The evaluation of statins as potential inhibitors of the LEDGF/p75-HIV-1 integrase interaction

Article abstract of DOI:10.1111/cbdd.12384

Lovastatin was identified through virtual screening as a potential inhibitor of the LEDGF/p75-HIV-1 integrase interaction. In an AlphaScreen assay, lovastatin inhibited the purified recombinant protein-protein interaction (IC₅₀ = 1.97 ± 0.45 μM) more effectively than seven other tested statins. None of the eight statins, however, yielded antiviral activity in vitro, while only pravastatin lactone yielded detectable inhibition of HIV-1 integrase strand transfer activity (31.65% at 100 μM). A correlation between lipophilicity and increased cellular toxicity of the statins was observed.

Full text of DOI:10.1111/cbdd.12384

Received Date : 16-Dec-2013
Revised Date : 03-May-2014
Accepted Date : 18-Jun-2014
Article type : Research Letter
The evaluation of statins as potential inhibitors of the LEDGF/p75 – HIV-1 integrase
interaction
Running title: Inhibition of the LEDGF/p75–HIV-1 IN interaction by statins
Angela T. Harrison1,2, Frederik H. Kriel1, Maria A. Papathanasopoulos2, Salerwe Mosebi1,
Shaakira Abrahams1,2, Raymond Hewer1,*
1 Biomed, Advanced Materials Division, Mintek, Private bag X3015, Randburg, 2125,
Johannesburg, South Africa.
2 HIV Pathogenesis Research Laboratory, Department of Molecular Medicine and
Haematology, University of Witwatersrand Medical School, 7 York Road, Parktown,
Johannesburg, 2193, South Africa.
*Corresponding author: Raymond Hewer, raymondh@mintek.co.za
Keywords: LEDGF/p75, HIV-1 integrase, lovastatin, statins, AlphaScreen
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
an 'Accepted Article', doi: 10.1111/cbdd.12384
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Abstract
Lovastatin was identified through virtual screening as a potential inhibitor of the
LEDGF/p75-HIV-1 integrase interaction. In an AlphaScreen assay, lovastatin inhibited the
purified recombinant protein-protein interaction (IC50 = 1.97 0.45 µM) more effectively
than seven other tested statins. None of the eight statins, however, yielded antiviral activity in
vitro while only pravastatin lactone yielded detectable inhibition of HIV-1 integrase strand
transfer activity (31.65% at 100 µM). A correlation between lipophilicity and increased
cellular toxicity of the statins was observed.
The virally-encoded enzyme, HIV-1 integrase (IN), catalyses the insertion of viral DNA into
the host chromation through a multi-step process that involves 3’-end processing (3-P) and
strand transfer (ST) activity. HIV-1 IN is fundamental to the life cycle of the virus and has
been successfully exploited as a target for therapeutic intervention. The IN ST inhibitors
(INSTIs) Raltegravir (RAL, Merck and Co, USA), Elvitegravir (EVG, Gilead Sciences,
USA) and Dolutegravir (DOL, GlaxoSmithKline, UK) have all been approved by the USA
Food and Drug Administration (FDA) and have demonstrated unequivocal clinical relevance.
The integration process is facilitated by a number of host and viral proteins sequestered to
HIV-1 IN to form the pre-integration complex (PIC). The interactions between proteins that
comprise the PIC represent attractive targets for therapeutic intervention, especially since
compounds that engage IN at sites distinct from the catalytic active site should lack cross-
resistance to INSTIs. Recent studies have demonstrated the potential of small molecule
modulators of the interaction between HIV-1 IN and the human lens epithelium-derived
growth factor/p75 (LEDGF/p75) (1-3). These targeted small molecules, categorised as
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allosteric IN inhibitors (ALLINIs) and exemplified by LEDGINs (for LEDGF/p75-IN
inhibitor) and NCINIs (for non-catalytic IN inhibitor), bind to the LEDGF/p75 binding site
formed through the IN catalytic core domain (CCD) dimer interface (reviewed in 4). In
addition to disruption of the LEDGF/p75- HIV-1 IN interaction, these inhibitors function
independently of LEDGF/p75 to induce multimerization and impair the catalytic activities of
HIV-1 IN. Interestingly, ALLINIs exert their full inhibitory activity in late phase of HIV-1
replication impeding particle core maturation and producing non-infectious progeny virus
(reviewed in 4). Finally, ALLINI’s have been shown to reduce HIV-1 replication in vitro at
nanomolar levels and present a distinct resistance profile to known INSTIs.
Owing to their relevance as a promising class of HIV-1 therapeutics, we sought novel
inhibitors of the LEDGF/p75–HIV-1 IN interaction. This brief study serves to describe the
identification of a known statin drug as a potential inhibitor of the protein-protein interaction
through virtual screening and the biological evaluation of this, and related, statins.
Methods and Materials
Molecular Modelling
Analysis of the protein-protein interaction between the CCD of HIV-1 IN with the IN-
binding domain (IBD) of LEDGF/p75 was conducted using Discovery StudioTM 3.1
(Accelrys, USA). As a preliminary to docking, the LEDGF/p75- HIV-1 IN complex (PDB
code 2B4J) was prepared utilizing a CHARMM based force field function contained in
Macromolecule’s Protein Prepare function to build missing loops, add hydrogens, assign
bond orders, and optimize bond lengths, bond angles, torsion angles, and non-bonded
interactions. The LEDGF/p75 protein was removed and the LEDGF/p75 binding site was
defined on the IN dimer using the Receptor-Ligand Interaction function. Small molecules for
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docking were prepared utilizing a CHARMM based force field function contained in Small
Molecules’ Prepare and Filter Ligands function to add hydrogens, assign bond orders, and
optimize bond lengths, bond angles, torsion, change ionization and generate isomers and
tautomers of the ligands. Compounds described in literature as disrupting the LEDGF/p75 –
HIV-1 IN interaction with varying potency were utilised as training molecules (1-5). Ligands
were docked into the predefined LEDGF/p75 binding site utilizing Libdock and CDOCKER
docking algorithms and scoring functions (LigScore1, LigScore2, PLP1, PLP2, Jain, PMF,
PMF04) contained in the Receptor-Ligand Interaction module were used to evaluate
goodness of fit of the docking runs. The top 1000 ligands from both the Libdock docking
function and the Jain scoring function were subjected to the CDOCKER docking function.
The top 100 results as defined by CDOCKER binding energy were analysed for compounds
successfully docked as well as number of times that isomers and tautomers of these specific
compounds appeared in the top 100 results.
Preparation of test compounds
Mevastatin was purchased in purified form (Santa Cruz Biotechnology, USA) and fluvastatin
(Lescol, Novartis Pharmaceuticals Inc., Switzerland) was cherry-picked from a compound
library (NIH Clinical Compound, NCC, Evotec, USA). Lovastatin (Mevacor, Merck and Co,
USA), simvastatin (Zocor, Merck & Co, Inc., USA), atorvastatin (Lipitor, Pfizer, USA) and
pravastatin (Pravachol, Bristol-Myers Squibb, USA) were prepared from whole tablets using
methods previously described (7-10). Briefly, each tablet was crushed separately until finely
ground and dissolved in distilled water (dH2O). The mixture was added to an extraction
vessel and methanol was passed through repeatedly with slight agitation. The combined
methanol solutions were filtered through a 0.22 µM filter following the addition of
ammonium sulphate to eliminate water residue and solvent was removed through
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evaporation. Lovastatin lactone was hydrolysed to the β-hydroxy acid form from purified
lovastatin as previously described (10). Briefly, purified lovastatin was dissolved in dH20 and
0.1 M NaOH and allowed to stir overnight; after which the solution was adjusted to pH 7.5. A
mixture of dH20, acetonitrile (ACN) and ethyl acetate (1:1:1) was used to precipitate the
lovastatin β-hydroxy acid product which was then filtered through a 22µM filter and dried.
Pravastatin acid was condensed to the lactone form by suspending purified pravastatin acid in
ethyl acetate, adding trifluoroacetic acid (TFA) and stirring overnight as per methods
previously described (9,10). The reaction was then dried and a mixture of dH20, ACN and
ethyl acetate (1:1:1) was used to precipitate the product which was then filtered through a
0.22 µM filter and dried. Each extracted product was analysed by nuclear magnetic resonance
(NMR, 400 MHz Bruker Avance Spectrometer at 298K equipped with a BBI 5 mm probe)
and mass spectroscopy (Dionex Ultimate 3000 Rapid Separation LC system equipped with a
C-18 pre-coated column and coupled to a microTOF QII Bruker mass spectrometer fitted
with an electrospray source) to confirm structure and purity. Purified compounds (> 95%
purity) were dissolved in dimethyl sulphate (DMSO) and stored at -20°C until used.
AlphaScreen Assay
The AlphaScreen assay was performed as previously described (1). Briefly, 300nM biotin-
labelled recombinant HIV-1 subtype B IN was incubated with CX05168 (Haoyan
Chemexpress Co, LTD, China) or test compounds (final concentrations of 100 µM for single-
dose experiments or 8 final concentrations ranging from 100 – 0.78 µM for dose-response
experiments) for 30 minutes at 26°C while shaking. Thereafter, 100nM HIS-tagged
LEDGF/p75 was added and the mixture was incubated for 60 minutes in assay buffer
(150mM NaCl, 25mM Tris, 2mM MgCl2, 1mM DTT, 0.01% Tween 20 and 0.02 % BSA, pH
7.4) at 26°C with gentle shaking. Streptavidin donor beads (Perkin Elmer, USA) and nickel
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chelate acceptor beads (Perkin Elmer, USA) at final concentrations of 10µg/ml each were
added to the mixture which was then incubated for 60 minutes at 30°C in the dark with gentle
shaking. The plate was read at 520 - 620nm using an EnSpire® Multimode Plate Reader
(Perkin Elmer, USA). Percentage inhibition was determined for single-dose experiments
while IC50 values were determined as the concentration required to reduce protein-protein
interaction by 50% and calculated using OriginPro® 8.0 software (Origin Lab Corporation,
USA). All inhibition values are the average of triplicate experiments.
Cytotoxicity assay
The following reagent was obtained through the NIH AIDS Reagent Program, Division of
AIDS, NIAID, NIH: MT-4 from Dr. Douglas Richman (catalogue number 120). Peripheral
blood mononuclear cells (PBMCs) were isolated from pooled buffy coats (South African
National Blood Service, SANBS, RSA) by means of the Ficol-Hypaque density gradient
separation method. The cytotoxicity assay was performed as per standard method and as
described previously (12). Briefly; MT-4 cells or 3 day PHA-stimulated PBMCs were plated
in 96-well microtiter plates at 2x105 cells/ml and allowed to stabilize for 2 hours at 37 ºC and
5% CO2. Thereafter, test compounds were added to the plate through serial dilution (two-
fold) to allow for 8 final compound concentrations ranging from 200 - 1.56 µM in a total
volume of 200 µl/well. The cells and compounds were then incubated for 96 hours at 37 ºC
and 5% CO2. To each well, 20 µl CellTiter 96® AQueous One Solution (Promega, USA) was
added; the plates were incubated for 4 hours and absorbance was read at 490 nm on a
multiplate reader (xMarkTM, Bio-Rad, USA). CC50 values were determined as the
concentration of the test compound required to reduce the cell viability by 50% and were
calculated using OriginPro® 8.0 software (Origin Lab Corporation, USA). The values
obtained were averages of at least three separate experiments.
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Antiviral activity
To determine antiviral activity, 50 µl of HIV-1NL4-3 virus was added to 2 x105 MT-4 cells/ml
at a multiplicity of infection of 0.1 and the mixture was spinoculated at 3000 xg for 90
minutes. After washing off unbound virus, cells were plated in 96 well microtiter plates at
100 µl/ well and allowed to stabilise for 1 hour at 37 ºC and 5% CO2. Thereafter, test
compounds were added to the plate through serial dilution (two fold) to allow for 8 final
compound concentrations ranging from 200 - 1.56 µM in a total volume of 200 µl/well. The
cells and compounds were then incubated for 96 hours at 37 ºC and 5% CO2. Cell-free
supernatants were collected from each well and p24 concentration was determined using the
Vironostika HIV-1 p24 Antigen ELISA (bioMérieux, France) as per manufacturer’s
instructions. EC50 values were determined as the concentration of the test compound required
to reduce p24 concentration by 50% and were calculated using OriginPro® 8.0 software
(Origin Lab Corporation, USA). The values obtained were averages of at least three separate
experiments. Selectivity index (SI) values were calculated as the ratio of CC50/EC50.
Strand transfer assay
The assay for the detection of HIV-1 IN ST inhibition was adapted from previously described
methods (13). Briefly, 0.15 µM double-stranded biotinylated donor DNA (5’-biotin-
GTGTGGAAAATCTCTAGCA-3’ and 5’-ACTGCTAGAGATTTTCCACAC-3’) was added
to the wells of streptavidin-coated 96-well microtiter plates (R&D Systems, USA). Following
incubation at room temperature for 60 minutes and a stringent wash step, 1 µM purified
recombinant HIV-1 subtype B IN (in the presence of MgCl2 and MnCl2) was assembled onto
the pre-processed donor DNA through incubation for 30 minutes at 22 ºC. Following a wash
step, the test compounds were titrated into individual wells either at a final concentration of
10 µM (for single-dose inhibition assays) or at a minimum of 8 different concentrations
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ranging from 50 – 0.39 µM (for dose-response inhibition assays). The microtiter plates were
incubated for 30 minutes at 37 ºC, washed and the ST reaction was initiated through the
addition
of
0.25
µM
double-stranded
FITC-labelled
target
DNA
(5’-
TGACCAAGGGCTAATTCACT-FITC-3’ and 5’-AGTGAATTAGCCCTTGGTCA-FITC-
3’) in Hepes buffer containing MgCl2 and MnCl2. After an incubation period of 60 minutes at
37 ºC, the plates were washed as before and an alkaline phosphatase (AP) - conjugated anti-
FITC secondary antibody (Sigma, USA) was added. Finally, the plates were washed and
chromogenic substrate (BluePhos, KPL, USA) was added to allow for photometric
measurement at 620nm using a microplate reader (xMarkTM, Bio-Rad, USA). Percentage
inhibition was calculated utilising the formula: % inhibition = (1 - (A620 Compound – A620
No IN control) / (A620 No inhibitor control – A620 No IN control))*100. IC50 values were
determined as the concentration required to reduce activity of HIV-1 IN by 50% and were
calculated using OriginPro® 8.0 software (Origin Lab Corporation, USA). All inhibition
values are the average of triplicate experiments.
Results and Discussion
Identification of lovastatin
Screening of virtual compound libraries through the LEDGF/p75-binding site on HIV-1 IN
identified several molecules of interest of which the top ten compounds, as defined by
CDOCKER binding energies, were selected for further investigation. These ten compounds
identified in silico were biologically evaluated in a previously described AlphaScreen assay
which had been validated with CX05168. Five of the ten compounds were observed to inhibit
the interaction between LEDGF/p75 and the HIV-1 IN complex at greater than 50% at a
single-dose concentration of 100 µM. These compounds were subjected to a dose-response
evaluation and the most potent molecule yielded an IC50 within the low micromolar range
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(IC50 = 1.97 0.45 µM, Table 1). Cross-referencing of this inhibitor, which appeared once in
top 100 results and produced a CDOCKER binding energy of -36.821 using the Jain scoring
function, identified the compound as the HMG-Coenzyme A reductase inhibitor and type 1
statin; lovastatin. Several previously published studies have examined the anti-HIV effect of
different statins in vitro and in vivo (14,15). For lovastatin, in particular, anti-HIV activity has
been attributed to pleiotropic actions, including the depletion of lipid rafts (16), the down
regulation of Rho activity (17), the suppression of LFA-1/ICAM-1 interaction (18) and the
inhibition of CCR5 coreceptor and RANTES expression (19) while other studies have
conversely reported no or negligible anti-HIV activity (20,21). Interestingly, Hu and co-
workers identified the type 2 statin, atorvastatin, as an inhibitor of the LEDGF/p75 – HIV-1
IN interaction via molecular docking and then determined an IC50 of 8.90 µM (22). In the
same study, they evaluated fluvastatin but found this type 2 statin to be lacking notable
inhibitory activity. To the best of our knowledge, no previous study has identified or
investigated lovastatin as a potential modulator of the LEDGF/p75 – HIV-1 IN interaction.
Preparation and biological evaluation of statins
For further evaluation of type 1 and type 2 statins, as well as of the hydroxy acid and lactone
forms we purified lovastatin, pravastatin, atorvastatin and simvastatin from whole tablets.
Mevastatin was purchased in purified form and fluvastatin was cherry-picked from a
compound library. Lovastatin was hydrolysed to its β-hydroxy acid form with NaOH while
pravastatin was condensed into its lactone form with TFA. All structures were validated
through nuclear magnetic resonance and mass spectroscopy and were shown to be > 95%
pure (results not shown).
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We then evaluated the activity of these additional statins in the AlphaScreen assay, with the
findings reported in Table 1. Briefly, for the type 1 statins, deviations from the lovastatin
structure served to reduce inhibitory activity. Pravastatin (lactone form), which differs from
lovastatin in that the six-alphamethyl group on the hexahydronaphthalene ring is replaced
with a hydroxy group, inhibited the interaction by 25.77% at a single-dose concentration of
100 µM. Simvastatin, which retains the alphamethyl group on the naphthalene ring but has an
additional methyl on the side chain, yielded 28.78% inhibition in the single-dose test.
Mevastatin, which differs from lovastatin only by the absence of the six-alphamethyl group,
inhibited the interaction by 13.25%. Variation from the lactone form to the hydroxy acid form
of lovastatin reduced the inhibition observed at 100 µM to 42.48%. Similarly, pravastatin in
its administered hydroxy acid form yielded a minimal 8.55% inhibition. From this
preliminary comparison of inhibition and statin structure it appears that a closed lactone ring
in addition to a methyl group is requisite for inhibitory activity, while minor alterations to the
side chain adversely affect inhibitory activity of type 1 statins within the described assay. For
the type 2 statins; atorvastatin inhibited the interaction by 54.26% in the 100 µM single-dose
evaluation and produced an IC50 of 10.22 0.80 µM. In contrast, fluvastatin inhibited the
interaction by 4.87% at 100 µM. The results obtained for the type 2 statins closely correlated
those obtained by Hu and co-workers also using an AlphaScreen assay (22).
In addition to blocking the LEDGF/p75- HIV-1 IN interaction, detailed mechanistic studies
have revealed that some LEDGINs also indirectly inhibit the strand transfer function of HIV-
1 IN (2,3, reviewed in 4). In our hands, the two pravastatin forms were most effective with
pravastatin lactone inhibiting activity at similar levels to CX05168 within a HIV-1 IN ST
ELISA-based assay. None of the other statins tested produced appreciable ST inhibition
(Table 1).
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We found lipophilicity of the statins to increase in vitro cellular toxicity, a phenomenon that
has been previously observed (23). Lovastatin yielded CC50 values of 5.31 µM and 6.50 µM
in the MT-4 cell line and in PBMCs respectively, as determined 96-hours following
compound addition. Studies conducted using the xCELLigence System (Roche Applied
Sciences, Germany) confirmed the toxic nature of this statin and revealed that lovastatin
initiated cell death within 2 hours of introduction to the HeLa cell-line and completed its
toxic effect within 24 hours (data not shown). Similarly, atorvastatin, simvastatin, lovastatin
acid, mevastatin and fluvastatin yielded low CC50 values (< 14 µM) in the MT-4 cell line
(Table 1). The two hydrophilic statins were observed to be less cytotoxic. Pravastatin lactone
yielded a CC50 value of 59.22 µM while no toxicity was observed for pravastatin within the
concentration limits defined for our assay. Irrespective of lipophilicity, none of the statins
yielded discernible SI values (Table 1). Cell-based antiviral activity could only be detected
within the toxic range of the statins and was attributed to cell death rather than to genuine
inhibition. In contrast, CX05168 effectively inhibited viral replication as described previously
(1,2) and yielded an SI value of 39.97 (Table 1).
Discussion
This report provides the first account of lovastatin as a potential useful inhibitor of the
LEDGF/p75-HIV-1 IN interaction. It also corroborates the previous identification of
atorvastatin as an inhibitor of the interaction and provides additional information for this and
other statins. Data compiled on all eight statins demonstrates the influence of lipophilicity on
the toxicity of the statins.
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Acknowledgements
The authors would like to thank Mintek and the Faculty of Health Sciences, University of
Witwatersrand for funding and permission to publish these findings. The Belgium Technical
Cooperation (BTC) is thanked for the fellowship provided to ATH.
Conflict of Interest
The authors have no conflict of interest to declare.
Table 1. Biological activity of eight statins and the known inhibitor, CX05168
c
d
LEDGF/P75 –
HIV-1 IN
EC50
SE (µM)
CC50
SE (µM)
SI e
Strand transfer
(ST) inhibition
MT-4
MT-4
(PBMCs)
% inhibition at
100µM SE a
(IC50 SE; µM)
% inhibition at
100µM SE
(IC50 SE ; µM)
b
91.37 6.85
(1.33 0.05)
1.78 0.28
71.15 2.33
39.97
0.56
31.73 4.89
(70.12 2.26)
CX05168
54.26 2.03
23.68 0.21
13.36 2.38
Enhanced activity
(10.22 0.80)
Atorvastatin
Simvastatin
Lovastatin
28.78 0.70
ND
12.70 0.66
-
9.43 1.22
7.82 5.61
75.51 6.89
(1.97 0.45)
6.95 0.21
5.31 0.19
(6.50 0.31)
0.76
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42.48 1.69
ND
6.53 0.77
-
8.60 2.81
Lovastatin
acid
13.25 3.66
4.87 1.18
6.70 2.20
3.72 0.25
0.55
0.63 14.12
11.51 4.60
Mevastatin
Fluvastatin
ND
10.64 1.14
-
25.77 5.53
29.55 0.56
59.22 1.66
2.00
31.65 6.63
18.54 1.02
Pravastatin
lactone
8.55 3.22
> 100
> 100
-
Pravastatin
a SE, Standard error. Standard error of the mean for at least three separate experiments.
b IC50, 50% inhibitory concentration. Calculated as the concentration of compound required to reduce the
LEDGF/p75-IN interaction by 50%.
c EC50, 50% effective concentration. Defined as the concentration of compound required to reduce HIV-1
replication by 50%.
d CC50, 50% cytotoxic concentration. Calculated as the concentration of compound required to reduce cell
viability by 50%.
e SI, selectivity index. Calculated as the ratio of CC50/EC50
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