Fluorescence polarization-based biochemical high throughput primary assay to identify inhibitors of sialic acid acetylesterase (SIAE)
Name: Fluorescence polarization-based biochemical high throughput primary assay to identify inhibitors of sialic acid acetylesterase (SIAE). ..more
BioActive Compounds: 2555
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute
Assay Provider: Shiv Pillai, Harvard Medical School
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: R01 AI090867
Grant Proposal PI: Shiv Pillai, Harvard Medical School
External Assay ID: SIAE_INH_FP_1536_1X%INH PRUN
Name: Fluorescence polarization-based biochemical high throughput primary assay to identify inhibitors of sialic acid acetylesterase (SIAE).
The key problem in HIV immunization is that many HIV epitopes that would be useful targets of neutralizing antibodies are weakly immunogenic in vivo. The only way high affinity neutralizing antibodies can be generated against some epitopes is by the induction of a high level of somatic hypermutation. Unfortunately no effective strategies exist today for reproducibly generating broadly neutralizing antibodies against HIV either in healthy volunteers or in experimental animals (1-4). A few monoclonal and phage display derived antibodies have been described that have potent neutralization activity (5-10). Similar neutralizing antibodies are not readily generated upon immunization. Although HIV infected individuals can generate broadly neutralizing antibodies, these antibodies are generated too late in the course of an infection to be of mitigating clinical value. Broadly neutralizing antibodies generated in the course of natural infection are characterized by extensive somatic mutation (4). Thus, HIV immunization strategies for generating effective neutralization activity should have the goal of generating a range of extensively somatically mutated antibodies against a number of conserved but weakly immunogenic gp120 epitopes.
We have recently discovered an important mechanism of B cell tolerance that appears to dampen the activation of naive B cells whose antigen receptors recognize self or foreign antigens with moderate or low affinity (11). A key player in this B cell intrinsic suppressive pathway is Sialic Acid Acetyl Esterase (SIAE). This pathway swings into action only in peripheral B cells.
Ongoing immunization studies in mice using adoptively transferred B cells deficient in SIAE strongly suggest that an SIAE inhibitor could be used to transiently lower the signaling threshold
during an immunization window in order to appropriately activate B cells that recognize certain relatively low avidity epitopes and allow the development of immune responses that result in extensive somatic mutation of antibodies with the potential to effectively neutralize pathogens and their toxins. SIAE is therefore a very attractive molecule from the viewpoint of identifying a "druggable" enzyme target that regulates humoral immunity at the level of setting the threshold for the activation of peripheral B cells. Such an enzyme could potentially be a very interesting target for the pharmacological induction of immune activation in order to #empower# B cells that are specific for #weak# epitopes, and to also enhance T cell memory responses.
1. Burton, D.R., R.L. Stanfield, and I.A. Wilson, Antibody vs. HIV in a clash of evolutionary titans. Proc Natl Acad Sci U S A, 2005. 102(42): p. 14943-8.
2. Forsell, M.N., et al., B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4. PLoS Pathog, 2008. 4(10): p.e1000171.
3. 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.
4. Scheid, J.F., et al., Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals. Nature, 2009. 458(7238): p. 636-40.
5. Alam, S.M., et al., The role of antibody polyspecificity and lipid reactivity in binding of broadly neutralizing anti-HIV-1 envelope human monoclonal antibodies 2F5 and 4E10 to glycoprotein 41 membrane proximal envelope epitopes. JImmunol, 2007. 178(7): p. 4424-35.
6. Calarese, D.A., et al., Dissection of the carbohydrate specificity of the broadly neutralizing anti-HIV-1 antibody 2G12. Proc Natl Acad Sci U S A, 2005. 102(38): p. 13372-7.
7. Cardoso, R.M., et al., Broadly neutralizing anti-HIV antibody 4E10 recognizes a helical conformation of a highly conserved fusion-associated motif in gp41. Immunity, 2005. 22(2): p. 163-73.
8. Haynes, B.F., et al., Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies. Science, 2005. 308(5730): p. 1906-8.
9. Ofek, G., et al., Structure and mechanistic analysis of the anti-human immunodeficiency virus type 1 antibody 2F5 in complex with its gp41 epitope. J Virol, 2004. 78(19): p. 10724-37.
10. Parren, P.W., et al., In vitro antigen challenge of human antibody libraries for vaccine evaluation: the human immunodeficiency virus type 1 envelope. J Virol, 1996. 70(12): p. 9046-50.
11. Srinivasan, G.V. and R. Schauer, Assays of sialate-O-acetyltransferases and sialate-O-acetylesterases. Glycoconj J, 2009. 26(8): p. 935-44.
PRUN, primary, SIAE, MGC87009, primary, sialic acid acetyl esterase, Sialate O-acetylesterase, sialic acid acetylesterase, serine hydrolase, HIV, immunization, humoral immunity, murine B cells, T cell memory, CD4, ABPP, activity-based protein profiling, FP-Rh, inhibitor, FP, fluorescence polarization, absorbance, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC inhibitor, fluorescence polarization, HTS, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this biochemical assay is to identify compounds that act as inhibitors of sialic acid acetylesterase (SIAE). In this assay, SIAE protein is incubated with test compounds and fluorophosphonate-rhodamine (FP-Rh) activity-based probe. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, test compounds that act as SIAE inhibitors will prevent SIAE-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds are tested in singlicate at a nominal test concentration of 9.66 micromolar.
Prior to the start of the assay, 3 microliters of assay buffer (1X DPBS and 0.01% Pluronic F-127) were dispensed into column 1 thru column 3 of 1536 microtiter plates. Next, 3 microliters of assay buffer containing 0.73uM of SIAE protein were dispensed into columns 4 thru 48. Then, 39 nL of test compound in DMSO or DMSO alone (0.97% final concentration) were added to the appropriate wells and incubated for 45 minutes at 25 degrees Celsius.
The assay was started by dispensing 1.0 microliter of 300 nM FP-Rh in assay buffer to all wells. Plates were centrifuged and after 120 minutes of incubation at 25 degrees Celsius, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525nm, emission = 598nm) for 25 seconds for each polarization plane (parallel and perpendicular).
Prior to further calculations, the following formula was used to calculate fluorescence polarization (FP):
FP = ( Raw1 - Raw2 ) / ( Raw1 + Raw2 )
Raw1 is defined as the S channel.
Raw2 is defined as the P channel.
The percent inhibition for each compound was calculated as follows:
100 *( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Test_Compound is defined as wells containing SIAE protein in the presence of test compound and FP-Rh.
High_Control is defined as wells containing DMSO, FP-Rh but, no SIAE protein.
Low_Control is defined as the median of the wells containing test compounds.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity 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 activity score range for active compounds is 100-2, for inactive 2-0.
List of Reagents:
SIAE protein (supplied by Assay Provider)
FP-Rh probe (supplied by Assay Provider)
DPBS (Mediatech, part 20-031-CV)
Pluronic F-127 (Invitrogen, part P6866)
1536-well plates (Greiner, part 789176)
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, 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.
** Test Concentration.
Data Table (Concise)