Inhibiting Nef: a novel drug target for HIV-host interactions
FP7 project iNEF, Grant
agreement number: 201412
Collaborative project, FP7-HEALTH-2007-2.3.2-1: HIV/AIDS Drug Discovery and Preclinical Development
Drug resistance and morbidity by side-effects limit current anti-retroviral therapy in HIV infected people. New targets, such as the interactions of accessory proteins with viral or host proteins are needed to overcome this. A wealth of evidence points to the direct pathogenetic function of the, HIV Nef protein. By high-throughput screening and with the molecular tools developed, the iNEF consortium identifies compounds that affect functions of Nef , such as, SH3 binding, NFAT activation, modulation of cell surface receptors like CD4 and viral replication.
HIV-1 Nef plays a major role in the progression to AIDS, above its contribution to viral replication. The protein facilitates immune evasion by its effect on infected and non-infected cells. Inhibition of the protein-protein interactions involved will be a new avenue of anti-retroviral therapy.
Partners in this consortium have already identified small molecule inhibitors targeting Nef function.
The first aim of this project is to validate the molecular events elicited by these molecules in both virus-free as well as in HIV infection in vitro assays. In a complementing approach, small compound libraries already available to the consortium will be used and adapted to screen for inhibitors of Nef induced modulation of cellular receptors, NFAT activation and the Nef SH3 binding domain, that likely contribute to the importance of Nef in HIV-1 pathogenicity.
In addition, functional screenings to discover druggable cellular Nef partners using RNA interference libraries will be performed. After validation of their importance in relation to the established host proteins co-interacting in the Nef cellular pathways, a selection will be additionally targeted by the developed small molecule inhibitors.
The consortium developed tools to evaluate Nef function, such as SH3 binding and NFAT activation assays, an exhaustive array of reporter viruses, shRNA libraries, etc. Compound, discovered to affect SH3 binding of Nef were further optimized, a consensus chemical structure was defined as a template for lead derivation (2 out of 50.000 screened), and distributed over the consortium partners performing the screenings. Apart from this, a new chemical library of about 5.000 molecules is screened in the effect on several Nef functions. E.g. more than 200 hits were found in this library that inhibit NFAT activation without being cytotoxic. When all molecules are tested in the different assays, a selection of the most promising candidates will be performed. Moreover, the library yielded 10 compounds with significant effect on HIV replication, the interaction with Nef will be analysed. Apart from replication, also cell-to-cell transfer will be studies and yielded our ultimate goal is to deliver a complementary portfolio of candidate drugs that target the most important parameters in the Nef-host interaction pathway.
The several interesting lead compounds found thus far are a first step into the development of agents that, by targeting critical cellular interaction partners much more conserved than viral enzymes, have the potential to inhibit disease progression without readily acquisition of drug resistance. Compounds like this are needed to cope with that are usually targeted in highly-active-antiretroviral therapy (HAART). Therefore, it is believed by the partners that the novel approach presented in this project proposal will yield compounds less likely to be subject to the occurrence of drug resistance
Project web-site: www.iNEF.UGent.be
Institution address: HIVlab, Department of Clinical Chemistry, Microbiology and Immunology
Ghent University, UZ Gent, De Pintelaan 185, B9000 Gent, Belgium
About the lab:
We study the role of HIV Nef protein in cellular trafficking, T cell function and development, but also in viral replication and fitness. We want to identify the cellular partner proteins of Nef that mediate Nef function.
Ghent University offers high-quality, research-based education in all academic disciplines. Today Ghent University attracts over 28.000 students, with a foreign student population of over 1.100 EU citizens and some 1.000 students from non-EU countries. With a view to cooperation in research and scientific service, numerous research groups, centres and institutes have been founded over the years. Several of them are renowned worldwide, in various scientific disciplines such as biotechnology, aquaculture, microelectronics, history,... Ghent University invests an annual amount of more than 175 million euro in research projects on behalf of public and private partners.
Institution address: Institute of Virology, University of Ulm, Albert-Einstein Allee 29, 89081 Ulm, Germany
About the lab
One goal of our research is to better understand which mechanisms allow primate lentiviruses to persist efficiently in their respective host and contribute to the destruction of the host immune system. We were able to show that a combination of Nef functions contributes to the maintenance of high viral loads associated with the development of immunodeficiency in the evolutionary recent human and the experimental macaque hosts of HIV-1 and SIVmac, respectively. Our analyses also demonstrated that Nef alleles from most SIVs that do not cause disease in their natural monkey hosts but not those of HIV-1 and its simian precursors, downmodulate TCR-CD3 to suppress T cell activation and programmed death. More recently, we found that inefficient downmodulation of CD3 by Nef correlates with loss of CD4+ T cells even in the natural hosts of SIV. Thus, the evolutionary loss of a specific Nef function may contribute to the high virulence of HIV-1 in humans.
Manipulation of T cell/APC interaction by primate lentiviral Nef proteins. Schematic presentation of (A) the interaction between uninfected APCs and CD4+ T cells and the effect of (B) HIV-1 and (C) SIVsmm or SIVagm infection on specific receptors expressed by these cell types. Receptors downmodulated by the nef alleles of the respective viruses are crossed out and erased if the effects are highly effective. Some effects remain to be demonstrated in primary cells from the respective primate species. (see article by Kirchhoff and coworkers, Cell Mol Life Sci. 2008).
About University of Ulm
The University of Ulm, Germany, enjoys an excellent reputation for innovative research, interdisciplinarity and successful training. Its four faculties support about 6400 students. The research profile is characterized by its focus on life sciences and medicine.
Institution address: Faculty of Medicine, Haartman Institute, Haartmaninkatu (PO Box 21) 3, 00014 Helsinki, Finland
About the lab
The main topic of our research is cellular signaling in virus-host interactions, in particular protein interactions involving viral non-structural proteins and host proteins with modular binding domains. Virus replication depends on the protein machinery of the host cell, which viruses have evolved to regulate and exploit in numerous and elaborate ways. Molecular understanding of these interactions is a key challenge in modern research in virology. In addition to revealing novel targets for antiviral drug development, studies in this area are likely to continue providing valuable insights into the basic mechanisms that regulate normal cellular behavior and become deregulated in various diseases.
The HIV Nef protein has been a key subject of our studies. Nef modifies host cell physiology to better support viral replication by binding to proteins involved in cellular signal structures via their SH3 domains. SH3 domains are the most ubiquitous class of modular protein binding units found in the nature. SH3 mediates many of the cellular effects of Nef, and therefore presents an interesting target for drug development.
It appears that many other viral proteins share the capacity of Nef to utilize binding to host cell SH3 proteins in order to manipulate host cell physiology. Elucidation and characterization of these interactions as well as normal cellular SH3 protein interaction are key goals of our current research.
HIV-1 Nef (blue) bound to an SH3 domains (red).
Lab homepage :
About University of Helsinki
The University of Helsinki (39.000 students) is involved in more than half of the National and Nordic Centres of Excellence in Research and it is also the only Finnish university invited to join the League of European Research Universities.
The Biocentrum Helsinki, a large umbrella organization at the University of Helsinki, with some 600 people being engaged in research in biotechnology and molecular biology. The member groups in Biocentrum Helsinki have been selected by international experts strictly on the basis of their scientific accomplishments, providing that the work of the group is related to the fields of biotechnology and molecular biology.
Koen Van Aken
Institution address: Ecosynth, Stationsstraat 123, B8400 Oostende, Belgium
EcoSynth is a dynamic discovery synthesis company located in Ostend, Belgium. It is specialized in robust and efficient reaction conditions for the synthesis of small (biologically active) molecules and the development of chemical processes.
The equipment (e.g. microwave, LC-MS/ELSD/UV, GC-MS, automated preparative chromatography, parrallel synthesizer, NMR access, etc...) is selected to carry out organic synthesis in a short turn-around time. Special attention is given to the use of reaction conditions which score high on the EcoScale1,2.This novel tool evaluates the quality of an organic preparation based on yield, cost, safety and ease of work-up.
An early evaluation of the scalability of a discovery chemistry synthesis, and the willingness to invest in route scouting and evaluation of potential long-term manufacturing options is very important for drug discovery companies. Too often, the synthesis protocol is acceptable for only the first 100 g. However, in the long term, scaling up a problematic discovery route, which does not deliver the required amount of drug substance on time and of the desired quality, can be much more expensive than devoting time to an investigation of alternative, convergent, scaleable, manufacturing-friendly and cost-effective syntheses using readily available reagents which are safe and environmentally friendly.
As such, EcoSynth is a reliable partner with a commercial and technical flexibility to supply novel target molecules with scaleable reaction protocols or IP-free focused libraries. Alternatively, an exclusive synthesis team can be set-up if no in-house chemistry capacity is present.
1. EcoScale, a semi-quantitative tool to select an organic preparation based on economical and ecological parameters. Koen Van Aken, Lucjan Strekowski and Luc Patiny, Beilstein Journal of Organic Chemistry 2006, 2:3.
2. The EcoScale as a post-reaction analysis tool to select reaction conditions for scale-up. Koen Van Aken, Lucjan Strekowski. Handbook of Green Chemistry, Oxford University Press (september 2008)
Institution address: Molecular Biology Research Unit, Clinical Research Institute of Montreal, Avenue des Pins Ouest 110, H2W 1R7, Montréal, Canada
About the lab
Our group has developed a small animal model of AIDS by expressing HIV-1 Nef in immature and mature CD4+ T cells and in cells of the macrophage/dendritic lineage of Tg mice. These Tg mice develop several phenotypes in the immune system: thymic atrophy, preferential depletion of CD4+ T cells, lack of helper function, activation of CD4+ and CD8+ T cells, enhanced B cell number, hypergammaglobulinemia, etc). In addition, these Tg mice develop other organ diseases [lung, kidney (HIVAN), and heart). All these phenotypes are very similar to those which have been described in AIDS patients and in macaques infected with SIV. We have shown that all these Tg phenotypes are Nef-mediated, Nef being necessary and sufficient for their development. This model should be instrumental for in vivo evaluation of therapeutic agents targeting Nef or its pathway.
About Clinical Research Institute of Montreal
The Clinical Research Institute of Montreal, a public institution founded in 1967 devoted to biomedical research, is affiliated with the University of Montreal. It is constituted by 37 independent laboratories (like that of P#6) working in fields as varied as medicinal chemistry, molecular biology, functional genomics, clinical research, biomedical engineering and bioethics. Near than 450 people work at the Institute. Each laboratory is directed by an independent active scientist. It is one of the biggest biomedical and top-performing research center in Canada.
Institution address: Virus and Immunity Group, Institut Pasteur, Rue du Docteur Roux 25-28, 75724 Paris, France
About Institut Pasteur
Institut Pasteur is a non-profit private foundation which contributes to the prevention and treatment of disease, primarily infectious diseases, through research, education, and public health activities. Its Parisian campus hosts almost 2500 individuals, within an independent international network of Pasteur institutes, which constitutes an original and autonomous body, the only one of its kind in the world.
Institution address: Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11
About the lab
The research interest is focused on the analysis of viral protein–host cell interactions. Naturally, viral invasion interferes with a broad range of cellular processes including the alteration of signal transduction pathways, the redirection of intracellular protein transport, stimulation of transcription and translation as well as remodeling of the cytoskeleton. The aim is to analyze structure function relationships of such processes by the means of biochemical, biophysical and structural methods. Currently, main topics include the myristoylated HIV-1 Nef protein and its interaction partners, the regulation of transcription elongation by P-TEFb, Hexim1 and 7SK snRNA and its stimulation by the Tat-TAR complex and formin mediated actin nucleation. Some of the projects we are currently working on are described below.
Binding kinetics of Nef to membranes
Nef mediated internalization of T cell receptor molecules from the surface of an infected cell is required for the pathogenicity of HIV and disease progression to AIDS. This function depends on the N-terminal myristoylation of Nef, a lipid modification which targets the protein to membranes. In an ongoing study the aim is to analyze how specific membrane features and sequence motifs within Nef determine the mutual protein–liposome interaction. Time-resolved fast kinetics using the stopped flow technique allow for the in vitro measurement of the association and dissociation of Nef with lipid membranes. Preliminary results show a charge and curvature dependence of the mutual interaction that correlates with specific sequence motifs within the protein.
About Max Planck Institute of Molecular Physiology
The Max Planck Institute of Molecular Physiology is one of 80 research institutes of the Max Planck Society for the Advancement of Science (German acronym: MPG). The focus of our work is on basic research, that is, the striving for new insights – without industrial/commercial orientation and for the benefit of all people.
A large part of the budget comes from public funding, as in all Max Planck institutes. Germany’s federal government and the state where the respective institute is located – in our case North Rhine-Westphalia – each contribute 50% to the budget.
Despite the extensive public funding, the Max Planck Society is not a public/state institution, but has the legal form of a registered association. It consists of more than 764 supporting members, honorary members and members by virtue of their office. In addition, the directors of the individual Max Planck Institutes who have been appointed to the Max Planck Society are also members of the association. Each of these directors is autonomous in his/her research activity.
Like every research institute of the Max Planck Society, the Max Planck Institute in Dortmund is subject to regular external evaluation: Every two years the Institute’s research performance is evaluated – and, if necessary, adjusted– by a team of external experts, the Scientific Advisory Board.
Institution address: Laboratoire d’Innovation Thérapeutique – Faculté de
Pharmacie Université de Strasbourgh, Route du Rhin 74, Illkirch 67401 France
About the lab
The Medicinal Chemistry laboratory Strasbourg University together with colleagues from the Illkirch Campus, developed novel strategies and technical platforms in order to rationalize and accelerate the discovery of ligands acting on genomic targets. This contributed to the development of the Strasbourg Génopole "from genes to drugs" and of national networks such as the Chimiothèque Nationale. One more compound is currently in Phase 1 clinical trials for Alzheimer's disease (Minozac®). The lab of gathers 120 scientists (33 with permanent positions) distributed in five teams with expertise in natural substances, chemistry, medicinal chemistry, molecular modelling and biology. Every team is headed by a renowned expert in its field with track records of publications and patents. The total surface is 2000 m², with state-of-the–art equipment (fume cupboards, LCMS, NMR, etc.). It is recurrently funded by the CNRS and the French research Ministry (200 000 €/year) and supported by French and European grants (2 M€ budget per year on average).
An open-access high throughput screening platform was set up and developed a historical collection of 5000 molecules produced by our scientists over the past 30 years. Seven start-up companies have been created by lab members since 2001.
About Strasbourg University
European by nature and international by design, the University's strengths and assets stem from its active involvement in virtually every discipline comprising the current body of knowledge. As a young university founded on an age-old tradition, it strives to attain cross-disciplinarity so that this mixing fosters new research opportunities and produces courses that meet society's need. The international dimension is fundamental for the University of Strasbourg and thanks to the worldwide reputation of its research teams, built on excellence and efficiency, it emerges among Europe's foremost research universities. Each of the University's main academic fields of instruction is based upon research sections that are the driving force of the institution, with over 2,600 professors and staff.
Solidly anchored in the European Higher Education Area, the University of Strasbourg, a beating heart of the Alsatian metropolis with its 42,000 students, has the potential to face the challenging international competition.
Institution address: INSERM UMRS 945, CHU Pitié Salpêtrière, Boulevard de l’Hôpital 91, Paris 75634, France
About the lab
The INSERM research lab is member of a federative research institute on immunity , cancer and infections. It is fully equiped with P2 and P3 facilities, animal facilities. Research members are trained for human and preclinical works. The team involved in the proposed project has a long experience in HIV research as well as other infectious diseases such as tuberculosis , CMV. In addition the research unit is part of University Piere and Marie Curie, and has access to genomic, and trancriptomic platforms and flow cytometry platforms. The research unit is situated in the Pitié-Salpêtrière Hospital, the largest one in Paris, member of assistance publique.