Ursula Dietrich

3D-epitope-explorer (3DEX) : Localization of conformational epitopes within three-dimensional structures of proteins

Neutralizing antibodies often recognize conformational, discontinuous epitopes. Linear peptides mimicking such conformational epitopes can be selected from phage display peptide libraries by screening with the respective antibodies. However, it is difficult to localize these “mimotopes” within the three‐dimensional (3D) structures of the target proteins. Knowledge of conformational epitopes of neutralizing antibodies would help to design antigens able to elicit protective immune responses. Therefore, we provide here a software that allows to localize linear peptide sequences within 3D structures of proteins. The 3D‐Epitope‐Explorer (3DEX) software allows to map conformational epitopes in 3D protein structures based on an algorithm that takes into account the physicochemical neighborhood of Cα‐ or Cβ‐atoms of individual amino acids. A given amino acid of a peptide sequence is localized within the protein and the software searches within predefined distances for the amino acids neighboring that amino acid in the peptide. Surface exposure of the amino acids can also be taken into consideration. The procedure is then repeated for the remaining amino acids of the peptide. The introduction of a joker function allows to map peptide mimotopes, which do not necessarily have 100% sequence homology to the protein. Using this software we were able to localize mimotopes selected from phage displayed peptide libraries with polyclonal antibodies from HIV‐positive patient plasma within the 3D structure of gp120, the exterior glycoprotein of HIV‐1. We also analyzed two recently published peptide sequences corresponding to known conformational epitopes to further confirm the integrity of 3DEX.

Targeting the viral nucleocapsid protein in anti-HIV-1 therapy.

The nucleocapsid protein (NC) plays seminal roles in HIV replication, thus representing a major drug target. NC functions rely on its two zinc-fingers and flanking basic residues. Zinc ejectors inhibit NC functions, but with limited specificity. New classes of molecules competing with NC or its viral nucleic acid and enzyme partners are reviewed here.

Application Of The EIIP/ISM Bioinformatics Concept in Development of New Drugs

The development of a new therapeutic drug is a complex, lengthy and expensive process. On average, only one out of 10,000 - 30,000 originally synthesized compounds will clear all the hurdles on the way to becoming a commercially available drug. The process of early and full preclinical discovery and clinical development for a new drug can take twelve to fifteen years to complete, and cost approximately 800 million dollars. The field of bioinformatics has become a major part of the drug discovery pipeline playing a key role in improvement and acceleration of this time and money consuming process. Here we reviewed the application of the EIIP/ISM bioinformatics concept for the development of new drugs. This approach, connecting the electron-ion interaction potential of organic molecules and their biological properties, can significantly reduce development time through (i) identification of promising lead compounds that have some activity against a disease by fast virtual screening of the large molecular libraries, (ii) refinement of selected lead compounds in order to increase their biological activity, and (iii) identification of domains of proteins and nucleotide sequences representing potential targets for therapy. Special attention is paid in this review to the application of the EIIP/ISM bioinformatics platform along with other experimental techniques (screening of a phage displayed peptide libraries, testing selected peptides and small molecules for antiviral activity in vitro) in development of HIV entry inhibitors, representing a new generation of the AIDS drugs.

Pseudotyping vesicular stomatitis virus with lymphocytic choriomeningitis virus glycoproteins enhances infectivity for glioma cells and minimizes neurotropism

ABSTRACT Vesicular stomatitis virus (VSV)-based oncolytic virotherapy has the potential to significantly improve the prognosis of aggressive malignancies such as brain cancer. However, VSVs inherent neurotoxicity has hindered clinical development so far. Given that this neurotropism is attributed to the glycoprotein VSV-G, VSV was pseudotyped with the nonneurotropic envelope glycoprotein of the lymphocytic choriomeningitis virus (LCMV-GP→VSV-GP). Compared to VSV, VSV-GP showed enhanced infectivity for brain cancer cells in vitro while sparing primary human and rat neurons in vitro and in vivo, respectively. In conclusion, VSV-GP has a much wider therapeutic window than VSV and is thus more suitable for clinical applications, especially in the brain.

Phage Display-directed Discovery of LEDGF/p75 Binding Cyclic Peptide Inhibitors of HIV Replication

The interaction between the human immunodeficiency virus (HIV) integrase (IN) and its cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is crucial for HIV replication. While recently discovered LEDGINs inhibit HIV-1 replication by occupying the LEDGF/p75 pocket in IN, it remained to be demonstrated whether LEDGF/p75 by itself can be targeted. By phage display we identified cyclic peptides (CPs) as the first LEDGF/p75 ligands that inhibit the LEDGF/p75-IN interaction. The CPs inhibit HIV replication in different cell lines without overt toxicity. In accord with the role of LEDGF/p75 in HIV integration and its inhibition by LEDGINs, CP64, and CP65 block HIV replication primarily by inhibiting the integration step. The CPs retained activity against HIV strains resistant to raltegravir or LEDGINs. Saturation transfer difference (STD) NMR showed residues in CP64 that strongly interact with LEDGF/p75 but not with HIV IN. Mutational analysis identified tryptophan as an important residue responsible for the activity of the peptides. Serial passaging of virus in the presence of CPs did not yield resistant strains. Our work provides proof-of-concept for direct targeting of LEDGF/p75 as novel therapeutic strategy and the CPs thereby serve as scaffold for future development of new HIV therapeutics.

Mimotopes selected with antibodies from HIV-1-neutralizing long-term non-progressor plasma.

A promising approach to identify HIV‐1 vaccine candidates is to dissect the natural immune response against the virus in persons controlling the infection over decades without any antiviral therapy. Here we focus on a group of such persons, eight long‐term non‐progressors (LTNP), in which we proved the presence of broadly neutralizing antibodies against HIV‐1 in the plasma as very likely cause for their LTNP status. The aim of this study was to identify the epitopes for these neutralizing antibodies, as these should represent immunogens potentially able to elicit neutralizing antibodies upon vaccination. We screened random peptide phage libraries with plasma antibodies from eight LTNP. After several rounds of positive and negative selection, about 700 HIV‐specific mimotopes were sequenced. The mimotope sequences were analyzed for homology to HIV‐1 Env, in particular for their capacity to represent conformational epitopes on the surface of the gp120 structure using our software 3DEX. Related phage groups were analyzed for crossreactivity with the LTNP plasma by ELISA as well as for their capacity to induce HIV‐1‐neutralizing antibodies in mice. Based on this study interesting mimotopes can now be selected for further immunization studies.

A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication

DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

Targeting the human DEAD-box polypeptide 3 (DDX3) RNA helicase as a novel strategy to inhibit viral replication.

Compounds currently used for the treatment of HIV-1 Infections are targeted to viral proteins. However, the high intrinsic mutation and replication rates of HIV-1 often lead to the emergence of drug resistant strains and consequent therapeutic failure. On this basis, cellular cofactors represent attractive new targets for HIV-1 chemotherapy, since targeting a cellular factor that is required for viral replication should help to overcome the problem of viral resistance. We and others have recently reported the identification of compounds suppressing HIV-1 replication by targeting the cellular DEAD-box helicase DDX3. These results provide a proof-of-principle for the feasibility of blocking HIV-1 infection by rendering the host cell environment less favorable for the virus. The rationale for such an approach and its implications in potentially overcoming the problem of drug resistance related to drugs targeting viral proteins will be discussed in the context of the known cellular functions of the DEAD-box helicase DDX3.

Re-engineering Vesicular Stomatitis Virus to Abrogate Neurotoxicity, Circumvent Humoral Immunity, and Enhance Oncolytic Potency

As cancer treatment tools, oncolytic viruses (OV) have yet to realize what some see as their ultimate clinical potential. In this study, we have engineered a chimeric vesicular stomatitis virus (VSV) that is devoid of its natural neurotoxicity while retaining potent oncolytic activity. The envelope glycoprotein (G) of VSV was replaced with a variant glycoprotein of the lymphocytic choriomeningitis virus (LCMV-GP), creating a replicating therapeutic, rVSV(GP), that is benign in normal brain but can effectively eliminate brain cancer in multiple preclinical tumor models in vivo. Furthermore, it can be safely administered systemically to mice and displays greater potency against a spectrum of human cancer cell lines than current OV candidates. Remarkably, rVSV(GP) escapes humoral immunity, thus, for the first time, allowing repeated systemic OV application without loss of therapeutic efficacy. Taken together, rVSV(GP) offers a considerably improved OV platform that lacks several of the major drawbacks that have limited the clinical potential of this technology to date.

Epidemic Transmission of Human Immunodeficiency Virus in Renal Dialysis Centers in Egypt

In 1993 an epidemic of human immunodeficiency virus (HIV) infection occurred among 39 patients at 2 renal dialysis centers in Egypt. The centers, private center A (PCA) and university center A (UCA) were visited, HIV-infected patients were interviewed, seroconversion rates at UCA were calculated, and relatedness of HIV strains was determined by sequence analysis; 34 (62%) of 55 patients from UCA and 5 (42%) of 12 patients from PCA were HIV-infected. The HIV seroconversion risk at UCA varied significantly with day and shift of dialysis session. Practices that resulted in sharing of syringes among patients were observed at both centers. The analyzed V3 loop sequences of the HIV strain of 12 outbreak patients were >96% related to each other. V3 loop sequences from each of 8 HIV-infected Egyptians unrelated to the 1993 epidemic were only 76%-89% related to those from outbreak strains. Dialysis patients may be at risk for HIV infection if infection control guidelines are not followed.