Liposomes and virosomes as delivery systems for antigens, nucleic acids and drugs

Lipid-based vesicles are a very promising approach to treat diseases such as cancer, chronic infections and auto-immunity. Modern drug encapsulation methods allow efficient packing of therapeutic substances inside liposomes, thereby reducing the systemic toxicity of the drugs. Specific targeting can enhance the therapeutic effect of the drugs through their accumulation at the diseased site. In the vaccine field, the integration of functional viral envelope proteins into liposomes has led to an antigen carrier and delivery system termed a virosome, a clinically proven vaccine platform for subunit vaccines with an excellent immunogenicity and tolerability profile.     

Protein—drug complexes important for immunoregulation and organ transplantation

Recently, two structures of the Ser/Thr phosphorylase calcineurin in complex with FK506 and its cognate immunophilin, FKBP12, have been reported, both solved by small pharmaceutical companies focused on structure-based drug design. A realization, however, that the toxicities associated with calcineurin-mediated immunosuppressants might be mechanism based has driven the current interest in alternative approaches to autoimmunity prophylaxis and preventing transplant rejection. Regulatory approval in 1995 of the immunosuppressant prodrug mycophenolate mofetil, whose active metabolite, mycophenolic acid, inhibits inosine monophosphate dehydrogenase, has focused attention on the potential significance of the de novo purine-biosynthesis pathway as a target for immunosuppressive drugs, leading ultimately to the solution of enzyme structure as a drug design target. As this and other clinically relevant targets are discovered, elaborated and refined via the activity of their cognate agents (as was the case for the phosphatase calcineurin via the activity of cyclosporin), a critical opportunity should ensue for structural biology to exert a profound effect on the future development of these therapies.     

Membrane-active host defense peptides--challenges and perspectives for the development of novel anticancer drugs

Although much progress has been achieved in the development of cancer therapies in recent decades, problems continue to arise particularly with respect to chemotherapy due to resistance to and low specificity of currently available drugs. Host defense peptides as effector molecules of innate immunity represent a novel strategy for the development of alternative anticancer drug molecules. These cationic amphipathic peptides are able to discriminate between neoplastic and non-neoplastic cells interacting specifically with negatively charged membrane components such as phosphatidylserine (PS), sialic acid or heparan sulfate, which differ between cancer and non-cancer cells. Furthermore, an increased number of microvilli has been found on cancer cells leading to an increase in cell surface area, which may in turn enhance their susceptibility to anticancer peptides. Thus, part of this review will be devoted to the differences in membrane composition of non-cancer and cancer cells with a focus on the exposure of PS on the outer membrane. Normally, surface exposed PS triggers apoptosis, which can however be circumvented by cancer cells by various means.Host defense peptides, which selectively target differences between cancer and non-cancer cell membranes, have excellent tumor tissue penetration and can thus reach the site of both primary tumor and distant metastasis. Since these molecules kill their target cells rapidly and mainly by perturbing the integrity of the plasma membrane, resistance is less likely to occur. Hence, a chapter will also describe studies related to the molecular mechanisms of membrane damage as well as alternative non-membrane related mechanisms. In vivo studies have demonstrated that host defense peptides display anticancer activity against a number of cancers such as e.g. leukemia, prostate, ascite and ovarian tumors, yet so far none of these peptides has made it on the market. Nevertheless, optimization of host defense peptides using various strategies to enhance further selectivity and serum stability is expected to yield novel anticancer drugs with improved properties in respect of cancer cell toxicity as well as reduced development of drug resistance.     

Functional microcircuitry in the accumbens underlying drug addiction: insights from real-time signaling during behavior

An understanding of the neurobiological basis of drug addiction requires examination of real-time (subsecond) cellular and chemical responses in the brain reward system during drug-seeking and drug-taking behavior. Electrophysiological and electrochemical studies in the rodent nucleus accumbens have examined changes in cell firing and rapid dopamine signaling during crucial periods of behavioral responding for drugs, and show the associative nature of those signals. These findings are considered with respect to the functional microcircuitry in the nucleus accumbens that underlies goal-directed behavior and the role of this circuit in drug addiction.     

Searching for new allosteric sites in enzymes

The discovery of new allosteric sites generates opportunities for the identification of novel pharmaceuticals and increases our understanding of basic biological processes. Increasingly, allosteric sites are being discovered in various families of proteins by several methods, paving the way for the development of entirely new classes of drugs with a wide range of chemotypes. New allosteric sites in enzymes have been discovered both incidentally and by directed means, and the mechanisms by which allosteric activation and inhibition occur at these sites have been investigated. By exploring recent structurally well-characterized examples, trends begin to emerge for both the modes of binding and mechanisms of inhibition.     

Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication

RNA viruses can rapidly mutate and acquire resistance to drugs that directly target viral enzymes, which poses serious problems in a clinical context. Therefore, there is a growing interest in the development of antiviral drugs that target host factors critical for viral replication, since they are unlikely to mutate in response to therapy. We recently demonstrated that phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) and its product phosphatidylinositol-4-phosphate (PI4P) are essential for replication of enteroviruses, a group of medically important RNA viruses including poliovirus (PV), coxsackievirus, rhinovirus, and enterovirus 71. Here, we show that enviroxime and GW5074 decreased PI4P levels at the Golgi complex by directly inhibiting PI4KIIIβ. Coxsackievirus mutants resistant to these inhibitors harbor single point mutations in the non-structural protein 3A. These 3A mutations did not confer compound-resistance by restoring the activity of PI4KIIIβ in the presence of the compounds. Instead, replication of the mutant viruses no longer depended on PI4KIIIβ, since their replication was insensitive to siRNA-mediated depletion of PI4KIIIβ. The mutant viruses also did not rely on other isoforms of PI4K. Consistently, no high level of PI4P could be detected at the replication sites induced by the mutant viruses in the presence of the compounds. Collectively, these findings indicate that through specific single point mutations in 3A, CVB3 can bypass an essential host factor and lipid for its propagation, which is a new example of RNA viruses acquiring resistance against antiviral compounds, even when they directly target host factors.     

Mathematical modeling of multi-drugs therapy: a challenge for determining the optimal combinations of antiviral drugs

In the current era of antiviral drug therapy, combining multiple drugs is a primary approach for improving antiviral effects, reducing the doses of individual drugs, relieving the side effects of strong antiviral drugs, and preventing the emergence of drug-resistant viruses. Although a variety of new drugs have been developed for HIV, HCV and influenza virus, the optimal combinations of multiple drugs are incompletely understood. To optimize the benefits of multi-drugs combinations, we must investigate the interactions between the combined drugs and their target viruses. Mathematical models of viral infection dynamics provide an ideal tool for this purpose. Additionally, whether drug combinations computed by these models are synergistic can be assessed by two prominent drug combination theories, Loewe additivity and Bliss independence. By combining the mathematical modeling of virus dynamics with drug combination theories, we could show the principles by which drug combinations yield a synergistic effect. Here, we describe the theoretical aspects of multi-drugs therapy and discuss their application to antiviral research.     

Identification of membrane associated drug targets in Borrelia burgdorferi ZS7- subtractive genomics approach

Lyme disease is an infectious disease caused by a spirochete Borrelia burgdorferi ZS7. This spirochete is most often spread by ticks. Single antibiotic therapy is sufficient for containment of the early stage progression of the disease but combinational therapy is more preferred in later stages. Research is in progress for the development of drugs against the pathogen, but till date no vaccines have been developed to effect the late stage infections. There is a rapid rise in the cases of antibiotic-resistant population which is more than 10% of the total infected individuals. In such condition vaccine becomes the sole alternative for prevention. Therefore effective treatment includes antibiotic combination and combination of antigenic surfaces (for vaccine preparation). Thus, a comprehensive list of drug targets unique to the microorganisms is often necessary. Availability of Borrelia burgdorferi ZS7 proteome has enabled insilico analysis of protein sequences for the identification of drug targets and vaccine targets. In this study, 272 essential proteins were identified out of which 42 proteins were unique to the microorganism. The study identified 15 membrane localized drug targets. Amongst these 15, molecular modeling and structure validation of the five membrane localized drug target proteins could only be achieved because of the low sequence identity of the remaining proteins with RCSB structures. These 3D structures can be further characterized by invitro and invivo studies for the development of novel vaccine epitopes and novel antibiotic therapy against Borrelia burgdorferi.     

Transmitting Plant Viruses Using Whiteflies

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species⁵, are vectors of many plant viruses. Several genera of these whitefly-transmitted plant viruses (Begomovirus, Carlavirus, Crinivirus, Ipomovirus, Torradovirus) include several hundred species of emerging and economically significant pathogens of important food and fiber crops (reviewed by^9,10,16). These viruses do not replicate in their vector but nevertheless are moved readily from plant to plant by the adult whitefly by various means (reviewed by^{2,6,7,9,10,11,17}). For most of these viruses whitefly feeding is required for acquisition and inoculation, while for others only probing is required. Many of these viruses are unable or cannot be easily transmitted by other means. Therefore maintenance of virus cultures, biological and molecular characterization (identification of host range and symptoms)^3,13, ecology^2,12, require that the viruses be transmitted to experimental hosts using the whitefly vector. In addition the development of new approaches to management, such as evaluation of new chemicals¹⁴ or compounds¹⁵, new cultural approaches^1,4,19, or the selection and development of resistant cultivars^7,8,18, requires the use of whiteflies for virus transmission. The use of whitefly transmission of plant viruses for the selection and development of resistant cultivars in breeding programs is particularly challenging⁷. Effective selection and screening for resistance employs large numbers of plants and there is a need for 100% of the plants to be inoculated in order to find the few genotypes which possess resistance genes. These studies use very large numbers of viruliferous whiteflies, often several times per year.Whitefly maintenance described here can generate hundreds or thousands of adult whiteflies on plants each week, year round, without the contamination of other plant viruses. Plants free of both whiteflies and virus must be produced to introduce into the whitefly colony each week. Whitefly cultures must be kept free of whitefly pathogens, parasites, and parasitoids that can reduce whitefly populations and/or reduce the transmission efficiency of the virus. Colonies produced in the manner described can be quickly scaled to increase or decrease population numbers as needed, and can be adjusted to accommodate the feeding preferences of the whitefly based on the plant host of the virus.There are two basic types of whitefly colonies that can be maintained: a nonviruliferous and a viruliferous whitefly colony. The nonviruliferous colony is composed of whiteflies reared on virus-free plants and allows the weekly availability of whiteflies which can be used to transmit viruses from different cultures. The viruliferous whitefly colony, composed of whiteflies reared on virus-infected plants, allows weekly availability of whiteflies which have acquired the virus thus omitting one step in the virus transmission process.     

A time-of-drug addition approach to target identification of antiviral compounds

Insight into the mode of action of newly discovered antiviral agents is now almost a prerequisite for clinical development. This protocol describes a method that provides information on the target of inhibitors of the human immunodeficiency virus (HIV); it can also be adapted to other viruses. The results from this experiment are available within 2 d. This time-based approach determines how long the addition of a compound can be postponed before losing its antiviral activity in cell culture. The target of an antiviral compound can be identified by comparing its relative position in the time scale to that of reference drugs. Therefore, it is more precise than, for example, in the case of HIV, a determination of pre- or postintegrational mode of action, and combines in one routine different assays for studying mechanisms of action.     

Effects of HIV aspartyl-proteinase inhibitors on Leishmania sp.

In this work, we have found an antiproliferative effect on Leishmania sp. promastigotes and axenic amastigotes by the human immunodeficiency virus (HIV) aspartyl-proteinase inhibitors, Ac-Leu-Val-Phenylalaninal, Saquinavir mesylate and Nelfinavir, the latter two being used as part of antiretroviral therapy. This effect appears to be the result of cell division blockage. In addition, these drugs induced in culture a decrease in the percentage of co-infected HIV/Leishmania monocytes and amastigotes of Leishmania per macrophage. The finding of a dose-dependent inhibition of Leishmania promastigotes aspartyl-proteinase activity by these drugs allows us to propose this activity as the drug parasite target. A direct action of these HIV aspartyl-proteinase inhibitors on the parasite, would be correlated with the effect that highly active antiretroviral therapy have had in the decrease of HIV/Leishmania coinfection, opening an interesting perspective for new drugs research development based on this novel parasite proteinase family.     

Nonstructural HIV proteins as targets for prophylactic or therapeutic vaccines

By the end of 2004, more than 20 HIV-1 vaccine candidates will have entered clinical testing in at least 30 trials worldwide. Almost half of these vaccines include nonstructural HIV-1 gene products. This represents an important innovation in the HIV vaccine field, because until 9 years ago not even preclinical testing in small animal models had been carried out with such immunogens. This review briefly discusses the experimental evidence that provides the rationale for the use of nonstructural HIV-1 gene products as vaccine antigens, and summarizes the current status and the future development of these novel vaccines.     

Cell cycle and differentiation

The development of multicellular organisms relies on the temporal and spatial control of cell proliferation and cell growth. The relationship between cell-cycle progression and development is complex and characterized by mutual dependencies. On the level of the individual cell, this interrelationship has implications for pattern formation and cell morphogenesis. On a supercellular level, this interrelationship affects meristem function and organ growth. Often, developmental signals not only direct cell-cycle progression but also set the frame for cell-cycle regulation by determining cell-type-specific cell-cycle modes. In other cases, however, cell-cycle progression appears to be required for the further differentiation of some cell types. There are also examples in which cell cycle and differentiation seem to be controlled at the same level and progress rather independently from each other or are linked by the same regulator or pathway. Furthermore, different relationships between cell cycle and differentiation can be combined in a succession of events during development, leading to complex developmental programs.     

Structural characterization of B and non-B subtypes of HIV-protease: insights into the natural susceptibility to drug resistance development

Although a majority of HIV-1 infections in Brazil are caused by the subtype B virus (also prevalent in the United States and Western Europe), viral subtypes F and C are also found very frequently. Genomic differences between the subtypes give rise to sequence variations in the encoded proteins, including the HIV-1 protease. The current anti-HIV drugs have been developed primarily against subtype B and the effects arising from the combination of drug-resistance mutations with the naturally existing polymorphisms in non-B HIV-1 subtypes are only beginning to be elucidated. To gain more insights into the structure and function of different variants of HIV proteases, we have determined a 2.1 A structure of the native subtype F HIV-1 protease (PR) in complex with the protease inhibitor TL-3. We have also solved crystal structures of two multi-drug resistant mutant HIV PRs in complex with TL-3, from subtype B (Bmut) carrying the primary mutations V82A and L90M, and from subtype F (Fmut) carrying the primary mutation V82A plus the secondary mutation M36I, at 1.75 A and 2.8 A resolution, respectively. The proteases Bmut, Fwt and Fmut exhibit sevenfold, threefold, and 54-fold resistance to TL-3, respectively. In addition, the structure of subtype B wild type HIV-PR in complex with TL-3 has been redetermined in space group P6(1), consistent with the other three structures. Our results show that the primary mutation V82A causes the known effect of collapsing the S1/S1' pockets that ultimately lead to the reduced inhibitory effect of TL-3. Our results further indicate that two naturally occurring polymorphic substitutions in subtype F and other non-B HIV proteases, M36I and L89M, may lead to early development of drug resistance in patients infected with non-B HIV subtypes.     

Virtual screening of phytochemicals to novel targets in Haemophilus ducreyi towards the treatment of Chancroid

Conventionally, drugs are discovered by testing chemically synthesized compounds against a battery of in vivo biological screens. Information technology and Omic science enabled us for high throughput screening of compound libraries against biological targets and hits are then tested for efficacy in cells or animals. Chancroid, caused by Haemophilus ducreyi is a public health problem and has been recognized as a cofactor for Human Immunodeficiency Virus (HIV) transmission. It facilitates HIV transmission by providing an accessible portal entry, promoting viral shedding, and recruiting macrophages as well as CD4 cells to the skin. So, there is a requirement to develop an efficient drug to combat Chancroid that can also diminish HIV infection. In-silico screening of potential inhibitors against the target may facilitate in detection of the novel lead compounds for developing an effective chemo preventive strategy against Haemophilus ducreyi. The present study has investigated the effects of approximately 1100 natural compounds that inhibit three vital enzymes viz. Phosphoenolpyruvate phosphotransferase, Acetyl-coenzyme A carboxylase and Fructose 1, 6-bisphosphatase of Haemophilus ducreyi in reference to a commercial drug Rifabutin. Results reveal that the lead compound uses less energy to bind to target. The lead compound parillin has also been predicted as less immunogenic in comparison to Rifabutin. Further, better molecular dynamics, pharmacokinetics, pharmacodynamics and ADME-T properties establish it as an efficient chancroid preventer.     

Exploiting natural peptide diversity: novel research tools and drug leads

During the course of evolution, nature has developed a vast number of peptides in all living and past species that display an exceeding diversity of structure and biological effects, such as hormonal and enzyme-controlling activity, communication between cells, and participation in host defence. Sensitive mass spectrometric technologies have been introduced and facilitate access to new natural peptides, even in trace amounts, and allow the quantitative determination of the peptide status of cells, organs and whole organisms (peptidomics). Among the large number of new biologically active peptides identified from an increasing variety of natural sources, regulators of ion channels, chemoattractants, protease inhibitors, metabolism-related hormones, cytotoxins, and antimicrobials have been found. These novel peptides serve as research tools and have potential as diagnostic biomarkers and for the development of peptide and peptidometic drugs.