Counterscreen for discovery of small molecules that bind to the HIV-1-gp120 binding antibody, PG9: TR-FRET-based biochemical high throughput dose response assay to identify small molecules that bind to the control antibody, PG126, which binds to a distinct site on the HIV envelope different from the PG9 binding site
Name: Counterscreen for discovery of small molecules that bind to the HIV-1-gp120 binding antibody, PG9: TR-FRET-based biochemical high throughput dose response assay to identify small molecules that bind to the control antibody, PG126, which binds to a distinct site on the HIV envelope different from the PG9 binding site. ..more
BioActive Compounds: 21
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: PG126_INH_TRFRET_1536_3XIC50 DCSRUN
Name: Counterscreen for discovery of small molecules that bind to the HIV-1-gp120 binding antibody, PG9: TR-FRET-based biochemical high throughput dose response assay to identify small molecules that bind to the control antibody, PG126, which binds to a distinct site on the HIV envelope different from the PG9 binding site.
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.
DCSRUN, dose response, titration, counterscreen, secondary, PG126, triplicate, 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, triplicate, 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 determine whether compounds identified as active in a set of previous experiments entitled, "TRFRET-based biochemical primary high throughput screening assay to identify small molecules that bind to the HIV-1-gp120 binding antibody, PG9" (AID 624416) are nonselective due to inhibition of binding of the PG126 antibody. This assay determines dose response curves for compounds. In this assay, test compounds are incubated with Europium-conjugated PGT126 (Eu-PGT126), followed by addition of a peptide complex comprised of biotinylated-gp120 (BG505 gp120) that has been reacted with XL665-conjugated streptavidin (SA) to form an Env-SA-APC complex. When PGT126 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 PGT126-Env complex will reduce the transfer of energy from Europium to APC, thereby inhibiting well FRET.
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 PG126 (Eu-PG126). When PG126 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 PG126-Env complex will reduce the transfer of energy from Eu to XL665, thereby inhibiting well FRET. Compounds are tested in triplicate using a dilution series starting at a maximum nomimal concentration of 67 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 (50 mM 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 0.044 uM 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 PG126 (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 EnVIsion microplate reader (PerkinElmer). Measurements were performed by exciting the plates at 320 nM, and monitoring well fluorescence at 615 nm (Eu) and 665 nm (XL665) with the EnVision 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 = ( I665nm / I615nm )
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-biotinylated and DMSO.
For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Accelrys Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 67 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 67 uM.
PubChem Activity Outcome and Score:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-87, and for inactive compounds 85-0.
List of Reagents:
PG126 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. The MLSMR was not able to provide all compounds selected for testing in this assay.
Assay: Dictionary: Version: 0.1
Assay: CurveFit : Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit : Mask: Excluded Points
* Activity Concentration. ** Test Concentration.
Data Table (Concise)