Design and testing of a new family of specific drug HIV Integrase inhibitors which do not interfere with V(D)J recombination
Autoritatea contractanta: Unitatea Executiva pentru Finantarea Invatamantului Superior, a Cercetarii, Dezvoltarii si Inovarii (UEFISCDI)
Numar / Data contract: PN-II-PT-PCCA-2013-4-0930
Program: Proiecte Parteneriate
Director proiect: Mihai Ciubotaru
Parteneri: P1-Apel Laser SRL, P2-Institutul de Biochimie al Academiei Romane, P3-Institute of Nuclear Physics and Engineering Horia Hulubei, Magurele(IFIN-HH).
Data incepere / finalizare proiect: 2014-07-01 / 2016-06-30
Valoarea proiectului: 1250000 RON
Summary: After birth newborn infants from HIV (Human Immunodeficiency Virus) infected mothers have to be subjected to prophylactic treatment against viral infection. HIV Integrase is the enzyme that mediates the viral genome integration into the host cells chromosomes and its catalytic prevention represents a major way to stop the viral infection. Despite its major clinical advantages (low toxicity compared to nucleotide analogs, lower effective IC50 and fewer adverse reactions than most protease inhibitors), the treatment with HIV Integrase inhibitors(INi) in newborns induces a paradoxical situation. Because HIV-IN resembles in its structure and reaction mechanism to Recombination Activating Gene protein RAG1, which assembles the lymphocytes antigen receptors, INi medication interferes with the ability of treated patients to develop a normal immune response. By reducing RAG activity 1, the currently used HIV-INi induce an iatrogenic immunodeficiency which mimics the onset of AIDS (acquired immune deficiency syndrome). This situation sometimes prompts at these treated patients a more aggressive therapy with higher medication doses, which in turn accentuates the aforementioned immunodeficiency. We propose to solve the major drawback of INi treatment that it cannot be addressed to newborns exposed to high risk acquiring HIV-1(type1) infections.The major goal of our proposal is to test new HIV specific Integrase inhibitory compounds which do not interfere with RAG somatic recombination. Our expertise with biophysical assays testing RAG activity is the key element ensuring the creativity and originality of this proposal. We propose to use a new strategy to separately screen for compounds inhibiting IN at each of its three integration steps: viral DNA-IN binding, catalytic 3′ DNA nicking and strand Transfer. Nowadays, in drug discovery and screening strategies just one of these IN activities is targeted for potential inhibition. Based upon our results from our work investigating critical RAG-DNA binding/bending activity2,3 in somatic recombination, we propose to develop rapid, low cost, efficient fluorescence resonance energy transfer (FRET) setups and screening assays some at single molecule level. With them we will high-throughput screen(HTPS) our in silico designed and derivatized library of HIV Integrase inhibitory compounds for their in vitro dual HIV Integrase inhibition and RAG activity interference effects. Dual screening for IN inhibition with no RAG activity interference is unprecedented and represents an entirely original approach for the development of this family of drugs. The FRET in vitro screened compounds will be quantitatively tested for their effect on RAG mediated DNA substrate cleavage reactions. The lead compounds will then be tested in a primary cell line culture screening assay. We will test their cell toxicity and their potential inhibitory effect on stage progression from pre B to immature B lymphocyte(which requires somatic recombination). This will be assessed by cell survival and preB/IgM surface markers display. The best candidate compounds screened by our assays will be further tested for their potential to interfere with antigen activation of B cells from biopsies of immunocompromised lymphoma patients. For this, the compounds will be added on lymphomatous B cells cultures with LPS (Gram Negative Bacterial Lipopolysaccharide) and Interleukin 4 (IL4) and their effect tested by proliferation tests directed against control cell cultures without drugs. Our selected lead compounds should not affect patients immunity, hence after future tests in animals and in patient clinical trial studies they should reach the highest marketing potential among all other drugs from their class.
Our project proposes five objectives:
- Building from the available HIV-IN data base domain structures a new model of the whole protein conformation on which to dock our designed chemical compounds in its key functional motifs. The best fitted configurations will be used for the synthesis of the compounds library.
- High-throughput screen the library of compounds for HIV-IN inhibition at various steps of its reaction mechanism: a)viral cDNA binding, b) 3’nicking and c)strand transfer.
- Build a newly designed Single Molecule Dual Lasers FRET multimodular setup able to detect high resolution fluorescence signals in a Total Internal Reflection optical system, for RAG interference drug screening.
- Test the best compounds resulted from HIV-IN inhibition screening for RAG interference using a high sensitive novel Single Molecule FRET assay.
5. The lead compounds will be tested for RAG enzymatic interference by coupled cleavage assays, whereas cell toxicity and nonspecific lymphoproliferation will be assessed on primary B cell lines and lymphoma cells from immunocompromised patients.
ETAPELE PROIECTULUI SI DATELE DE PREDARE
1. Etapa unica an 2014 (2014-12-15)
2. Etapa unica an 2015 (2015-12-15)
3. Etapa unica an 2016 (2016-06-30)
ECHIPA DE CERCETARE SI GRADUL DE IMPLICARE AL TINERILOR CERCETATORI