Drugs in Development

Drugs in Development

OyaGen seeks to induce transient and beneficial changes in the protein expression and function in human tissues by involving the editing enzymes in targeting biomedically relevant pathways.

OyaGen’s platform technology encompasses a family of fourteen (14) editing enzymes and the proteins that modulate their activity. The functions of editing enzymes have broad biomedical implications many of these enzymes are unexplored. The enzymes that have been studied are known to affect a range of important functions including antiviral, cancer, immunoglobulin gene rearrangements, inflammation, ion channel function, LDL reduction, neuro-cognitive functions and red blood cell expression in fetal liver.

Intellectual property obtained from several universities have been developed by OyaGen as the basis for assays used in high throughput screening for small molecule drug candidates.

OyaGen is strategically positioned to pursue novel therapeutics using assays for highthroughput screening on five targets affecting Vif and APOBEC3G.———

 

1. Vif Dimerization Antagonist

OyaGen holds an exclusive license from Thomas Jefferson University (TJU) covering the targeting of the Vif dimerization sequence for the development of anti-HIV drugs.   OyaGen has validated the availability of the Vif dimerization domain in living cells and the requirement of Vif dimerization for APOBEC3G distruction and viral infectivity.13   A highthroughput assay in mammalian cells has been developed to identify compounds that inhibit Vif dimerization.  An E coli assay based on Vif dimerization has also been established for HTS of Vif dimerization antagonists.

2. & 3. Vif interaction with APOBEC3G

OyaGen holds an exclusive license from the University of Rochester and nonexclusive licenses from Oregon Health Science Center on the interaction of Vif with APOBEC3G and the targeting of this interaction in the pursuit of anti-HIV compounds.  A highthroughput assay in mammalian cells has been developed to identify compounds that disrupt Vif:APOBEC3G complexes by binding to either Vif or APOBEC3G..  An  E coli assay based on Vif:APOBEC3G interaction has also been established for HTS of Vif:APOBEC complex formation antagonists.

4. Vif dependent polyubiquitination of APOBEC3G

OyaGen holds an exclusive license from the University of Rochester and nonexclusive licenses from Oregon Health Science Center on technology for the expression of functional Vif and its interaction with the ubiquitination machinery. Inhibiting this interaction with small molecules identified through high throughput screening will prevent Vif-dependent polyubiquitination of APOBEC3G and of Vif. This novel intervention will preserve APOBEC3G host anti-viral host defense.

5.  APOBEC3G activators

OyaGen holds an exclusive license from the University of Rochester and nonexclusive licenses from Oregon Health Science Center on the interaction of APOBEC3G and RNA and the targeting of this interaction in the pursuit of  compounds that will disrupt APOBEC 3G binding to RNA and thereby reactivate APOBEC3G deaminase activity and host defense activity.  A high throughput assay based on recombinant macromolecules has been developed to identify these compounds.

The HIV life cycle can be broken down into early and late phases during which viral functions are expressed and/or carried out that are critical for host cell infection and virion production.  These functionalities have been targeted by drug discovery efforts and have given rise to the important therpeutics used to treat HIV/AIDS. Cells that are not permissive to HIV infection express APOBEC3G  and maintain the enzyme as low molecular mass complex primarily consisting of C-terminal dimers.1-3

The early phase of the HIV life cycle involves viral particles binding to surface receptors on permissive cells and replication of the proviral DNA from the viral RNA genome through reverser transcription.  Integration of proviral DNA into the host cell chromosomal DNA is a prerequisite to the expression of virions during the late phase of the HIV life cycle.  APOBEC3G in nonpermissive cells binds to nascent single strand proviral genomes during their replication and catalyze dC to dU mutation that will template dG to dA mutations in the coding strand of the HIV genome.  These point mutations affect the protein coding capacity of the HIV genome and or the dU transitions lead to degradation of the proviral genomes prior to their integration. 4-6

Vif is expressed in the late phase of the HIV life cycle. Vif binds to Elongin B/C and recruits Culin5 along with the E2 ubiquitin ligase to APOBEC3G. APOBEC3G is degraded along with Vif via the proteosome thereby creating a biological APOBEC3G knockdown.9-12 OyaGen has shown that disrupting Vif dimerization disables Vif’s ability to bind to and destroy APOBEC3G.  This enables APOBEC3G to be package with virions as they are assembling by binding to Gag and HIV RNA genomes. 13-15

 

Activation of T cells makes them permissive to HIV infection and under this circumstance APOBEC3G deaminase activity is inactivated by binding to cellular RNA1-2, 5-6 allowing the incoming viruses to replicate, integrate and express viral proteins. OyaGen high throughtput screening has identified compounds that antagonize APOBEC3G binding to cellular RNAs. These compounds activate A3G deaminase activity in permissive cells and thereby inhibit viral replication of incoming virus.

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References

1 Wedekind J.E., Gillilan, R., Janda, A., Krucinska, J., Salter, J.D., Bennett, R.P., Raina, J., Smith, H.C. Nanostructures of APOBEC3G support a hierarchical assembly model of high molecular mass ribonucleoprotein particles from dimeric subunits.  J. Biol. Chem. 281, 38122-126 (2006).
2 Bennett, R.P., Salter J.D., Liu, X., Wedekind, J.E., Smith, H.C. APOBEC3G subunits self-associate via the C-terminal deaminase domain. JBC 283:33329-36 2008 (2008).
3 Salter, J.D. Krucinska, J., Raina, J., Smith, H.C., Wedekind, J.E. A hydrodynamic analysis of APOBEC3G reveals a monomer-dimer-tetramer self-association that has implications for anti-HIV function. Biochemistry. 48:10685-7 (2009).
4 Yu, Q., R. Konig, S. Pillai, K. Chiles, M. Kearney, S. Palmer, D. Richman, J.M. Coffin, and N.R. Landau. Single-strand specificity of APOBEC3G accounts for minus-strand deamination of the HIV genome. Nat Struct Mol Biol 11:435-442 (2004). & Chelico, L., P. Pham, P. Calabrese, and M.F. Goodman. 2006. APOBEC3G DNA deaminase acts processively 3′ –> 5′ on single-stranded DNA. Nat Struct Mol Biol 13:392-399.
5 Chiu, Y.L., Soros, V.B., Kreisberg, J.F., Stopak, K., Yonemoto, W., Greene, W.C. Cellular APOBEC3G restricts HIV-1 infection in resting CD4+ T cells. Nature 435:108-14 (2005).
6 Chiu, Y.L., Greene, W.C. The APOBEC3 cytidine deaminases: an innate defensive network opposing exogenous retroviruses and endogenous retroelements. Annu Rev Immunol 26:317-53 (2008).
7 Gallois-Montbrun, S., B. Kramer, C.M. Swanson, H. Byers, S. Lynham, M. Ward, and M.H. Malim. 2007. Antiviral protein APOBEC3G localizes to ribonucleoprotein complexes found in P bodies and stress granules. J Virol 81:2165-2178.
8 Sheehy, A.M., Gaddis, N.C., Choi, J.D., Malim, M.H.  Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418:646-650 (2002).
9 Svarovskaia, E.S., Xu, H., Mbisa, J.L., Barr, R., Gorelick, R.J., Ono, A., Freed, E.O., Hu, W.S. and Pathak, V.K.  Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is incorporated into HIV-1 virions through interactions with viral and nonviral RNAs. J Biol Chem. 279: 35822-8  (2004).
10 Yu, Y., Xiao, Z., Ehrlich, E.S., Yu, X. and Yu, X.F.  Selective assembly of HIV-1 Vif-Cul5-ElonginB-ElonginC E3 ubiquitin ligase complex through a novel SOCS box and upstream cysteines. Genes Dev. 18: 2867-72  (2004).
11 Conticello, S.G., Harris, R.S. and Neuberger, M.S.   The Vif protein of HIV triggers degradation of the human antiretroviral DNA deaminase APOBEC3G. Curr Biol. 13: 2009-13 (2003).
12 Marin, M., Rose, K.M., Kozak, S.L. and Kabat, D.   HIV-1 Vif protein binds the editing enzyme APOBEC3G and induces its degradation. Nat Med. 9: 1398-403 (2003).
13 Miller, H.-J. Presnyak, V., Smith, H.C. The dimerization domain of HIV-1 viral infectivity factor Vif is required to block virion incorporation of APOBEC3G. Retrovirology 4:81-92 (2007).
14 Zennou, V., Perez-Caballero, D., Gottlinger, H. and Bieniasz, P.D.   APOBEC3G incorporation into human immunodeficiency virus type 1 particles. J Virol. 78: 12058-61 (2004).
15 Cen, S., Guo, F., Niu, M., Saadatmand, J., Deflassieux, J. and Kleiman, L.  The interaction between HIV-1 Gag and APOBEC3G. J Biol Chem. 279: 33177-84 (2004).