PF74 and its novel derivatives stabilize hexameric lattice of HIV-1 mature-like particles

Article abstract of DOI:10.3390/molecules25081895

A major structural retroviral protein, capsid protein (CA), is able to oligomerize into two different hexameric lattices, which makes this protein a key component for both the early and late stages of HIV-1 replication. During the late stage, the CA prote

Full text of DOI:10.3390/molecules25081895

molecules
Article
PF74 and Its Novel Derivatives Stabilize Hexameric
Lattice of HIV-1 Mature-Like Particles
1
2
1
1
1
Alžb eˇ ta Dostálková , Kryštof Škach , Filip Kaufman , Ivana K rˇ ížová , Romana Hadravová ,
2
3
, Richard Hrabal 4 and Michaela Rumlová *
1,
Martin Flegel , Tomáš Ruml
1
Department of Biotechnology, University of Chemistry and Technology, 166 28 Prague, Czech Republic;
dostalkl@vscht.cz (A.D.); kaufmanf@vscht.cz (F.K.); krizovaa@vscht.cz (I.K.);
romana.hadravova@uochb.cas.cz (R.H.)
Department of Chemistry of Natural Compounds, University of Chemistry and Technology, 166 28 Prague,
Czech Republic; K.Skach@seznam.cz (K.Š.); martin.leglef@gmail.com (M.F.)
Department of Biochemistry and Microbiology, University of Chemistry and Technology, 166 28 Prague,
Czech Republic; tomas.ruml@vscht.cz
2
3
4
NMR Laboratory, University of Chemistry and Technology, 166 28 Prague, Czech Republic; hrabalr@vscht.cz
Correspondence: michaela.rumlova@vscht.cz; Tel.: +420-220-444-177
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 19 March 2020; Accepted: 15 April 2020; Published: 20 April 2020
Abstract: A major structural retroviral protein, capsid protein (CA), is able to oligomerize into
two different hexameric lattices, which makes this protein a key component for both the early and
late stages of HIV-1 replication. During the late stage, the CA protein, as part of the Gag polyprotein
precursor, facilitates protein–protein interactions that lead to the assembly of immature particles.
Following protease activation and Gag polyprotein processing, CA also drives the assembly of the
mature viral core. In the early stage of infection, the role of the CA protein is distinct. It controls
the disassembly of the mature CA hexameric lattice i.e., uncoating, which is critical for the reverse
transcription of the single-stranded RNA genome into double stranded DNA. These properties make
CA a very attractive target for small molecule functioning as inhibitors of HIV-1 particle assembly
and/or disassembly. Of these, inhibitors containing the PF74 scaffold have been extensively studied.
In this study, we reported a series of modifications of the PF74 molecule and its characterization
through a combination of biochemical and structural approaches. Our data supported the hypothesis
that PF74 stabilizes the mature HIV-1 CA hexameric lattice. We identified derivatives with a higher
in vitro stabilization activity in comparison to the original PF74 molecule.
Keywords: HIV-1 CA inhibitor; PF74 derivatives; uncoating; disassembly
1
. Introduction
Capsid protein (CA) is the main structural protein of retroviral particles. CA consists of two
helical domains, the N-terminal (CA-NTD) and C-terminal (CA-CTD) domains, which are connected
by a short linker. As a part of structural polyprotein Gag, CA mediates intra- as well as inter-molecular
interactions that lead to the formation of a hexameric lattice of immature particle. Later on, following
retroviral protease activation, the Gag polyprotein is processed to individual structural proteins and
released as CA molecules re-assembled into mature hexameric lattice that form a viral core or capsid.
Despite a low amino acid sequence similarity, both CA domains share similar secondary and tertiary
structures among various retroviral genera. However, the quaternary structures of CA domains
within the immature lattices of various retroviruses, such as Mason-Pfizer monkey virus, Human
immunodeficiency virus 1 (HIV-1), Rous sarcoma virus, and Murine leukemia virus, are different [
1–4].
In contrast to a purely hexameric, immature lattice, which can be bended and closed only due to
Molecules 2020, 25, 1895; doi:10.3390/molecules25081895
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1895
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several openings (gaps) in the structure [5], the properly assembled mature hexameric lattice can form
fully closed fullerene or multilayered structures [1]. This is enabled by the accommodation of twelve
pentamers in the hexameric structure of mature retroviral cores [ ]. Researchers have proposed
1,6–9
that a variety of retroviral core architectures (e.g., conical, tubular, or pleiomorphic) are determined by
the position of pentamers [10–12].
In addition to the key role of CA during the assembly of both immature and mature types of
retroviral particles, CA is also critical during virus uncoating. Uncoating is a poorly understood
post-entry event in the early stage of the retroviral life cycle, comprising controlled disassembly of the
core upon entering the cytoplasm. Even though this process has been intensively studied, the exact
subcellular location, mechanism, and timing of retroviral uncoatings are unclear and the data are
controversial [13
CA lattice it is plausible that the viral core persists largely intact upon entry to the cytoplasm [22
However, whether the uncoating is accomplished in the cytoplasm or at the nuclear pore remains
unclear [13 15 16 18 20 24 25]. A recent study demonstrated that multiple CA proteins can direct the
viral genome into the nucleus of the infected cell [26]. HIV-1 capsid disassembly is tightly connected to
reverse transcription as its inhibition delays the uncoating [19 27 28]. HIV-1 uncoating is also linked
to binding of a variety of cellular proteins and small cofactor molecules [29 38]. Due to the above
mentioned importance of CA in various steps of the HIV-1 replication cycle, mutations in CA are
very often fatal for the virus [13 39 40]. Therefore, researchers expected that the mutations within CA
–
21]. Based on several studies showing that the host cell proteins bind to the assembled
,
23].
,
, , – , ,
,
,
–
,
,
developed against CA-targeting drugs might compromise the fitness of the virus [39].
The importance of CA in many essential functions, such as the assembly of immature particles,
the formation of the mature core, and the controlled disassembly, makes it a highly attractive
pharmacological target [41]. Indeed, studies reported several capsid-binding small molecules that
inhibited HIV-1 replication, for review see [42–44]. Recent studies suggested that these drugs blocking
viral nuclear import by targeting the viral CA could be a successful therapeutic approach [45]. Based
on all the above mentioned recent knowledge, the development of new compounds that can directly
and specifically block the early steps of viral infection, and probably also CA assembly, are highly
in demand.
The PF-3450074 (PF74) molecule (Figure 1), binding to CA lattice is one of the most studied and
most promising inhibitors of HIV-1 [45
reported to inhibit HIV-1 replication [48
the stability of mature capsid. However, whether PF74 stabilizes or destabilizes the hexameric lattice is
still unclear [47 49 51 53]. The structure of PF74 consists of two components: a phenylalanine part
Figure 1, red) and an indole substituent (Figure 1, green), which are connected by a linker (Figure 1,
blue). PF74 binds by its phenylalanine core to the pocket within the CA-NTD formed by the helices H3,
H4, H5, and H7, while the indole ring interacts with the CA-CTD of the adjacent subunit [22 47 51 54].
,
46]. The PF74 molecule, first identified by Pfizer, USA [47], was
–
50]. PF74 binds to the HIV-1 CA hexameric lattice and affects
,
, –
(
,
,
,

Molecules 2020, 25, 1895
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Figure 1. PF74 and its derivatives. Chemical structures of PF74 and its indole derivatives used in
this study.
To improve the poor therapeutic qualities and poor metabolic stability [55], several efforts to
modify PF74 were reported [45 46 55 56]. In this work, we report several modifications of the indole
,
, ,
and linker part of the PF74 molecule. To quantify the effect of PF74 derivatives on the stability of the
viral capsid, we used a combination of in vitro, cell-based, and NMR structural assays. The data gained
by our recently developed in vitro disassembly inhibitor test for HIV (DITH) [57] strongly supported
the hypothesis that the mechanism of PF74 is to stabilize, not to destabilize, the mature HIV-1 CA
hexameric lattice. Moreover, the higher in vitro stabilization activity of the D9 and D10 derivatives in
comparison to the original PF74 pointed toward the nature of further possible modifications.
2
. Results and Discussion
2
.1. The Design and Synthesis of PF74 Derivatives
To enhance the inhibiting capability of PF74, we designed and synthesized a series of modified
PF74 derivatives comprising the indole substituent and connected linker (Figure 1: green and blue,
respectively). The capability to interact with the PF74 binding sites of CA was tested in silico by
the docking program Glide Schrödinger, USA). In all cases, the HIV-1 CA N-terminal binding part
(
(
Figure 1: red) remained intact and the modifications targeted only the C-terminal binding indole part
Figure 1: green) and the linker (extended by one carbon in D11).
2
.2. Analysis of PF74 Derivatives Activity Using in Vitro Protein-Based Methods
2
.2.1. Fast Assembly Inhibitor Test for HIV (FAITH) Analysis
To analyze the effects of the PF74 derivatives on the HIV-1 core assembly, disassembly, and stability
in vitro, we first employed the Fast Assembly Inhibitor Test for HIV (FAITH) [58]. Using recombinant
HIV-1 capsid-nucleocapsid (CANC) protein and dually labeled TaqMan-like oligonucleotide (tqON),
this assay provides quantitative information regarding the efficiency of the assembly of HIV-1
mature-like particles. During the three-hour assembly reaction, HIV-1 CANC and tqON formed

Molecules 2020, 25, 1895
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mature-like structures. Inside these assemblies were hidden the tqON molecules, which were bound
by the NC domains and packaged into the forming particles. When Exonuclease I was added to the
reaction mixture, it degraded those tqON that were not packaged inside the particles and remained
free in the solution. During tqON degradation, a reporter dye-fluorescein (FAM) was separated from
its quencher molecule-black hole quencher (BHQ), and the FAM fluorescence emission was measured
(Figure 2a, purple curve).
Figure 2. Quantification of the in vitro assembly of HIV-1 CANC in the presence of PF74 and its
derivatives by Fast Assembly Inhibitor Test for HIV (FAITH). HIV-1 CANC (18
with tqON (1.8 M) CAI, and PF74 or its derivatives to a final concentration 10 M. Following the
assembly reaction, Exonuclease I was added to each sample to degrade free, non-incorporated tqON.
) Fluorescence released from degraded tqON was monitored for tqON itself (purple), CANC without
µM) was mixed
µ
µ
(a
inhibitor (black) and from the sample containing. DMSO without inhibitor (black) and from the sample
containing CAI (green), PF74 (red), D1 (light green), D2 (grey), D4R (yellow), D7 (pink), D8S (dark
green), D8 (light blue), D9 (cyan), D10 (violet), and D11 (azure). (b) Assembly efficacy was calculated
from the difference in relative fluorescence between the control represented by free tqON (panel a,
purple curve) and the assembled CANC particles in the absence of an inhibitor (panel a, black curve)
or in presence of inhibitors. The relative percentage of the efficacy of HIV-1 CANC assembly in the
presence of PF74 and its derivatives was compared to that of CANC, which was considered as 100%.
(
c) Following FAITH, in vitro assembled HIV-1 CANC in the presence of indicated inhibitors were
negatively stained and analyzed by TEM. Bar represents 200 nm.

Molecules 2020, 25, 1895
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As the fluorescence is released from the non-incorporated, free tqONs but not from those packaged
and protected by the CANC assembled particles, the amount is proportional to the efficiency of
the CANC assembly. To analyze the effects of the PF74 derivatives on the assembly of mature-like
HIV-1 particles, we mixed the tested inhibitors with HIV-1 CANC protein and, following a one hour
incubation, the assembly reaction was initiated by tqON addition. Next, Exonuclease I was added,
and the fluorescence release, due to the degradation of free, non-incorporated tqONs, was measured
(
Figure 2a). The level of assembly efficiency was then calculated from three independent measurements
as a difference in the relative fluorescence between the control represented by free tqONs (Figure 2a,
purple curve) and the assembled CANC particles in dimethyl sulfoxide (DMSO) in the absence of
inhibitor (CANC, black curve) or in presence of inhibitors (Figure 2a). The relative percentage of the
efficacy of the HIV-1 CANC assembly in the presence of PF74 and its derivatives was then compared
to that of the CANC without inhibitor, which was considered as 100% (Figure 2b).
As expected, in contrast to the control peptide inhibitor CAI [59] that completely abolished the
CANC assembly (Figure 2), no effect of PF74 and its derivatives on the assembly of mature-like CANC
particles was observed by FAITH (Figure 2a,b). In accord with that, transmission electron microscopic
analysis of selected negatively stained assembled samples showed the presence of typical tubular and
cone-like structures in the tested samples (Figure 2c), confirming the observation that none of the PF74
derivatives affected the in vitro assembly of HIV-1 mature particles. In the absence of an inhibitor,
CANC typically assembled into long, tubular structures. The same phenotype was observed for CANC
assembled in the presence of the D7 derivative. However, the predominant formation of cone-like and
shorter tubular structures was observed for HIV-1 CANC particles assembled in the presence of PF74
and D10 inhibitors (Figure 2c).
2
.2.2. DITH Analysis
In contrast to the assembly of immature particles, PF74 was reported to affect the stability of the
hexameric lattice of HIV-1 mature particles during disassembly. To quantify and compare the effects
of PF74 and its derivatives on the stabilization/destabilization of the CA hexameric lattice of CANC
tubular structures, we next applied the DITH assay [57]. Similarly to FAITH, this method was based
on the measurement of fluorescence released from the tqON. DITH uses preassembled CANC-tqON
complexes that are incubated under disassembly conditions in the presence or absence of PF74 and its
derivatives. The amount of fluorescence signal released from tqON is then proportional to the level
of HIV-1 CANC disassembly. The stabilization or destabilization effect of tested compounds can be
then calculated. We assembled CANC tubes in assembly buffer in the presence of tqON. Following
1
h incubation of the CANC tubes with DMSO (control sample) or PF74 and its derivatives (final
concentration 10 M), the particles were diluted into disassembly buffer and incubated overnight
µ
at laboratory temperature. Immediately following the Exonuclease I addition, the fluorescence was
measured (Figure 3a) and the relative percentage of CANC stability was calculated for each sample
(Figure 3b).

Molecules 2020, 25, 1895
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Figure 3. Disassembly inhibitor test for HIV (DITH) analysis of the effect of PF74 and its derivatives
on the stability of HIV-1 CANC. ( ) Graph showing the fluorescence emission curves demonstrating
a
the kinetics of tqON release from preassembled CANC particles incubated in assembly buffer (black
curve) and disassembly buffer (red curve) in the absence of PF74 derivatives or in the disassembly
buffer containing: PF74 red, D1 light green, D2 grey, D4R yellow, D7 pink, D8S dark green, D8R light
blue, D9 cyan, D10 violet, and D11 azure at the final concentration 10 µM. The stabilization effect of
PF74 and its derivatives was calculated as the difference between the relative fluorescence of tqON
at 90 min in the disassembly and assembly reactions according to the calculation: relative percent of
stabilization = 100*
particles incubated in the disassembly buffer in the presence of PF74 and its derivatives, measured and
calculated as described in ( ). The relative stability of CANC in the absence of inhibitor in disassembly
buffer was considered as 0%. ( ) Following DITH, the in vitro assembled HIV-1 CANC in the presence
∆2/∆1. (b) DITH quantification of the relative stability of preassembled CANC
a
c
of indicated inhibitors were incubated in disassembly buffer, negatively stained, and analyzed by TEM.
Bar represents 200 nm.
The relative percentage of stability of HIV-1 CANC mature particles in the presence of PF74
and its derivatives was determined as the difference between fluorescence of degraded tqON at
9
0 min in disassembly and assembly reactions for the CANC and PF74 derivatives treated CANC
assemblies (Figure 3a, 1 and 2, respectively). The relative percentage of PF74 derivatives-mediated
stabilization was determined using the formula: 100* 2/ 1 and compared to that of the wild type in
∆
∆
∆
∆
the disassembly buffer whose stability was considered as 0% (Figure 3b). The stability of PF74-treated
HIV-1 CANC assemblies in disassembly buffer was about 60% higher compared to the control CANC

Molecules 2020, 25, 1895
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sample. Compounds D9 and D10 turned out to be more potent stabilizers than PF74, increasing the
CANC assembly stability to 80% and 90%, respectively.
The D1 compound was only slightly less active in stabilization than PF74. All other tested PF74
derivatives (D2, D4R, D7, D8R, D8S, and D11) showed a lower effect on HIV-1 CANC stability than
PF74 (Figure 3b). TEM analysis of the samples following DITH (Figure 3c) confirmed the presence of
the intact CANC particles in the samples containing PF74 or compounds that increased the CANC
particle stability in the DITH assay (D9 and D10). In contrast, TEM analysis of the CANC particles
disassembled in the absence of PF74 or in the presence of the non-active inhibitors (D2, D4R, D7, D8R,
D8S, and D11) showed mainly disassembled material. These data clearly support the observation that
a PF74 inhibitor stabilized the mature hexameric lattice and thus inhibited the processes connected to
uncoating. A similar conclusion was reported based on the measurement of HIV-1 CA cores stiffness,
which was enhanced in the presence of PF74 [52].
2
.3. Analysis of PF74 Derivatives Using Cell-Based Methods
2
.3.1. Effect of PF74 Derivatives on HIV-1 Infectivity
First, using a resazurin assay, we tested the cytotoxicity of PF74 derivatives (Table 1). None of the
tested inhibitors were cytotoxic, with the exception of D10 with cytotoxic concentration CC50 value
M and D4R CC50 27 M. Then we verified the impact of PF74 derivatives on HIV-1 infectivity
10
µ
µ
using a single-round HIV infectivity assay. HIV-1 particles pseudotyped with vesicular stomatitis
virus (VSV) glycoproteins that were produced in human embryonic kidney (HEK 293) cells. At 48 h
post-transfection, the content of HIV-1 CA protein in the culture media was quantified by ELISA,
and normalized amounts of VSV-G pseudotyped HIV-1 containing green fluorescent protein (GFP)
were used to infect fresh HEK 293 cells. Immediately after infection, various concentrations of PF74
or its derivatives were added to the cells (DMSO was used for the control). At 48 h post-infection,
the HIV-1 infectivity was determined by the quantification of GFP-positive cells using flow cytometry,
and the 50% inhibition concentration (IC ) value for each inhibitor was calculated. The IC was
50
50
defined as the concentration of compound that reduced the HIV-1 infectivity by 50% compared to the
DMSO-treated control. The D10 derivative revealed a better inhibitory activity against HIV-1 than
PF74. The activity of three other derivatives D1, D8R, and D9 was only slightly weaker than that of
PF74. These data nicely correlated with an in vitro stabilization assay (Figure 3b).
Table 1. Cytotoxicity and inhibitory effect of PF74 and its derivatives.
Compound
CC50 (µM)
IC50 (µM)
PF74
>40
D1
>40
D2
>40
D4R
27 ± 3.5
>20
D7
>40
>20
D8S
>40
>20
D8R
>40
D9
>40
D10
D11
>40
10 ± 1.6
0.5 ± 0.3
1.75 ± 0.5
3.5 ± 0.6
8.0 ± 1.2
4.5 ± 1.5
3.4 ± 0.9
15 ± 3
2
.3.2. Cyclosporin A (CsA)-Washout Assay
The DITH results suggested that three of the PF74 derivatives (D1, D9, and D10) stabilized the
HIV-1 CA lattice in vitro more efficiently than the PF74 molecule. To verify these in vitro data and
evaluate the DITH assay in comparison with a cell-based method, we also tested the inhibitors using
the CsA-washout assay. This assay monitored the HIV-1 capsid core uncoating (or disassembly)
within infected cells [19
expressed—e.g., in owl monkey kidney (OMK)—HIV-1 restriction factor, tripartite motif-containing
protein 5 (TRIM5 )-Cyclophilin A (CypA), specifically binds to the CA hexameric lattice of the mature
,60]. The CsA-washout assay is based on the fact that the endogenously
α
HIV-1 core and blocks viral infection. However, in the presence of CsA, HIV-1 loses sensitivity to its
restriction factor. In the CsA-washout assay, CsA is gradually washed out of the cells, thus enabling
TRIM5
in this assay, we first tested the cytotoxic effect of the PF74 compounds on OMK cells. Unfortunately,
the promising D10 derivative showed a CC50 below 3 M which prevented its further testing in
α-CypA binding to the CA lattice and blocking HIV-1 infection [19]. To test the PF74 derivatives
µ

Molecules 2020, 25, 1895
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the CsA-washout analysis. To test the other PF74 derivatives, the OMK cells were transfected with
an ELISA-normalized amount of VSV-G pseudotyped GFP-HIV-1 virions in the presence of CsA and
inhibitors or DMSO (as a control).
Following the CsA-washout at various time intervals, the percentages of GFP-positive (i.e., infected)
cells were determined by flow cytometry, normalized to the non-drug control reaction (in DMSO), by
setting the percentage at 5 h to 100%, and used in the graph (Figure 4). PF74 and D1 derivative showed
the strongest impact on HIV-1 infectivity in OMK cells at the early stage of HIV-1 infection. All other
PF74 derivatives appeared to affect the uncoating, and D8R had a major effect. The D9 derivative
appeared to be highly active during the first two hours post infection, then its activity dropped.
Figure 4. CsA-washout assay. Owl monkey kidney (OMK) cells were infected by spinoculation
with an ELISA-normalized amount of VSV-G pseudotyped HIV-1 in the presence of CsA and DMSO,
PF74 and its derivatives. Inhibitors were added to the HIV-1 sample before spinoculation at a final
concentration of 10 µM. At the indicated times post-infection, the CsA-containing medium was replaced
with fresh, CsA-free culture medium. The percentage of GFP-positive OMK cells was determined
by flow cytometry using a BD FACS Aria III (Becton Dickinson) flow cytometer and the data were
analyzed with Diva 8 software. The number of GFP-positive cells measured at the indicated time
was then calculated and normalized to the DMSO-containing non-drug control reaction, which was
considered as 100%.
2
.4. NMR Analysis of the Binding Mode of D10
As the derivative D10 was the most active stabilizer in in vitro analysis, we determined its
1
15
binding mode by measuring a series of 2D H- N heteronuclear single quantum correlation (HSQC)
experiments on 15N-labeled CA-NTD with increasing concentration of the inhibitor and comparing the
results with the data for PF74. Figure 5 shows the histograms of chemical shift index values (CSI) for
all amino acid residues of CA-NTD with bound PF74 and D10.

Molecules 2020, 25, 1895
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Figure 5. The chemical shift index of binding of PF74 and D10 to CA-NTD. Chemical shift changes,
caused by DMSO, were subtracted from the experimental changes caused by the addition of inhibitors
dissolved in DMSO. The horizontal red lines indicate the cut off, which was calculated as the average
chemical shift index values (CSI) of all amino acids plus one standard deviation. The red ovals highlight
the areas of substantial CSIs corresponding to the amino acids of the binding site.
CSIs were calculated from the experimental data according to the formula
r
h
ꢀ
ꢁi
1
2
2
2
N
d =
δ + α × δ
(1)
H
1
where
δ
H
and
δ
are H and 15N; respectively, chemical shift changes and
α
is a weighting factor (0.2
N
in this case).
The most affected amino acid residues in both molecules were Thr 54, Leu56, Asn57, Gln63, Ala64,
Met66, Gln67, Leu69, Lys70, Asn74, Glu75, Glu76, Asp103, Ile104, Gly106, Thr107, and Thr108. This is
in accordance with the binding site of PF74 to CA-NTD, as it was previously determined by X-ray
crystallography [51]. In silico docking of D10 resulted in the same binding mode, which was confirmed
by highly similar histograms. The comparison of the structures of D10 and PF74 is shown in Figure 6.

Molecules 2020, 25, 1895
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Figure 6. The structures of PF74 and D10. PF74 is in khaki and D10 in pale green. Hydrogen atoms
are omitted for simplification. The most important residues of CA-NTD ( ) and CA-CTD ( ) for the
A
B
inhibitor binding are labeled. Created in Flare (Cresset Inc., New Cambridge House, Bassingbourne
Road, Littlington, Cambridgeshire SG8 0SS, UK).
The published data concerning the effects of PF74 are rather contradictory. Some experiments
proved that PF74 decreased the stability of the HIV-1 core and thus accelerated its disassembly [49].
However, another paper documented that PF74 strengthened the stability of the HIV-1 CA cores,
and thus slowed down the disassembly process [51,52,57]. Our data, obtained using an in vitro
stabilization assay (DITH) supported the model in which PF74 acted as a stabilizer of the HIV-1 mature
hexameric lattice [51]. Two of the PF74 derivatives with a modified indole moiety, D9 and D10, revealed
higher in vitro stabilization activity when compared to PF74. In order to diminish the D10 cytotoxicity,
further modifications are currently under our investigation.
3
. Materials and Methods
3
.1. Expression Vector Preparation
HIV-1 CANC, encoding CA, SP1, and the NC fusion protein was prepared as described
earlier [58,61]. Three vectors: the packaging psPAX2, encoding HIV-1 Gag, Pol, Tat, and Rev;
the reporter/transfer pWPXLd-GFP, encoding long terminal repeat (LTR), rev-response element (RRE),
and GFP and envelope pHEF-VSV-G, encoding vesicular stomatitis virus Env (VSV-G) were used for
the production of pseudotyped VSV-G HIV-1 particles. The psPAX2 vector [62] was kindly provided
by Dr. Jeremy Luban, UMASS Medical School, Worcester, MA, USA) and the pWPXLd-GFP and
pHEF-VSV-G vectors were purchased from Addgene (Cambridge, MA, USA).
3
.2. Expression and Purification of HIV-1 CA-Derived Proteins
The HIV-1 CANC protein was purified as previously described [58,61,63]. Briefly, the HIV-1 CANC
protein was expressed in Escherichia coli (E.coli) BL21 (DE3) and following cell lysis, polyethyleneimine
to a final concentration of 0.15% (w/v) was added to the cell lysate and the nucleic acids were removed
◦
by ultracentrifugation (Beckman (Brea, CA, USA), TI 90, 55,000 rpm, 3 h, 4 C). The HIV-1 CANC
protein was purified by a combination of ion-exchange chromatography using HiPrep™SP FF 16/10

Molecules 2020, 25, 1895
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column (GE Healthcare, Chicago, IL, USA) and gel-filtration chromatography using HiLoad
Superdex™ column. The HIV-1 CANC protein was concentrated to 4 mg/mL and stored at −80 C.
™
26/600
◦
3
.3. Fast Assembly Inhibitor Test for HIV (FAITH)
The assay was used to quantify the effect of PF74 derivatives on the assembly of mature-like
HIV-1 particles, as described in [58]. In 96-well plate, we pre-incubated the HIV-1 CANC protein
60 g/100 L) with the tested inhibitor (final concentration 10 M) and kept it on ice for 1 h. To start
the assembly reaction, 3 g of dually labelled oligonucleotide (tqON) was added to the CANC protein
and the volume of the reaction mixture was adjusted to 100 L using the assembly buffer (50 mM Tris,
pH 8.0, 1 M ZnCl , 340 mM NaCl). Following a 3-h incubation at room temperature, Exonuclease I
ExoI) 6 mM Mg2 was added and the fluorescence of the fluorophore released from degraded tqON
was measured using a Tecan M200Pro plate reader.
(
µ
µ
µ
µ
µ
µ
2
+
(
3
.4. Stabilization Fast Assembly Inhibitor Test for HIV (DITH)
This assay was used as described in [57]. Tested inhibitors were added at a final concentration
of 10
The mixture of CANC protein and tqON was incubated for 3 h at room temperature. Then, inhibitors
were added and incubation continued for 1 h. Next, we added 100 L of disassembly buffer (50 mM
Tris, pH 7.0, 1 M ZnCl ) to the each sample in the plate, and incubation, under moderate agitation,
µM to HIV-1 CANC tubular structures in a 96-well plate, assembled as described for FAITH.
µ
µ
2
2
+
continued overnight at room temperature. ExoI and Mg ions were added 16 h later, and the
fluorescence was measured as for FAITH.
3
.5. Determination of Cytotoxicity Using Resazurin Assay
5
The human embryonic kidney cells (HEK 293T) were seeded in a 48 well plate (3
×
10 cells/mL) in
◦
DMEM medium supplemented with 10% FBS in 5% CO atmosphere at 37 C. The next day, the cells
2
were treated with various concentrations of PF74 and its derivatives, ranging from 10–50
µM. We added
resazurin of a final concentration of 25 g/mL, 24 h later, to each sample. The cells were incubated
µ
◦
for an additional 4 h, in 5% CO atmosphere at 37 C in the dark. The metabolic activity of the cells
2
was then measured using a microplate reader (Tecan M200Pro) at 560 nm excitation/590 nm emission
wavelength. All experiments were done in triplicates.
3
.6. Production of VSV-G Pseudotyped HIV-1 Particles
HIV-1 particles were obtained from HEK 293 cells co-transfected with a combination of three
vectors: psPAX2 pWPXLd-GFP and pHEF-VSV-G. HEK 293 cells were grown in Dulbecco’s modified
Eagle medium (DMEM, Sigma, St. Louis, MI, USA) supplemented with 10% fetal bovine serum
◦
(
Sigma) and 1% L-glutamine (Sigma) at 37 C under 5% CO . The day before transfection, the cells
2
5
were plated at a concentration of 3
×
10 cells per well. The following day, the cells were transfected
with the appropriate vectors (0.4 mg each) using polyethylenimine (PEI, 1 mg/mL) at a 2:1 PEI:DNA
ratio. Four hours after transfection, the culture medium was replaced with fresh DMEM. At 48 h
post-transfection, the culture media containing released virions were harvested, filtered through
0.45-
µm pore membranes and used for the immunochemical quantification and characterization by
ELISA and western blot using rabbit anti-HIV-1 CA antibody.
3
.7. Enzyme-Linked Immunosorbent Assay (ELISA)
To normalize the amount of VSV-G pseudotyped HIV-1 particles used for the infection of HEK
93 cells, the CA content of the particles was determined by sandwich ELISA. The calibration curve was
2
measured using recombinant purified HIV-1 CA protein [64] in the concentration range of 1.4–8.0 ng/mL
in 0.01 M PBS containing 0.05% Tween-20. All samples were analyzed in triplicates in a 96-well plate,
which was coated with 100 µL of rabbit anti-HIV-1 CA polyclonal antibody (1:4000 dilution) in 0.05 M

Molecules 2020, 25, 1895
12 of 22
◦
carbonate-bicarbonate buffer, pH 9.6, overnight at 4 C. The plate was then washed three times with
.01 M PBS containing 0.05% Tween-20 for 10 min at room temperature. The culture medium containing
VSV-G pseudotyped HIV-1 particles was filtered through a 0.45 M filter (Amicon) and diluted (1:100)
with 0.01 M PBS. The viral particles were lysed in 0.01 M PBS containing 1% Triton X-100 for 20 min at
0
µ
room temperature. Aliquots (100
were added to the wells and incubated for 1 h at 37 C. The plate was then washed three times with
µ
L) of the lysed HIV-1 particles or recombinant HIV-1 CA protein
◦
0
.01 M PBS containing 0.05% Tween-20 for 10 min at room temperature, and 100 µL of rabbit anti-HIV-1
CA antibody conjugated with horseradish peroxidase (HRP; 1:1,000 dilution in 0.01 M PBS containing
◦
0
.05% Tween-20) was added. After 1 h incubation at 37 C, the plate was washed four times, and 100
µL
0
0
of the substrate (1 mg of 3,3 ,5,5 -tetramethylbenzidine from Sigma-Aldrich diluted in 1 mL.
DMSO, 0.006% hydrogen peroxide, 0.05 M phosphate-citrate buffer, pH 5.0, from Sigma-Aldrich)
was added. The reaction was stopped by addition of 50 L of 2 M sulfuric acid and the absorbance of
µ
the samples was measured at 450 nm using a Tecan M200 Pro. The HIV-1 CA amount was calculated
using linear regression equation in the linear part of the calibration curve.
3
.8. Single-Round Infectivity Assay
Infectivity was determined similarly as described for Mason-Pfizer monkey virus [65–71]. Briefly,
culture media from HEK 293 cells transfected with psPAX2, pWPXLd-GFP, and pHEF-VSV-G vectors
in a 1:1:1 ratio in the presence of test compounds were collected 48 h post-transfection and filtered
through a 0.45-µm filter. HIV-1 CA content was determined by ELISA. Freshly seeded HEK 293 cells
were infected with ELISA-normalized amounts of VSV-G pseudotyped HIV-1 particles, tested PF74
derivatives or DMSO (as a control) were added at various concentrations and incubated for 48 h.
The cells were fixed with 2% paraformaldehyde and transferred into a fluorescent activated cell
sorting (FACS) tube. Quantification of GFP-positive cells was performed using a BD FACS Aria III
flow cytometer.
3
.9. Flow Cytometry
The sample preparation and measurements were carried out as described elsewhere [68,70,71].
The VSV-G GFP-HIV infected cells were analyzed with a BD FACS Aria III flow cytometer (Becton
Dickinson) with the excitation at 488 nm and the emission separated by a 530/30 band pass filter.
The obtained data were analyzed with Diva 8 software. In total, 10,000 events were evaluated per
sample. First, forward (FSC-A) and side scatter (SSC-A) parameters were used to eliminate dead
cells and debris and to yield population P1. The SSC-A and FSC-H parameters were used to analyze
only single cells and thus obtain population P2. Using a mock (non-infected, GFP-negative) sample,
a proper gating of the GFP-positive cell population was performed for all samples.
3
.10. Synthesis of PF74 Derivatives
The PF74 molecule consisted of three synthons: 2-(2-methyl-1H-indol-3-yl)acetic acid,
L-phenylalanine and N-methylaniline. All compounds were substituted at the site of the 2-(2-methyl-
H-indol-3-yl)acetamide. Phenylalanine N-methylanilide was connected to the carboxylic group of
1
the synthons (D7,D8 (R),D9) replacing an indole-like structure in the PF74 analogs. Formation of the
®
amide bond was carried out with the aid of the coupling agent T3P . The resulting compounds were
purified by column chromatography on silica gel and analytically characterized by NMR and mass
spectrometry. The thin-layer chromatography (TLC) was performed using (TLC Silica gel 60 F254,
Merck, Darmstadt, Germany). TLC detection (254 nm). The column chromatography was performed
using silica gel (100-160
The NMR spectra were acquired using device from Agilent Technologies with a working frequency
of 400 MHz, a chemical shift in ppm ( ), and J-constants in Hz. The orientation LC-MS spectra were
µm, Merck) and a glass column with 2 cm in diameter and 12 cm in height.
δ
acquired using HP series 1100 with quadrupole ionization (Agilent Technologies 6130) with a C18
column. The mass spectra of the final compounds were measured using LC-MS TSQ Quantum Access

Molecules 2020, 25, 1895
13 of 22
Max (Thermo). The final products were identified via H NMR, 13C NMR, Correlation spectroscopy
1
(COSY), HMQC, and MS.
Due to the commercial inaccessibility of the corresponding indole substituents, the following
structures were synthesized (Figure 7):
Figure 7. Syntheses of corresponding indole substituents. (a) Synthesis of 4 and 6, (b) synthesis of 8,
(c) synthesis of 9. i: triphosgene/NaHCO , ii: AlCl , iii: aq. 5% triethylamine, iv: MeI/NaH, v: ammonium
3 3
formate, formic acid, vi: Raney nickel, hydrazine hydrate, vii: H PO /polyphosphoric acid.
3
4
3
.10.1. Methyl (S)-2-isocyanato-3-phenylpropanoate (1)
To a suspension of methyl l-phenylalaninate (700 mg, 3.9 mmol) in CH Cl (10 mL) at 0 C we
◦
2
2
added saturated aqueous NaHCO (10 mL) and triphosgene (387 mg, 1.3 mmol) in a single portion with
3
◦
vigorous stirring. The reaction mixture was stirred at 0 C for 1 h and then poured into a separatory
funnel. The organic phase was washed with brine, dried over Na SO , filtered, and the solvent was
2
4
evaporated to give product 1 as a colorless oil (0.6462 g, 81%) which was used in the next step without
1
further purification. H NMR (CDCl , 400 MHz):
δ 7.37–7.24 (m, 3H,), 7.23–7.17 (m, 2H), 4.27 (dd,
3
J = 7.8, 4.6 Hz, 1H), 3.81 (s, 3H), 3.16 (dd, J = 13.8, 4.6 Hz, 1H), 3.03 (dd, J = 13.8, 4.6 Hz, 1H).
3
.10.2. Methyl (S)-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (2)
To a solution of compound (0.640 g, 3.11 mmol) in dry CH Cl (10 mL) AlCl (0.837 mg,
.25 mmol) was added and the resulting mixture was refluxed for 3 h. The reaction mixture was
1
2
2
3
6
cooled to room temperature and then placed in an ice-water bath. Water (8 mL) was slowly added
and the mixture was stirred for 30 min. The organic layer was separated and washed with brine.
The organic phase was then dried over Na SO . Compound 2 was isolated by evaporation was
2
4
purified via column chromatography (silica gel, hexane/ethyl acetate (EtOAc), 1/1). Yield 348.8 mg,
1
5
5%. TLC (hexane:EtOAc, 1:1 v/v): Rf = 0.21; H NMR (CDCl , 400 MHz):
δ
7.97 (dd, J = 7.7, 1.1 Hz,
3

Molecules 2020, 25, 1895
14 of 22
1
H), 7.38–7.21 (m, 3H), 7.13 (d, J = 7.5 Hz, 1H), 4.34 (ddd, = 8.2, 5.5, 2.5 Hz, 1H), 3.64 (s, 3H), 3.25
+
(
dd, J = 15.8, 5.5 Hz, 1H), 3.12 dd, J = 15.8, 5.5 Hz, 1H); MS: for C H NO (M+H ) m/z 206.1 found;
11 12 3
2
06.1 calculated.
3
.10.3. Triethylammonium Salt of (S)-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (3)
We dissolved 105 mg (0.51 mmol) of compound in 8 mL of 5% N-triethylamine in water
2
and stirred for 2 h. The reaction mixture was freeze dried. Compound 4 was isolated as
a triethylammonium salt without any further purification assuming quantitative conversion (0.150 g,
1
~
100%). TLC (CH Cl :MeOH, 5:1 v/v): Rf = 0.58; H NMR (CD OD, 400 MHz):
δ
7.96 (dd, J = 7.7,
2
2
3
1.1 Hz, 1H), 7.35 (td, J = 7.5, 1.4 Hz, 1H), 7.28–7.20 (m, 1H), 7.17 (d, J = 7.5 Hz, 1H), 6.78 (s, 1H), 4.09
(
ddd, J = 12.9, 4.4, 1.0 Hz, 1H), 3.30–3.16 (m, 2H), 3.09–2.91 (m, 6H), 1.21 (t, J = 7.3 Hz, 9H); MS: for
+
C H NO (M + H ) m/z 192.1 found; 192.1 calculated.
10
10
3
3
.10.4. Methyl 2-methyl-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (4)
NaH (60% dispersion in mineral oil; 56.5 mg, 1.42 mmol) was slowly added to a stirred solution of
2
(116 mg, 0.57 mmol) in DMF (10 mL). MeI (160 mg, 70.4
µ
L, 1.13 mmol) was added subsequently.
◦
◦
The mixture was stirred at 80 C for 1 h. The reaction was quenched with water (8 mL) at 0 C and
extracted with CH Cl . The combined extracts were washed with water and brine and dried over
2
2
Na SO . The solvent was removed. The crude product was purified by column chromatography
2
4
(
silica gel, hexane/EtOAc, 1/1) to give compound 4. Yield 82.3 mg, 66%. TLC (hexane:EtOAc, 1:1 v/v):
1
Rf = 0.33; H NMR (CDCl , 400 MHz):
δ 8.02 (dd, J = 7.7 1.1 Hz, 1H), 7.35 (td, J = 7.5, 1.4 Hz, 1H),
3
7
.38–7.32 (m, 1H), 7.08 (dd, J = 14.9, 5.4 Hz, 1H), 4.21 (dd, J = 6.8, 2.0 Hz, 1H), 3.58 (s, 3H), 3.43 (dd,
+
J = 16.1,6.8 Hz, 1H), 3.26–3.18 (m, 1H), 3.13 (s, 3H); MS: for C H NO (M + H ) m/z 220.1 found;
12
14
3
2
20.1 calculated.
3
.10.5. Triethylammonium Salt of 2-methyl-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (5)
We dissolved 82.3 mg (0.38 mmol) of compound in 8 mL of 5% triethylamine in water and stirred
4
for 2 h. The reaction mixture was freeze dried. Compound 5 was isolated as a triethylammonium
salt without any further purification assuming quantitative conversion. Yield 113.9 mg, 99%. TLC
1
(
CH Cl :MeOH, 5:1 v/v): Rf = 0.72; H NMR (CD OD, 400 MHz):
δ
7.98 (dd, J = 7.7 1.1 Hz, 1H), 7.32
2
2
3
(td, J = 7.5, 1.4 Hz, 1H), 7.36-7.30 (m, 1H), 7.06 (dd, J = 14.9, 5.4 Hz, 1H), 6.52 (s, 1H), 4.18 (dd, J = 6.8,
2
7
.0 Hz, 1H), 3.42 (dd, J = 16.1,6.8 Hz, 1H), 3.24-3.16 (m, 1H), 3.09 (s, 3H), 3.08–2.92 (m, 6H), 1.24 (t, J =
+
.3 Hz, 9H); MS: for C H NO (M+H ) m/z 206.8 found; 206.8 calculated.
11
12
3
3
.10.6. 3-Amino-3-(2-nitrophenyl)propanoic Acid (6)
2
-Nitrobenzaldehyde (2 g, 13.2 mmol), formic acid (85%, 2.5 mL, 37.8 mmol) and malonic acid
◦
(
1.8 g, 17.3 mmol) were stirred at 45 C for half an hour. Then, ammonium formate (2.08 g, 33 mmol)
was added, the reaction temperature was raised to 70 C. We stirred the mixture for 1 h, and then
stirred at 95 C for another 4 h. Concentrated HCl was added (8 mL in 5 min) and the mixture
◦
◦
was further stirred, maintaining this temperature for 1 h. After mixture cooling, 5 mL of water was
added and extracted with EtOAc. The aqueous phase was adjusted to a pH of 4 with 50% NaOH
solution. A slightly yellow solid was obtained. The product was dried over NaOH to obtain 662.4 mg
1
of compound 6 (yield 24%). TLC (CH Cl :MeOH, 10:1 v/v): Rf = 0.35; H NMR (CDCl , 400 MHz):
2
2
3
δ
7.19–7.23 (m, 1H), 6.78–6.68 (m, 3H), 4.41 (q, J = 5.4 Hz, 1H), 2.21 (dd, J = 12.6, 5.7 Hz, 2H); MS: for
+
C H N O (M+H ) m/z 210.1 found; 210.1 calculated.
9
11
2
4
3
.10.7. 2-(1. H-indazol-3-yl)acetic Acid (7)
The compound 6 (502 mg, 2.4 mmol) was dissolved in 2.8 mL of aqueous solution of 5% NaOH
◦
and then 98% hydrazine hydrate (160
µ
L) was added. The reaction was heated to 80 C, and Raney

Molecules 2020, 25, 1895
15 of 22
nickel (5 mg) reduction was carried out. After 30 min, the reaction mixture was cooled and adjusted
to a pH of 2 with concentrated HCl. The precipitated solid was filtered off and dried over NaOH in
1
desiccator to obtain the compound 8 (329.9 mg, 78%). TLC (CH Cl :MeOH, 3:1 v/v): Rf = 0.53; H NMR
2
2
(
3
CD OD, 400 MHz):
.98 (s, 2H); MS: for C H N O (M + H ) m/z 176.1 found; 176.1 calculated.
δ
7.76–7.71 (m, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.39-7.33 (m, 1H), 7.12 (t, J = 7.5 1H),
3
+
9
9
2
2
3
.10.8. (S)-1-(1H-benzo[d]imidazol-2-yl)ethan-1-amine (8)
o-Phenylenediamine (50 mg, 0.46 mmol), l-alanine (42 mg, 0.47 mmol), polyphosphoric acid
◦
(
(
10 mg), and H PO (300
µ
L, 5.6 mmol) were heated at 150 C in the microwave reactor for 2 h
3
4
normal absorption mode). The mixture was diluted with 500 µL of water and the pH was adjusted to
approximately 9 by a saturated aqueous solution of NaOH. The reaction mixture was diluted with 5 mL
of EtOH, cooled to room temperature, and filtered. The solvent from the filtrate was evaporated and
the crude product was purified with the aid of column chromatography (silica gel, DCM/MeOH, 5:1,).
1
3
3.2 mg of compound 8 with 45% yield was obtained. TLC (CH Cl :MeOH, 5:1 v/v): Rf = 0.34; H NMR
2
2
(
3
CD OD, 400 MHz):
δ
11.82 (s, 1H), 7.49–7.45 (m, 2H), 7.12-7.08 (m, 2H), 4.35 (dd, J = 11.2, 8.4 Hz, 1H),
3
+
.25 (s, 1H), 1.89 (d, J = 13.2 Hz, 3H); MS: for C H N (M + H ) m/z 162.2 found; 162.2 calculated.
9
12
3
Pathway to final compounds (Figure 8).
®
®
Figure 8. Synthetic pathway of final derivatives. i: T3P , pyridine, ii: trifluoroacetic acid, iii: Dowex
-
®
1
×
8 in OH form, iv: T3P , pyridine, R-COOH, v: triphosgene, N,N-diisopropylethylamine, R-NH .
2
3
.10.9. Tert-butyl (S)-(1-(methyl(phenyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (DX).
Boc-l-phenylalanine (100 mg, 0.38 mmol) was dissolved in a solution of pyridine and EtOAc
(
5 mL, 3:1, v/v). N-methylaniline (61.3 µ µL, 0.75 mmol) were added.
L, 0.57 mmol) and T3P® (449
The reaction was stirred for 5 h or overnight. The reaction mixture was diluted with EtOAc (5 ml)
and transferred to the separation funnel. The extraction was carried out with 0.5 M HCl and with
0
.5 M NaHCO . The organic phase was dried over dry Na SO and evaporated. The crude product
3 2 4
was then purified using column chromatography (silica gel, hexane/EtOAc, 2:1). 104.2 mg, 77% yield.
1
TLC (hexane:EtOAc, 5:1 v/v): Rf = 0.37; H NMR (CDCl , 400 MHz):
δ 7.34-7.14 (m, 2H), 7.15–6.99 (m,
3
3
H), 6.92-6.62 (m, 5H), 5.47 (d, J = 8.8 Hz, 1H), 4.46 (dd, J = 15.1, 7.2 Hz, 1H), 3.10 (s, 3H), 2.87–2.55 (m,
+
1
H), 1.26 (s, 9H); MS: for C H N O (M + H ) m/z 355.4 found; 355.4 calculated.
21 27 2 3
3
.10.10. (S)-2-amino-N-methyl-N,3-diphenylpropanamide (DX2)
DX (100 mg, 0.28 mmol) was dissolved in 2.5 mL of CH Cl and 2.5 mL of trifluoroacetic acid
2
2
was added while stirred. Trifluoroacetic acid was evaporated and after 30 min, the product was
®
dissolved in CH Cl and subsequently stirred with preactivated Dowex
1
×
8 in hydroxide form to
2
2
remove trifluoroacetic anion. The obtained product was used directly for the next reaction step. TLC
CH Cl :MeOH, 100:1 v/v): Rf = 0.19; MS: for C H N O (M + H ) m/z 255.1 found; 255.1 calculated.
+
(
2
2
16 19
2

Molecules 2020, 25, 1895
16 of 22
3
.10.11. (S)-2-(2-(1H-indol-3-yl)acetamido)-N-methyl-N,3-diphenylpropanamide (D1)
The condensations of compound DX2 (35.9 mg, 0.14 mmol) and 2-(1H-indol-3-yl)acetic acid
16.4 mg, 0.09 mmol) were carried out according to the general method (pg. 13). The compound was
(
purified using column chromatography (silica gel, hexane/EtOAc, 1:1). It was obtained at 34.7 mg,
1
yield 60%. TLC (hexane:EtOAc, 1:1 v/v): Rf = 0.17; H NMR (CDCl , 400 MHz):
δ 8.44 (s, 1H), 7.45 (d,
3
J = 7.9 Hz, 1H), 7.34 (ddd, 6.75, 5.9, 5.0 Hz, 4H), 7.24–7.18 (m, 1H), 7.16–7.14 (m, 1H), 7.13 (d, J = 1.8 Hz
,
1
H), 7.11 (d, J = 3.1 Hz, 2H), 7.08 (s, 1H), 7.06 (s, 1H), 6.98 (d, J = 2.2 Hz, 1H), 6.70 (d, J = 7.1 Hz, 2H),
6
.30 (d, J = 8.3 Hz, 1H), 4.82 (dd, J = 15.3, 7.1 Hz, 1H), 3.66 (d, J = 1.7 Hz, 2H), 3.17 (s, 3H), 2.76 (dd,
171.36, 170.76,
42.40, 136.35, 136.05, 129.73, 129.17, 128.26, 128.11, 127.29, 127.04, 126.68, 123.67, 122.35, 119.82, 118.68,
J = 13.3, 7.1 Hz, 1H), 2.57 (dd, J = 13.3, 7.1 Hz, 1H); 13C NMR (CDCl , 400 MHz):
δ
3
1
1
4
+
11.32, 108.64, 51.10, 38.81, 37.60, 33.28; HRMS/ESI: for C H N O (M + H ) m/z 412.20226 found;
12.20195 calculated, for C H N O Na (M + Na ) m/z 434.18420 found; 434.18390 calculated.
26
26
3
2
+
26
25
3
2
3
.10.12. (S)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)-N-methyl–N,3-
diphenylpropanamide (D2)
The condensations of compound DX2 (46.6 mg, 0.18 mmol) and indomethacin (43.6 mg,
0.12 mmol) were carried out according to general method. The compound was purified using
column chromatography (silica gel, hexane/EtOAc, 2:1). It was obtained at 62.9 mg, yield 58%.
1
TLC (hexane:EtOAc, 1:1 v/v): Rf = 0.47; H NMR (CDCl , 400 MHz):
δ 7.66–7.62 (m, 2H), 7.50–7.45
3
(
(
1
m, 2H), 7.41–7.32 (m, 3H), 7.12 (ddd, J = 6.3, 3.7, 1.3 Hz, 1H), 7.09–7.03 (m, 2H), 7.01 (s, 1H), 6.98
d, J = 6.3 Hz, 2H), 6.84 (d, J = 2.5 Hz, 1H), 7.66–7.62 (m, 1H), 6.70–6.66 (m, 2H), 6.25 (d, J = 8.1 Hz,
H), 4.81 (dd, J = 14.9, 6.9 Hz, 1H), 3.80 (s, 3H), 3.55 (d, J = 5.1 Hz, 2H), 3.21 (s, 3H), 2.78 (dd, J = 13.4,
.6 Hz, 1H), 2.58 (dd, J = 13.4, 7.0 Hz, 1H), 2.4 (s, 3H); 13C NMR (CDCl , 400 MHz):
171.02, 169.13,
6
δ
3
168.27, 156.24, 142.42, 139.26, 135.96, 135.86, 133.80, 131.22, 130.96, 130.30, 129.92, 129.15, 129.10, 128.27,
1
28.18, 127.31, 126.75, 115.10, 112.81, 112.31, 100.85, 55.72, 51.15, 38.52, 37.67, 32.11, 13.41; HRMS/ESI:
+
for C H ClN O (M+H ) m/z 594.21566 found; 594.21541 calculated, for C H ClN O Na (M +
Na ) m/z 616.19782 found; 616.19736 calculated, for C H ClN O K (M + K ) m/z 632.17092 found;
35
33
3
4
35 32
3
4
+
+
35
32
3
4
6
32.17129 calculated.
3
.10.13. (S)-2-((R)-2-(4-isobutylphenyl)propanamido)-N-methyl-N,3-diphenylpropanamide (D4(R))
The condensations of compound DX2 (57.4 mg, 0.23 mmol) and racemic ibuprofen (31 mg,
0.15 mmol) were carried out according to the general method. The compound was purified using
column chromatography (silica gel, hexane/EtOAc, 5:1). The yields of both diastereomers were 68%
67.6 mg). The yield of D4(R) was 30% (29.8 mg). The product was identified by comparison with
(
an optically pure standard prepared in the same procedure using the R-ibuprofen. TLC (hexane:EtOAc,
1
3
:1 v/v): Rf = 0.15; H NMR (CDCl , 400 MHz):
δ
7.35 (tdd, J = 7.45, 5.9, 1.7 Hz, 2H), 7.18–7.12 (m, 1H),
3
7.09 (t, J = 7.3 Hz, 3H), 7.05 (s, 4H), 6.97 (d, J = 6.3 Hz, 2H), 6.67 (d, J = 7.1 Hz, 2H), 6.03 (s, 1H), 4.84
(
dd, J = 15.3, 7.0 Hz, 1H), 3.48 (q, J = 7.1 Hz, 1H), 3.21 (s, 3H), 2.74 (dd, J = 13.4, 6.6 Hz, 1H), 2.54 (dd,
J = 13.4, 6.6 Hz, 1H), 2.45 (d, J = 7.2 Hz, 2H), 1.85 (tt, J = 13.5, 6.7 Hz, 1H), 1.40 (d, J = 7.1 Hz, 3H),
.90 (d, J = 6.6 Hz, 6H); 13C NMR (CDCl , 400 MHz):
173.41, 171.44, 142.44, 140.44, 138.48, 136.08,
29.80, 129.48, 129.16, 128.23, 128.11, 127.29, 126.62, 50.91, 46.43, 45.04, 38.70, 37.62, 30.17, 22.39, 18.34;
0
1
δ
3
+
HRMS/ESI: for C H N O (M + H ) m/z 443.26922 found; 443.26930 calculated, for C H N O Na
29
35
2
2
29 34
2
2
+
+
(
M + Na ) m/z 465.25132 found; 465.25125 calculated, for C H N O K (M + K ) m/z 481.22491 found;
29 34 2 2
4
81.22519 calculated.
3
.10.14. (S)-N-((S)-1-(methyl(phenyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-oxo-1,2,3,4-
-
tetrahydroisoquinoline-3-carboxamide (D7)
The condensations of compound DX2 (35.9 mg, 0.14 mmol) and compound 3 (17.9 mg,
0
.09 mmol) were carried out according to the general method. Compound was purified using column
chromatography (silica gel, hexane/EtOAc, 3:2). It was obtained 56.8 mg, yield 90%. TLC (EtOAc):

Molecules 2020, 25, 1895
17 of 22
Rf = 0.26; 1H NMR (CDCl , 400 MHz):
δ
8.04 (dd, J = 7.6, 1.1 Hz, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.61
3
(s, 1H), 7.43–7.38 (m, 1H), 7.15–7.11 (m, 1H), 7.10–7.08 (m, 2H), 7.06–7.04 (m, 1H), 7.00–6.93 (m, 2H),
6
3
.73–6.68 (m, 2H), 4.79 (dd, J = 14.7, 8.0 Hz, 1H), 4.29 (td, J = 6.2, 3.6 Hz, 1H), 3.26 (t, J = 6.0 Hz, 2H),
13
.20 (s, 3H), 2.83 (dd, J = 13.4, 6.4 1H), 2.68 (dd, J = 13.4, 6.4 Hz, 1H); C NMR (CDCl , 400 MHz):
δ
3
1
1
71.68, 170.24, 165.52, 142.25, 136.57, 136.12, 132.50, 129.82, 129.02, 128.33, 128.03, 127.52, 127.28, 126.65,
+
25.94, 124.32, 54.06, 51.75, 38.46, 37.80, 30.94; HRMS/ESI: for C H N O Na (M + Na ) m/z 450.17911
26
25
3
3
+
found; 450.17881 calculated, for C H N O K (M + K ) m/z 466.15254 found; 466.15275 calculated.
26
25
3
3
3
.10.15. (R)-2-methyl-N-((S)-1-(methyl(phenyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-oxo–1,2,3,4-
tetrahydroisoquinoline-3-carboxamide (D8(R))
The condensations of compound DX2 (39.5 mg, 0.15 mmol) and the compound 5 (21.2 mg,
0
.10 mmol) were carried out according to general method. The compound was purified using column
chromatography (silica gel, hexane/EtOAc, 3:2). Both obtained diastereomers were further separated.
The yields of both diastereomers were 60% (43.2 mg). Yield of D8(R) 35% (25.2 mg). TLC (hexane:EtOAc,
1
3
:2 v/v): Rf = 0.38; H NMR (CDCl , 400 MHz):
δ
8.04 (dd, J = 7.5, 1.4 Hz, 1H), 7.39–7.28 (m, 5H, H-8,
3
H10), 7.09–7.01 (m, 3H), 7.00-6.90 (m, 4H), 6.45 (d, J = 7.2 Hz, 2H), 4.72 (td, J = 8.2, 5.9 Hz, 1H), 4.07 (dd,
J = 7.1, 2.2 Hz, 1H), 3.35 (dd, J = 16.2, 7.2 Hz, 1H), 3.23 (s, 3H), 3.20 (s, 3H), 2.63 (dd, J = 13.6, 5.8 Hz,
13
1
H), 2.37 (dd, J = 13.6, 5.8 Hz, 1H); C NMR (CDCl , 400 MHz):
δ
170.95, 169.50, 164.29, 142.24, 135.64,
3
1
3
35.15, 132.05, 129.87, 128.68, 128.32, 128.25, 127.97, 127.33, 127.26, 126.60, 61.98, 51.27, 38.47, 37.70,
+
5.12, 30.67; HRMS/ESI: for C H N O Na (M + Na ) m/z 464.19472 found; 464.19446 calculated, for
27 27 3 3
+
C H N O K (M + K ) m/z 480.16794 found; 480.16840 calculated.
27
27
3
3
3
.10.16. (S)-2-methyl-N-((S)-1-(methyl(phenyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-oxo–1,2,3,4-
tetrahydroisoquinoline-3-carboxamide (D8(S))
The condensations of compound DX2 (39.5 mg, 0.15 mmol) and the compound 5 (21.2 mg,
0.10 mmol) were carried out according to the general method. The compound was purified using
column chromatography (silica gel, hexane/EtOAc, 3:2). Both obtained diastereomers were further
separated. The yields of both diastereomers were 60% (43.2 mg). The yield of D8(S) was 25% (18 mg).
1
TLC (hexane:EtOAc, 3:2 v/v): Rf = 0.22; H NMR (CDCl , 400 MHz):
δ 8.07 (dd, J = 7.6, 1.3 Hz, 1H), 7.39
3
(
tt, J = 6.1, 3.0 Hz, 1H), 7.36–7.29 (m, 1H), 7.29–7.24 (m, 3H), 7.21–7.14 (m, 3H), 7.08 (d, J = 7.4 Hz, 1H),
6.82 (dd, J = 7.1, 2.3 Hz, 2H), 6.71–6.65 (m, 2H), 6.60–6.53 (m, 1H), 4.65 (dd, J = 15.3, 7.3 Hz, 1H), 4.05
(
1
dd, J = 6.8, 2.3 Hz, 1H), 3.14 (dd, J = 16.1, 2.5 Hz, 1H), 3.10 (s, 3H), 3.07 (s, 3H), 2.84 (dd, J = 13.3, 7.2 Hz,
13
H), 2.61 (dd, J = 13.3, 7.2 Hz, 1H); C NMR (CDCl , 400 MHz):
δ
170.70, 169.16, 164.53, 142.15, 135.98,
3
1
3
34.95, 131.86, 129.77, 129.17, 128.40, 128.17, 128.11, 127.40, 127.05, 126.86, 61.81, 51.23, 38.60, 37.56,
+
4.94, 31.16; HRMS/ESI: for C H N O Na (M + Na ) m/z 464.19473 found; 464.19446 calculated, for
27 27 3 3
+
C H N O K (M + K ) m/z 480.16840 found; 480.16840 calculated.
27
27
3
3
3
.10.17. (S)-2-(2-(1H-indazol-3-yl)acetamido)-N-methyl-N,3-diphenylpropanamide (D9)
The condensations of compound DX2 (37.3 mg, 0.15 mmol) and compound 7 (17.1 mg,
.10 mmol) were carried out according to the general method. The compound was purified using
0
column chromatography (silica gel, hexane/EtOAc, 2:1). It was obtained at 36.2 mg, yield 57%.
1
TLC (hexane:EtOAc, 2:1 v/v): Rf = 0.23; H NMR (CDCl , 400 MHz):
δ 8.13 (d, J = 8.5 Hz, 1H), 7.73 (d,
3
J = 8.1 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.34 (dd, J = 8.0, 7.2 Hz, 1H), 7.30–7.24 (m, 3H), 7.15–7.01 (m,
4
8
H), 6.85-6.73 (m, 4H), 4.88 (dd, J = 15.2, 8.0 Hz, 1H), 4.09-3.91 (m, 2H), 3.21 (s, 3H), 2.90 (dd, J = 13.2,
.0 Hz, 1H), 2.70 (dd, J = 13.2, 8.0 Hz, 1H); 13C NMR (CDCl , 400 MHz):
δ
172.00, 168.90, 142.21, 141.00,
3
1
3
40.27, 136.20, 129.61, 129.25, 128.21, 128.07, 127.25, 126.71, 126.66, 122.18, 120.55, 120.38, 110.18, 51.39,
+
9.15, 37.73, 35.35; HRMS/ES: for C H N O Na (M + Na ) m/z 435.17920 found; 435.17915 calculated,
25
24
4
2
+
for C H N O K (M + K ) m/z 451.15289 found; 451.15308 calculated.
27
27
3
3

Molecules 2020, 25, 1895
18 of 22
3
.10.18. (S)-2-(3-((1H-benzo[d]imidazol-2-yl)methyl)ureido)-N-methyl-N,3-diphenylpropanamide
(
D10)(General procedure of urea-linked derivatives preparation)
◦
The compound DX2 (45.4 mg, 0.18 mmol) was dissolved in 5 mL of CH Cl and cooled to 0 C.
2
2
N,N-diisopropylethylamine (311 µL, 1.78 mmol) and triphosgene (17.5 mg, 0.06 mmol) were then
added. The (1H-benzo[d]imidazol-2-yl)methanamine dihydrochloride (59.4 mg, 0.27 mmol) was added
after 30 min. The mixture was stirred at room temperature overnight. The solvent was evaporated
and obtained compound D10 was crystallized from acetonitrile. We obtained 21.6 mg, yield 28%.
1
TLC (CH Cl :MeOH, 10:1 v/v): Rf = 0.59; H NMR (CD OD, 600 MHz):
δ 7.53 (dd, J = 5.8, 3.2 Hz,
2
2
3
2
H), 7.43–7.36 (m, 2H, H-8), 7.22 (dd, J = 6.0, 3.2 Hz, 2H), 7.20–7.16 (m, 3H), 7.09 (s, 2H), 6.88 (dd,
J = 6.4 2.7 Hz, 2H), 4.87 (s, 2H), 4.62–4.55 (m, 2H), 4.50 (d, J = 16.4 Hz, 1H), 3.24 (s, 3H), 2.94 (dd,
J = 13.4 171.30, 157.82,
6.5 Hz, 1H), 2.70 (dd, J = 13.4, 8.1 Hz, 1H); 13C NMR (CD OD, 600 MHz):
53.43, 143.40, 137.95, 129.96, 129.35, 128.56, 128.19, 127.96, 126.81, 122.32, 118.96, 115.93, 52.40, 38.68,
,
,
δ
3
1
3
+
8.13, 37.58; HRMS/ESI: for C H N O (M + H ) m/z 428.20815 found; 428.20810 calculated, for
25
26
5
2
+
+
C H N O Na (M + Na ) m/z 450.19001 found; 450.19005 calculated, for C H N O K (M + K ) m/z
25
25
5
2
25 25
5
2
4
66.16356 found; 466.16398 calculated.
3
.10.19. (S)-2-(3-((S)-1-(1H-benzo[d]imidazol-2-yl)ethyl)ureido)-N-methyl-N,3-
-
diphenylpropanamide (D11)
The connection of DX2 (46.6 mg, 0.18 mmol) to the compound 8 (43.5 mg, 0.27 mmol) was done
through a urea bridge according to the general procedure (see above). It was purified using column
chromatography (silica gel, DCM/MeOH, 30:1). We obtained 25 mg, yield 33%. TLC (CH Cl :MeOH,
2
2
1
1
0:1 v/v): Rf = 0.63; H NMR (DMSO-d , 600 MHz):
δ
7.68 (s, 2H), 7.46-7.32 (m, 3H), 7.18 (d, J = 6.6 Hz
H), 7.13-7.03 (m, 3H), 6.83 (d, J = 5.0 Hz, 2H), 6.76 (d, J = 7.0 Hz, 2H), 6.61 (d, J = 8.2 Hz, 1H),
.05–4.98 (m, 1H), 4.18–4.13 (m, 1H), 3.52 (s, 2H), 3.15 (s, 3H), 2.79 (dd, J = 13.4, 4.5 Hz, 1H), 2.53 (dd,
172.14, 157.32, 157.01,
43.30, 141.23, 137.76, 130.00, 129.31, 128.52, 128.24, 127.92, 126.77, 124.95, 114.71, 52.33, 43.93, 38.63,
,
6
2
5
J = 13.4, 4.5 Hz, 1H), 1.54 (d, J = 6.8 Hz, 3H); 13C NMR (DSMO-d , 600 MHz):
δ
6
1
3
+
7.58, 19.38; HRMS/ESI: for C H N O (M + H ) m/z 422.22388 found; 422.22375 calculated, for
26
28
5
2
+
+
C H N O Na (M + Na ) m/z 464.20559 found; 464.20570 calculated, for C H N O K (M + K ) m/z
26
27
5
2
26 27
5
2
4
80.17923 found; 480.17963 calculated.
3
.11. The Cyclosporin A (CsA) Washout Assay
Owl monkey kidney (OMK) cells were seeded in a 48-well plate at a concentration of 15,000 cells
per well. The next day, Eagle’s Minimum Essential Medium (EMEM) was replaced with medium
containing 2.5 M CsA and polybrene (5 ng/ L), and the OMK cells were spinoculated for 1 h with
a normalized amount of HIV-1 particles in the presence or absence of PF74 and D7, D8(R), and D9
derivatives (10 M). Two hours after infection, the medium was replaced with fresh medium containing
.5 M CsA. At various times, the cultivation medium containing CsA was removed and replaced with
µ
µ
µ
2
µ
fresh medium without CsA. After 48 h, the cells were fixed with 2% formaldehyde and GFP-positive
cells were counted using flow cytometry (BD FACS AriaIII).
3
.12. NMR Titration
The NMR sample contained 0.6 mM CA-NTD in phosphate buffer, 100 mM NaCl (pH 6.0),
and 10% D O. The NMR spectra were acquired at 25 deg on Bruker Avance III 600 MHz equipped with
2
15
13
1
N/ C/ H triple resonance, cryogenically cooled probe, and were analyzed in Sparky. The inhibitors
were added in DMSO and the ratios were (inhibitor:protein) 1:8, 1:4, 1:2, 1:1, and 2:1. The NMR data
were processed in Topspin 3.6 and analyzed in Sparky.
Author Contributions: A.D. was engaged in the FAITH, DITH, ELISA, and CsA washout assay; F.K. participated
in the CANC and CA-NTD protein expression/purification and initial NMR analysis; K.Š. prepared and purified the
PF74 derivatives; I.K. contributed the single round VSV-G HIV infectivity assay and flow cytometry measurement;
RoH performed the TEM analysis; M.F. and R.H. designed the PF74 derivatives; R.H. performed the NMR analysis;

Molecules 2020, 25, 1895
19 of 22
T.R. participated in the research design and manuscript preparation; M.R. did the research and experiment design
and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.
ˇ
Funding: This research was funded by GA CR(CZ) 17-25602S and 20-19906S.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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Sample Availability: Samples of all compounds are available from the authors.
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