Inhibition of Human Immunodeficiency Virus-1 Integrase by β-Diketo Acid Coated Gold Nanoparticles

Article abstract of DOI:10.1021/acsmedchemlett.9b00648

Gold nanoparticles (GNPs) have been proposed as carriers for drugs to improve their intrinsic therapeutic activities and to overcome pharmacokinetic problems. In this study, novel nanosystems constituted by a model β-diketo acid (DKA) grafted to the surface of GNPs were designed and synthesized following the "multivalent high-affinity"binding strategy. These first nanoscale DKA prototypes showed improved inhibition of HIV-1 integrase (HIV-1 IN) catalytic activities as compared with free DKA ligands.

Full text of DOI:10.1021/acsmedchemlett.9b00648

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Letter
Inhibition of Human Immunodeficiency Virus-1
Integrase by #-Diketo Acid Coated Gold Nanoparticles
Vanna Sanna, Mohamed Fathy Youssef, Nicolino Pala, Dominga Rogolino, Mauro
Carcelli, Pankaj Kumar Singh, Tino Sanchez, Nouri Neamati, and Mario Sechi
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00648 • Publication Date (Web): 20 Mar 2020
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Inhibition of Human Immunodeficiency Virus-1 Integrase by β-
Diketo Acid Coated Gold Nanoparticles
Vanna Sanna,† Mohamed Fathy Youssef,§,‡ Nicolino Pala,§ Dominga Rogolino,# Mauro Carcelli,#
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Pankaj Kumar Singh,§ Tino Sanchez,° Nouri Neamati,^,* and Mario Sechi§,*
§
†Nanomater s.r.l., c/o Porto Conte Ricerche, Alghero, Italy; Department of Chemistry and Pharmacy, Laboratory of Drug
‡
Design and Nanomedicine, University of Sassari, Italy; Chemistry Department, Faculty of Science, Suez Canal University,
Ismailia, Egypt; #Department of Chemical, Life Science and Environmental Sustainability, University of Parma, Parco Area
delle Scienze, Parma, Italy; °National Center for Advancing Translational Sciences – NIH, Rockville, MD, USA;
^Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI, USA.
KEYWORDS: HIV-1 Integrase Inhibitors; -Diketo Acids; Gold Nanoparticles; Multivalent Ligands.
ABSTRACT: Gold nanoparticles (GNPs) have been proposed as carriers for drugs to improve their intrinsic therapeutic activities
and to overcome pharmacokinetic problems. In this study, novel nanosystems constituted by a model β-diketo acid (DKA) grafted
to the surface of GNPs were designed and synthesized following the “multivalent high-affinity” binding strategy. These first
nanoscale DKA prototypes showed improved inhibition of HIV-1 integrase (HIV-1 IN) catalytic activities as compared with free
DKA ligands.
HIV-1 integrase (HIV-1 IN) has been validated as a target
for developing novel antiretroviral agents.1-3 It catalyzes the
insertion of retro-trascribed viral cDNA into the host genome
(Chart 1, A) to form a stable provirus through two coordinated
reactions, 3′-processing (3’-proc) and strand transfer (ST).4-6
Because of its vital role in the viral replication cycle, with no
human counterpart of the enzyme known, the addition of an
IN inhibitor to existing components of antiretroviral therapy
has improved the outcome of therapy by potential synergism,
without exacerbating toxicity.1,2,7
ligands, which were able to produce simultaneous and
reversible molecular recognition interactions.18-20
In the past decade, several prototypes as multimeric ligands
targeting tumor/viral receptors have been exploited, and
multivalency can be envisaged as a way to improve the
binding performance of small molecule–drug construct, thus
expecting to reach a similar efficiency of the antibody–drug
conjugates.18,21-23
O
A)
B)
OH
Among a plethora of compounds with diverse structural
features reported as IN inhibitors,7 a class of compounds
bearing a β-diketo acid (DKA, Chart 1, B) functionality has
emerged and validated as the most effective chemical space in
anti-IN drug discovery, and some compounds belonging to
this family have had clinical success.8-13 Several DKA
congeners selectively inhibit the strand transfer reaction, and
in cell-based assays they inhibit integration without affecting
earlier phases of the HIV-1 replication cycle.8 Mechanistically,
DKA pharmacophoric motif is involved in a functional
sequestration of divalent metal ions (Chart 1, C), which are
critical cofactors at the enzyme catalytic site.7,12-15 This would
subsequently block the transition state of the IN-DNA
complex.2,7,13,14 Despite the outstanding advances in the HIV-
IN drug discovery, it was of paramount importance to explore
further possibilities to optimize the action of DKA-based
compounds as IN inhibitors by the investigation of newer
chemical entities.16,17
Host DNA-
OH
O
O
Base
Host DNA
O
O
O
Virus DNA
DKA (I)
O
O
H
O
O
O
P
O
O
O
Base
C)
Base(A)
X
O
O
Y
O
O
O
O
E152
D64
D116
Chart 1. A) HIV-1 IN catalytic mechanism; B) Representative
DKA and pharmacophoric motif (in red); C) DKA-based
chemotype chelates metal ions (orange spheres) cofactors on
the active site.
On the other hand, the application of nanotechnology in
drug design is having an impact on diagnostics and drug
delivery and, more recently, in drug discovery.24-26 Therefore,
the concept of multivalency, by nanoparticle (NP)-based
platforms with multiple ligands coated on their surface, has
emerged as suitable strategy to enhance the binding affinity of
Starting from the complexity of biological systems to
exploit widespread advantage of multivalency, i.e. the
combination of several entities involved in individual
interactions or binding events, this characteristic has been
followed as suitable strategy in the generation of high-affinity
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simple monovalent ligands. Nanoparticles (NPs) can be
Page 2 of 6
The preparation of DKA-coated GNPs (DKA-GNPs, 1) was
achieved in a two-step reaction by reduction of chloroaurate
with NaBH4 in the presence of the lipoic acid-tailed
diketoester (DKE-GNPs, 2), followed by an alkaline
hydrolysis (Scheme 1).
1
2
3
4
5
6
7
8
synthetically engineered to present multiple high-affinity
molecules on their surface to tune binding affinity over several
orders of magnitude. Moreover, such nanosystems may be
effective in disrupting key protein/protein interactions
involved in several pathogenic events. NP-based prototypes
possessing multivalent ligands are also expected to produce a
high local concentration of binding molecules which,
consequently, can statistically promote formation of more
ligand-receptor interactions, thus enhancing affinity and
selectivity at the biological target.
Scheme 1. Preparation of DKE- and DKA-GNPs
prototypes 1 and 2
O
9
OH
O
O
OH
S
Au
S
COOCH3
S
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S
S
OCH3
S
O
In this context, gold nanoparticles (GNPs) are extensively
investigated for various biomedical applications due to their
large surface area to volume ratio and thermal stability, as well
as their less toxicity, synthetic accessibility and amenability of
functionalization.27-29 Some gold nanosystems have been
proposed as carriers for drugs, to overcome pharmacokinetic
problems and to improve their intrinsic therapeutic activities.30
S
O
S
S
HAuCl4.4H2
O
N
S
H
S
S
DKE-GNPs (2)
NaBH4, DMSO /
r.t., 2 h
9
S
S
S
2N NaOH
MeOH, r.t., 5 h
S
S
S
S
S
O
S
O
S
OH
COOH
S
Au
S
S
The demonstration that GNPs convert a weak interaction of
active small ligands into multivalent more effective and
biologically active therapeutics have been offered by several
seminal reports, which include, for example, inhibition of HIV
fusion and antiretroviral delivery,31,32 activation/inhibition of
carbonic anhydrase,33,34 and tumor targeting.35,36
S
S
S
DKA-GNPs (1)
The key synthone 9 was synthesized by coupling lipoic acid
with the 4-amino-DKE 6 in the presence of EDCI/DMAP, as
outlined in Scheme 2.
In this scenario, we sought to explore the development of
novel GNPs that could effectively inhibit viral enzymes such
as HIV-1 IN (Figure 1). Therefore, a nanoparticulate carrier
was designed simultaneously with a model DKA inhibitor.
Scheme 2. Preparation of the synthon 9
O
OH
COOH
O
OH
OCH3
OCH3
+
O
O
EDCI, DMAP
DMA / r.t., 1 h
O
N
H2N
S
H
S
S
S
6
9
Synthetic approaches for the preparation of the key
intermediate 6, and for the other free ligands 7 and 8 and their
precursors are depicted in Scheme 3.
Scheme 3. Synthetic routes for the preparation of free
ligands and intermediates
O
O
OH
O
OCH3
O
O
BOC anhydride
THF, reflux, 5 h
Dimethyl oxalate
O
O
N
O
N
Figure 1. a) Graphical representation of a GNP coated with
DKA ligands on its surface; b) virtual interaction of GNPs
with the HIV-1 IN enzyme; c) interaction of a conjugated
DKA ligand with the metal ions on the catalytic site.
H2N
MeOCH3, THF /
reflux, 2 h
H
H
3
4
5
2N NaOH,
r.t., 5 h
Formic acid,
r.t., 1 h
O
OH
O
O
OH
OH
OH
OCH3
OH
4M HCl / dioxane
r.t., 30 min
O
O
O
O
O
N
H2N
H2N
H
7
6
8
In this work, we developed ~3.5 nm diameter DKA-coated
GNPs as a nanoscale platform to construct novel HIV-1 IN
multivalent therapeutics. These particles were designed
starting from a model derivative of the simple 2-hydroxy-4-
oxo-4-phenylbut-2-enoic acid (I, Chart 1); it was coupled with
lipoic acid to have both a) a flexible linker between the ligand
and the gold surface, and b) the appropriate thiolic terminal
groups needed for anchoring such ligands to the NP surface
(Figure 1).
Briefly, 6 was obtained by following the Boc protection of
the commercially available 4-amino-acetophenone 3 to give
the 4-Boc-amino-acetophenone 4, which was converted to the
diketoester derivative
5 by Claisen condensation with
dimethyl oxalate and sodium methoxide, using a procedure
previoulsy reported by us, with slight modification.11
Subsequent Boc deprotection of 5 by formic acid in mild
conditions gave 6 in good yield. The acids 7 and 8 were
obtained upon acid or alkaline hydrolysis of diketoesters 6 and
5, respectively (Scheme 3), for comparison purpose.
The obtained gold nanosystems resulted in precise and
monodisperse nanoscale constructs, which were tested for
anti-HIV-1 IN activities.
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All small molecules have been characterized by means of
NMR, IR, mass spectrometry (ESI and FAB) and elemental
analysis.
(Supporting information). Inhibition of IN catalytic activities,
3’-proc and ST, were evaluated using oligonucleotide-based
assays and the results are reported in Table 1 (see below for
schematic of IN activity, and in Supporting information for
experimental details).
1
2
3
4
5
6
7
8
GNPs 1 and 2 were characterized by transmission electron
microscopy (TEM) (Figure 2). These particles are
monodispersed, roughly spherical in shape, having an average
particle size of ~3.7 nm; the number of gold atoms is
calculated as ~1430 (for DKEs-GNPs 1 and DKAs-GNPs 2).27
Elemental analysis and ICP-OES measurements indicated that
the number of ligands grafted on the NP’ surface resulted 193
for both systems (considering the Au/S w/w ratio).27,33,34,37
With the exception of the amino-DKEs 6 and 9, all tested
compounds showed anti-IN activity in (low) concentration
ranges (IC50s from 0.96  0.34 to 44  6 μM), with more
potency exhibited towards the catalyzed ST process for free
ligands 5 and 7 versus 3’-proc, as evidenced by their
selectivity indexes. The ligands differ in activities as
previously reported, thus confirming that the nature of the
substituents on the aromatic ring significantly influence the
potency.
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DKA-GNPs (1)
(1)
Table 1. In vitro HIV-1 integrase inhibition and MTT
assay
!
DKE-GNPs (2)
(2)
3’-Processing Strand Transfer
Cpds
bSI
cCC50
aIC50 (µM)
aIC50 (µM)
!
5
>100
>2.3
~1
-
-
Figure 2. Left: TEM images of the GNPs 1 and 2 (scale bar = 50
nm); right, the corresponding nanoparticle size distribution.
44  6
8
-
15  1
>100
14  1
>100
Therefore, the obtained GNPs were consistent with a
proposed empirical formula of [Au1434(C18H21O5NS2)193] and
[Au1434(C19H23O5NS2)193], for 1 and 2, respectively.
6
-
7
5.3
-
-
74  6
>50
14  3
IR spectrum of GNPs-DKE 2 is given in Figure 3A. The
C=O stretching bands of the COOCH3/COOH functionalities
were found at approximately 1700 cm-1 in the spectrum of
lipoic acid-tailed DKE 9, used for comparison.
9
>50
-
>10
>10
-
DKAs-GNPs (1)
DKEs-GNPs (2)
dLA-GNPs
eHL2
0.8
~1
-
0.96  0.34
2.5  0.12
>5
1.2  0.85
2.4  0.1
>5
Moreover, Figure 3B shows the UV–Vis spectrum of DKE-
and DKA-capped GNPs 1 and 2. A strong absorption at about
540 nm, a resonance corresponding to excitation of surface
plasmon vibrations in the GNPs,27 was observed for both gold
nanosystems.
71.4
-
10  8
0.14  0.03
aIC50: inhibitory concentration 50% (average data for
inhibition of 3’-processing and strand transfer of purified IN),
data is presented as mean ± SD from three independent
experiments. bSI: Selectivity Index. cCC50: Cytotoxic
concentration 50% in MT-4 cells, determined by MMT assays.
dLipoic acid-coated GNPs. eData from ref. 38.
9
DKE-GNPs 2
DKA-GNPs 1
A)
B)
DKA-GNPs 2
Surprisingly, with respect to that showed from compounds 5
and 7, as well as from the reference compound, the Boc-
protected DKA 8 shared a similar inhibition profile for both
catalytic activities (IC50s = 15  1 and 14  1 μM for 3’proc
and ST, respectively).
Figure 3. A) FT-IR spectra of DKEs-GNPs 2 and DKEs free
ligand 9. B) UV-Vis spectra of DKE-GNPs 2 and DKA-GNPs 1.
GNPs 1 and 2, and ligands 5-8 were tested for their ability
to inhibit IN catalytic activity in in vitro assays employing
purified enzyme, using the raltegravir-based derivative (HL2)38
as reference compound, and LA-GNPs, used as a control
This behavior was translated to the GNPs, which exhibited
inhibition profiles against both catalytic processes.
Specifically, GNP-DKA 1 was the most potent compound
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Author Contributions
(IC50 values of the 1.2  0.85 μM and 0.96  0.34 μM for ST
1
2
3
4
5
6
7
8
and 3’-proc, respectively). While the lipoic acid-coated GNPs
(LA-GNPs) were not active (at >5 μM) as expected, GNPs-
DKE 2 was approximately 2-fold less potent than the
corresponding diketoacid 1, thus demonstrating that the acid-
and ester- terminal functionalities of the nanosystems similarly
inhibited HIV-IN functions. Interestingly, these GNPs (i.e. 1
and 2) did not display any selectivity for IN reactions, and did
not show cytotoxicity in human MT-4 cells at 10 μM (Table
1).
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Funding Sources
This work was partially supported by FAR2019 - UniSS.
Notes
The authors dedicate this work to the memory of the Prof.
Maurizio Botta. The authors declare no competing financial
interest.
9
In this study, we proposed the first application of small
molecule (β-DKE/DKA)-coated GNPs as effective HIV-1 IN
inhibitors. These nanoscale anti-IN prototypes demonstrated
an improved IN inhibition potency against purified enzyme
with respect to the free compounds. Analysis of preliminary
results seems to support the hypothesis that these
nanoprotopypes could act by interfering with both catalytic
processes, thus competing with IN and DNA. Both the volume
of the nanogold and the presence of multiple conjugated
ligands in proximity of the enzyme active site can play a role
in chelating the metal ions and in interfering with
protein/DNA complex. Work is in progress to clarify the
mechanism of action of these gold nanosystems, which can be
useful for development of powerful nanogold-based
antiretrovirals.
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ACKNOWLEDGMENT
The authors are grateful to the technicians of the Remote
Microscopy Laboratory, Sardinia District, for their assistance with
TEM analysis, and of the Laboratorio di Microanalisi – University
of Florence, for ICP-AES and ESEM analyses.
ABBREVIATIONS
HIV-1, Human Immunodeficiency Virus-1; IN, Integrase; DKAs,
β-diketo acids; NP, nanoparticles; GNPs, gold nanoparticles;
cDNA; dexoxyribonuleic acid; TEM, transmission electron
microscopy;
EDCI,
N-(3-Dimethylaminopropyl)-N’-
ethylcarbodiimide; DMAP, 4-(Dimethylamino)pyridine.
ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the
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Experimental methods. Synthesis of DKA/DKE-GNPs (1 and 2)
and LA-GNPs, TEM analyses, synthesis and characterization
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(MTT) assays. (PDF)
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integration: Mechanism of viral DNA cleavage and DNA
strand transfer Cell 1991, 67, 1211– 1221
AUTHOR INFORMATION
Corresponding Authors
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integration. Virology 2011, 411, 194– 205
X.; Krishnan,
L.; Cherepanov,
P.; Engelman,
DNA
*(M.S.) Phone: +39 079-228-753. Fax: +39 079-229-559.
E-mail: mario.sechi@uniss.it.
*(N.N.) Phone: +1 734-647-2732. Fax: +1 734-763-8152.
E-mail: neamati@umich.edu
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ORCID:
Mario Sechi: 0000-0003-2983-6090
Nouri Neamati: 0000-0003-3291-7131
Pankaj Kumar Singh: 0000-0002-4774-6358
Mauro Carcelli: 0000-0001-5888-4556
Dominga Rogolino: 0000-0003-2295-5783
Nicolino Pala: 0000-0002-9681-5233
Vanna Sanna: 0000-0001-9068-349
Present Addresses
§,‡ M.F.Y. worked at the Department of Chemistry and
Pharmacy, University of Sassari, and his current affiliation is at
the Chemistry Department, Faculty of Science, Suez Canal
University, Ismailia, Egypt.
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Graphical representation of a GNP decorated with DKA ligands on its surface. DKA-coated GNPs as a nanoscale platform to
construct novel HIV-1 IN multivalent therapeutics have been developed. These anti-IN prototypes demonstrated an improved
IN inhibition potency against purified enzyme as compared with free DKA ligands.
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Inhibition of Human Immunodeficiency Virus-1 Integrase by β-diketo acid Coated Gold Nanoparticles
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