TRFRET-based biochemical primary high throughput screening assay to identify small molecules that bind to the HIV-1-gp120 binding antibody, PG9
Name: TRFRET-based biochemical primary high throughput screening assay to identify small molecules that bind to the HIV-1-gp120 binding antibody, PG9. ..more
BioActive Compounds: 1546
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Michael J. Caulfield , International Aids Vaccine Initiative
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: DA033177-01
Grant Proposal PI: Michael J. Caulfield , International Aids Vaccine Initiative
External Assay ID: HIV-PG9-GP120_INH_TRFRET_1536_1X%INH PRUN
Name: TRFRET-based biochemical primary high throughput screening assay to identify small molecules that bind to the HIV-1-gp120 binding antibody, PG9.
Development of a vaccine to prevent AIDS is the best hope for controlling the epidemic that has led to infection of more than 30 million people with the HIV-1 virus worldwide (1). A vaccine approach that reduces viral load would certainly be beneficial, but one that elicits sterilizing immunity would be preferred (2). The HIV envelope glycoprotein (Env) is key to vaccine development since it is the only target for neutralizing antibodies (3-5). The Env consists of the gp120 surface glycoprotein and gp41 transmembrane protein associated in a trimer of gp120-gp41 heterodimers. The presence of broadly neutralizing sera from some HIV-1 infected individuals (3, 6-9) and the protection in monkeys by passive transfer of several neutralizing monoclonal antibodies (mAbs) (10) suggest that if a suitable antibody response to Env can be obtained, then protection from infection will be possible.
Conventional vaccine approaches based on delivery of HIV-1 envelope (Env) proteins or peptides derived from Env sequences have failed to generate broadly neutralizing antibodies (bNAbs) to the virus, which mutates rapidly to escape from the immune response (11). Recently, new potent and broad neutralizing antibodies against the CD4 binding site (CD4Bs) (VRC01 and VRCPG04) and a novel site on HIV-1 Env trimer consisting of conserved elements on the variable loops V1/2 stem, V2 and V3 on the Env (PG9, PG16) have been defined (12, 13) . These new bNAbs neutralize a broad panel of HIV-1 viruses at concentrations that are an order of magnitude lower than the neutralizing concentrations of "first generation" bNAbs (b12, 2G12, 2F5 and 4E10). The availability of these new bNAb reagents enhances the prospect of designing the corresponding immunogens to evaluate as vaccine candidates. Recently published results (14) document that antibody-binding small molecules could be selected by probing a large chemical library with D5, a human mAb that binds to a pre-hairpin fusion intermediate on gp41 of HIV-1. The importance of this finding is that such molecules can be rendered immunogenic by conjugation with a carrier protein thereby forming the basis for development of AIDS vaccine candidates. Since the D5 mAb used in published studies is not very potent and lacks sufficient breadth, we propose to utilize the new more potent bNAbs to test the hypothesis that individual bNAbs can bind specifically to diverse chemical entities that can be selected in a high throughput screen of a large chemical collection. The objective of this proposal is to develop and implement screening assays to utilize existing human mAbs with broad neutralizing activity (bNAbs) to screen a large and diverse small molecule library to identify haptens that bind specifically to the antibody combining site of the bNAb. These small molecule mimetics of the HIV-1 envelope protein could be useful in development of an AIDS vaccine.
This project will exploit the antigen-binding property of newly discovered broad and potent neutralizing antibodies to select complementary small molecule leads that can be used as building blocks for the development of a vaccine to prevent infection with the HIV-1 virus.
1. Burton, D.R., R.C. Desrosiers, R.W. Doms, W.C. Koff, P.D. Kwong, J.P. Moore, G.J. Nabel, J. Sodroski, I.A. Wilson, and R.T. Wyatt, HIV vaccine design and the neutralizing antibody problem. Nat Immunol, 2004. 5(3): p. 233-6.
2. Karlsson Hedestam, G.B., R.A. Fouchier, S. Phogat, D.R. Burton, J. Sodroski, and R.T. Wyatt, The challenges of eliciting neutralizing antibodies to HIV-1 and to influenza virus. Nat Rev Microbiol, 2008. 6(2): p. 143-55.
3. Stamatatos, L., L. Morris, D.R. Burton, and J.R. Mascola, Neutralizing antibodies generated during natural HIV-1 infection: good news for an HIV-1 vaccine? Nat Med, 2009. 15(8): p. 866-70.
4. Haynes, B.F. and D.C. Montefiori, Aiming to induce broadly reactive neutralizing antibody responses with HIV-1 vaccine candidates. Expert Rev Vaccines, 2006. 5(4): p. 579-95.
5. Srivastava, I.K., J.B. Ulmer, and S.W. Barnett, Role of neutralizing antibodies in protective immunity against HIV. Hum Vaccin, 2005. 1(2): p. 45-60.
6. Binley, J.M., E.A. Lybarger, E.T. Crooks, M.S. Seaman, E. Gray, K.L. Davis, J.M. Decker, D. Wycuff, L. Harris, N. Hawkins, B. Wood, C. Nathe, D. Richman, G.D. Tomaras, F. Bibollet-Ruche, J.E. Robinson, L. Morris, G.M. Shaw, D.C. Montefiori, and J.R. Mascola, Profiling the specificity of neutralizing antibodies in a large panel of plasmas from patients chronically infected with human immunodeficiency virus type 1 subtypes B and C. J Virol, 2008. 82(23): p. 11651-68.
7. Sather, D.N., J. Armann, L.K. Ching, A. Mavrantoni, G. Sellhorn, Z. Caldwell, X. Yu, B. Wood, S. Self, S. Kalams, and L. Stamatatos, Factors associated with the development of cross-reactive neutralizing antibodies during human immunodeficiency virus type 1 infection. J Virol, 2009. 83(2): p. 757-69.
8. Carotenuto, P., D. Looij, L. Keldermans, F. de Wolf, and J. Goudsmit, Neutralizing antibodies are positively associated with CD4+ T-cell counts and T-cell function in long-term AIDS-free infection. AIDS, 1998. 12(13): p. 1591-600.
9. Li, Y., S.A. Migueles, B. Welcher, K. Svehla, A. Phogat, M.K. Louder, X. Wu, G.M. Shaw, M. Connors, R.T. Wyatt, and J.R. Mascola, Broad HIV-1 neutralization mediated by CD4-binding site antibodies. Nat Med, 2007. 13(9): p. 1032-4.
10. Hart, M.K., T.J. Palker, T.J. Matthews, A.J. Langlois, N.W. Lerche, M.E. Martin, R.M. Scearce, C. McDanal, D.P. Bolognesi, and B.F. Haynes, Synthetic peptides containing T and B cell epitopes from human immunodeficiency virus envelope gp120 induce anti-HIV proliferative responses and high titers of neutralizing antibodies in rhesus monkeys. J Immunol, 1990. 145(8): p. 2677-85.
11. Emiliani, S., C. Van Lint, W. Fischle, P. Paras, Jr., M. Ott, J. Brady, and E. Verdin, A point mutation in the HIV-1 Tat responsive element is associated with postintegration latency. Proc Natl Acad Sci U S A, 1996. 93(13): p. 6377-81.
12. Euler, Z., E.M. Bunnik, J.A. Burger, B.D. Boeser-Nunnink, M.L. Grijsen, J.M. Prins, and H. Schuitemaker, Activity of Broadly Neutralizing Antibodies, Including PG9, PG16, and VRC01, against Recently Transmitted Subtype B HIV-1 Variants from Early and Late in the Epidemic. J Virol, 2011. 85(14): p. 7236-45.
13. Doores, K.J. and D.R. Burton, Variable loop glycan dependency of the broad and potent HIV-1-neutralizing antibodies PG9 and PG16. J Virol, 2010. 84(20): p. 10510-21.
14. Caulfield, M.J., V.Y. Dudkin, E.A. Ottinger, K.L. Getty, P.D. Zuck, R.M. Kaufhold, R.W. Hepler, G.B. McGaughey, M. Citron, R.C. Hrin, Y.J. Wang, M.D. Miller, and J.G. Joyce, Small molecule mimetics of an HIV-1 gp41 fusion intermediate as vaccine leads. J Biol Chem, 2010. 285(52): p. 40604-11.
PRUN, HIV, HIV-1, AIDs, human acquired immunodeficiency virus, env, gp120, glycoprotein, BG505, V2, V3, V2/V3, PG9, XL665, europium, Eu, biotin, biotinylated, streptavidin, SA, antibody, antigen, hapten, mimetic, binding, virus, protein, peptide, protein-protein interaction, interaction, inhibit, inhibitor, APC, Allophycocyanin, FRET, TRFRET, TR-FRET, time resolved fluorescence resonance energy transfer, fluorescence, primary, singlicate, HTS, 1536, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify compounds that bind to PG9, an antibody that binds to the V2/V3 loop of the HIV-1 gp120 envelope glycoprotein. gp120 is the viral coat protein responsible for HIV entry into cells, and forms the spikes sticking out of a HIV virus particle. Its main function is to bind to CD4 in human cells. One spike is composed of three gp120 and gp41 subunits. Specifically, compounds should inhibit the interaction of Eu-PG9 antibody and BG505 gp120 peptide.
In this assay, test compounds are incubated with a peptide complex comprised of biotinylated-gp120 (BG505 gp120) that has been reacted with XL665-conjugated streptavidin (SA) to form an Env-SA-Allophycocyanin (APC) complex, followed by addition of Europium-conjugated PG9 (Eu-PG9). When PG9 binds to Env gp120, it brings the Eu into close proximity with the APC substrate, resulting in TR-FRET from Eu to XL665. As designed, compounds that prevent or inhibit formation of the PG9-Env complex will reduce the transfer of energy from Eu to XL665, thereby inhibiting well FRET. Compounds are tested in singlicate at a nominal concentration of 6.7 uM.
Prior to the start of the assay, 2 uL of a mixture containing 0.036 uM gp120 (non-biotinylated) and 0.044 uM SA-XL665 in Assay Buffer (50mM Sodium Phosphate, 0.4 M Potassium Fluoride, 0.1%BSA, 0.1% Tween-20, pH 7.00), filtered at 0.22 um was dispensed into columns 1 to 3 of 1536-well assay plates. A mixture of 0.036 uM gp120 (biotynilated) and 0.044uM SA-XL665 in Assay Buffer, filtered at 0.22 um, was dispensed into wells 4-48.
Next, 27 nl of compounds or DMSO alone (0.5% final concentration) were distributed into appropiate wells. The plates were incubated for 20 minutes at 25 C. The assay started by the addition of 2 ul of a mixture containing 0.0024 uM PG9 (Europium-conjugated) in Assay Buffer( filtered at 0.22 um), to all wells and plates were centrifuged. The plates were incubated for 1 hr at 25 C, after which TR-FRET was measured by the ViewLux microplate reader (PerkinElmer). Measurements were performed by exciting the plates at 340 nM, and monitoring well fluorescence at 618 nm (Eu) and 671 nm (XL665) with the ViewLux microplate reader .
To normalize data, values measured from both fluorescence emission wavelengths were used to calculate a ratio for each well, according to the following mathematical expression:
Ratio = ( I671nm / I618nm )
I represents the measured fluorescence emission intensity at the enumerated wavelength in nm.
The percent inhibition was calculated from the median ratio as follows:
%_Inhibition = 100 * ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) )
Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing gp120 non-biotinylated and DMSO.
Low_Control is defined as wells containing gp120-biotynilated and DMSO.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-8, and for inactive compounds 8-0.
List of Reagents:
PG9 Eu-conjugated antibody (supplied by Assay Provider)
BG505 gp120 antigen (supplied by Assay Provider)
Streptavidin-XL665 (Cisbio, part 610SAXLB)
Potassium Fluoride (Sigma, part 402931)
Bovine Serum Albumin (Sigma, part A7906)
Tween 20 (Fisher, part BP337)
Dithiothreitol (Acros, part1 6568-0250)
Sodium phosphate monobasic (Fisher, part BP329-500)
Sodium phosphate dibasic (Fisher, part BP332-500)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)