Identification of novel 2-benzoxazolinone derivatives with specific inhibitory activity against the HIV-1 nucleocapsid protein

In this report, we present a new benzoxazole derivative endowed with inhibitory activity against the HIV-1 nucleocapsid protein (NC). NC is a 55-residue basic protein with nucleic acid chaperone properties, which has emerged as a novel and potential pharmacological target against HIV-1. In the pursuit of novel NC-inhibitor chemotypes, we performed virtual screening and in vitro biological evaluation of a large library of chemical entities. We found that compounds sharing a benzoxazolinone moiety displayed putative inhibitory properties, which we further investigated by considering a series of chemical analogues. This approach provided valuable information on the structure-activity relationships of these compounds and, in the process, demonstrated that their anti-NC activity could be finely tuned by the addition of specific substituents to the initial benzoxazolinone scaffold. This study represents the starting point for the possible development of a new class of antiretroviral agents targeting the HIV-1 NC protein

Inhibition of Hedgehog-dependent tumors and cancer stem cells by a newly identified naturally occurring chemotype

Hedgehog (Hh) inhibitors have emerged as valid tools in the treatment of a wide range of cancers. Indeed, aberrant activation of the Hh pathway occurring either by ligand-dependent or -independent mechanisms is a key driver in tumorigenesis. The smoothened (Smo) receptor is one of the main upstream transducers of the Hh signaling and is a validated target for the development of anticancer compounds, as underlined by the FDA-approved Smo antagonist Vismodegib (GDC-0449/Erivedge) for the treatment of basal cell carcinoma. However, Smo mutations that confer constitutive activity and drug resistance have emerged during treatment with Vismodegib. For this reason, the development of new effective Hh inhibitors represents a major challenge for cancer therapy. Natural products have always represented a unique source of lead structures in drug discovery, and in recent years have been used to modulate the Hh pathway at multiple levels. Here, starting from an in house library of natural compounds and their derivatives, we discovered novel chemotypes of Hh inhibitors by mean of virtual screening against the crystallographic structure of Smo. Hh functional based assay identified the chalcone derivative 12 as the most effective Hh inhibitor within the test set. The chalcone 12 binds the Smo receptor and promotes the displacement of Bodipy-Cyclopamine in both Smo WT and drug-resistant Smo mutant. Our molecule stands as a promising Smo antagonist able to specifically impair the growth of Hh-dependent tumor cells in vitro and in vivo and medulloblastoma stem-like cells and potentially overcome the associated drug resistance

Gli1/DNA interaction is a druggable target for Hedgehog-dependent tumors

Hedgehog signaling is essential for tissue development and stemness, and its deregulation has been observed in many tumors. Aberrant activation of Hedgehog signaling is the result of genetic mutations of pathway components or other Smo-dependent or independent mechanisms, all triggering the downstream effector Gli1. For this reason, understanding the poorly elucidated mechanism of Gli1-mediated transcription allows to identify novel molecules blocking the pathway at a downstream level, representing a critical goal in tumor biology. Here, we clarify the structural requirements of the pathway effector Gli1 for binding to DNA and identify Glabrescione B as the first small molecule binding to Gli1 zinc finger and impairing Gli1 activity by interfering with its interaction with DNA. Remarkably, as a consequence of its robust inhibitory effect on Gli1 activity, Glabrescione B inhibited the growth of Hedgehog-dependent tumor cells in vitro and in vivo as well as the self-renewal ability and clonogenicity of tumor-derived stem cells. The identification of the structural requirements of Gli1/DNA interaction highlights their relevance for pharmacologic interference of Gli signaling

50th Rocky Mountain Conference on Analytical Chemistry

Final program, abstracts, and information about the 50th annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-endorsed by the Colorado Section of the American Chemical Society and the Rocky Mountain Section of the Society for Applied Spectroscopy. Held in Breckenridge, Colorado, July 27-31, 2008

INHIBITING HEPATITIS B VIRUS GENE EXPRESSION WITH HAMMERHEAD RIBOZYMES THAT TARGET THE HBx OPEN READING FRAME

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2002Hepatitis B virus (HBV) infection is endemic to several populous regions and is often complicated by cirrhosis and hepatocellular carcinoma (HCC). Present treatment of chronic HBV infection is usually ineffective and novel therapeutic approaches are an important medical objective. The X open reading frame (ORF) of HBV, HBx, is a conserved sequence that overlaps with the polymerase ORF and viral c/'s-elements, and is present within all viral transcripts. In addition, the HBx ORF encodes a 17 kDa transactivator protein, HBx, which is required for the establishment of viral infection and has been implicated in HBV-associated hepatocarcinogenesis. The HBx sequence thus represents a compelling target for applying nucleic acid hybridisation-based therapeutic agents for the inhibition of HBV gene expression and replication.IT201

Computer-aided design, synthesis and evaluation of novel antiviral compounds

RNA viruses are a major cause of disease that in the last fifteen years counted for frequent outbreaks, infecting both humans and animals. Examples of emerging or ri-emerging viral pathogens are the Foot-and- Mouth disease virus (FMDV) for animals, Chikungunya virus (CHIKV), Coxsackie virus B3 (CVB3) and Respiratory Syncytial virus (RSV) for humans, all responsible for infections associated with mild to severe complications. Although both vaccines and small-molecule compounds are at different stages of development, no selective antiviral drugs have been approved so far, therefore for all four these viruses improved treatment strategies are required. Promising targets are the viral non-structural proteins, which are commonly evaluated for the identification of new antivirals. Starting from the study of different viral proteins, several computer-aided techniques were applied, aiming to identify hit molecules first, and secondly to synthesise new series of potential antiviral compounds. The available crystal structures of some of the proteins that play a role in viral replication were used for structure- and ligand-based virtual screenings of commercially available compounds against CVB3, FMDV and RSV. New families of potential anti-CHIKV compounds were rationally designed and synthesized, in order to establish a structureactivity relationship study on a lead structure previously found in our group. Finally, a de-novo drug design approach was performed to find a suitable scaffold for the synthesis of a series of zinc-ejecting compounds against RSV. Inhibition of virus replication was evaluated for all the new compounds, of which different showed antiviral potential

Function and dynamics of aptamers: A case study on the malachite green aptamer

Aptamers are short single-stranded nucleic acids that can bind to their targets with high specificity and high affinity. To study aptamer function and dynamics, the malachite green aptamer was chosen as a model. Malachite green (MG) bleaching, in which an OH- attacks the central carbon (C1) of MG, was inhibited in the presence of the malachite green aptamer (MGA). The inhibition of MG bleaching by MGA could be reversed by an antisense oligonucleotide (AS) complementary to the MGA binding pocket. Computational cavity analysis of the NMR structure of the MGA-MG complex predicted that the OH- is sterically excluded from the C1 of MG. The prediction was confirmed experimentally using variants of the MGA with changes in the MG binding pocket. This work shows that molecular reactivity can be reversibly regulated by an aptamer-AS pair based on steric hindrance. In addition to demonstrate that aptamers could control molecular reactivity, aptamer dynamics was studied with a strategy combining molecular dynamics (MD) simulation and experimental verification. MD simulation predicted that the MG binding pocket of the MGA is largely pre-organized and that binding of MG involves reorganization of the pocket and a simultaneous twisting of the MGA terminal stems around the pocket. MD simulation also provided a 3D-structure model of unoccupied MGA that has not yet been obtained by biophysical measurements. These predictions were consistent with biochemical and biophysical measurements of the MGA-MG interaction including RNase I footprinting, melting curves, thermodynamic and kinetic constants measurement. This work shows that MD simulation can be used to extend our understanding of the dynamics of aptamer-target interaction which is not evident from static 3D-structures. To conclude, I have developed a novel concept to control molecular reactivity by an aptamer based on steric protection and a strategy to study the dynamics of aptamer-target interaction by combining MD simulation and experimental verification. The former has potential application in controlling metabolic reactions and protein modifications by small reactants and the latter may serve as a general approach to study the dynamics of aptamer-target interaction for new insights into mechanisms of aptamer-target recognition

Enhancement of HIV-1 neutralisation by modulation of the virus envelope

Despite over 25 years of research, the goal of producing an HIV-1 vaccine able to prevent infection has not been realised. Due to the limited immunogenicity of HIV-1 glycoproteins in vivo, this thesis explores the use of cholesterol depletion in increasing the antigenicity and immunogenicity of an whole-inactivated HIV-1 immunogen. The primary aim was to demonstrate that cholesterol depletion of HIV-1 could enhance antibody binding and viral neutralisation. The laboratory-adapted strain, HIV-1MN, was utilised to demonstrate effective cholesterol depletion of virus using the compound methyl-β-cyclodextrin (MBCD). A range of MBCD concentrations were explored to assess the effect of removing cholesterol on viral infectivity, morphology and protein composition of the virus. A 1mM MBCD concentration, which effected a 50% reduction in viral envelope cholesterol with little impact on viral structure and protein composition whilst retaining viral infectivity, was chosen to explore the effect of cholesterol depletion on virus antibody binding and neutralisation. Removal of cholesterol from the viral envelope increased antibody binding and neutralisation using a number of monoclonal antibodies, soluble CD4 (sCD4) and HIV-1 positive patient antisera. This concept was then extended to a pseudotyped primary isolate of HIV-1. Cholesterol depletion of this isolate demonstrated a more restricted enhancement of antibody binding and neutralisation of virus by monoclonal antibodies, sCD4 and homologous and heterologous HIV-1 infected patient antisera. A number of inactivating agents were then explored in the aim of creating a cholesteroldepleted, whole inactivated HIV-1MN immunogen that retained key conformational gp160 epitopes. This immunogen was then tested in a mouse model to investigate whether cholesterol depletion could enhance the immunogenicity of whole-inactivated HIV-1MN. Although measurement of antibody responses from pooled mouse sera indicated significant enhancement of humoral responses to cholesterol-depleted, wholeinactivated HIV-1MN, analysis of individual mouse responses to immunisation yielded more variable results