SAR analysis of compounds that inhibit Human Immunodeficiency Virus Fusion. - BioAssay Summary
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 ..
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 Tested Compounds
 Tested Compounds
All(41)
 
 
Active(41)
 
 
 Tested Substances
 Tested Substances
All(41)
 
 
Active(41)
 
 
 Related BioAssays
 Related BioAssays
Table of Contents
AID: 434967
Data Source: Burnham Center for Chemical Genomics (BCCG-A370-GP41-Inhibitors-DryPowder)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-06-03
Hold-until Date: 2011-06-01
Modify Date: 2011-06-01

Data Table (Complete):           Active    All
Target
BioActive Compounds: 41
Depositor Specified Assays
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AIDNameTypeComment
1986uHTS fluorescence assay for the identification of Human Immunodeficiency Virus Fusion Inhibitors.confirmatory
1991Summary assay for the identification of Human Immunodeficiency Virus Fusion Inhibitors.summary
435031SAR analysis of compounds that inhibit Human Immunodeficiency Virus Fusion, cytoxicity assayconfirmatory
435029SAR analysis of compounds that inhibit Human Immunodeficiency Virus Fusion, cell-cell fusion assayconfirmatory
Description:
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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

A metallopeptide-based fluorescence assay has been developed to detect HIV-1 fusion inhibitors, by the screening of small molecules that bind to the hydrophobic pocket of gp41. The assay involves two peptides representing the inner N-terminal-heptad-repeat (HR1) coiled coil and the outer C-terminal-heptad-repeat (HR2) helical domains of the gp41 six-helix bundle which forms prior to fusion. The HR1 peptide is modified with a metal-ligated dye complex, which maintains structural integrity and permits association with a fluorescein-labeled HR2 peptide concomitant with fluorescence quenching. Compounds able to disrupt six-helix bundle formation can act as fusion inhibitors, and can be detected in the assay from an increase in the fluorescence that is correlated with compound's efficacy.

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.
Protocol
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Assay materials:
1) Env2.0 peptide
Seq: Ac-MTWBEWDREIBNYTSLIC(FAM)-NH2
2) C18-Aib(FL) peptide
Seq: bpy-GQAVEAQQHLLQLTVWGIKQLQARILAVEKK-NH2
3) Assay Buffer: 25 mM Tris-acetate pH 7.0, 0.01% Tween-20
4) The Fe(II) complex [Fe(II)(env2.0)3]2+ is prepared by addition of a one-third stoichiometry of freshly prepared ferrous ammonium sulfate to peptide in 25 mM Tris-acetate pH 7.0

Procedure
Mix equal parts of the Env2.0/Fe complex and C18-Aib(FL) (60 uM) just prior to the assay
1) Using a Labcyte Echo, DMSO and test compounds are transferred to wells of a black, Corning 1536 well assay plate. DMSO only is transferred to columns 1-3 and 46-48(Control wells), while varying volumes of test compounds are transferred to columns 4-45 to achieve the desired test concentrations. Compounds are transferred from a 2 mM stock to give the stated final concentration. Test compound wells in the assay plate are back-filled with DMSO to equalize final assay concentrations.
2) Immediately following Echo Transfer, 2 ul of assay buffer is added to columns 1 and 2.
3) Add 2 ul of 60 uM C18-Aib(FL) into columns 3-48of a black, Corning (#2725) 1536 well assay plate.
4) Add 2 ul of 4 uM Env2.0/Fe complex into all columns (1-48)
5) Incubate lidded plate for 30 minutes at room temp.
6) Read plate on a BMG PHERAstar at 485/520nm in Fluorescence Intensity mode
i. Positioning delay = 0.0
ii. Flashes/well = 10
Comment
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Compounds were tested one or more times in this assay over one or more ranges.

Range1 0-100 uM

All samples that resolved to an IC50 value were averaged. Compounds with a demonstrated IC50_Mean <= 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
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.
Result Definitions
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Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_Mean_QualifierThis qualifier is to be used with the next TID, IC50_Mean. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
2IC50_Mean*IC50 value determined using sigmoidal dose response equationFloatμM
3IC50_Qualifier_1_Range1This qualifier is to be used with the next TID, IC50_1. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
4IC50_1_Range1IC50 value determined using sigmoidal dose response equationFloatμM
5Std.Err(IC50)_1_Range1Standard Error of IC50 valueFloatμM
6nH_1_Range1Hill coefficient determined using sigmoidal dose response equationFloat
7Excluded_Points_first_point_Range1Flags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
8% inhibition at 100 uM_first_point_Range1 (100μM**)% Inhibition at a given concentrationFloat%
9% inhibition at 50 uM_first_point_Range1 (50μM**)% Inhibition at a given concentrationFloat%
10% inhibition at 25 uM_first_point_Range1 (25μM**)% Inhibition at a given concentrationFloat%
11% inhibition at 12.5 uM_first_point_Range1 (12.5μM**)% Inhibition at a given concentrationFloat%
12% inhibition at 6.25 uM_first_point_Range1 (6.25μM**)% Inhibition at a given concentrationFloat%
13% inhibition at 3.125 uM_first_point_Range1 (3.125μM**)% Inhibition at a given concentrationFloat%
14% inhibition at 1.5625 uM_first_point_Range1 (1.5625μM**)% Inhibition at a given concentrationFloat%
15% inhibition at 0.78125 uM_first_point_Range1 (0.78125μM**)% Inhibition at a given concentrationFloat%
16% inhibition at 0.390625 uM_first_point_Range1 (0.390625μM**)% Inhibition at a given concentrationFloat%
17% inhibition at 0.1953125 uM_first_point_Range1 (0.195312μM**)% Inhibition at a given concentrationFloat%
18% inhibition at 0.09765625 uM_first_point_Range1 (0.0976562μM**)% Inhibition at a given concentrationFloat%
19% inhibition at 0.04882812 uM_first_point_Range1 (0.0488281μM**)% Inhibition at a given concentrationFloat%
20Excluded_Points_second_point_Range1Flags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
21% inhibition at 100 uM_second_point_Range1 (100μM**)% Inhibition at a given concentrationFloat%
22% inhibition at 50 uM_second_point_Range1 (50μM**)% Inhibition at a given concentrationFloat%
23% inhibition at 25 uM_second_point_Range1 (25μM**)% Inhibition at a given concentrationFloat%
24% inhibition at 12.5 uM_second_point_Range1 (12.5μM**)% Inhibition at a given concentrationFloat%
25% inhibition at 6.25 uM_second_point_Range1 (6.25μM**)% Inhibition at a given concentrationFloat%
26% inhibition at 3.125 uM_second_point_Range1 (3.125μM**)% Inhibition at a given concentrationFloat%
27% inhibition at 1.5625 uM_second_point_Range1 (1.5625μM**)% Inhibition at a given concentrationFloat%
28% inhibition at 0.78125 uM_second_point_Range1 (0.78125μM**)% Inhibition at a given concentrationFloat%
29% inhibition at 0.390625 uM_second_point_Range1 (0.390625μM**)% Inhibition at a given concentrationFloat%
30% inhibition at 0.1953125 uM_second_point_Range1 (0.195312μM**)% Inhibition at a given concentrationFloat%
31% inhibition at 0.09765625 uM_second_point_Range1 (0.0976562μM**)% Inhibition at a given concentrationFloat%
32% inhibition at 0.04882812 uM_second_point_Range1 (0.0488281μM**)% Inhibition at a given concentrationFloat%
33Excluded_Points_third_point_Range1Flags to indicate which of the third dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
34% inhibition at 100 uM_third_point_Range1 (100μM**)% Inhibition at a given concentrationFloat%
35% inhibition at 50 uM_third_point_Range1 (50μM**)% Inhibition at a given concentrationFloat%
36% inhibition at 25 uM_third_point_Range1 (25μM**)% Inhibition at a given concentrationFloat%
37% inhibition at 12.5 uM_third_point_Range1 (12.5μM**)% Inhibition at a given concentrationFloat%
38% inhibition at 6.25 uM_third_point_Range1 (6.25μM**)% Inhibition at a given concentrationFloat%
39% inhibition at 3.125 uM_third_point_Range1 (3.125μM**)% Inhibition at a given concentrationFloat%
40% inhibition at 1.5625 uM_third_point_Range1 (1.5625μM**)% Inhibition at a given concentrationFloat%
41% inhibition at 0.78125 uM_third_point_Range1 (0.78125μM**)% Inhibition at a given concentrationFloat%
42% inhibition at 0.390625 uM_third_point_Range1 (0.390625μM**)% Inhibition at a given concentrationFloat%
43% inhibition at 0.1953125 uM_third_point_Range1 (0.195312μM**)% Inhibition at a given concentrationFloat%
44% inhibition at 0.09765625 uM_third_point_Range1 (0.0976562μM**)% Inhibition at a given concentrationFloat%
45% inhibition at 0.04882812 uM_third_point_Range1 (0.0488281μM**)% Inhibition at a given concentrationFloat%
46IC50_Qualifier_2_Range1This qualifier is to be used with the next TID, IC50_2. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
47IC50_2_Range1IC50 value determined using sigmoidal dose response equationFloatμM
48Std.Err(IC50)_2_Range1Standard Error of IC50 valueFloatμM
49nH_2_Range1Hill coefficient determined using sigmoidal dose response equationFloatμM
50Excluded_Points_fourth_point_Range1Flags to indicate which of the fourth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
51% inhibition at 100 uM_fourth_point_Range1 (100μM**)% Inhibition at a given concentrationFloat%
52% inhibition at 50 uM_fourth_point_Range1 (50μM**)% Inhibition at a given concentrationFloat%
53% inhibition at 25 uM_fourth_point_Range1 (25μM**)% Inhibition at a given concentrationFloat%
54% inhibition at 12.5 uM_fourth_point_Range1 (12.5μM**)% Inhibition at a given concentrationFloat%
55% inhibition at 6.25 uM_fourth_point_Range1 (6.25μM**)% Inhibition at a given concentrationFloat%
56% inhibition at 3.125 uM_fourth_point_Range1 (3.125μM**)% Inhibition at a given concentrationFloat%
57% inhibition at 1.5625 uM_fourth_point_Range1 (1.5625μM**)% Inhibition at a given concentrationFloat%
58% inhibition at 0.78125 uM_fourth_point_Range1 (0.78125μM**)% Inhibition at a given concentrationFloat%
59% inhibition at 0.390625 uM_fourth_point_Range1 (0.390625μM**)% Inhibition at a given concentrationFloat%
60% inhibition at 0.1953125 uM_fourth_point_Range1 (0.195312μM**)% Inhibition at a given concentrationFloat%
61% inhibition at 0.09765625 uM_fourth_point_Range1 (0.0976562μM**)% Inhibition at a given concentrationFloat%
62% inhibition at 0.04882812 uM_fourth_point_Range1 (0.0488281μM**)% Inhibition at a given concentrationFloat%
63Excluded_Points_fifth_point_Range1Flags to indicate which of the fifth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
64% inhibition at 100 uM_fifth_point_Range1 (100μM**)% Inhibition at a given concentrationFloat%
65% inhibition at 50 uM_fifth_point_Range1 (50μM**)% Inhibition at a given concentrationFloat%
66% inhibition at 25 uM_fifth_point_Range1 (25μM**)% Inhibition at a given concentrationFloat%
67% inhibition at 12.5 uM_fifth_point_Range1 (12.5μM**)% Inhibition at a given concentrationFloat%
68% inhibition at 6.25 uM_fifth_point_Range1 (6.25μM**)% Inhibition at a given concentrationFloat%
69% inhibition at 3.125 uM_fifth_point_Range1 (3.125μM**)% Inhibition at a given concentrationFloat%
70% inhibition at 1.5625 uM_fifth_point_Range1 (1.5625μM**)% Inhibition at a given concentrationFloat%
71% inhibition at 0.78125 uM_fifth_point_Range1 (0.78125μM**)% Inhibition at a given concentrationFloat%
72% inhibition at 0.390625 uM_fifth_point_Range1 (0.390625μM**)% Inhibition at a given concentrationFloat%
73% inhibition at 0.1953125 uM_fifth_point_Range1 (0.195312μM**)% Inhibition at a given concentrationFloat%
74% inhibition at 0.09765625 uM_fifth_point_Range1 (0.0976562μM**)% Inhibition at a given concentrationFloat%
75% inhibition at 0.04882812 uM_fifth_point_Range1 (0.0488281μM**)% Inhibition at a given concentrationFloat%
76Excluded_Points_sixth_point_Range1Flags to indicate which of the sixth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
77% inhibition at 100 uM_sixth_point_Range1 (100μM**)% Inhibition at a given concentrationFloat%
78% inhibition at 50 uM_sixth_point_Range1 (50μM**)% Inhibition at a given concentrationFloat%
79% inhibition at 25 uM_sixth_point_Range1 (25μM**)% Inhibition at a given concentrationFloat%
80% inhibition at 12.5 uM_sixth_point_Range1 (12.5μM**)% Inhibition at a given concentrationFloat%
81% inhibition at 6.25 uM_sixth_point_Range1 (6.25μM**)% Inhibition at a given concentrationFloat%
82% inhibition at 3.125 uM_sixth_point_Range1 (3.125μM**)% Inhibition at a given concentrationFloat%
83% inhibition at 1.5625 uM_sixth_point_Range1 (1.5625μM**)% Inhibition at a given concentrationFloat%
84% inhibition at 0.78125 uM_sixth_point_Range1 (0.78125μM**)% Inhibition at a given concentrationFloat%
85% inhibition at 0.390625 uM_sixth_point_Range1 (0.390625μM**)% Inhibition at a given concentrationFloat%
86% inhibition at 0.1953125 uM_sixth_point_Range1 (0.195312μM**)% Inhibition at a given concentrationFloat%
87% inhibition at 0.09765625 uM_sixth_point_Range1 (0.0976562μM**)% Inhibition at a given concentrationFloat%
88% inhibition at 0.04882812 uM_sixth_point_Range1 (0.0488281μM**)% Inhibition at a given concentrationFloat%
89IC50_Qualifier_3_Range1This qualifier is to be used with the next TID, IC50_3. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
90IC50_3_Range1IC50 value determined using sigmoidal dose response equationFloatμM
91Std.Err(IC50)_3_Range1Standard Error of IC50 valueFloatμM
92nH_3_Range1Hill coefficient determined using sigmoidal dose response equationFloatμM
93Excluded_Points_seventh_point_Range1Flags to indicate which of the seventh dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
94% inhibition at 100 uM_seventh_point_Range1 (100μM**)% Inhibition at a given concentrationFloat%
95% inhibition at 50 uM_seventh_point_Range1 (50μM**)% Inhibition at a given concentrationFloat%
96% inhibition at 25 uM_seventh_point_Range1 (25μM**)% Inhibition at a given concentrationFloat%
97% inhibition at 12.5 uM_seventh_point_Range1 (12.5μM**)% Inhibition at a given concentrationFloat%
98% inhibition at 6.25 uM_seventh_point_Range1 (6.25μM**)% Inhibition at a given concentrationFloat%
99% inhibition at 3.125 uM_seventh_point_Range1 (3.125μM**)% Inhibition at a given concentrationFloat%
100% inhibition at 1.5625 uM_seventh_point_Range1 (1.5625μM**)% Inhibition at a given concentrationFloat%
101% inhibition at 0.78125 uM_seventh_point_Range1 (0.78125μM**)% Inhibition at a given concentrationFloat%
102% inhibition at 0.390625 uM_seventh_point_Range1 (0.390625μM**)% Inhibition at a given concentrationFloat%
103% inhibition at 0.1953125 uM_seventh_point_Range1 (0.195312μM**)% Inhibition at a given concentrationFloat%
104% inhibition at 0.09765625 uM_seventh_point_Range1 (0.0976562μM**)% Inhibition at a given concentrationFloat%
105% inhibition at 0.04882812 uM_seventh_point_Range1 (0.0488281μM**)% Inhibition at a given concentrationFloat%
106Excluded_Points_eighth_point_Range1Flags to indicate which of the eighth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
107% inhibition at 100 uM_eighth_point_Range1 (100μM**)% Inhibition at a given concentrationFloat%
108% inhibition at 50 uM_eighth_point_Range1 (50μM**)% Inhibition at a given concentrationFloat%
109% inhibition at 25 uM_eighth_point_Range1 (25μM**)% Inhibition at a given concentrationFloat%
110% inhibition at 12.5 uM_eighth_point_Range1 (12.5μM**)% Inhibition at a given concentrationFloat%
111% inhibition at 6.25 uM_eighth_point_Range1 (6.25μM**)% Inhibition at a given concentrationFloat%
112% inhibition at 3.125 uM_eighth_point_Range1 (3.125μM**)% Inhibition at a given concentrationFloat%
113% inhibition at 1.5625 uM_eighth_point_Range1 (1.5625μM**)% Inhibition at a given concentrationFloat%
114% inhibition at 0.78125 uM_eighth_point_Range1 (0.78125μM**)% Inhibition at a given concentrationFloat%
115% inhibition at 0.390625 uM_eighth_point_Range1 (0.390625μM**)% Inhibition at a given concentrationFloat%
116% inhibition at 0.1953125 uM_eighth_point_Range1 (0.195312μM**)% Inhibition at a given concentrationFloat%
117% inhibition at 0.09765625 uM_eighth_point_Range1 (0.0976562μM**)% Inhibition at a given concentrationFloat%
118% inhibition at 0.04882812 uM_eighth_point_Range1 (0.0488281μM**)% Inhibition at a given concentrationFloat%
119Excluded_Points_ninth_point_Range1Flags to indicate which of the ninth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
120% inhibition at 100 uM_ninth_point_Range1 (100μM**)% Inhibition at a given concentrationFloat%
121% inhibition at 50 uM_ninth_point_Range1 (50μM**)% Inhibition at a given concentrationFloat%
122% inhibition at 25 uM_ninth_point_Range1 (25μM**)% Inhibition at a given concentrationFloat%
123% inhibition at 12.5 uM_ninth_point_Range1 (12.5μM**)% Inhibition at a given concentrationFloat%
124% inhibition at 6.25 uM_ninth_point_Range1 (6.25μM**)% Inhibition at a given concentrationFloat%
125% inhibition at 3.125 uM_ninth_point_Range1 (3.125μM**)% Inhibition at a given concentrationFloat%
126% inhibition at 1.5625 uM_ninth_point_Range1 (1.5625μM**)% Inhibition at a given concentrationFloat%
127% inhibition at 0.78125 uM_ninth_point_Range1 (0.78125μM**)% Inhibition at a given concentrationFloat%
128% inhibition at 0.390625 uM_ninth_point_Range1 (0.390625μM**)% Inhibition at a given concentrationFloat%
129% inhibition at 0.1953125 uM_ninth_point_Range1 (0.195312μM**)% Inhibition at a given concentrationFloat%
130% inhibition at 0.09765625 uM_ninth_point_Range1 (0.0976562μM**)% Inhibition at a given concentrationFloat%
131% inhibition at 0.04882812 uM_ninth_point_Range1 (0.0488281μM**)% Inhibition at a given concentrationFloat%
* Activity Concentration. ** Test Concentration.
Additional Information
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Grant Number: 1R21NS059403-01

Data Table (Concise)
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