SAR analysis of compounds that inhibit Human Immunodeficiency Virus Fusion, cell-cell fusion assay
The fusion-active conformation of the envelope protein gp41 of HIV-1 consists of an N-terminal trimeric a-helical coiled coil domain, and three anti-parallel C-terminal helices which fold down the grooves of the coiled coil to form a six-helix bundle. Disruption of the six-helix bundle is considered to be a key component of an effective non-peptide fusion inhibitor. This structure forms as a more ..
BioActive Compounds: 40
Data Source: Burnham Center for Chemical Genomics (BCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: 1R21NS059403-01
Assay Provider: Dr. Miriam Gochin, Touro University-California, Vallejo, CA
The fusion-active conformation of the envelope protein gp41 of HIV-1 consists of an N-terminal trimeric a-helical coiled coil domain, and three anti-parallel C-terminal helices which fold down the grooves of the coiled coil to form a six-helix bundle. Disruption of the six-helix bundle is considered to be a key component of an effective non-peptide fusion inhibitor. This structure forms as a result of a conformational change in gp41, triggered by gp120 and co-receptor binding to host cell receptors. Prevention of six-helix bundle formation has been recognized as an important mechanism for viral fusion inhibition
This confirmatory, concentration-response assay has been developed and performed to confirm the hits originally identified in "uHTS fluorescence assay for the identification of Human Immunodeficiency Virus Fusion Inhibitors" (AID1986) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.
Cell-cell fusion was measured following a published procedure(1), and using cell lines obtained from the NIH Research and Reference Reagents Program. TZM-bl cells expressing CD4, CCR5 and CXCR4(2), and containing an integrated reporter gene for firefly luciferase under control of HIV-1 LTR(3) were used as target cells. They were grown overnight in 96 well plates in DMEM supplemented with 10% FBS, using 25,000 cells per well. The following day, the medium was exchanged with 50 ul reduced serum medium (Gibco - Invitrogen, USA), and 1 ul of compound was added to each well, using 6 serial dilutions to obtain dose response curves. HL2/3 effector cells which produce HIV-1 HXB2 Env, Tat and Rev(4) were added in reduced serum medium, using 50,000 cells per well, to a total well volume of 100 ul. After 6 hours incubation, luciferase expression was measured using Luciferase Assay Reagent (Promega, WI, USA). Controls containing 1ul DMSO with and without HL2/3 cells were measured for each compound, and experiments were performed in duplicate.
1. Wexler-Cohen, Y. Shai, Y. Faseb J 2007, 21, 3677.
2. Platt, E. J. Wehrly, K. Kuhmann, S. E. Chesebro, B. Kabat, D. J Virol 1998, 72, 2855.
3. Wei, X. Decker, J. M. Liu, H. Zhang, Z. Arani, R. B. Kilby, J. M. Saag, M. S. Wu, X. Shaw, G. M. Kappes, J. C. Antimicrob Agents Chemother 2002, 46, 1896.
4. Ciminale, V. Felber, B. K. Campbell, M. Pavlakis, G. N. AIDS Res Hum Retroviruses 1990, 6, 1281.
Compounds with a demonstrated IC50 < 100 uM are defined as actives in this assay.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data and is not applicable in this assay.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in this assay demonstrates a Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account the items discussed above is
Score = 82 + 3*(pIC50 - 3)*QC,
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 85 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)