enantiomer and elimination of metabolic liabilities is presented. Improvement of the pharmacokinetic profile of the series translated to exceptional oral efficacy in the P. berghei infected malaria mouse model where full cure was achieved in four of five mice with three daily doses of 30 mg/kg.

A novel family of 1H-imidazol-2-yl-pyrimidine-4,6-diamines has been identified with potent activity against the erythrocyte-stage of Plasmodium falciparum (Pf), the most common causative agent of malaria. A systematic SAR study resulted in the identification of compound 40 which exhibits good potency against both wild-type and drug resistant parasites and exhibits good in vivo pharmacokinetic properties., (2010 Elsevier Ltd. All rights reserved.)

Screening our in-house compound collection using a cell based Plasmodium falciparum proliferation assay we discovered a known pan-kinase inhibitor scaffold as a hit. Further optimization of this series led us to a novel benzamide scaffold which was devoid of human kinase activity while retaining its antiplasmodial activity. The evolution of this compound series leading to optimized candidates with good cellular potency against multiple strains as well as decent in vivo profile is described in this Letter.

The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of approximately 1.7 million compounds, we identified a diverse collection of approximately 6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 microM). Most known antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities.

Many companies possess a compound collection consisting of purified compounds and of unpurified products from combinatorial libraries. Using commercial and proprietary compounds as examples, this report provides clear examples of the significant impact purification can have on the activity observed for a compound and highlights the need to retest the purified compounds prior to creating structure-activity relationships. Crude mixtures made with commercial compounds led to an increase in the number of false positives in the SXR-GAL4 assay as compared with their pure and purified counterparts. An examination of proprietary compounds in an HIV assay resulted in the purification of 61 active crude synthetic mixtures. Of these 61 compounds, 32 were 5-fold less active and 2 were 5-fold more active after purification. This report details a semiautomated process developed and implemented for cherry-picking, tracking, and selectively purifying compounds found active in high-throughput screening campaigns.

The synthesis and preliminary structure-activity relationship of a series of pyrrolidinones are described. These pyrrolidinones have been characterized as novel non-nucleoside reverse transcriptase inhibitors (NNRTIs) which are highly potent against wild-type and drug-resistant human immunodeficiency viruses (HIV-1).

HIV-1 integrase (HIV-IN) is a well-validated antiviral drug target catalyzing a multistep reaction to incorporate the HIV-1 provirus into the genome of the host cell. Small molecule inhibitors of HIV-1 integrase that specifically target the strand transfer step have demonstrated efficacy in the suppression of virus propagation. However, only few specific strand transfer inhibitors have been identified to date, and the need to screen for novel compound scaffolds persists. Here, the authors describe 2 homogeneous time-resolved fluorescent resonance energy transfer-based assays for the measurement of HIV-1 integrase 3'-processing and strand transfer activities. Both assays were optimized for high-throughput screening formats, and a diverse library containing more than 1 million compounds was screened in 1536-well plates for HIV-IN strand transfer inhibitors. As a result, compounds were found that selectively affect the enzymatic strand transfer reaction over 3beta processing. Moreover, several bioactive molecules were identified that inhibited HIV-1 reporter virus infection in cellular model systems. In conclusion, the assays presented herein have proven their utility for the identification of mechanistically interesting and biologically active inhibitors of HIV-1 integrase that hold potential for further development into potent antiviral drugs.

Ribozymes are small, catalytic RNA molecules that can be engineered to down-regulate gene expression by cleaving specific mRNA. Here we report the selection of hairpin ribozymes that inhibit human immunodeficiency virus (HIV) replication from a combinatorial ribozyme library. We identified a total of 17 effective ribozymes, each capable of inhibiting HIV infection of human CD4(+) cells. These ribozymes target diverse steps of the viral replication cycle, ranging from entry to transcription. One ribozyme suppressed HIV integration and transcription by inhibiting the expression of the Ku80 subunit of the DNA-activated protein kinase. Another ribozyme specifically inhibited long terminal repeat transactivation, while two additional ones blocked a step that can be bypassed by vesicular stomatitis virus G-protein pseudotyping. The function of Ku80 in HIV replication and its mechanism of action were further confirmed using short interfering RNA. Identification of the gene targets of these and other selected ribozymes may reveal novel therapeutic targets for combating HIV infection.

Dendritic cells hold promise as adjuvant for immunotherapy for cancer and infectious diseases. We demonstrate that a significant number of cryopreserved peripheral blood CD34(+) cells from HIV-infected subjects can be transduced with a replication-incompetent lentiviral vector expressing HIV antigens. In addition, untransduced and transduced CD34(+) cells from HIV-infected individuals were able to differentiate into dendritic cells with strong T-cell stimulatory capacity. Thus, cryopreserved CD34(+) cells from HIV-infected subjects may prove useful for immunotherapy for HIV/AIDS., (Copyright 2003 Wiley-Liss, Inc.)

Recent findings show that human immunodeficiency virus (HIV)-1 protease inhibitors designed to specifically inhibit the aspartic protease of HIV-1 nonetheless exert various effects on immune cell function in vitro and in vivo. Dendritic cells (DC), central players of the immune system, express several aspartic proteases that are important for DC function. In the present study, we demonstrate that all of the HIV-1 protease inhibitors tested affect DC maturation. In addition, saquinavir had a strong inhibitory effect on the T-cell stimulatory capacity of mature DC. In contrast, indinavir had only a slight effect on DC induced T-cell proliferation and allowed efficient transduction of DC with a replication-incompetent HIV-1 vector designed for DC-based immunotherapy. HIV-1 protease inhibitors that have little or no effect on DC function may be preferable for combination with immunotherapy for HIV/AIDS.

Dendritic cells (DCs) genetically modified to continually express and present antigens may be potent physiologic adjuvants for induction of prophylactic or therapeutic immunity. We have previously shown that an env and nef deleted HIV-1 vector (HIV-1 delta EN) pseudotyped with VSV-G transduced monocyte-derived macrophages as well as CD34(+) precursors of DCs. Here we extended these findings with HIV-1 delta EN to highly differentiated human DCs derived in culture from circulating monocytes (DCs). In addition, a new vector derived from HIV-1 delta EN but further deleted in its remaining accessory genes vif, vpr, and vpu (HIV-1 delta EN V(3)) was also tested. Both vectors efficiently transduced DCs. Transduction of DCs did not significantly alter their viability or their immunophenotype when compared with untransduced DCs. Furthermore, the phagocytic potential of immature DCs, as well as their ability to differentiate into mature DCs capable of stimulating T-cell proliferation, was not affected. Finally, DCs transduced by the HIV-1 delta EN vector were able to elicit a primary antiviral cytotoxic T-cell response in autologous CD8 T cells. These results suggest that HIV-1-based vectors expressing viral antigens may be useful for in vivo active immunization as well as ex vivo priming of cytotoxic T cells for adoptive T-cell therapy. (Blood. 2000;96:1327-1333)

The double-stranded RNA-dependent protein kinase PKR is an interferon-inducible enzyme that possesses antiviral and antiproliferative activities. We examined expression of PKR transcripts in human placenta tissue and cultured human amnion U cells. Alternative exon 2 structures were identified and characterized that possess different functional activities. Cloning and sequence analyses of 5'-RACE cDNAs from human placenta established a linkage between exon 1 and three alternative exon 2 structures that constitute, together with part of exon 3, the 5'-untranslated region of the PKR mRNA. The alternative splice variants of exon 2 were designated Ex2alpha (83 nucleotides), Ex2beta (167 nucleotides), and Ex2gamma (401 nucleotides). All three exon 2 variants were present in placenta tissue. However, only the Ex2alpha and Ex2beta forms were detectable in the amnion U cell line. Nuclease protection analysis revealed that the Ex2beta form was slightly more abundant than the Ex2alpha form, in both placenta tissue and U cells. Interferon treatment of U cells increased the level of both Ex2alpha and Ex2beta RNA by approximately 5-fold. The translational activities, measured in a luciferase reporter assay, of RNA transcripts possessing the Ex2alpha and Ex2beta forms of the PKR 5'-UTR were comparable to each other and more efficient than those with the Ex2gamma form., (Copyright 1999 Academic Press.)

The PKR protein kinase is an important regulator of viral mRNA translation. A approximately 50-kb gene (Pkr) encodes the human PKR protein that is inducible by interferon (IFN). The Pkr promoter region has a novel 15-bp DNA element designated as KCS required for transcriptional activity that is located 4 bp upstream of a 13-bp IFN-stimulated response element (ISRE) that confers inducibility by type I IFN. We have carried out a systematic analysis of the 5' flanking region of the human Pkr gene to define how the novel KCS element acts to affect basal as well as IFN-inducible transcription. Electrophoretic mobility shift analyses (EMSA) revealed that nuclear proteins bound selectively to the KCS element in a manner that was not dependent upon either IFN treatment or protein binding at the adjacent ISRE element. KCS protein binding activity in vitro correlated with activation of transcription in vivo in transient transfection assays. Competitionsupershift EMSA assays revealed that multiple proteins were involved in bandshift complex formation with KCS, one of which was identified as factor Sp1. In addition to the positive regulatory domain containing the KCSISRE elements, a negative regulatory domain (NRD) was identified within a 40-bp region positioned approximately 400-bp upstream of the KCSISRE elements. Deletion and substitution mutations indicated that the NRD negatively affected Pkr transcription by a mechanism dependent upon the KCS element. These results define novel positivenegative regulatory domains within the Pkr promoter that function through the KCS element to affect basalIFN-inducible transcription of Pkr., (Copyright 1999 Academic Press.)

Lentiviruses are associated with chronic diseases of the hematological and neurological systems in animals and man. In particular, human immunodeficiency virus type 1 (HIV-1) is the etiological agent of the global AIDS epidemic. The genomes of lentiviruses are complex, encoding a number of regulatory and accessory proteins not found in other retroviruses. This complexity is reflected in their replication cycle, which reveals intricate regulatory pathways and unique mechanisms for viral persistence. In this review, we highlight some of these unique features for HIV-1, with particular focus on the transcriptional and posttranscriptional control of gene expression. Although our understanding of the biology of HIV-1 is far from complete, the knowledge gained thus far has already led to novel strategies for both virus intervention and exploiting the lentiviruses for therapeutic applications.

RNA-dependent protein kinase PKR is an important regulator of gene expression in interferon (IFN)-treated and virus-infected cells. The 50-kb gene encoding human PKR kinase (pkr) is inducible by IFN. Transfection analyses, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5'-flanking fragments of the human pkr gene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription. Sequence determination and mutational analysis of the pkr promoter region revealed, in addition to a functional copy of the IFN-stimulated response element (ISRE) responsible for inducibility by type I IFN, a novel 15-bp element required for optimal promoter activity mediated by the ISRE. This element (5' GGGAAGGCGGAGTCC 3'), designated KCS for kinase-conserved sequence, is exactly conserved between the human and mouse pkr promoters in sequence and position relative to the ISRE. We have now carried out an extensive mutational analysis of the 15-bp KCS element. Site-directed mutagenesis was performed, whereby every base pair position within the KCS element was replaced by each of the other three alternatives. Forty-five substitution mutants were analyzed for promoter activity by transient transfection analysis of untreated and IFN-treated human cells. The results establish 5' NNRRRGG(C,A,T)GGRGYYN 3', where R stands for purine and Y stands for pyrimidine, as the consensus sequence for the KCS element, both for basal and for IFN-inducible promoter activity. KCS-binding proteins were detected by electrophoretic mobility shift analysis (EMSA). Competition EMSA established that constitutively expressed nuclear proteins bound the KCS element selectively; KCS protein binding activity correlated with promoter activity in the transient transfection reporter assay.

Post-translational modifications such as protein phosphorylation provide an important mechanism by which the functional activity of proteins can be controlled and, hence, biological processes regulated. Interferons (IFN) are a multigene family of cytokines that can profoundly affect a wide variety of functions in animal cells including virus replication, cell growth and differentiation, and the immune response. Changes in protein phosphorylation mediated by the IFN-inducible, RNA-dependent protein kinase (PKR) are implicated in the control of cell proliferation mediated by IFNs. Our knowledge of the structure, regulation and function of PKR will be summarized in this brief review, with focus on those aspects of protein phosphorylation and interferon action involving PKR that are central to the roles of the enzyme in the control of cell growth and proliferation.

The RNA-dependent protein kinase (PKR) is inducible by interferon (IFN) and is implicated in the antiviral and antiproliferative actions of IFN. We have now isolated human genomic clones that contain the promoter region required for transcription of the Pkr gene. Transient transfection analyses, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5'-flanking fragments of the Pkr gene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription of CAT. Sequence determination and deletion analysis of the promoter region revealed an element (5'GGAAAACGAAACT3') involved in IFN inducibility that corresponds to the consensus sequence of the IFN-stimulated response element (ISRE). Comparison of the promoter sequence of the human Pkr gene to that of the mouse homolog identified a novel element (5'GGGAAGGCGGAGTCC3') immediately upstream of the ISRE element which so far is unique to the human and mouse Pkr gene promoters. We have designated this new motif as KCS, for kinase conserved sequence. Deletion and substitution mutants of the Pkr promoter region showed that the ISRE element was required for transcriptional induction by type I IFN, whereas the KCS motif increased promoter activity mediated by the ISRE. Additional potential regulatory cis-elements were identified in the human Pkr promoter that are commonly associated with growth control regulation and differentiation. Other than the ISRE and novel KCS elements, the overall organization of potential binding sites for transcription factors was not well conserved between the IFN-inducible promoters of the human and mouse Pkr genes. The strict conservation of sequence, distance, and position of KCS, relative to ISRE, together with mutagenesis results, suggest an important functional role for the newly recognized KCS motif.

The gene encoding the interferon-inducible, RNA-dependent protein kinase (PKR) was isolated as lambda phage and P1 phage clones from human genomic DNA libraries and characterized by Southern blot and nucleotide sequence analyses. Southern blot analyses were consistent with a single PKR gene, and genomic clones colocalized by fluorescence in situ hybridization to human chromosome 2p. Sequence analysis demonstrated that the human PKR gene consists of 17 exons and spans about 50 kb. The AUG translation initiation site for the 551-amino-acid PKR protein was located in exon 3; exon 17 was the largest exon and included the UAG translation termination site, AUUAAA polyadenylation signal, and putative C(A) 3' cleavage site. Two RNA-binding motifs, RI and RII, were present in exons 4 and 6, respectively, and the codon phasing of these exon junctions was conserved between them. The organization of the regulatory and catalytic subdomains of the PKR protein was remarkably preserved between the human and the mouse PKR genes; the amino acid junction positions for 13 of the 15 protein coding exons were exactly conserved.

The effects of the simple phospholipids phosphatidic acid (PA) and lysophosphatidic acid (LPA) on the growth and function of Madin Darby Canine Kidney (MDCK) cells has been studied. We observed that PA and LPA not only stimulated the growth of MDCK cells (at 20 microM), but also stimulated the growth of normal rabbit kidney cells in serum free medium (albeit at a lower dosage of 5 microM). Evidence was obtained that PA interacts synergistically with insulin so as to elicit a growth stimulatory effect. Recently, extracellular PA and LPA were proposed to stimulate mitogenesis in several types of animal cells by binding to particular sites on the plasma membrane which are coupled to signaling mechanisms such as adenylate cyclase via a pertussis toxin sensitive, inhibitory guanosine triphosphate binding protein (Gi protein) (15). However, even when the pertussis toxin dosage was increased to 50 ng/ml, LPA still had a dramatic growth stimulatory effect on MDCK cells. In the absence of LPA pertussis toxin was slightly growth stimulatory to MDCK cells. Phospholipids such as PA and LPA have been observed to prevent prostaglandin-induced increases in adenylate cyclase activity in other cell types via their effects on such a pertussis toxin sensitive Gi protein. If PA and LPA act on MDCK cells in this manner, then these phospholipids may possibly prevent the effect of PGE1 on the growth of normal MDCK cells. However PGE1 was still growth stimulatory to normal MDCK cells. The effects of PA on PGE1 independent variants of MDCK cells, which have elevated intracellular cyclic AMP levels (22), were also examined. In the presence of PA, PGE1 remained growth inhibitory, rather than growth stimulatory to the PGE1 independent cells. However, the PA dosage required to elicit an optimal growth response (5 microM) was dramatically reduced, as compared with normal MDCK cells (20 microM). This altered dosage requirement could be explained by the elevated intracellular cyclic AMP levels in the PGE1 independent variants. Like PGE1 and 8-bromocyclic AMP, PA and LPA also significantly increased the initial rate of Rb+ uptake by confluent monolayers of MDCK cells. The increase in the initial rate of Rb+ uptake could be explained by an increase in the ouabain-sensitive component of Rb+ uptake. An increase in the initial rate of ouabain-insensitive Rb+ uptake was also observed in LPA treated MDCK cell cultures.