Design, synthesis and characterization of HIV-1 ca-targeting small molecules: Conformational restriction of PF74

Article abstract of DOI:10.3390/v13030479

Small molecules targeting the PF74 binding site of the HIV-1 capsid protein (CA) confer potent and mechanistically unique antiviral activities. Structural modifications of PF74 could further the understanding of ligand binding modes, diversify ligand chemical classes, and allow identification of new variants with balanced antiviral activity and metabolic stability. In the current work, we designed and synthesized three series of PF74-like analogs featuring conformational constraints at the aniline terminus or the phenylalanine carboxamide moiety, and characterized them using a biophysical thermal shift assay (TSA), cell-based antiviral and cytotoxicity assays, and in vitro metabolic stability assays in human and mouse liver microsomes. These studies showed that the two series with the phenylalanine carboxamide moiety replaced by a pyridine or imidazole ring can provide viable hits. Subsequent SAR identified an improved analog 15 which effectively inhibited HIV-1 (EC₅₀ = 0.31 μM), strongly stabilized CA hexamer (?Tm = 8.7^?C), and exhibited substantially enhanced metabolic stability (t_1/2 = 27 min for 15 vs. 0.7 min for PF74). Metabolic profiles from the microsomal stability assay also indicate that blocking the C5 position of the indole ring could lead to increased resistance to oxidative metabolism.

Full text of DOI:10.3390/v13030479

viruses
Article
Design, Synthesis and Characterization of HIV-1 CA-Targeting
Small Molecules: Conformational Restriction of PF74
1
, Raquel Diana-Rivero , Sanjeev Kumar V. Vernekar , Lei Wang , Haijuan Du 2,3,
1
1
1
Rajkumar Lalji Sahani
2
,3
2,3
4
2,3
2,3
Huanchun Zhang , Andres Emanuelli Castaner , Mary C. Casey , Karen A. Kirby
Jiashu Xie , Stefan G. Sarafianos
, Philip R. Tedbury
,
1
2,3
1,
and Zhengqiang Wang *
1
2
Center for Drug Design, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA;
rsahani@umn.edu (R.L.S.); raquel.diana.rivero@gmail.com (R.D.-R.); vvsanjeev99@gmail.com (S.K.V.V.);
leiwang@dlut.edu.cn (L.W.); jxie@umn.edu (J.X.)
Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine,
Atlanta, GA 30322, USA; haijuan.du@emory.edu (H.D.); huanchun.zhang@emory.edu (H.Z.);
andres.emanuelli@emory.edu (A.E.C.); karen.kirby@emory.edu (K.A.K.); philip.tedbury@emory.edu (P.R.T.);
stefanos.sarafianos@emory.edu (S.G.S.)
3
4
Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA
Department of Molecular Microbiology and Immunology, School of Medicine, Christopher S. Bond Life
Sciences Center, University of Missouri, Columbia, MO 65211, USA; mcc6x2@mail.missouri.edu
Correspondence: wangx472@umn.edu; Tel.: +1-612-626-7025
*
Abstract: Small molecules targeting the PF74 binding site of the HIV-1 capsid protein (CA) confer
potent and mechanistically unique antiviral activities. Structural modifications of PF74 could further
the understanding of ligand binding modes, diversify ligand chemical classes, and allow identification
of new variants with balanced antiviral activity and metabolic stability. In the current work, we
designed and synthesized three series of PF74-like analogs featuring conformational constraints
at the aniline terminus or the phenylalanine carboxamide moiety, and characterized them using
a biophysical thermal shift assay (TSA), cell-based antiviral and cytotoxicity assays, and in vitro
metabolic stability assays in human and mouse liver microsomes. These studies showed that the
two series with the phenylalanine carboxamide moiety replaced by a pyridine or imidazole ring can
provide viable hits. Subsequent SAR identified an improved analog 15 which effectively inhibited
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Sahani, R.L.; Diana-Rivero,
R.; Vernekar, S.K.V.; Wang, L.; Du, H.;
Zhang, H.; Castaner, A.E.; Casey,
M.C.; Kirby, K.A.; Tedbury, P.R.; et al.
Design, Synthesis and
Characterization of HIV-1
CA-Targeting Small Molecules:
Conformational Restriction of PF74.
Viruses 2021, 13, 479. https://
doi.org/10.3390/v13030479
◦
HIV-1 (EC50 = 0.31 µM), strongly stabilized CA hexamer (∆Tm = 8.7 C), and exhibited substantially
enhanced metabolic stability (t1/2 = 27 min for 15 vs. 0.7 min for PF74). Metabolic profiles from the
microsomal stability assay also indicate that blocking the C5 position of the indole ring could lead to
increased resistance to oxidative metabolism.
Academic Editor: Gilda Tachedjian
Received: 11 February 2021
Accepted: 10 March 2021
Published: 15 March 2021
Keywords: HIV-1; capsid protein; PF74; conformational constraint; metabolic stability
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1
. Introduction
The capsid protein (CA) of human immunodeficiency virus type 1 (HIV-1), expressed
as part of the viral Gag polyprotein, plays a critical role in multiple steps of the viral
replication cycle [ ]. In the late stage, CA–CA interactions drive the assembly of Gag
polyproteins, and the reassembly of released CAs, toward immature and mature viral
capsids, respectively [ ]. In the early stage, productive HIV-1 infection entails numerous
CA-mediated post-entry events [ ] up to the completion of integration, including uncoating,
1
2
1
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
cytoplasmic trafficking, reverse transcription, nuclear import, and integration site targeting.
These events typically involve CA binding by host proteins, such as nucleoporin 153
(
NUP153) [
which are required for the nuclear import of HIV-1 pre-integration complexes (PICs). CPSF6
is also required for viral integration site targeting [ ]. In addition, the CA-cyclophilin
A (CypA) interaction allows HIV-1 to evade restriction by host factor tripartite motif-
3] and cleavage and polyadenylation specificity factor 6 (CPSF6) [4,5], both of
6,7
4
.0/).
containing protein 5 alpha (TRIM5a) [
8]. Therefore, CA-targeting small molecules could
Viruses 2021, 13, 479. https://doi.org/10.3390/v13030479
https://www.mdpi.com/journal/viruses

Viruses 2021, 13, 479
2 of 17
inhibit viral replication via two distinct mechanisms of action: by disrupting CA–CA
interactions, they could alter the overall core stability and impact viral assembly and
uncoating; by competing against NUP153 and CPSF6 for CA binding, they could block
viral nuclear entry and/or productive integration. These mechanisms, along with CA as an
antiviral target, have been strongly validated with a few chemically distinct CA-targeting
small molecule chemotypes [9], including mainly BI-2 [10], BM-4, BD-3 [11], CAP-1 [12],
PF74 [13], GS-6207 (lenacapavir) [14
,15]. Structurally, the CA monomer contains two
primarily helical domains: the N-terminal domain (CANTD) comprised of seven alpha-
helices H1-7, and the C-terminal domain (CACTD) consisted of four alpha-helices H8-
1
1 [16,17]. Reported CA-binding compounds predominantly target the CANTD where
multiple binding sites have been identified. Amongst these, the PF74 binding pocket [17] is
particularly well-characterized and significant. PF74 binds to the interface formed between
the H3 and H4 of CANTD and the H8 and H9 of the adjacent CACTD within a CA hexamer
(Figure 1A) [13,17]. In this binding pocket, PF74 interacts with N57 of H3 through the
carbonyl-O4 and N(2)-H of the phenylalanine moiety (Figure 1C), K70 and Q63 of H4
through the other carbonyl-O3 and the indole N(1)-H, and several other residues, N53,
M66, A105, T107, and Y130, though hydrophobic interactions (Figure 1A). Two other
CA-targeting chemotypes, BI-2 [10] and GS-6207 [14,15], also bind to the same pocket,
with very similar backbone binding (Figure 1B), despite their hugely different structural
complexities (Figure 1C). Furthermore, the PF74 binding site also accommodates CPSF6
and NUP153, the host factors required for nuclear entry and integration site targeting of
viral PICs (Figure 1D) [18]. Therefore, small molecules targeting this binding pocket can
interfere with both early and late events of viral replication, and confer unique antiviral
profiles with a bimodal mechanism of action, as manifested with PF74 and GS-6207 [19].
Although GS-6207 is far superior in antiviral potency to PF74, it lacks sufficient
aqueous solubility to be an oral drug. Furthermore, mutations conferring resistance to
GS-6207 have been selected in vitro [14,15]. One of the mutations that appeared in vitro,
Q67H, was reported in recent clinical trials to be present in a patient sample, after nine
days of treatment with GS-6207 [14]. On the other hand, PF74 suffers from extremely low
metabolic stability [20–23]. These deficiencies necessitate continued medicinal chemistry
efforts in identifying additional chemotypes targeting the PF74 pocket. For this purpose, the
structural simplicity of PF74 offers a distinct synthetic chemistry advantage over GS-6207
for chemical modifications. We have previously pursued the design and synthesis of PF74-
like compounds and have identified several subtypes with improved antiviral potency
and/or enhanced metabolic stability [20–23]. The current work studies the impact of
overall molecular rigidity on CA targeting and metabolic stability. The primary rationale is
that binding to a protein typically results in the conformational/configurational restriction
of a ligand, and hence, an entropy loss that needs to be compensated for, the amount of
which will be reduced if the ligand is conformationally restricted to begin with [24]. The
importance of rigidity in small molecule inhibitor design has been demonstrated [25
In addition, PF74 features an aniline N-methyl group, which is a well-known metabolic
handle [27 28] and a likely culprit of the poor metabolic stability of PF74. In our redesign of
,26].
,
PF74 (Figure 2), rigidity is introduced either to the aniline moiety by capping the N-methyl
and the ortho–phenyl end of PF74 (modification A, chemotype 1), or to the phenylalanine
amide moiety by capping the N-methyl and the amide carbonyl (modification B, chemotype
2
and chemotype 3). The subsequent structure–activity relationship (SAR) also explores
previously reported [20
,
22] key interactions, such as halogen bonding conferred by the
1
para-halogen (Figure 2, R ) of the aniline phenyl to N74, hydrophobic interaction by the
4
3
indole N-Et (R ), and H-bonding by the indole C5-OH (R ) to K182 of the adjacent CACTD.

Viruses 2021, 13, 479
3 of 17
Figure 1. Structure of HIV-1 capsid protein (CA) and key CA-ligand and -host binding pocket. (
capsid protein (PDB code: 4XFZ [17]). PF74 occupies a binding pocket formed around alpha-helices H3 and H4 in CANTD
grey) and H8 and H9 in adjacent CACTD (orange). Two other CA-targeting small molecule chemotypes, BI-2 [10] and
GS-6207 [14 15], as well as host proteins CPSF6 and NUP153 [18], also occupy this same pocket; ( ) superimposition of PF74,
BI-2, and GS-6207 in the same binding pocket; ( ) chemical structures of PF74, BI-2 and GS-6207, and amino acid sequence
of host protein CPSF6 derived peptide. The boxed (cyan) phenylalanine residue corresponds with the phenylalanine moiety
A) Binding of PF74 on HIV-1
(
,
B
C
of PF74 in the binding pocket; (
binding pocket.
D) superimposition of PF74 and CPSF6 derived peptide (PDB code: 4U0A [18]) in the same
Figure 2. Design of conformationally constrained chemotypes based on PF74.

Viruses 2021, 13, 479
4 of 17
2. Materials and Methods
2.1. Chemistry
All tested analogs were synthesized as described in Schemes 1–3, and were fully
1
13
characterized with H and C NMR, and HRMS. Synthetic procedures and compound
characterization data are included in Supplementary Materials. The general synthetic
strategies for major analogs (1–25) tested in this work are described here (Schemes 1–3) and
the synthesis of other intermediates is outlined in supporting information (Supplementary
Schemes S1–S3).
Scheme 1. Synthesis of analogs 1–3. Reagents and conditions: (a) L-phenylalanine methyl ester
hydrochloride, HATU, DIPEA, DMF, rt, 12 h; (
b
) LiOH, THF, rt, 12 h; (c) amine, PyOAP, DIPEA, DMF,
rt, 12 h.
◦
Scheme 2. Synthetic strategy for analogs
then MeOH, rt, 2 h; ( ) DIBAL-H, THF,
) benzyl-magnesium chloride, DCM,
4
–
19. Reagents and conditions: (
d
) (COCl) , cat. DMF, DCM, 0 C
−
rt, 2 h,
2
◦
e
−
78 C, 3 h; (
◦
f
) (R)-(+)-2-methyl-2-propanesulfinamide, CuSO , DCM, rt, 3 h;
4
◦
(
g
−
78 C
−
rt, 5 h; (
h
) boronic acid, Pd(PPh ) Cl , K CO , DME, MW, 120 C 45 min.;
3
2
2
2
3
(
i) 4N HCl/dioxane, MeOH, rt, 1 h; (j) acid, HATU or T P, DIPEA, DMF, rt, 12 h.
3

Viruses 2021, 13, 479
5 of 17
◦
Scheme 3. Synthetic Strategy for Analogs 20
–
25. Reagents and conditions: (
k
) glyoxal, NH (g), MeOH,
−
78 C
−
rt, 4 days;
3
(
l
) boronic acid, Cu(OAc) , DIPEA, DCM, 4Å MS, rt, 4 days; (
m) TFA, DCM, rt, 12 h; (
n
) acid, HATU, DIPEA, DMF, rt, 12 h.
2
Chemotype 1 was synthesized according to procedures describe in Scheme 1 [20].
Commercially available indole-acetic acids 26 were treated with commercially available
L-Phenylalanine methyl ester hydrochloride under a well-established strategy using HATU
in the presence of DIPEA to afford 27. LiOH hydrolysis of intermediates 27 afforded acid
intermediates 28, which were further reacted with corresponding indoline or tetrahydro-
quinoline to give the desired compounds 1–3.
The synthesis of intermediates 30
a route described in Scheme 2 [29 30]. Commercially available 3-bromopicolinic acid
was converted to 3-bromopicolinaldehyde 30 through oxalyl chloride/DMF assisted
esterification, followed by DIBAL-H reduction. Intermediate 30 was then treated with
–37 and major analogs (4–19) was achieved using
,
29
commercially available (R)-(+)-2-Methyl-2-propanesulfinamide to form sulfonimine 31
which was subjected to Grignard addition of benzyl-magnesium chloride to produce two
diastereomers 32 and 33 in 3:1 ratio. Subsequently, intermediate 32 was subjected to Suzuki
coupling with various boronic acids to produce intermediates 34, which upon deprotection
furnished intermediates 35. Finally, intermediates 35 were reacted with corresponding
,
acids with HATU in the presence of DIPEA to afford compounds
4–8. Similarly, the
synthesis of analogs 19 was accomplished from intermediate 33, with the exception
9–
of using T P in place of HATU in the final amide-coupling reaction for the synthesis of
3
compound 15.
Analogs 20
intermediates 39
–
–
25 were synthesized as described in Scheme 3 [30]. The synthesis of
41 is discussed in detail in the supporting information (Scheme S3).
Commercially available N-boc phenyl-alaninal 38 and glyoxal were treated with ammonia
to construct imidazole ring intermediate 39, which was subjected to copper catalyzed Chan-
Lam reaction with various boronic acids to produce intermediate 40. TFA deprotection of
Boc group resulted in intermediate 41, which was reacted with various indole-acetic acids
using HATU in the presence of DIPEA to afford compounds 20–25.
2
.2. Cells
TZM-GFP cells are a modified version of TZM-bl cells and contain an integrated
nlsGFP reporter gene under the transcriptional control of the HIV-1 long terminal repeat
LTR) [31 32]; TZM-GFP cells produce GFP following infection with HIV-1. TZM-GFP cells
(
,
were kindly provided by Dr. Marc Johnson (University of Missouri-Columbia, Columbia,
MO, USA) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS;
Hyclone, Logan, UT, USA). HEK293-FT cells were cultured in DMEM supplemented with
1
0% FBS. MT-2 cells were grown in RPMI supplemented with 10% heat-inactivated FBS. All
◦
cells were grown and maintained in humidified atmosphere containing 5% CO at 37 C.
2
2.3. Method Details
2
.3.1. Thermal Shift Assays (TSAs) to Screen Compounds for Effect on HIV-1 CA
Hexamer Stability
Compounds were screened for binding using purified covalently-crosslinked hexam-
A14C/E45C/W184A/M185A
eric CA
(CA121). CA121 cloned in a pET11a expression plasmid was
kindly provided by Dr. Owen Pornillos (University of Virginia, Charlottesville, VA, USA).
Protein was expressed in E. coli BL21(DE3)RIL and purified as reported previously [33].
The TSA has been previously described [20
–23]. Briefly, the TSA was conducted on the
PikoReal Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) or the

Viruses 2021, 13, 479
6 of 17
QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific, Waltham MA, USA). Each
reaction contained 7.5
µ
M final concentration CA121, 1
×
Sypro Orange Protein Gel Stain
(Life Technologies, Carlsbad, CA, USA) in 50 mM sodium phosphate buffer (pH 8.0) and
1
9
% DMSO (control) or 20
5 C with a heating rate of 0.2 C/10 s. The fluorescence intensity was measured with
µ
M compound in 1% DMSO. The plate was heated from 25 to
◦
◦
an Ex range of 475–500 nm and Em range of 520–590 nm. The differences in the melting
temperature ( Tm) of CA hexamer in DMSO (T ) verses in the presence of compound (Tm)
were calculated using the following formula: ∆Tm = Tm − T0.
∆
0
2.3.2. Virus Production
The wild-type laboratory HIV-1 strain, HIV-1NL4-3 [34], was produced using a pNL4-3
vector that was obtained through the NIH AIDS Reagent Program, Division of AIDS,
NIAID, NIH. HIV-1NL4-3 was generated by transfecting HEK 293FT cells in a 10 cm
tissue culture dish with 10 µg of the pNL4-3 vector and X-tremeGENE HP DNA Trans-
fection Reagent (Roche, Indianapolis, IN, USA), following the manufacturer’s protocol.
Supernatant was harvested 48–72 h post-transfection. The viral supernatant was then
concentrated using Lenti-X Concentrator (Takara, Mountain View, CA, USA), following
the manufacturer’s protocol. The resulting viral-containing pellet was concentrated 10-fold
◦
by resuspension in DMEM without FBS and stored at −80 C.
2.3.3. Anti-HIV-1 and Cytotoxicity Assays
Anti-HIV-1 activity of PF74 and PF74-related analogs were examined in TZM-GFP
cells. The potency of HIV-1 inhibition by a compound was based on its inhibition of HIV-1
infection of TZM-GFP cells compared with that of compound-free (DMSO) controls. Briefly,
4
TZM-GFP cells were plated at a density of 1
×
10 cells per well in a 96-well plate. 24 h
later, medium was replaced with medium containing compound dilutions. 24 h post
treatment, cells were exposed to HIV-1NL4-3 (MOI = 0.1). After incubation for 48 h, anti-
HIV-1 activity was assessed by counting the number of GFP positive cells on a Cytation
5
Imaging Reader (BioTek, Winooski, VT, USA) and 50% effective concentration (EC )
50
values were determined.
Cytotoxicity of each compound was also determined in TZM-GFP cells. Cells were
4
plated at a density of 1
×
10 cells per well in a 96-well plate and were continuously
exposed to medium containing compound dilutions for 72 h. Cell viability was determined
using a Cell Proliferation Kit II (XTT), and 50% cytotoxicity concentration (CC ) values
5
0
were determined. All the cell-based assays were conducted with technical duplicates and
with at least two independent experiments and the average values were determined.
For the EC50 and CC50 dose responses, values were plotted in GraphPad Prism 5 and
analyzed with the log (inhibitor) vs. normalized response–variable slope equation. Final
values were calculated in each independent assay and the average values were determined.
Statistical analysis (calculation of standard deviation) was performed using Microsoft Excel.
2.4. Metabolic Stability Assay
Microsomal stability. The in vitro microsomal stability assay was conducted in trip-
licate in mouse and human liver microsomal systems, which were supplemented with
nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. Briefly, a compound
(1 µM final concentration) was spiked into the reaction mixture containing liver microsomal
protein (0.5 mg/mL final concentration) and MgCl (1 mM final concentration) in 0.1 M
2
potassium phosphate buffer (pH 7.4). The reaction was initiated by addition of 1 mM
◦
NADPH, followed by incubation at 37 C. A negative control was performed in parallel
without NADPH to reveal any chemical instability or non-NADPH dependent enzymatic
degradation for each compound. At various time points (0, 5, 15, 30 and 60 min), 1 volume
of reaction aliquot was taken and quenched with 3 volumes of acetonitrile containing 0.1%
formic acid. The samples were then vortexed and centrifuged at 15,000 rpm for 5 min

Viruses 2021, 13, 479
7 of 17
◦
at 4 C. The supernatants were collected and analyzed by LC/MS/MS to determine the
remaining percentage and in vitro metabolic half-life (t1/2).
2.5. Molecular Modeling
Molecular modeling was performed using the Schrödinger small molecule drug
discovery suite 2019-4 [35]. The crystal structure of PF74 [17] in complex with native
HIV-1 capsid protein was retrieved from the protein data bank (PDB code: 4XFZ [17]).
Selected structures were evaluated for their interaction with native HIV-1 capsid protein
using Maestro [36] (Schrödinger; LLC: New York, NY, USA) by subjecting to a docking
protocol, involving preparing protein of interest, grid generation, ligand preparation, and
docking. Post processing of each docked pose was done by PyMOL [37] (Schrödinger;
LLC: New York, NY, USA). The native HIV-1 capsid protein/PF74 crystal structure was
refined using the protein preparation wizard [38] (Schrödinger; LLC: New York, NY, USA)
in which missing hydrogen atoms, side chains, and loops were added using Prime; waters
beyond 5 Å were deleted, and minimized using the OPLS3e force field [39] to optimize
the hydrogen bonding network and converge the heavy atoms to an RMSD of 0.3 Å. The
active site around the native ligand PF74 was defined by the receptor grid generation tool
in Maestro (Schrödinger; LLC: New York, NY, USA) covering all the residues within 12 Å.
All the compounds were drawn using 2D-sketcher in Maestro and subjected to LigPrep
to generate conformers and possible protonation at pH of 7
±
2. All the dockings were
performed using Glide XP [40] (Glide, version 8.2) with the van der Waals radii of nonpolar
atoms for each of the ligands scaled by a factor of 0.8 to decrease penalties for close contacts.
All docked poses were subjected to post-docking minimization (PMD) to minimize a small
number of poses within the field of the receptor to produce better poses. The residue
numbers of HIV-1 capsid protein used in the discussion and the figures were based on the
native HIV-1 capsid protein.
3. Results
Analogs synthesized for each series were first tested in a biophysical thermal shift
assay (TSA), which measures how compounds affect the stability of covalently crosslinked
CA hexamers. TSA results are presented as the change of protein melting temperature in
the presence of a compound compared to the protein melting temperature in the presence
of DMSO control (
a left shift (negative
tested in cell-based assays to determine anti-HIV-1 activity and cytotoxicity. The initial
antiviral screening was conducted at 20 M, from which compounds showing significant
∆
Tm). A right shift (positive
∆Tm) denotes a stabilizing effect, whereas
∆
Tm) indicates a destabilizing effect. All final compounds were also
µ
inhibition were further tested in a dose-response assay to determine antiviral EC50 values.
CC50 values were determined by cytotoxicity assay. The parent PF74 was used as a control
◦
in these assays (
∆
Tm = 6.9 C, EC50 = 0.70
µM, CC50 = 76
µM). Selected compounds were
also tested for metabolic stability in liver microsomes. Molecular modeling was performed
for a few analogs to corroborate the binding modes and understand the SAR.
3.1. The Indoline and Quinoline Analogs (Chemotype 1)
This short series sought to explore the effect of rigidifying the terminal N-methyl
aniline. As shown in Table 1, cyclization of the N-methyl to the ortho position of the phenyl
ring via a five-membered or six-membered ring is not tolerated as the resulting analogs
1–3
showed no significant antiviral activity or CA-stabilizing/destabilizing effect. This is likely
to be due to the loss of an important van der Waals interaction of the N-methyl group with
the side chain of N53 [20].

Viruses 2021, 13, 479
8 of 17
Table 1. Anti-HIV-1 activity, cytotoxicity, and CA hexamer stability profiles of chemotype 1.
EC50 (µM) a
CC50 (µM) b
TSA ∆Tm ( C)
◦
c
Cpd
Structure
PF74
0.70 ± 0.07
76 ± 9
6.9 ± 0.5
1
2
>20
>20
>100
>100
−0.5
−1.0
3
>20
>50
1.8 ± 1.2
a
Concentration of compound inhibiting HIV-1 replication by 50%, expressed as the mean
±
standard deviation from at least two
b
independent experiments. Concentration of compound causing 50% cell death, expressed as the mean
±
standard deviation from at least
Tm: change of CA crosslinked hexamer melting temperature in the presence of
compound minus melting temperature of CA crosslinked hexamer in the presence of DMSO (control).
two independent experiments. c TSA: thermal shift assay.
∆
3.2. SAR of the Pyridine Series (Chemotype 2)
This chemotype features a key backbone conformational constraint conceived via
building the phenylalanine carboxamide into a pyridine ring (Figure 2, modification B).
Incidentally, such a design resulted in a phenylalanine mimic similar to the core of GS-6207
(
Figure 1C). The prototype of this chemotype, analog
9
(Table 2), considerably inhibited
◦
HIV-1 (EC50 = 2.6
the enantiomer of
µM) and strongly stabilized CA hexamer (
∆
Tm = 4.5 C). In stark contrast,
9
, the R-phenylalanine derived analog
4
, was completely inactive in both
the antiviral assay and the TSA (Table 2). This dramatic SAR trend was largely retained
as additional R-configured analogs (5–8) showed neither CA-stabilizing nor destabilizing
effect, and they lacked significant antiviral activity, whereas S-configured analogs 10–19
all inhibited HIV-1 at low to sub-micromolar concentrations (Table 2). The stereochemical
requirement of the S configuration (Figure 2) is not unexpected since the phenyl ring of
the phenylalanine serves as an anchor for ligand and host factor binding [41]. Among
the S-configured analogs, the ones with an indazole (10) or a pyrazole (11) in place of an
indole only weakly stabilized CA hexamers, and both inhibited HIV-1 at low micromolar
EC50 concentrations. By contrast, with the exception of 12, all indole analogs (13
–
19
)
◦
strongly stabilized CA-hexamers (
∆
Tm = 5.9–8.7 C). Notably, having a chlorine atom
at the para position of the terminal phenyl ring (13
antiviral activity and the CA-stabilizing effect, which may reflect halogen bonding with
the CA-protein [22 23]. The OH group at the indole C5 position also conferred improved
–15) appeared to benefit both the
,
potency and CA-stabilizing effect (15 vs. 13), though the OH effect was not as prominent
as observed with PF74 analogs [22]. In the latter case, OH is believed to form an H-bond
with the K182 of the adjacent CACTD. When the indole N is ethylated, the resulting analogs
were considerably less active (16 vs. 13
trend compared to PF74 analogs [20]. Interestingly, significant difference was not observed
between Br and Cl (19 vs. 16 18 vs. 17). Finally, compounds with a halogen (13 19) of
this series were all somewhat cytotoxic. Overall, 15 was the most potent compound of this
series (EC50 = 0.31 M, Table 2 and Figure 3), with a better selectivity index (SI) over the
, 17 vs. 14), which amounts to a reversed SAR
,
–
µ

Viruses 2021, 13, 479
9 of 17
parent PF74 (SI = 142 for 15 vs. 109 for PF74). A Wilcoxon rank-sum test was performed to
determine the significance of the EC50 value of compound 15 compared to the EC50 value
of the parent PF74 compound (EC50 = 0.7 µM). The p-value = 0.029, which demonstrates
that difference between these two samples is statistically significant.
Table 2. Anti-HIV-1 activity, cytotoxicity, and CA hexamer stability profiles of chemotype 2.
Cpd d
EC50 (µM) a
CC50 (µM) b
◦
c
Structure
TSA ∆Tm ( C)
PF74
0.70 ± 0.07
76 ± 9
6.9 ± 0.5
4
5
>20
>20
<50
<50
0.1 ± 0.1
0.1 ± 0.07
6
7
8
>15
>20
>20
>50
>50
>50
0.3 ± 0.1
0 ± 0
0.1 ± 0.1
9
2.6 ± 0.4
8.8 ± 0.4
5.1 ± 0.7
<50
<50
>50
4.5 ± 0.0
1.0 ± 0.07
1.1 ± 0.07
1
1
0
1

Viruses 2021, 13, 479
10 of 17
Table 2. Cont.
EC50 (µM) a
Cpd d
CC50 (µM) b
◦
c
Structure
TSA ∆Tm ( C)
1
1
2
3
>20
>50
−0.2 ± 0.07
7.7 ± 0.2
0.79 ± 0.25
22 ± 2
1
4
1.4 ± 0.7
39 ± 0.2
7.8 ± 0.3
1
5 d
0.31 ± 0.07
2.7 ± 0.2
3.6 ± 0.2
44 ± 1
41 ± 0.1
48 ± 6
8.7 ± 0.3
6.6 ± 0.2
5.9 ± 0.2
1
6
7
1
1
1
8
9
2.1 ± 0.8
3.2 ± 0.3
40 ± 4
41 ± 2
6.3 ± 0.6
6.3 ± 0.1
a
Concentration of compound inhibiting HIV-1 replication by 50%, expressed as the mean
±
standard deviation from at least two
standard deviation from at least
b
independent experiments. Concentration of compound causing 50% cell death, expressed as the mean
two independent experiments. TSA: thermal shift assay.
compound minus melting temperature of CA crosslinked hexamer in the presence of DMSO (control). SI (selectivity index = CC50/EC50):
±
c
∆
Tm: change of CA crosslinked hexamer melting temperature in the presence of
d
1
09 (PF74), 142 (15).

Viruses 2021, 13, 479
11 of 17
Figure 3. Dose-response curve of the best compound, 15. Values are represented as mean of
four independent experiments, with standard error bars shown. Data were analyzed as nonlinear
regression, sigmoidal dose-response (variable slope) curve fit in GraphPad Prism.
3.3. SAR of the Imidazole Series (Chemotype 3)
This series was designed by converting the phenylalanine carboxamide moiety into an
imidazole ring for achieving backbone conformational constraint. Similar to the pyridine
series (chemotype 2), the PF74-derived prototypical imidazole analog 20 displayed signif-
◦
icant antiviral activity (EC50 = 3.7
µM) and strong CA-stabilizing effect (
∆
Tm = 3.9 C).
However, subsequent SAR did not yield improved compounds, with analogs 21
,
23–25 all
showing similar activity profiles (Table 3). The single exception is compound 22 featuring a
para-tetrazole group on the terminal phenyl ring, which conferred no antiviral activity. In-
terestingly, halogenated analogs within this series (23
than the pyridine series (13–19).
–25) were substantially less cytotoxic
Table 3. Anti-HIV-1 activity, cytotoxicity, and CA hexamer stability profiles of chemotype 3.
EC50 (µM) a
CC50 (µM) b
TSA ∆Tm ( C)
◦
c
Cpd
Structure
PF74
0.70 ± 0.07
76 ± 9
6.9 ± 0.5
2
2
2
0
1
2
3.7 ± 0.8
6.6 ± 0.4
>20
>50
>50
>50
3.9 ± 0.4
2.2 ± 0.0
−0.3 ± 0.1

Viruses 2021, 13, 479
12 of 17
Table 3. Cont.
EC50 (µM) a
CC50 (µM) b
93 ± 2
◦
c
Cpd
Structure
TSA ∆Tm ( C)
2
2
2
3
4
5
3.3 ± 0.03
3.5 ± 0.3
3.3 ± 0.2
4.0 ± 0.3
2.8 ± 0.3
2.7 ± 0.1
>100
>100
a
Concentration of compound inhibiting HIV-1 replication by 50%, expressed as the mean
±
standard deviation from at least two
standard deviation from at least
b
independent experiments. Concentration of compound causing 50% cell death, expressed as the mean
two independent experiments. TSA: thermal shift assay.
±
c
∆Tm: change of CA crosslinked hexamer melting temperature in the presence of
compound minus melting temperature of CA crosslinked hexamer in the presence of DMSO (control).
3.4. Metabolic Stability
A major deficiency of PF74 as an antiviral compound is the extremely low metabolic
stability [42
–
44], with half-life (t1/2) less than 1 min in both human liver microsomes
23]. Our previous work also revealed
(HLMs) and mouse liver microsomes (MLMs) [20
–
that this is mainly due to the poor resistance to cytochrome P450 3A4 (CYP3A4)-mediated
oxidative metabolism, as the half-life was drastically elongated in the presence of a CYP3A4
inhibitor [20–23]. To assess the impact of conformational restriction on the susceptibility
toward CYP3A4, we tested seven selected analogs in the microsomal stability assay. Overall,
very poor microsomal stability was observed with these analogs (Table 4), suggesting that
conformationally constrained analogs remain good substrates [45] for CYP3A4, as are
many peptidomimetics, including PF74. A notable exception was analog 15 (t1/2 = 27 min)
which contains a OH group at the C5 position of the indole ring.
Table 4. Phase I metabolic stability in liver microsomes t1/2 (min).
MLMs b
Cpd
HLMs a
Metabolite (s)
PF74
9
0.7 c
0.6 c
–
–
15
–
1.2 ± 0.04
1.7 ± 0.02
27.2 ± 0.5
1.4 ± 0.02
1.3 ± 0.01
0.9 ± 0.004
1.3 ± 0.01
1.0 ± 0.01
0.9 ± 0.004
3.1 ± 0.3
14
15
16
17
20
22
1.4 ± 0.02
1.3 ± 0.005
0.7 ± 0.01
0.9 ± 0.01
15
15 and 14
–
–
a
HLMs: human liver microsomes; b MLMs: mouse liver microsomes. c Data from ref [21].
3.5. Molecular Modeling
To analyze the observed SAR for pyridine and imidazole based chemotype 2 and
chemotype 3, molecular modeling of key ligands was performed on the crystal structure of
PF74 bound to native HIV-1 capsid protein (PDB code: 4XFZ [17]). Both (S)-configured-
pyridine and -imidazole based analogs
pocket, superimpose well with PF74, and interact with similar amino acid residues of the
HIV-1 capsid protein (Figure 4A). The inhibitory activities of compounds (EC : 2.6 M)
9 and 20 were predicted to occupy the PF74 binding
9
µ
5
0

Viruses 2021, 13, 479
13 of 17
and 20 (EC : 3.7
µM) can be attributed to their backbone resemblance to, and common
5
0
key interactions with PF74 (EC : 0.7
µM), including (i) H-bonding between indole NH of
50
compounds
9,
20 and PF74 with Q63; (ii) cation- interaction of the indole ring of the three
π
compounds with protonated K70; (iii) H-bonding between the indole acetic acid carbonyl
group and K70; and (iv) H-bonding between NH and carbonyl group of phenylalanine
moiety of PF74 and terminal amide group of N57. The absence of phenylalanine carbonyl-
O4 and N57 hydrogen-bonding in compounds 9 and 20 is likely to be partially compensated
for by the bonding between the nitrogen atom of pyridine and imidazole rings and terminal
amide of N57 (Figure 4A). A complete loss of potency was observed in the case of (R)-
configured-pyridine based analog
4
(EC50
µ
M: >20 for
4
vs. 2.6 for its (S)-configured analog
(circled) is expected to
9). This is presumably because the benzyl group of compound
4
point away from the PF74-cavity, resulting in loss of hydrophobic interactions with M66
and other hydrophobic amino acid residues (Figure 4B). That, in turn, may lead to loss of
the key H-bonding interaction between pyridine ring nitrogen atom and N57 (Figure 4B).
Halogen-bonding was evident in current SAR as compound 13 showed a four-fold increase
in potency compared to its non-halogen analog
addition, the 5-hydroxy substitution on the indole ring of the pyridine analog 15 further
enhanced antiviral potency 12- and 2-fold in comparison with compounds 13, and PF74
EC50 M: 0.31 for 15 vs. 0.7 for PF74 vs. 0.79 for 13 vs. 2.6 for ). This increased potency
9 (EC50 µM: 0.79 for 13 vs. 2.6 for 9). In
9,
(
µ
9
can be attributed to the potential H-bonding by the OH of compound 15 to residue K182
on the adjacent CACTD (Figure 4C).
Figure 4. Docking poses of key ligands based on native HIV-1 capsid protein bound to PF74 (PDB code: 4XFZ [17]).
) Superimposition of PF74 and compounds and 20 depicting binding similarity to PF74 and the fitting of compounds
and 20 within the PF74 binding pocket. Glide scores (kcal/mol): 5.6 (PF74); 6.1 ( ); 5.3 (20); ( ) predicted binding mode
of in which the benzyl group (circled) of the phenylalanine fragment points away from the hydrophobic pocket around
residue M66. Glide score (kcal/mol): 3.7; ( ) predicted binding of analog 15 within the preferred PF74 binding cavity
showing additional H- and halogen-bonding with K182 and K70, respectively. Glide score (kcal/mol): 7.7; ( ) chemical
structures of compounds 15 and 20. H-bond, halogen-bond and cation- interactions are depicted as black dotted lines,
(
A
9
9
−
−
9
−
B
4
−
C
−
D
4,
9,
π
pink dotted line and double headed arrow, respectively. CANTD around H3 and H4, and CACTD around H8 and H9 are
colored green and orange, respectively. The nitrogen, oxygen, sulfur, and chlorine atoms are colored blue, red, yellow, and
green, respectively.

Viruses 2021, 13, 479
14 of 17
4. Discussion
PF74 is a phenylalanine-derived peptidomimetic with a well-defined binding mode
to CA and sub-micromolar antiviral activity. Although mechanistically unique and syn-
thetically highly accessible, PF74 is far less potent than GS-6207 and exceedingly unstable
toward CYP-mediated oxidative metabolism. The three series of analogs (chemotypes 1-3,
Figure 2) designed and synthesized in this study feature different forms of conformational
restrictions of the PF74 backbone to improve antiviral potency and increased metabolic sta-
bility. Notably, both modifications (cyclization patterns A and B, Figure 2) involved altering
the aniline N-methyl of PF74, which forms a key van der Waals interaction with the N53
required for the potency of the parent PF74 as per previously reported SAR [20]. Consistent
with this SAR observation, modification A yielded compounds (chemotype 1) without
appreciable antiviral activity or CA-stabilizing/destabilizing effect (Table 1). By contrast,
modification B (Figure 2) is apparently tolerated as the resulting pyridine (chemotype 2)
and imidazole (chemotype 3) series both contain compounds with significant antiviral
activity and strong CA-stabilizing effect (Tables 2 and 3), suggesting that the loss of the
van der Waals interaction can be compensated for with modification B (chemotype 2 and
chemotype 3) but not modification A (chemotype 1). As for modification B, while the SAR
of the imidazole series (Table 3) was largely flat with the exception of the inactive analog
22, the pyridine series displayed prominent SAR trends where both the antiviral potency
and the CA-stabilizing effect were highly dependent on the S-configured phenylalanine
moiety as well as the indole ring (Table 2).
Another important aim of the current work is to identify and avoid structural features
with potential metabolic liability to improve metabolic stability. As mentioned earlier, the
aniline N-methyl group of PF74 is highly susceptible toward oxidative metabolism. However,
designing out this N-methyl group using a pyridine (
) ring did not lead to significantly improved metabolic stability (Table 4). In the meantime,
we have previously shown that replacing the electron-rich and easily oxidizable indole ring
with less electron-rich ring systems could largely mitigate the metabolic liability [22 23].
9, 14, 16–17, Table 4) or imidazole (20,
22
,
Intriguingly, when an electron-donating hydroxyl group was introduced onto the C5 position
of the indole ring, the resulting analog 15 showed drastically improved metabolic stability,
despite being more electron-rich. This is presumably because the major oxidative metabolic
pathway of the indole ring features a hydroxylation at the C5 position [46] as exemplified by
the well-known conversion of tryptophan to serotonin [47]. Consistent with this metabolic
pathway, 15 was also observed as a stable metabolite of 14
metabolic pathways are described in Figure 5. The conversion of 16 to 15 likely entails a
C5 hydroxylation and a de-ethylation at the indole N via -carbon hydroxylation, followed
by the collapse of the hemiaminal in 16b. A similar hydroxylation of 17 could generate 14
which was indeed observed as a metabolite of 17. The de-methylation of the OMe group in
is likely via a hemiacetal intermediate 14a produced by the -carbon hydroxylation of the
OMe. It is noteworthy that CYP-mediated de-alkylation is well-known [48].
, 16 and 17. The proposed
α
,
14
α

Viruses 2021, 13, 479
15 of 17
Figure 5. Proposed metabolic pathways of 17
include oxidative O-de-methylation and N-de-ethylation, and 5 -hydroxylation of the indole ring.
, 14 and 16 toward 15. Major cyclophilin- (CYP)-mediated oxidative reactions
0
5. Conclusions
To identify novel small molecules targeting the PF74 binding site of HIV-1 CA, we de-
signed, synthesized and characterized three chemotypes featuring different conformational
constraints. Through subsequent SAR, we identified analog 15 which displayed potent an-
◦
tiviral activity against HIV-1 (EC50 = 0.31
µM), strong CA-stabilizing effect (
∆
Tm = 8.7 C),
and drastically improved metabolic stability (t1/2 = 27 min). The observed metabolic
pathways also suggest that blocking the C5 position of the indole ring may represent an
important approach to metabolically stable PF74 analogs.
Supplementary Materials: The following are available online at https://www.mdpi.com/1999-4
915/13/3/479/s1, Scheme S1: synthesis of intermediates 27
–28 and compounds
1–
3, Scheme S2:
synthesis of intermediates 30–37 and compounds
4–
19, Scheme S3: synthesis of intermediates 39–
41
and compounds 20–25.
Author Contributions: S.G.S. and Z.W. conceptualized the research. R.L.S. and R.D.-R. designed,
synthesized and characterized all compounds, with the help of S.K.V.V. and L.W., R.L.S. conducted
all modeling. J.X. performed the microsomal stability assays. H.D., H.Z., A.E.C., M.C.C., K.A.K., and
P.R.T. performed and/or evaluated data from TSA, antiviral, and cytotoxicity assays. R.L.S. and Z.W.
wrote the manuscript. K.A.K. and P.R.T. edited the manuscript. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by the National Institute of Allergy and Infectious Diseases
(NIAID) at the National Institutes of Health (NIH), grant number R01AI120860 (to S.G.S. and Z.W.).
S.G.S. acknowledges funding from the Nahmias-Schinazi Distinguished Chair in Research.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are Supplementary Materials.
Acknowledgments: We thank the Minnesota Supercomputing Institute (MSI) at the University of
Minnesota for providing molecular modeling resources.
Conflicts of Interest: The authors declare no conflict of interest.

Viruses 2021, 13, 479
16 of 17
References
1
.
Novikova, M.; Zhang, Y.; Freed, E.O.; Peng, K. Multiple Roles of HIV-1 Capsid during the Virus Replication Cycle. Virol. Sin.
019, 34, 119–134. [CrossRef]
2
2
3
.
.
Freed, E.O. HIV-1 assembly, release and maturation. Nat. Rev. Microbiol. 2015, 13, 484–496. [CrossRef]
Buffone, C.; Martinez-Lopez, A.; Fricke, T.; Opp, S.; Severgnini, M.; Cifola, I.; Petiti, L.; Frabetti, S.; Skorupka, K.;
Zadrozny, K.K.; et al. Nup153 Unlocks the Nuclear Pore Complex for HIV-1 Nuclear Translocation in Nondividing
Cells. J. Virol. 2018, 92, e00648-18. [CrossRef]
4
.
Chin, C.R.; Perreira, J.M.; Savidis, G.; Portmann, J.M.; Aker, A.M.; Feeley, E.M.; Smith, M.C.; Brass, A.L. Direct Visualization of
HIV-1 Replication Intermediates Shows that Capsid and CPSF6 Modulate HIV-1 Intra-nuclear Invasion and Integration. Cell Rep.
2
015, 13, 1717–1731. [CrossRef]
5
.
.
Bejarano, D.A.; Peng, K.; Laketa, V.; Börner, K.; Jost, K.L.; Lucic, B.; Glass, B.; Lusic, M.; Müller, B.; Kräusslich, H.-G. HIV-1 nuclear
import in macrophages is regulated by CPSF6-capsid interactions at the nuclear pore complex. eLife 2019, 8, e41800. [CrossRef]
Sowd, G.A.; Serrao, E.; Wang, H.; Wang, W.; Fadel, H.J.; Poeschla, E.M.; Engelman, A.N. A critical role for alternative polyadeny-
lation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin. Proc. Natl. Acad. Sci. USA 2016, 113,
E1054–E1063. [CrossRef] [PubMed]
6
7
8
.
.
.
Achuthan, V.; Perreira, J.M.; Sowd, G.A.; Puray-Chavez, M.; McDougall, W.M.; Paulucci-Holthauzen, A.; Wu, X.; Fadel, H.J.;
Poeschla, E.M.; Multani, A.S.; et al. Capsid-CPSF6 Interaction Licenses Nuclear HIV-1 Trafficking to Sites of Viral DNA Integration.
Cell Host Microbe 2018, 24, 392–404. [CrossRef] [PubMed]
Kim, K.; Dauphin, A.; Komurlu, S.; McCauley, S.M.; Yurkovetskiy, L.; Carbone, C.; Diehl, W.E.; Strambio-De-Castillia, C.;
Campbell, E.M.; Luban, J. Cyclophilin A protects HIV-1 from restriction by human TRIM5
CrossRef] [PubMed]
Carnes, S.K.; Sheehan, J.H.; Aiken, C. Inhibitors of the HIV-1 capsid, a target of opportunity. Curr. Opin. HIV AIDS 2018, 13,
59–365. [CrossRef]
α. Nat. Microbiol. 2019, 4, 2044–2051.
[
9
1
3
0. Lamorte, L.; Titolo, S.; Lemke, C.T.; Goudreau, N.; Mercier, J.-F.; Wardrop, E.; Shah, V.B.; von Schwedler, U.K.; Langelier, C.;
Banik, S.S.R.; et al. Discovery of Novel Small-Molecule HIV-1 Replication Inhibitors That Stabilize Capsid Complexes. Antimicrob.
Agents Chemother. 2013, 57, 4622–4631. [CrossRef] [PubMed]
1
1. Lemke, C.T.; Titolo, S.; von Schwedler, U.; Goudreau, N.; Mercier, J.-F.; Wardrop, E.; Faucher, A.-M.; Coulombe, R.; Banik, S.S.R.;
Fader, L.; et al. Distinct Effects of Two HIV-1 Capsid Assembly Inhibitor Families That Bind the Same Site within the N-Terminal
Domain of the Viral CA Protein. J. Virol. 2012, 86, 6643. [CrossRef] [PubMed]
1
1
1
1
2. Kelly, B.N.; Kyere, S.; Kinde, I.; Tang, C.; Howard, B.R.; Robinson, H.; Sundquist, W.I.; Summers, M.F.; Hill, C.P. Structure of the
Antiviral Assembly Inhibitor CAP-1 Complex with the HIV-1 CA Protein. J. Mol. Biol. 2007, 373, 355–366. [CrossRef] [PubMed]
3. Blair, W.S.; Pickford, C.; Irving, S.L.; Brown, D.G.; Anderson, M.; Bazin, R.; Cao, J.; Ciaramella, G.; Isaacson, J.; Jackson, L.; et al.
HIV Capsid is a Tractable Target for Small Molecule Therapeutic Intervention. PLoS Pathog. 2010, 6, e1001220. [CrossRef]
4. Link, J.O.; Rhee, M.S.; Tse, W.C.; Zheng, J.; Somoza, J.R.; Rowe, W.; Begley, R.; Chiu, A.; Mulato, A.; Hansen, D.; et al. Clinical
targeting of HIV capsid protein with a long-acting small molecule. Nature 2020, 584, 614–618. [CrossRef] [PubMed]
5. Bester, S.M.; Wei, G.; Zhao, H.; Adu-Ampratwum, D.; Iqbal, N.; Courouble, V.V.; Francis, A.C.; Annamalai, A.S.; Singh, P.K.;
Shkriabai, N.; et al. Structural and mechanistic bases for a potent HIV-1 capsid inhibitor. Science 2020, 370, 360. [CrossRef]
6. Pornillos, O.; Ganser-Pornillos, B.K.; Yeager, M. Atomic-level modelling of the HIV capsid. Nature 2011, 469, 424–427. [CrossRef]
7. Gres, A.T.; Kirby, K.A.; KewalRamani, V.N.; Tanner, J.J.; Pornillos, O.; Sarafianos, S.G. X-ray crystal structures of native HIV-1
capsid protein reveal conformational variability. Science 2015, 349, 99. [CrossRef] [PubMed]
1
1
1
8. Price, A.J.; Jacques, D.A.; McEwan, W.A.; Fletcher, A.J.; Essig, S.; Chin, J.W.; Halambage, U.D.; Aiken, C.; James, L.C. Host
Cofactors and Pharmacologic Ligands Share an Essential Interface in HIV-1 Capsid That Is Lost upon Disassembly. PLoS Pathog.
2
014, 10, e1004459. [CrossRef]
1
2
9. Kleinpeter, A.B.; Freed, E.O. HIV-1 Maturation: Lessons Learned from Inhibitors. Viruses 2020, 12, 940. [CrossRef]
0. Wang, L.; Casey, M.C.; Vernekar, S.K.V.; Do, H.T.; Sahani, R.L.; Kirby, K.A.; Du, H.; Hachiya, A.; Zhang, H.; Tedbury, P.R.; et al.
Chemical profiling of HIV-1 capsid-targeting antiviral PF74. Eur. J. Med. Chem. 2020, 200, 112427. [CrossRef]
1. Vernekar, S.K.V.; Sahani, R.L.; Casey, M.C.; Kankanala, J.; Wang, L.; Kirby, K.A.; Du, H.; Zhang, H.; Tedbury, P.R.; Xie, J.; et al.
Toward Structurally Novel and Metabolically Stable HIV-1 Capsid-Targeting Small Molecules. Viruses 2020, 12, 452. [CrossRef]
2. Wang, L.; Casey, M.C.; Vernekar, S.K.V.; Sahani, R.L.; Kankanala, J.; Kirby, K.A.; Du, H.; Hachiya, A.; Zhang, H.; Tedbury, P.R.; et al.
Novel HIV-1 capsid-targeting small molecules of the PF74 binding site. Eur. J. Med. Chem. 2020, 204, 112626. [CrossRef]
3. Wang, L.; Casey, M.C.; Vernekar, S.K.V.; Sahani, R.L.; Kirby, K.A.; Du, H.; Zhang, H.; Tedbury, P.R.; Xie, J.; Sarafianos, S.G.; et al.
Novel PF74-like small molecules targeting the HIV-1 capsid protein: Balance of potency and metabolic stability. Acta Pharm. Sin.
B 2020. [CrossRef]
2
2
2
2
2
4. Chang, C.E.A.; Chen, W.; Gilson, M.K. Ligand configurational entropy and protein binding. Proc. Natl. Acad. Sci. USA 2007
04, 1534. [CrossRef]
5. Lawson, A.D.G.; MacCoss, M.; Heer, J.P. Importance of Rigidity in Designing Small Molecule Drugs To Tackle Protein–Protein
,
1
Interactions (PPIs) through Stabilization of Desired Conformers. J. Med. Chem. 2018, 61, 4283–4289. [CrossRef] [PubMed]

Viruses 2021, 13, 479
17 of 17
2
6. Borsari, C.; Rageot, D.; Dall’Asen, A.; Bohnacker, T.; Melone, A.; Sele, A.M.; Jackson, E.; Langlois, J.-B.; Beaufils, F.;
Hebeisen, P.; et al. A Conformational Restriction Strategy for the Identification of a Highly Selective Pyrimido-pyrrolo-oxazine
mTOR Inhibitor. J. Med. Chem. 2019, 62, 8609–8630. [CrossRef]
2
2
2
3
3
3
7. Kedderis, G.L.; Hollenberg, P.F. Peroxidase-catalyzed N-demethylation reactions: Deuterium solvent isotope effects. Biochemistry
1
985, 24, 6158–6163. [CrossRef] [PubMed]
8. Hollenberg, P.F.; Miwa, G.T.; Walsh, J.S.; Dwyer, L.A.; Rickert, D.E.; Kedderis, G.L. Mechanisms of N-demethylation reactions
catalyzed by cytochrome P-450 and peroxidases. Drug Metab. Dispos. 1985, 13, 272. [PubMed]
9. Bondy, S.S.; Cannizzaro, C.E.; Chou, C.-H.; Halcomb, R.L.; Hu, E.Y.; Link, J.O.; Liu, Q.; Schroeder, S.D.; Tse, W.C.; Zhang, J.R.
Compounds for the treatment of HIV. Patent WO 2013/006738 A1, 1 October 2013.
0. Belema, M.; Bender, J.A.; Beno, B.; Gentles, R.G.; Li, G.; Meanwell, N.A.; Pendri, A.; Yang, Z.; Zhu, S. Inhibitors of Human
Immunodeficiency Virus Replication. U.S. Patent 2017/0304239 A1, 27 November 2017.
1. Lange, M.J.; Lyddon, T.D.; Johnson, M.C. Diphtheria Toxin A-Resistant Cell Lines Enable Robust Production and Evaluation of
DTA-Encoding Lentiviruses. Sci. Rep. 2019, 9, 8985. [CrossRef]
2. Rosa, A.; Chande, A.; Ziglio, S.; De Sanctis, V.; Bertorelli, R.; Goh, S.L.; McCauley, S.M.; Nowosielska, A.; Antonarakis, S.E.;
Luban, J.; et al. HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation. Nature 2015, 526, 212–217.
[CrossRef]
3
3. Pornillos, O.; Ganser-Pornillos, B.K.; Kelly, B.N.; Hua, Y.; Whitby, F.G.; Stout, C.D.; Sundquist, W.I.; Hill, C.P.; Yeager, M. X-Ray
Structures of the Hexameric Building Block of the HIV Capsid. Cell 2009, 137, 1282–1292. [CrossRef]
3
4. Adachi, A.; Gendelman, H.E.; Koenig, S.; Folks, T.; Willey, R.; Rabson, A.; Martin, M.A. Production of acquired immunodeficiency
syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol. 1986
9, 284. [CrossRef]
,
5
3
3
3
3
5. Schrödinger. Schrödinger Small-Molecule Drug Discovery Suite 2019-4; Schrödinger, LLC: New York, NY, USA, 2019.
6. Schrödinger. Schrödinger Release 2019-4: Maestro; Schrödinger, LLC: New York, NY, USA, 2019.
7. Schrödinger. The PyMOL Molecular Graphics System; Version 2.0; Schrödinger, LLC: New York, NY, USA, 2019.
8. Sastry, G.M.; Adzhigirey, M.; Day, T.; Annabhimoju, R.; Sherman, W. Protein and ligand preparation: Parameters, protocols, and
influence on virtual screening enrichments. J. Comput. Aided Mol. Des. 2013, 27, 221–234. [CrossRef] [PubMed]
9. Jorgensen, W.L.; Maxwell, D.S.; Tirado-Rives, J. Development and Testing of the OPLS All-Atom Force Field on Conformational
Energetics and Properties of Organic Liquids. J. Am. Chem. Soc. 1996, 118, 11225–11236. [CrossRef]
3
4
0. Friesner, R.A.; Murphy, R.B.; Repasky, M.P.; Frye, L.L.; Greenwood, J.R.; Halgren, T.A.; Sanschagrin, P.C.; Mainz, D.T. Extra
Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein Ligand Complexes. J. Med.
−
Chem. 2006, 49, 6177–6196. [CrossRef]
4
4
4
1. Singh, K.; Gallazzi, F.; Hill, K.J.; Burke, D.H.; Lange, M.J.; Quinn, T.P.; Neogi, U.; Sönnerborg, A. GS-CA Compounds: First-In-Class
HIV-1 Capsid Inhibitors Covering Multiple Grounds. Front. Microbiol. 2019, 10. [CrossRef]
2. Xu, J.P.; Francis, A.C.; Meuser, M.E.; Mankowski, M.; Ptak, R.G.; Rashad, A.A.; Melikyan, G.B.; Cocklin, S. Exploring Modifications
of an HIV-1 Capsid Inhibitor: Design, Synthesis, and Mechanism of Action. J. Drug Des. Res. 2018, 5, 1070. [PubMed]
3. Sun, L.; Huang, T.; Dick, A.; Meuser, M.E.; Zalloum, W.A.; Chen, C.-H.; Ding, X.; Gao, P.; Cocklin, S.; Lee, K.-H.; et al. Design,
synthesis and structure-activity relationships of 4-phenyl-1H-1,2,3-triazole phenylalanine derivatives as novel HIV-1 capsid
inhibitors with promising antiviral activities. Eur. J. Med. Chem. 2020, 190, 112085. [CrossRef] [PubMed]
4
4. Wu, G.; Zalloum, W.A.; Meuser, M.E.; Jing, L.; Kang, D.; Chen, C.-H.; Tian, Y.; Zhang, F.; Cocklin, S.; Lee, K.-H.; et al. Discovery of
phenylalanine derivatives as potent HIV-1 capsid inhibitors from click chemistry-based compound library. Eur. J. Med. Chem.
2
018, 158, 478–492. [CrossRef] [PubMed]
4
4
4
4
5. Wacher, V.J.; Silverman, J.A.; Zhang, Y.; Benet, L.Z. Role of P-Glycoprotein and Cytochrome P450 3A in Limiting Oral Absorption
of Peptides and Peptidomimetics. J. Pharm. Sci. 1998, 87, 1322–1330. [CrossRef] [PubMed]
6. Pavlova, T.; Vidova, V.; Bienertova-Vasku, J.; Janku, P.; Almasi, M.; Klanova, J.; Spacil, Z. Urinary intermediates of tryptophan as
indicators of the gut microbial metabolism. Anal. Chim. Acta 2017, 987, 72–80. [CrossRef] [PubMed]
7. Höglund, E.; Øverli, Ø.; Winberg, S. Tryptophan Metabolic Pathways and Brain Serotonergic Activity: A Comparative Review.
Front. Endocrinol. 2019, 10. [CrossRef] [PubMed]
8. Hesse, L.M.; Venkatakrishnan, K.; von Moltke, L.L.; Shader, R.I.; Greenblatt, D.J. CYP3A4 Is the Major CYP Isoform Mediating
the in Vitro Hydroxylation and Demethylation of Flunitrazepam. Drug Metab. Dispos. 2001, 29, 133. [PubMed]