Microplate-based assay for identifying small molecules that bind a specific intersubunit interface within the assembled HIV-1 capsid

Article abstract of DOI:10.1128/AAC.00646-15

Despite the availability of>30 effective drugs for managing HIV-1 infection, no current therapy is curative, and long-term management is challenging owing to the emergence and spread of drug-resistant mutants. Identification of drugs against novel HIV-1 targets would expand the current treatment options and help to control resistance. The highly conserved HIV-1 capsid protein represents an attractive target because of its multiple roles in replication of the virus. However, the low antiviral potencies of the reported HIV-1 capsid-targeting inhibitors render them unattractive for therapeutic development. To facilitate the identification of more-potent HIV-1 capsid inhibitors, we developed a scintillation proximity assay to screen for small molecules that target a biologically active and specific intersubunit interface in the HIV-1 capsid. The assay, which is based on competitive displacement of a known capsid-binding small-molecule inhibitor, exhibited a signal-to-noise ratio of>9 and a Z factor of>0.8. In a pilot screen of a chemical library containing 2,400 druglike compounds, we obtained a hit rate of 1.8%. This assay has properties that are suitable for screening large compound libraries to identify novel HIV-1 capsid ligands with antiviral activity.

Full text of DOI:10.1128/AAC.00646-15

AAC Accepted Manuscript Posted Online 15 June 2015
Antimicrob. Agents Chemother. doi:10.1128/AAC.00646-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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A Microplate-Based Assay for Identifying Small Molecules that Bind a Specific
Intersubunit Interface Within the Assembled HIV-1 Capsid
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Running Title: Screening Assay for HIV-1 Capsid Inhibitors
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Upul D. Halambage,1 Jason P. Wong,1 Bruce J. Melancon,2 Craig W. Lindsley,2,3 Christopher
Aiken1
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1Department of Pathology, Microbiology and Immunology, Vanderbilt University School of
Medicine, Nashville, Tennessee, 37232
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2Department of Pharmacology, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt
University Medical Center, Nashville, Tennessee, 37232
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3Department of Chemistry, Vanderbilt University, Nashville, Tennessee, 37232
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*Address correspondence to:
Christopher Aiken
Department of Pathology, Microbiology and Immunology
Vanderbilt University School of Medicine
A-5301 Medical Center North
Nashville, TN 37232-2363
Tel: 615-343-7037
Fax: 615-343-7392
Email: chris.aiken@vanderbilt.edu

26 Abstract
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Despite the availability of more than 30 effective drugs for managing HIV-1 infection, no current
therapy is curative and long-term management is challenging owing to the emergence and spread
of drug-resistant mutants. Identification of drugs against novel HIV-1 targets would expand the
current treatment options and help to control resistance. The highly conserved HIV-1 capsid
protein represents an attractive target owing to its multiple roles in replication of the virus.
However, the low antiviral potencies of the reported HIV-1 capsid- targeting inhibitors render
them unattractive for therapeutic development. To facilitate the identification of more potent
HIV-1 capsid inhibitors, we developed a scintillation proximity assay to screen for small
molecules that target a biologically active and specific inter-subunit interface in the HIV-1
capsid. The assay, which is based on competitive displacement of a known capsid-binding small
molecule inhibitor, exhibited a signal-to-noise ratio above 9 and a Z-factor above 0.8. In a pilot
screen of a chemical library containing 2400 drug-like compounds, we obtained a hit rate of
1.8%. This assay has properties that are suitable for screening of large compound libraries to
identify novel HIV-1 capsid ligands with antiviral activity.
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42 Introduction
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HIV/AIDS continues to represent a major global health problem, with about 1.5 million
HIV-related deaths and more than 2 million new HIV infections reported annually. Currently
estimated 35 million people live with HIV (1). However, the number of HIV-related deaths is
declining, owing to the increased availability and improvements of antiretroviral therapy (ART).
The current arsenal of FDA-approved anti-HIV drugs used in ART are based on 28 molecular
entities (2). ART has been fairly successful in containing the HIV/AIDS epidemic, but the side
effects of long-term ART and the emergence of drug-resistant HIV-1 mutants warrants discovery
and development of anti-HIV drugs. Because existing regiments predominantly target the three
viral enzymes and are not curative, the development of drugs against novel targets is an
important goal.
The HIV-1 capsid is a relatively unexploited therapeutic target and has gained attention
owing to improved understanding of its structural and mechanistic roles in the HIV-1 replication
cycle. The capsid, which encases the viral genome and associated proteins, is critical for
efficient reverse transcription, nuclear entry, and possibly integration. In addition, the capsid
appears to cloak the reverse transcribed viral genome, thus promoting innate immune evasion by
limiting access to cytosolic DNA sensors (3). The capsid is composed of a lattice of capsid
protein (CA) hexamers that are stabilized by three major intersubunit interfaces and are
connected through interactions between the carboxyl-terminal domain of CA (4, 5). The conical
shape of the HIV-1 capsid is a result of the asymmetric distribution of CA pentamers in the
lattice, which promote curvature necessary for closure of the ends (6-8).
Several HIV-1 capsid targeting small molecule antiviral compounds have been reported
(9-13); however, their relatively low potencies and/or chemical properties have rendered them
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unsuitable for clinical development. Nonetheless, HIV-1 capsid-targeting inhibitors have been
useful as probes in mechanistic and biochemical studies of HIV-1 capsid function (14, 15). The
small molecule PF-3450074 (PF74) inhibits HIV-1 infection at submicromolar concentrations,
and binds to a pocket in the amino-terminal domain of the capsid protein (CA) at the
intrahexamer interface formed by the carboxyl-terminal domain of an adjacent subunit (10, 16,
17). The PF74 capsid-binding pocket has been identified as an interaction site for several host
proteins that play important roles in the HIV-1 infection cycle (16, 18). This region of CA is
highly conserved among HIV-1 isolates (19), and resistance of HIV-1 to PF74 appears to require
multiple mutations in CA (10, 20). These observations suggest that the genetic barrier to clinical
resistance to inhibitors that bind to the PF74 binding pocket will be high in vivo, which is
attractive from the perspective of therapeutic durability. Although the HIV-1 capsid represents
an attractive drug target, compounds with sufficient antiviral potency for clinical promise have
yet to be identified.
It was recently shown that PF74 binds with higher affinity to the assembled CA hexamer
in vitro than to monomeric CA protein, consistent with its functional target being the intersubunit
interface (16, 17). Based on this observation, we developed a novel target-based assay for
unbiased identification of compounds that interact with the PF74 binding pocket in the HIV-1
capsid. The radiometric assay detects the ability of compounds to competitively inhibit the
binding of PF74 to the assembled CA hexamer in vitro. CA hexamers are immobilized on
scintillation proximity assay (SPA) beads, which emit light upon binding of 3H-PF74. Unlabeled
compounds that bind competitively to the PF74 binding pocket will compete for PF74 binding,
thus reducing the signal. This endpoint assay is mix-and-read, scalable to a 384 well format, and
sufficiently robust for high throughput screening.
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88 Materials and Methods
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Chemicals and reagents. Streptavidin-coated polyvinyltoluene SPA beads were
purchased from Perkin Elmer and suspended in STE buffer (100 mM NaCl, 10 mM Tris-HCl, 1
mM EDTA, pH 7.4) at 25 mg/ml. Detergents were purchased from Thermo Scientific (Pierce
Surfact-Amps Detergent Sampler). PF74, diiodo-PF74, PF74 analog compounds and BI-2 were
synthesized and purified in the Chemical Synthesis Core, Vanderbilt Institute of Chemical
Biology. The structures of these compounds have been previously reported (10, 13). Working
stocks of these chemicals were prepared by dissolution in DMSO to 10 mM concentrations.
3H-PF74 was produced by 3H-I exchange reaction with diiodo-PF74 to a specific radioactivity of
52 Ci/mmol by Moravek Biochemicals and Radiochemicals (La Brea, CA). A compound library
(Spectrum Collection) for pilot screening was obtained from Microsource Discovery, Inc. and
accessed through the Vanderbilt High Throughput Screening Facility, Vanderbilt Institute of
Chemical Biology.
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Preparation of biotinylated HIV-1 CA hexamer. HIV-1 CA hexameric protein (hCA)
was produced by assembly of recombinant CA with cysteine and alanine substitutions to
facilitate the formation of stable hexameric units, as previously reported (21). Biotinylated
HIV-1 CA hexameric protein (bhCA) used in the assay was prepared either by (i) direct
biotinylation of hexameric CA (bhCA-1) or (ii) by co-assembling of CA and biotinylated CA
(bhCA-2). Biotinylation of CA was carried out using Pierce EZ-Link Sulfo-NHS-Biotin (Thermo
Scientific) according to the manufacturer’s recommendation, and the conditions were adjusted to
limit the reaction to link 9 to 12 biotin molecules per hexamer, as quantified with a HABA assay
(Pierce Biotin Quantitation Kit). To produce bhCA-2, a 1:2 molar ratio mixture of bhCA-1 and
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hCA was first disassembled and then co-assembled into the hexamers as follows. First, the
hexamer mixture was dialyzed into 50 mM Tris-HCl buffer (pH 8.0) in the presence of 100 mM
β-mercaptoethanol for 16h to achieve disassembly of the hexamers into individual CA subunits.
Then the buffer was exchanged with 50mM Tris-HCl buffer (pH 8.0) with 1.0 M NaCl for 24h to
promote co-assembly of biotinylated and unbiotinylated CA and crosslinking of the subunits
within hexamers in the lattice. The reaction was then dialyzed into 50 mM Tris-HCl (pH 8.0) for
24h to disassemble the tubes into disulfide crosslink-stabilized hexamers. All the dialysis steps
were performed at 4°C. Final product was flash frozen in liquid nitrogen and stored in aliquots at
-80°C.
Method for the SPA-based PF74 binding competition assay. Various quantities of
SPA beads and detergents were mixed in STE buffer and incubated for 10 minutes. Separately,
various amounts of bhCA and 3H-PF74 were mixed in STE buffer and incubated for 10 minutes.
The detergent reduced the nonspecific binding of 3H-PF74 to the beads, and it proved necessary
to mix beads with the detergent prior to adding bhCA to limit nonspecific binding. Following the
incubation, the two mixtures were combined and dispensed into 96-well or 384-well white
opaque scintillation counting plates (Perkin Elmer OptiPlate). The total volume of the assay mix
was 100 µL and 25 µL per well for 96-well and 384-well plates, respectively. The assay plates
were incubated at room temperature for 16 h, and scintillation was quantified on a Perkin Elmer
Topcount NXT instrument with at least 30 s counting time. To establish the background signal of
the assay, the assay mixture was prepared without bhCA. Testing of the competing compounds
was performed by dispensing the appropriate amounts of the compounds into the assay plate
wells prior to the addition of the assay mixture.
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The assay was optimized to achieve a satisfactory signal to background ratio (S/B) with a
minimum reagent cost by adjusting the amounts of the beads, the detergents, the receptor protein,
and 3H-PF74 in the assay mixture. The optimal assay mixture contained 1.25 mg/ml SPA beads,
0.2% (w/vol) Brij 58, 0.3 µM bCA, and 0.1 µM 3H-PF74. The competence of the assay in 384-
well plates was evaluated by preparing a checkerboard plate, in which the assay mixture with
bhCA (positive samples) and without bhCA (for background binding), are distributed in
alternating wells.
Testing of PF74 and analogs with the assay. Unlabeled PF74 and its analog compounds
were dissolved in DMSO and dispensed into 96-well plates to yield the desired concentrations
(from 0 to 10 µM) in the final assay volume. The DMSO concentrations of all wells were kept at
a constant value below 2%. The assay mixture was prepared and dispensed into the wells.
Following incubation for 16 h incubation at room temperature, the plate was read.
Testing of the assay for high-throughput screening. Stock solutions of the compounds
in the Spectrum Collection (2400 compounds) were available at 10 mM concentrations in
DMSO, and 25 nL of the each compound and appropriate controls were dispensed into empty
384-well plates using a Labcyte Echo dispenser. The assay mixture containing SPA beads,
protein, and [3H]-PF74 was prepared and 25 μl dispensed into each well with a Multidrop Combi
(Thermo Scientific) reagent dispenser. The assay contents were mixed by shaking the plates on
an orbital shaker for 5 minutes, and incubated at room temperature for 16 h, and scintillation was
quantified with the TopCount instrument.
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157 Results and Discussion
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Design of the assay. To identify novel compounds that interact with the PF74 binding
site in the HIV-1 capsid, we adopted an approach based on the scintillation proximity assay
(SPA) (Figure 1). Disulfide-stabilized HIV-1 CA hexamers were prepared by assembly of
recombinant CA protein containing engineered Cys substitutions at positions 14 and 45 in the
protein sequence. The CA hexamers were biotinylated in vitro, then immobilized onto
streptavidin-coated SPA beads. Upon addition of [3H]-PF74, binding of the compound to the
hCA on the surface of the beads results in excitation of the scintillant within the beads by emitted
β particles, resulting in emission of light. Emissions from unbound ligand molecules are
quenched by the solution, owing to the low energy of the tritium emission. Thus, SPA allows
detection of bound radioligand in the presence of unbound ligand, thus obviating the need for a
washing step. In principle, the assay may be used for screening of compound libraries for
compounds that compete with PF74 for binding to hCA.
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Optimization of the biotinylated CA hexamer. We employed PVT beads coated with
streptavidin thus permitting the facile immobilization of the biotinylated CA hexamer. In initial
studies in which we employed equlibrium dialysis to quantify PF74 affinity for CA hexamer, we
observed that biotinylation of hCA reduced its affinity for PF74, and the reduction of the affinity
correlated with the amount of biotin incorporated into the protein (data not shown). Therefore, to
ensure adequate biotinylation for bead capture, while minimizing the loss of binding affinity, the
biotinylation was controlled by changing the reaction time and biotin concentration in order to
achieve nine to twelve biotin molecules per hexamer.
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We optimized the assay to achieve a high signal-to-background ratio (S/B) and low
reagent consumption. Although the assay was ultimately intended for 384-well plates, the initial
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steps of the assay development were performed in 96-well plates because of the convenience of
handling relatively higher reagent volumes compared to 384-well plates. Each assay condition
was tested in duplicate and processed in parallel in the same plate.
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The first objective of the assay development was to reduce of the background due to
binding of the relatively hydrophobic radioligand 3H-PF74 to hydrophobic PVT beads. The high
background resulted in S/B < 3 in all the assay conditions tested. To reduce the background
binding, we tested several detergents, bovine serum albumin (BSA), and polyethylene glycols to
the assay mixture. Among them, only the detergents Brij 35, Brij 58, Tween 80, and Igepal CA-
630 showed a substantial reduction in the background signal resulting a higher S/B ratio (Figure
2A). Brij 58 at 0.2% w/vol concentration was chosen for further development of the assay. In
the preparation of the assay mixture, we found it was important to mix the beads with the
detergent prior to the addition of bhCA in order to achieve a low background signal. Therefore,
the order of addition was: beads, detergent, bhCA, and [3H]-PF74.
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We next optimized the 3H-PF74 concentration. We observed that concentrations between
0.025 to 0.2 µM resulted the highest S/B ratios (Figure 2B) for both bhCA-1 and bhCA-2. To
ensure a sufficient level of scintillation from an assay well while conserving the radioligand, 0.1
µM 3H-PF74 was chosen for the assay. Between the two bhCA proteins, bhCA-2 generated a
higher S/B ratio. This might be due to the reduction of the affinity of bhCA-1 for PF74 compared
to bhCA-2 due to the differences in the amount of biotinylation (all subunits of bhCA-1 hexamer
are biotinylated, whereas the assembly of bhCA-2 was controlled to have on average two
biotinylated subunits per hexamer). Because of its greater S/B, bhCA-2 was selected for the
further development of the assay. Concentrations of bhCA-2 and the beads were optimized in
tandem, and it revealed that the beads concentrations of 0.5 mg/mL and bhCA-2 concentration of
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0.05 µM resulted the highest S/B (Figure 2, panels C and D). Thus, the final optimized assay
mixture contained 0.5 mg/mL streptavidin-coated PVT SPA beads, 0.2% W/V Brij 58, 0.3 µM
bhCA-2 (quantified as CA subunit), and 0.1 µM 3H-PF74. Upon the mixing of the assay
ingredients, the assay signal reached equilibrium within onr hour and remained unchanged for 24
hours (Figure 2E).
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Assay validation. To test whether the SPA-based assay detects specific binding to the
PF74 binding pocket in CA, we employed hCA protein derived from the PF74-resistant 5Mut
virus. 5Mut hexamers, which contain 5 substitutions in CA, exhibit low PF74 affinity relative to
WT CA hexamers (20, 22). Use of 5Mut-bhCA-1 in the assay resulted in a signal close to the
background (Figure 2E), confirming the specificity of the assay.
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To determine whether the assay is capable of detecting differences in compound binding
affinity, we performed a competition assay with various concentrations of unlabeled PF74 and
BI-2 as competitors. BI-2 is another capsid-targeting HIV-1 inhibitor that interacts with the same
PF74-binding pocket, yet with lower affinity (16). At 10 µM concentration, unlabeled PF74
reduced the signal to nearly background level (Figure 3A). By contrast, BI-2 had only weak
inhibitory effect (~20%) at concentrations of up to 20 μM. These results confirm that the SPA
based competition assay is capable of detecting differences in relative binding affinity between
two compounds that interact with the PF74 binding site.
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To determine whether the assay can be miniaturized to 384 well plates for high-
throughput screening (HTS), we performed a checkerboard assay in a 384 well plate. The assay
yielded a S/B ratio of 8.5 and a Z-factor of 0.81 (Figure 3B). Assays with a Z-factor above 0.5
are considered as excellent for HTS (23, 24). Therefore, the PF74 competition assay has
statistical properties that are desirable for HTS.
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To further validate the assay as a screen for compounds with antiviral activity, we tested
a number of PF74 derivative compounds that were designed to have various minor modifications
to the PF74 backbone structure (Table S1). The synthesized compounds were dissolved in
DMSO and, as a result, it was unavoidable to have some level of DMSO in the assay mixture.
Therefore a 1% DMSO concentration was maintained in all wells. In additional studies, we
observed that DMSO concentrations up to 3% vol/vol did not significantly affect the assay signal
(data not shown). At a 10 μM concentration of each compound, the assay showed a range of
signal reduction. Evaluation of antiviral potency of the PF74 derivatives against wild type
HIV-1 revealed that many were less potent inhibitors than PF74 (Figure 4 and Table S1). None
of the compounds showed an IC50 value higher than that of PF74, and several of them did not
exhibit any detectable anti-HIV-1 activity. Interestingly, the IC50 values of the PF74 derivatives
showed a significant correlation with their extent of inhibition observed in the SPA (Spearman
one-tailed r = -0.93, p < 0.0001) (Figure 4). The results from compound BI-2, which is
structurally different from PF74, also fell on the curve. Collectively, these results indicate that
results from the SPA-based competition assay are likely to predict the antiviral potency of
compounds identified during HTS.
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Testing of the assay in HTS. To determine the utility of the assay in HTS, we performed
a small-scale pilot screen of the Spectrum Collection compound library at 10 µM compound
concentration. The stock compounds were available at 10 mM concentration in DMSO, thereby
resulting 0.1% v/v DMSO concentration in the final assay mixture. Results of the screen showed
that 43 compounds reduced the PF74 binding signal to a value below the threshold set at 3σ
below from the mean signal value of the all compounds analyzed (Figure 5). This represented a
1.8% hit rate, a value that seems appropriate for HTS.
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The screening assay we have developed is a biochemical target-based assay based on
competition of binding of a known antiviral compound to the HIV-1 CA hexamer. The assay has
properties desirable for HTS, including a simple mix-and-read endpoint format, a stable
endpoint, relatively low reagent cost, and excellent statistical properties. It is radiometric, which
may be undesirable to some laboratories owing to regulatory requirements for handling
radioisotopes. The radiometric design of the assay also renders it susceptible to false positives
owing to the potential for quenching by colored compounds. This limitation may be overcome by
using an instrument with an advanced quench correction method, (e.g. Perkin-Elmer Microbeta
Trilux), or by screening with an imaging-based instrument using SPA beads that are less
susceptible to color quenching. Additionally, nonradiometric direct binding approaches such as
surface plasmon resonance and biolayer interferometry may be used as orthogonal assays to test
hits.
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For convenience, our competition-binding assay employs a CA protein hexamer that is
stabilized by an engineered disulfide bond to ensure the preservation of the PF74-binding
interface within the hexamer. We and others have shown that PF74 binds specifically and with
higher affinity to the disulfide-stabilized CA hexamer than the isolated CA subunit (16, 17).
While the crosslinking ensures efficient and specific PF74 binding, the substitutions could alter
some structural or biological properties of the viral capsid. Specifically, the A14C and E45C
substitutions could affect the intrinsic stability of the capsid as well as its interactions of the viral
capsid with host factors. Additionally, biotinylation of the CA hexamer may reduce its affinity
for PF74. Notwithstanding these issues, the SPA-based assay we have developed should be
useful for screening large compound libraries for identifying novel chemical scaffolds that
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interact with the specific binding site for PF74 in the HIV-1 capsid, thus expanding the chemical
space for development of capsid-targeting antiviral drugs.
273 Acknowledgments
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We thank David Weaver, Paige Vinson, and Rey Redha of the Vanderbilt High Throughput
Screening Core Facility for advice and technical support. The Vanderbilt High Throughput
Screening Core Facility is an institutionally supported core. This work was supported by grants
R01 AI089401, R01 AI114339, and 1U54MH084659-01 from the National Institutes of Health.
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17

372 Figure Legends
373
374
375
376
377
378
379
380
381
Figure 1. Diagram of the assay strategy. Streptavidin-coated SPA beads are first treated with a
detergent to prevent non-specific binding of the radioligand to the beads. Next, biotinylated hCA
and 3H-PF74 are added to the mixture. Because of the limited penetration of the low energy 3H β
particles in aqueous media (<1.5µm), β particles from unbound 3H-PF74 are unlikely to excite
the scintillator in the SPA beads. Binding between hCA and 3H-PF74 retains a large fraction of
3H-PF74 molecules at the bead surface, thereby promoting the interaction between the β particles
emitted from 3H-PF74 and the scintillators in the SPA beads, resulting in light emission (assay
signal). The assay signal drops in the presence of a molecule that competes with 3H-PF74 for
binding to hCA.
382
383
384
385
386
387
388
389
390
391
392
Figure 2. Optimization of the SPA competition assay. (A) Detergent optimization of S/B ratio.
Shown are the results from Brij 58 titration in reactions containing 1.25 mg/mL beads, 0.125 µM
bhCA1, 0.125 µM 3H-PF74 (Assay signal) or the equivalent lacking bhCA (Background). (B)
Optimization of 3H-PF74 concentration. Two biotinylated CA hexamer protein preparations
(bhCA1 and bhCA2) were tested at 0.125 µM concentration in reactions containing 1.25 mg/ml
beads and 0.2% w/v Brij 58. (C) Optimization of the concentrations of the SPA beads. (D)
Optimization of bhCA2 concentration. Reactions in C and D contained 0.2% w/v Brij 58 and 0.1
µM 3H-PF74. (E) Specificity of the assay signal and stability of the assay with time. Background
was established with assay mixture lacking bhCA (No bhCA). 5Mut-bhCA1, which exhibits low
affinity for PF74, was prepared by direct biotinylation of the 5Mut CA hexamer.
393
18

394
395
396
397
398
399
Figure 3. Validation of the assay with unlabeled competitor compounds and determination of Z-
factor. Assay conditions included 0.5 mg/ml beads, 0.2 % (wt/vol) Brij 58, 0.3 µM bhCA2, and
0.1 µM 3H-PF74. (A) Test of the response of the assay to unlabeled PF74 and BI-2 at various
concentrations in 96-well plates. Reactions contained 1% v/v DMSO. (B) Results of the
checkerboard evaluation of the assay in 384-well plates. Assay mixtures (25 µl) were manually
pipetted into the the wells.
400
401
402
403
Figure 4. The correlation between the assay response (signal reduction at 10 μM concentration)
and IC50 of the PF74 derivatives relative to IC50 of PF74. The two parameters were
significantly correlated (Spearman r = -0.93, p < 0.0001).
404
405
406
407
408
409
Figure 5. Results of the screening of the Spectrum Compound Collection (2400 compounds).
Reactions (25 μl) were performed in 384 well plates and included 0.5 mg/ml beads, 0.2%
(wt/vol) Brij 58, 0.3 µM bhCA2, and 0.1 µM 3H-PF74. The screening scored 43 hits that
produced an assay signal below three standard deviations from the average signal from the all the
screened compounds.
19

Biotin
Streptavidin
SPA
bead
Detergent
molecules
Biotinylated
CA hexamer
[3H]-PF74

A
C
B
12
60000
50000
40000
30000
20000
10000
0
Assay signal
Background
S/B =3.5
10
8
S/B = 7.0
6
S/B =8.7
S/B =10.0
4
S/B =9.7
bhCA-1
bhCA-2
2
0
0.3
0.1
0.2
0.4
0.5
0.00
0.05
0.10
0.20
0.30
3H-PF74 (μM)
Brij58 concentration (% wt/vol)
D
15000
12500
10000
7500
5000
2500
0
15000
12500
10000
7500
5000
2500
0
S/B= 15.7
S/B= 13.5
S/B= 1.4
Assay signal
Background
S/B= 11.8
S/B= 1.9
S/B= 2.2
S/B= 9.1
S/B= 3.5
S/B= 6.2
S/B= 6.2
S/B= 2.9
S/B= 1
0.5
1
2
3
5
0.00 0.05 0.1 0.2 0.3 0.5 1.0
SPA beads (mg/mL)
bhCA-2 (µM)
bhCA-2
bhCA-1
E
5Mut-bhCA-1
No bhCA
10
8
6
4
2
0
0
5
10
15
20
25
Time (h)

A
B
12500
10000
7500
PF74
BI-2
Background
4000
3000
2000
1000
0
No competition
Background
Z-factor = 0.81
5000
2500
0
0
100
200
Well Number
300
400
0
5
10
15
20
Concentration (µM)

100
80
60
40
20
0
BI-2
-20
0
10
20
30
40
IC50/ IC50PF74

7000
6000
5000
4000
3000
2000
Sample
average
3σ
1000
0
Background
average
0
500
1000
1500
2000
2500
Compound number