Chemical optimization of macrocyclic HIV-1 inactivators for improving potency and increasing the structural diversity at the triazole ring

Article abstract of DOI:10.1039/c7ob01448a

HIV-1 entry inhibition remains an urgent need for AIDS drug discovery and development. We previously reported the discovery of cyclic peptide triazoles (cPTs) that retain the HIV-1 irreversible inactivation functions of the parent linear peptides (PTs) and have massively increased proteolytic resistance. Here, in an initial structure-activity relationship investigation, we evaluated the effects of variations in key structural and functional components of the cPT scaffold in order to produce a platform for developing next-generation cPTs. Some structural elements, including stereochemistry around the cyclization residues and Ile and Trp side chains in the gp120-binding pharmacophore, exhibited relatively low tolerance for change, reflecting the importance of these components for function. In contrast, in the pharmacophore-central triazole position, the ferrocene moiety could be successfully replaced with smaller aromatic rings, where a p-methyl-phenyl methylene moiety gave cPT 24 with an IC₅₀ value of 180 nM. Based on the observed activity of the biphenyl moiety when installed on the triazole ring (cPT 23, IC₅₀ ～ 269 nM), we further developed a new on-resin synthetic method to easily access the bi-aryl system during cPT synthesis, in good yields. A thiophene-containing cPT AAR029N2 (36) showed enhanced entropically favored binding to Env gp120 and improved antiviral activity (IC₅₀ ～ 100 nM) compared to the ferrocene-containing analogue. This study thus provides a crucial expansion of chemical space in the pharmacophore to use as a starting point, along with other allowable structural changes, to guide future optimization and minimization for this important class of HIV-1 killing agents.

Full text of DOI:10.1039/c7ob01448a

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Chemical Optimization Of Macrocyclic HIV-1 Inactivators For Improving
Potency and Increasing the Structural Diversity at The Triazole Ring
Adel A. Rashad†, Kriti Acharya, Ann Haftl, Rachna Aneja#, Alexej Dick, Andrew P.
Holmes and Irwin Chaiken†
Department of Biochemistry & Molecular Biology, Drexel University College of
Medicine, Philadelphia, Pennsylvania 19102
# Current address: The Scripps Research Institute, 130 Scripps Way, Jupiterꢀ33458,
Florida
AUTHOR INFORMATION†
†
Corresponding Author: IC, Email: ichaiken@drexelmed.edu: Phone: (215) 762ꢀ
4197. Fax: (215) 762ꢀ4452.
†Email for AAR: adel.ahmed@drexelmed.edu
Abstract
HIVꢀ1 entry inhibition remains an urgent need for AIDS drug discovery and
development. We previously reported the discovery of cyclic peptide triazoles (cPTs)
that retain the HIVꢀ1 irreversible inactivation functions of the parent linear peptides
(PTs) and have massively increased proteolytic resistance. Here, in an initial
structureꢀactivity relationship investigation, we evaluated the effects of variations in
key structural and functional components of the cPT scaffold in order to produce a
platform for developing nextꢀgeneration cPTs. Some structural elements, including
stereochemistry around the cyclization residues and Ile and Trp side chains in the
1

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gp120ꢀbinding pharmacophore, exhibited relatively low tolerance for change,
reflecting the importance of these components for function. In contrast, in the
pharmacophoreꢀcentral triazole position, the ferrocene moiety could be successfully
replaced with smaller aromatic rings, where a pꢀmethylꢀphenyl methylene moiety
gave cPT 24 with an IC50 value of 180 nM. Based on the observed activity of the
biphenyl moiety when installed on the triazole ring (cPT 23, IC50 ~ 269 nM), we
further developed a new onꢀresin synthetic method to easily access the biꢀaryl
system during cPT synthesis, in good yields. A thiopheneꢀcontaining cPT AAR029N2
(36) showed enhanced entropically favored binding to Env gp120 and improved
antiviral activity (IC50 ~ 100 nM) compared to the ferroceneꢀcontaining analogue.
This study thus provides a crucial expansion of chemical space in the
pharmacophore to use as a starting point, along with other allowable structural
changes, to guide future optimization and minimization for this important class of
HIVꢀ1 killing agents.
Introduction
Despite advancements in drug discovery and structureꢀbased design of HIVꢀ1
inhibitors, Acquired Immune Deficiency Syndrome (AIDS) is still a major global
health problem with no curative agents nor protective vaccines available.1, 2 Highly
Active Antiretroviral Therapy (HAART) has significantly improved survival rates3 and
has also decreased the incidence of AIDS associated complications among the HIVꢀ
infected population, improving the patients' quality of life.4, 5 However, HAART does
not cure HIV infection, because the virus hides by integrating proviral DNA into cells
that can form long living reservoirs, and already formed viruses can persist in
lymphatic tissues attached to follicular dendritic cells where they cannot be killed by
2

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current therapy.6, 7 The ability of HAART to bring the acute HIV infection into a
chronic status has been associated with other chronic health problems caused by
the side effects of the HAART drug cocktail. Moreover, recent global reports indicate
a significant increase in HAART resistant population, increasing the demand to find
novel drugs with new modes of action.
HIVꢀ1 entry into the host cell remains an underꢀutilized target of the current therapy.8
This process is entirely mediated by the Env protein machinery, which functions
through proteinꢀprotein interactions (PPI) with host cell receptors.9 The two
components of HIVꢀ1 Env, namely gp120 and gp41, represent attractive targets for
the discovery of novel entry inhibitors. Enfuvirtide (T20), a 36 amino acid peptide, is
the only FDA approved fusion inhibitor targeting the gp41 NHR region.10, 11 However,
T20 is only used as a second line of treatment and has suffered from short duration
of action, fast development of viral resistance and inconvenient route of
administration. There are no approved gp120 inhibitors up to date, though the
prodrug of BMSꢀ626529 (1, Figure 1) is currently in phase III clinical trials as an
attachment inhibitor.12 Studies have shown that the mode of action of BMSꢀ626529
is to reversibly block the CD4 induced conformational changes in Env and
consequently inhibit entry.13 Small molecule CD4 mimetics (2, Figure 1) are another
class of entry inhibitors.14 They act by directly competing with CD4 for binding to the
Phe43 cavity on the gp120 units. They have been shown to be useful in increasing
the sensitivity of Env to antibodyꢀdependent cellꢀmediated cytotoxicity (ADCC)
response.15
Generally, macrocyclic compounds have been found to be very effective inhibitors
for poorly druggable targets such as proteinꢀprotein interactions (PPIs).16 Among all
human proteins, only a relatively few can be targeted by small molecules,17
3

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highlighting the need for novel PPI inhibitors. In fact, macrocyclic inhibitors are able
to effectively inhibit PPIs by covering large hot spots between the two interacting
proteins that traditional small molecules cannot reach, highlighting their importance
in developing inhibitors for PPIs.18, 19 The example of HIVꢀ1 Env and host cell
receptors is an opportune PPI to target with macrocyclic inhibitors.
In this context, we have previously reported a class of small cyclic peptide triazoles
(cPTs, represented by the first lead AAR029b20, 3, Figure 1) that is able to
irreversibly inactivate HIVꢀ1 virions, even before host cell encounter.20 They exert
this unique mode of inactivation by hijacking the metastability of the Env protein
complex,21 converting the gp120 into an inactive conformation that leads to gp120
shedding off the Env from the virion surface.20 The resultant gp41 coated virions
become nonꢀinfectious. Interestingly, the binding site of cPTs seems to overlap both
binding sites of BMSꢀ626529 and the CD4 small molecule mimetics.22 Given the
effectiveness of the initial cPTs to inactivate HIVꢀ1 Env, we undertook a series of
chemical explorations around the scaffold of cPT in order to better understand the
structural requirements for function and to identify new methodologies to derive cPT
compounds with an increased capacity for structural variation. We found in this SAR
study that variations in many cPT structural elements were tolerated, though only
within a narrow range of possibilities. In contrast, significant variations were tolerated
in the substituted triazole component, leading to chemical space expansion,
improved potency and molecularꢀstructural properties, and establishment of a
platform to guide future cPT development.
4

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Fig.1 Chemical structures of small molecule and small macrocyclic gp120
targeting inhibitors. The conformational blocker attachment inhibitor BMSꢀ626529
(1), small molecule CD4 mimetic (+)ꢀBNMꢀIVꢀ147 (2) and the conformational
entrapping HIVꢀ1 inactivator cPT AAR029b (3).
Results
I. Changing stereocenters at Lys and Asp centers
It has been previously shown that the stereocenters of the active core (Ileꢀ
triazoloProꢀTrp) in the linear peptide triazoles have to be in the S configuration for
optimal activity, whereas the R configuration can only be tolerated at the most N and
C terminal residues.23 We examined here the effect of modifying the stereocenters at
both the Asp and Lys residues, used for the cyclization (sideꢀchain to sideꢀchain), to
investigate whether changes of these positions could tune the activity. As shown in
(Figure 2), the newly introduced R configuration can only be tolerated at the Asp
chiral center (center 1, cPT 5), with 7ꢀfold decrease in activity compared to the lead
cPT 3. In contrast, changing the configuration of the Lys chiral center (center 2, cPT
4) from S to R had a more devastating effect (> 50 fold decrease) on the activity.
Therefore, the S configuration at both the Asp and Lys centers was judged to be
optimal for cPT functions, though there is a degree of allowable tolerance at the Asp
chiral center that can be an optimization point.
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Fig.2 Structure of cPTs with altered stereochemistry at the Asp and Lys chiral
centers. Each cPT was synthesized starting from pure L or D amino acids. The
cutoff used in the infection inhibition assay was 20 ꢁM, after which the compound
was considered weak and no longer pursued.
II. Side chain variations (Ile secꢀbutyl and Trp indole) in the pharmacophore region
We had previously shown the importance of the pharmacophore (IXW, where X =
arylꢀtriazoleꢀPro) for the functions of the parent linear PTs.24 Here, we sought to
identify nonꢀnatural replacements for this pharmacophore to allow: a) exploration of
the chemical space around this pharmacophore and b) nonꢀnatural replacements
which will allow functionalization and derivatization at each pharmacophore site.
Figure 3 shows the variations we made for both the indole and the secꢀbutyl side
chains of the pharmacophore. As expected, the unꢀsubstituted indole ring could be
nicely replaced with the isostere25, 26 benzo[b]thiophene (14, IC50 ~ 170 nM, Figure
3). This observation was also previously seen in a linear hexapeptide analogue.22
Additions on the indole ring with a methoxy group resulted in very weak activity (13,
Figure 3), arguing that this added group might prevent docking of the indole ring
onto its target site on gp120 protein. The same weakening effect was also observed
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when benzonitrile, as an aromatic ring carrying a Hꢀbond acceptor, was evaluated as
a side chain (cPT 15). Along with the previous findings, it was evident that unꢀ
substituted indole and benzo[b]thiophene are both optimal for the activity at that
pharmacophore site.
We then moved to the Ile secꢀbutyl side chain, as it has not been previously
explored. Interestingly, the branched nature of the secꢀbutyl group was found
important where a methyl shuffle to the terminal carbon (7, Figure 3) resulted in
massive reduction in activity. A similar effect was observed when the two terminal
methyl groups were tethered in a cyclopropyl form (8, Figure 3). These weaker
activities argued that increasing the bulkiness/branching of the hydrocarbon at this
position might improve the activity. We therefore tried the bulky cyclohexyl group,
which actually showed retained activity (9, IC50 ~ 240 nM, Figure 3) compared to 3
(IC50 ~ 237 nM). We next asked whether adding a polar group on this hydrophobic
hydrocarbon would affect the activity. The hypothesis was that the hydrocarbon side
chain might occupy a hydrophobic protein pocket and adding an electronegative
atom may be able to abstract a Hꢀbond from the protein backbone amide NHs, if
they were close enough to the protein surface. In addition, adding a polar atom on
these hydrocarbon groups might improve aqueous solubility. We found that adding
an oxygen atom to the original secꢀbutyl side chain (as in 12, Figure 3) and to the
cyclohexyl (as in 11, Figure 3) resulted in very weak activities. The same weakening
effect also was observed when we changed the secꢀbutyl group with a smaller group
containing unmasked OH group (10, Figure 3). The overall conclusion from this
polar atom addition is that increasing the total polar surface area (tPSA) of these
hydrocarbon moieties at the secꢀbutyl position leads to a massive reduction in
functions.
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Fig.3 Structural changes in the indole (red) and sec-butyl (bue) moieties of
cPT. Activities shown are from HIVꢀ1Bal.01 infection inhibition assay. The designation
weak means the compound showed weak inhibition (less than 50%) at 10 ꢁM, which
was used as a cutoff point where the compound was no longer pursued.
III. Changes in the aryl group on the triazole moiety
The main challenge in this work was to find alternatives for the ferrocene moiety,
which has been shown to give optimal activities with both linear PTs27 as well as
cPTs.20 The chemical nature of ferrocene does not allow exploration of the chemical
space of triazole ring substituents. Hence, from a medicinal chemistry perspective, it
was necessary to find a more acceptable moiety to replace the ferrocene ring and
ultimately enhance drug development. As shown in Table 1, we used both
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commercially available alkynes and also synthesized additional key alkynes
(Scheme 1), to identify triazole substituents that would enhance exploration of the
chemical space on the triazole ring. The new triazole derivatives retained the dual
hostꢀcell receptor antagonism signature of the ferroceneꢀcontaining lead cPT 3
(Supplementary Table S1).
Table 1. Structure activity relationship around the triazole moiety. A general
structure is shown with the R group specified in the table, each with the HIVꢀ1Bal.01
infection inhibition IC50 value. Asterisk (*) indicates that the cPT was made “in house”
using a synthetic alkyne that was incorporated in the click reaction during the cPT
synthesis.
Compound/
R group
16
HIVꢀ1 IC50
(nM)
Compound/
R group
HIVꢀ1 IC50
(nM)
17
2018 ± 9520
1400 ± 200
Inactive
18
19
438 ± 12
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21
20
22
1200 ± 500
900 ± 45
23
350 ± 25
269 ± 32
25*
24*
180 ± 9
5100 ± 220
302 ± 50
27*
26*
28*
220 ± 42
29*
2000 ± 390
8000 ± 250
30
31
5000 ± 850
1800 ± 500
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32*
33*
7300 ± 740
6000 ± 450
We synthesized several alkynes starting from the commercially available benzyl
bromides (Scheme 1). The prepared alkynes were relatively unstable and hence
were utilized immediately for synthesizing cPTs (Table 1).
Scheme 1. Chemical synthesis of alkynes a9-a16. The final alkynes were volatile
and unstable and hence were used immediately for the click reaction with the resinꢀ
bound azidoꢀcPT to produce the triazole variant denoted “*” in Table 1.
III.1. Structure activity relationship (SAR) of the triazole variant:
The HIVꢀ1 cell infection inhibition activities of the new triazole derivatives were
evaluated (Table 1 and Supplementary Figure S1). The hydrophobicity and
aromaticity of the R group on the triazole ring were found to be important, as well as
the spacing atoms between the triazole and the aryl group. Interestingly, using the
basic Nꢀmethyl imidazole in cPT 17 gave the only triazole derivative was completely
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inactive. Comparing cPT 16 and 20, bringing the phenyl closer to the triazole ring
slightly improved the activity (IC50 ~ 1.2 ꢁM for 20 compared to IC50 ~ 2 ꢁM for 16).
Building up on 20 by adding extra methyl group(s) further improved the activity as
shown for the isopropyl group (21, IC50 ~ 0.9 ꢁM) and the tertꢀbutyl group (22, IC50 ~
0.35 ꢁM). This observation indicated the importance of increasing the bulkiness at
the paraꢀ position of the phenyl ring. Replacing the tertꢀbutyl group (in 22) with a
phenyl group further confirmed this hypothesis where the biphenyl containing cPT 23
exhibited an IC50 value of 269 nM. Introducing a bulky, nonꢀaromatic moiety resulted
in an active cPT (18, IC50 ~ 1.3 ꢁM), though less active than the aromatic biphenyl
system (23). We also evaluated the isoquinoline moiety (30, IC50 ~ 5 ꢁM), which was
not as active as the biphenyl system (23). We noticed that a single atom spacer
between the triazole and the phenyl was missing in our prior PT28 and cPT20
optimization studies, and therefore evaluated the phenyl methyl moiety (in 19).
Interestingly, the activity improved (IC50 ~ 438 nM for 19) compared to the twoꢀ
spacer atoms (as in 16) and the directly attached phenyl (as in 20). This enhanced
potency indicated the importance of a single spacer atom in directing the phenyl ring
to an optimal position. We then synthesized (Scheme 1) a series of alkynes to
investigate the SAR around the phenyl of 19. Introducing an electron donating
methyl group at the paraꢀ position further improved potency (24, IC50 ~ 180 nM
versus IC50 ~ 438 nM for 19). A methyl shuffle from the paraꢀ position to the metaꢀ
position was well tolerated (26, IC50 ~ 220 nM). However, when introduced at the
orthoꢀ position, activity was greatly decreased (25, IC50 ~ 5.1 ꢁM, about 28ꢀfold lower
potency compared to 24). Surprisingly, adding a paraꢀmethyl group to 25 restored
the activity (27, IC50 ~ 302 nM). This added paraꢀmethyl may have enabled a contact
with a hot spot on gp120 that was not reached in the case of 25. A combination of
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either paraꢀmethyl and metaꢀmethyl groups (in 29) or two metaꢀmethyl groups (in 28)
resulted in decreased antiꢀHIV activity (8 ꢁM for 29 and 2 ꢁM for 28). These results
argue that increasing the bulkiness around the closer sides of the paraꢀ position may
have negative effects on correctly positioning the phenyl ring on its target site on the
gp120 protein, though addition of methyl far from the paraꢀ position could be
tolerated (orthoꢀ position as in 27). Introducing electronꢀwithdrawing halogens on the
phenyl ring of 19 resulted in decreased activities (31 with IC50 ~ 1.8 ꢁM, 32 with IC50
~ 7.3 ꢁM and 33 with IC50 ~ 6 ꢁM). We considered the possibility that the
hydrophobicity of these halogens may act similarly to the methyl groups, in addition
to the possible halogen bonding with the protein carbonyl groups. However, the
electronꢀwithdrawing nature of the halogens may have reduced the electron density,
on the phenyl ring, which may be required for essential πꢀ π interactions with gp120
residues.
Based on the aboveꢀdescribed variations (Figure 3 and Table 1), we envisioned that
a combination of the pharmacophore side chain substituents would enhance the
potency. We synthesized cPT 34 which has benzo[b]thiophene, cyclohexyl and
ethylꢀphenyl moieties, as hydrophobic groups, (Figure 4) to evaluate the activity of
these combined variations. However, this cPT variant had poor solubility, and IC50
determination was challenging because of precipitation problems during the infection
inhibition assay. In an attempt to increase the aqueous solubility of 34, we
synthesized 35 (Figure 4) in which we replaced the amide side chain of Asn with an
amine using a Dab amino acid during the cPT synthesis. As expected, 35 had better
solubility because of the newly introduced amino group. However this cPT was found
to be inactive. This reflects the importance of the amide functionality of the Asn
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residue or the unfavored positioning of the positively charged amine functionality
within gp120.
Fig.4 Attempted modification in cPT scaffold to improve solubility of cPT 34.
Each cPT was synthesized starting from the corresponding amino acids: Asn for 34
and Dab for 35. Designation of “poor” and “good” solubility was based on behavior of
cPT in PBS buffer or PBS buffer with 5% DMSO.
IV. Onꢀresin synthesis of biꢀaryl containing cPT
Based on the observation that the biꢀphenyl containing cPT 23 (Table 1) has a
comparable potency (IC50 value of 269 nM) to the parent ferrocene containing cPT 3
(IC50 value of 237 nM), we decided to explore the biꢀaryl system as a potential
activityꢀtuning moiety. We therefore designed a facile onꢀresin microwaveꢀassisted29ꢀ
31
synthetic pathway that will allow exploring different biꢀaryl systems (Scheme 2).
Briefly, after assembling the cPT sequence and onꢀresin cyclization, click reaction
with the bromoꢀalkyne was achieved. The bromine handle was then used for an onꢀ
resin crossꢀcoupling reaction using a boronic acid in presence of a palladium catalyst
and a base. Mild reaction conditions were enough to achieve the complete formation
of the biꢀaryl system (Scheme 2). Interestingly, replacing the terminal phenyl ring of
23 (Table 1, IC50 value of 269 nM) with its isostere25, 26 thiophene32, 33 ring resulted in
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> 2ꢀfold improvement in potency (36, IC50 value of 105 nM, Figure 5). This argues
that the lipophilic nature of the aryl moiety in this part of the molecule is important for
the activity. SPR competition assays showed that the newly derived thiopheneꢀ
phenyl system retained the dual hostꢀcell receptor antagonism signature of cPTs: 36
inhibited gp120 binding to both CD4 and 17b, the latter used as a coꢀreceptor
surrogate34 (Figure 5). However, replacing the middle phenyl ring (between the
triazole and the thiophene of 36) with another thiophene ring (a biꢀthiophene
containing cPT 37) resulted in reduced activity (IC50 value of 405 nM for 37). This
change in activity could be related to the change in the orientation of the long arm
triazoleꢀthiopheneꢀthiophene relative to the gp120ꢀbinding site. In this view, the
terminal thiophene of 37 installed in position 4 of the middle thiophene (Figure 5)
could have resulted in slight bending of this long triazole arm compared to the
straight arm in 36. Installing the terminal thiophene at position 5 of the middle
thiophene is yet to be investigated. Cell viability was assessed for 36 and 37; no
cytotoxicity was detected at the highest concentrations used (100 ꢁM, Figure 5).
Therefore, the selectivity index (SI) for 36 can be estimated to be >> 1000 based on
the IC50 and the CC50 values, indicating a good cellular safety profile for this new
lead compound.
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Scheme 2. On-resin synthesis of the bi-aryl system on cPT. Reaction conditions
and reagents are shown on the arrows.
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36 IC50 ~ 105 ± 4 nM
37 IC50 ~ 405 ± 11 nM
H2N
H2N
A
B
C
O
O
O
O
5
O
O
S
S
NH HN
NH HN
4
H2N
H2N
O
S
N
N
O
O
O
O
N
N
N
N
N
N
O
NH
HN
NH
HN
HN
O
HN
O
O
NH2
O
NH2
N
N
H
H
36
37
IC50 ~ 100 nM
IC50 ~ 400 nM
Conc. [nM] of cPT
D
E
F
36 CC50 > 100 μM
37 CC50 > 100 μM
IC50 = 66 ± 4 nM
IC50 = 56 ± 5 nM
Conc. [µM] of cPT
Conc. [nM] of 36
Conc. [nM] of 36
Fig.5 Structures and functional properties of cPTs 36 & 37. (A) cPT 36, (B) cPT
37, (C) infection inhibition curves against HIVꢀ1Bal.01 for 36 & 37, (D) cell viability
assay for 36 & 37. SPR competition assay were used to determine the ability of 36 to
inhibit gp120 binding to both 17b (E) and CD4 (F).
V. Isothermal titration calorimetry (ITC) of
3 versus 36
To better explain the differences in antiviral potencies between cPTs 3 and 36, we
determined their binding affinities and thermodynamic profiles by isothermal titration
calorimetry (ITC). Binding was measured in the monomeric gp120ꢀYUꢀ2 context.
Glycoprotein gp120 YUꢀ2 bound 3 with an affinity of 415 nM and a stoichiometry of
one cPT per gp120 molecule (Figure 6, A). The binding mode was mainly enthalpy
driven, with a ∆H value of ꢀ5.96 kcal/mol (Figure 6, C). The new cPT 36 showed
improved binding affinity with dissociation constant of 350 nM and was bound by one
gp120 molecule, comparable to 3 in the binding comparison (Figure 6, B).
Interestingly, the thermodynamic profile of 36 indicated a slight increase towards an
entropically favored (ꢀT∆S = ꢀ4.74 kcal/mol) binding mechanism compared to 3 (ꢀ
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T∆S = ꢀ2.75 kcal/mol, Figure 6, C). The effect of peptide cyclization can be
observed in the thermodynamic signature by comparing cPTs and PTs. In this study,
both 3 and 36 showed positive
ΔS values, suggesting an entropically favored
binding mechanism. In contrast, the thermodynamic signature for linear PTs
previously showed negative ∆S values,23 reflecting a large unfavorable entropy
change.
Fig.6 cPT binding and thermodynamic profile of monomeric wild type gp120
YU-2 for cPTs 3 (A and C) and 36 (B and C). Compound solution, 390 µM (for 3) or
200 µM (for 36), was titrated in 8 µL steps into 30 µM of YU2 fullꢀlength gp120 at
25°C in the reaction chamber of a VPꢀITC instrument using 1x PBS buffer at pH 7.3.
The resulting heat changes were integrated, and the values obtained were fitted to a
quadratic binding equation (one site binding model). The following KD and
thermodynamic values were derived: 3 (A and C): KD = 415 ± 98 nM, n = 1.06 ±
0.02, ∆H = ꢀ5.96 ± 0.13 kcal/mol, ∆S = 9.22 cal/mol; 36 (B and C): KD = 350 ± 62 nM,
n = 1.06 ± 0.01, ∆H = ꢀ4.07 ± 0.06 kcal/mol, ∆S = 15.9 cal/mol.
VI. Binding model of 36 with HIVꢀ1 Env gp120 unit (pdb: 5FUU35)
PTs and cPTs bind to an inactive state of the conformationally dynamic36, 37 HIVꢀ1
Env that lacks a formed bridging sheet, possibly by inserting a hydrophobic part of
the molecule under the β20/21 loop of the bridging sheet.38, 39 To investigate the
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binding mode of new cPT 36 with the dynamic HIVꢀ1 Env gp120, we used Glide
InducedFit Extended Sampling to allow flexibility of the protein at the active site.
Multiple conformers of 36 were generated (Schrödinger Macrocycle Conformational
Sampling)40, 41 and clustered using heavy atoms. This yielded 30 clusters, for each of
which one conformer was selected as a cluster representative. The 30 cluster
representatives were docked (using Schrödinger InducedFit Extended Sampling),
and the returning pose/protein complexes were sorted according to their Glide
gscores. Examination of the top scored poses was based on the selection criteria
(Table 2) that were extracted from structure activity relationships in prior work20, 22, 27,
28 as well as from the current study. The first pose that meets all the selection criteria
(pose 4 from Table 2) was selected as a representative putative binding mode
(Figure 7) for 36 within gp120. The overall ligandꢀprotein interaction energy for this
pose was calculated using SZYBKI (v1.8.0.1: OpenEye Scientific Software, Santa
Fe, NM. http://www.eyesopen.com) and found to be ꢀ27.13 kcal/mol, indicating high
stability within this binding conformation. Interestingly, the pose shows a similar
orientation to what has been previously illustrated for different cPTs.20, 38 The newly
introduced thiopheneꢀphenyl on the triazole nicely penetrates through the preꢀ
identified subꢀsite 222 within gp120 located under the α1ꢀhelix and β20/21 loop (yellow
shading in Figure 7), and the Trp indole buries itself in subꢀsite 122 in proximity to
residue Thr257 and the α5ꢀhelix residue Asn478 (light blue shading in Figure 7).
The biꢀaromatic system stabilizes itself in the gp120 pocket through two possible Tꢀ
shaped and edgeꢀtoꢀface stacking interactions. The thiophene stacks to the
important residue Trp112 indole ring,22 whereas the phenyl (between the triazole and
thiophene) stacks with residue Phe382 (Figure 7). Such interactions could be the
reason for activity retention by the biꢀaryl systems comparable to the bulkier
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ferrocene moiety. The putative model also shows the secꢀbutyl moiety of the cPTꢀIle
buried between the β20/21 loop and the CD4 binding loop (Figure 7). This finding
might explain the importance of this branched/bulky moiety compared to less
branched or tethered moieties (Figure 3). In addition, this model is consistent with
the finding that adding polar atoms to this cPTꢀIle side chain moiety/substituent
would cause solvation of this part of the molecule, therefore could be pulling the
molecule out into the solvent and preventing the proper docking onto the active site.
Table 2. Selection criteria of a putative binding pose of 36 with gp120 (pdb
code 5FUU35). Green “correct” marks mean that the pose fits the criterion, the red
“cross” marks mean that the pose doesn’t fit the criterion. The first pose, which
meets all criteria, is highlighted in yellow.
Contact with
Thr257/Ser375
cluster22
Contact with
Glide
Trp/Ile/arylꢀ
C terminal
N terminal
Trp112
gscore
triazoloPro buried amide exposed amine exposed
cluster22
Pose 1
Pose 2
Pose 3
Pose 4
ꢀ15.494
ꢀ14.388
ꢀ13.938
ꢀ13.741
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Fig.7 Putative binding pose 4 (from Table 2) of cPT 36 docked onto gp120 from
the pdb 5FUU35. cPT 36 is shown in cyan sticks, Hꢀbonds are shown as black
dashed lines and πꢀπ stacking interactions are shown in red dashed lines.
VII. Effect of removing of the primary amine group from 36.
The aboveꢀdescribed model (Figure 7) showed that the N terminal of 36 is solvent
exposed. We investigated the potential of removing this primary NH2 from the cPT
scaffold. This would allow exploring the importance of this part of the scaffold, which
acts as a connecting bridge to the important cPT pharmacophore. Additionally, this
NH2 removal would reduce the total polar surface area of the scaffold and increase
the lipophilicity. Controlled lipophilicity42 could be a step towards optimizing the
scaffold for enhanced oral bioavailability. We designed a synthetic route to achieve
the cyclization using a nonꢀchiral, NH2ꢀfree linker (Scheme 3). The cyclization
intermediate 4ꢀ((9Hꢀfluorenꢀ9ꢀyl)methoxy)ꢀ4ꢀoxobutanoic acid (a19) was synthesized
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as previously reported starting from 9ꢀfluorenylmethanol (FmOH, a17) and succinic
anhydride (a18).43 The resultant cPT 38 showed HIVꢀ1 IC50 value of ~ 882 ± 44 nM
(Supplementary Figure S2), with about 8ꢀfold decrease in potency compared to the
NH2ꢀcontaining analogue 36 (IC50 ~ 100 nM). Interestingly, this 8ꢀfold decrease in
potency is comparable to that observed with changing the chirality of the Asp residue
in cPT 5 from the S to R configuration (Figure 2). This potency decrease could
therefore be related to disturbing the cPT conformation. In this view, losing the
chirality by removing the NH2 results in more flexibility, and therefore a less ordered
conformation of cPTs, leading to decreased potency. As expected by the binding
model, the role of NH2 in interaction with gp120 could then be ruled out. Supporting
this hypothesis, we previously showed that this amino terminal could be extended
through amide bond formation with additional residues without potency decrease.20
However, other possible electrostatic interactions by the amide bond cannot be
precluded and should be investigated. This is an important result, which will need
further confirmation to direct further chemical optimization and designs at this part of
the cPT scaffold, where we envision that rigidification will be important.
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Scheme 3. Synthesis of the cPT 38, with no free amine group. The cyclization
linker a19 (shown in blue) was synthesized using FmOH (a17) and succinic
anhydride (a18)43 and was incorporated in the cPT synthesis.
Discussion
We have previously discovered small cPTs that are metabolically stable and have
great potential to be developed as HIVꢀ1 therapeutics.20 Moreover, we have recently
shown that gold nanoparticles coated with linear PTs are not only able to efficiently
kill HIVꢀ1 virions but also to kill HIVꢀ1 infected cells via irreversible cell lysis.44 As
cPTs have all the phenotypes of the parent linear PTs, the important infectedꢀcellꢀ
killing ability can also be recapitulated with the metabolically stable cPTs,
highlighting the potential of formulating these agents as a possible HIV cure.
The primary goal in this work was to carry out a first SAR investigation of the
chemical space around the cPT scaffold in order to identify paths for chemical and
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potency optimization. This was achieved by examining the effects of chemical
variations in different parts of the cPT scaffold, including the pharmacophore IleꢀXꢀ
Trp (where X is the arylꢀtriazoloꢀPro) and the other half of the molecule (the
cyclization bridge). We found that, within the pharmacophore IleꢀXꢀTrp, the aryl
moiety on the triazole can act as an activity tuner, whereas the SAR around the Ile
and Trp side chains was more limited and changes were less permissible.
The major finding in this work was the successful replacement of the less drugꢀlike
ferrocene moiety (on the triazole ring) with other moieties that retained similar
activities. Previously, the bulky ferrocene moiety on the triazole ring was found, after
a series of optimizations,27, 28 to be optimal for activity. However, since varying the
chemical substituents around the ferrocene is limited, this hindered exploration of
different chemical environments in that part of the pharmacophore. Here, we
evaluated new triazole substituent moieties that had not been previously explored
and found hits that can allow further chemical optimization.
We found that installing a long planer arm on the triazole ring that contains aromatic
electron system(s) not only can preserve the activity previously observed with the
bulky ferrocene moiety, but also can tune the activity to improve potency and obtain
more drugꢀlike cPTs. A first promising moiety was the pꢀmethylꢀphenyl methylene
moiety, where we observed slight improvement in activity (cPT 24, IC50 ~ 180 nM)
compared to the lead cPT 3. The second promising system found in this work was
the biꢀaryl system (cPTs 23 and 36), wherein we first observed the activity retention
with the biꢀphenyl moiety. Looking at cPTs 24 and 36, an obvious future effort would
be to hybridize these two cPTs by introducing a methylene atom between the
thiopheneꢀphenyl moiety of 36 and the triazole ring; this could potentially improve the
activities of 36. One advantage of cPTs as an emerging class of HIVꢀ1 inhibitors is
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the facile short solid phase mediated synthesis, an important feature in inhibitor
development. We therefore developed an onꢀresin method to install different biꢀaryl
systems on the triazole ring, keeping the ease of synthesis unchanged.
Since newly discovered triazole substituent components come from synthetic
building blocks, rather than the amino acids, it can also be helpful to tune the
physiochemical properties of the molecule. One example was replacing the terminal
phenyl ring in cPT 23 with a thiophene isostere, in cPT 36, which resulted in
improved antiꢀHIV potency. Similar approaches therefore could be utilized to improve
aqueous solubility, by introducing heteroatoms on selected positions of the scaffold,
and to increase metabolic stability, by introducing metabolic blocking groups or even
locking the conformation of the biꢀaryl system that may have favorable effects on
activity and/or physiochemical properties.
Overall, findings from optimizing the pharmacophore could suggest simultaneous
multiple changes in the pharmacophore IleꢀXꢀTrp as a future strategy to improve
potency. The challenge here will be selection of moieties that can be combined
together to maintain a balance between compound hydrophobicity and solubility.
Utilizing the “activity tuner” as mentioned above could be a strategy to improve
solubility while allowing combinations of structural changes to be made.
In the current work, we also found that the other half of the cPT scaffold, the
cyclization bridge, greatly affects the antiꢀHIV activity. This could occur not through
the direct effect of losing or gaining contacts with the target gp120 protein by this
part of the molecule, but rather through correctly/incorrectly directing the
pharmacophore IleꢀXꢀTrp within the active binding site on gp120. This part of the
scaffold, therefore, can be referred to as a “conformational tuner”. We previously
found that varying the length of this cyclization bridge has powerful impact on
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activities.20 Here, we further confirmed this fact by observing the changes in activities
in response to changes in the cyclization bridge. Changing the chirality of cPTAsp
residue from the S to R configuration was more tolerated (7ꢀfold change) than the
same change in the cPTLys residue. One explanation for that could be the proximity
of the cPTLys residue to the pharmacophoreꢀTrp, whereas the cPTAsp residue is oneꢀ
residue apart from the pharmacophoreꢀIle. Therefore, conformational changes at the
cPTAsp residue could be more easily overcome than those caused by cPTLys residue
disruption. Interestingly, complete removal of the chiral center at the cPTAsp residue
resulted in comparable 8ꢀfold decrease in activity observed with the S to R
conversion. And since an N terminal extension is tolerated in cPTs, the direct role of
+
the missing NH3 as a binding group can be ruled out. The results argue instead that
+
removing the chirality by removing the NH3 group had the same conformational
disturbance as did the S to R conversion. For future optimization, it must be taken
into consideration where optimizing this cyclization bridge will benefit from modifying
rigidification, yet retaining functional conformation. The scheme in Figure 8
summarizes an overall SAR perspective that can be extracted from this study to
guide future synthetic modifications.
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Branched & bulky
No polar atoms
Pharmacophore
Amide func, onality
cri, cal
Reversing chirality
or
removing NH2
tolerated
S configura, on
cri, cal
Cri, cal Unsubs, tuted indole
NH could be replaced by S
cPT 36
MW: 978.14; tPSA: 324;
No of HBDs: 9; No of HBAs: 13
No of rotatable bonds: 10
clogP: ꢀ0.04; clogD7.4: ꢀ0.2
Fig.8 Overall structure activity relationship of cPTs depicted with cPT 36.
Molecular properties of 36 were calculated using MarvinSketch software (version
15.11.9.0, 2015, ChemAxon, http://www.chemaxon.com).
Generally, macrocyclic drugs, both naturally derived and synthetic, have received
increasing attention in the past years owing to their excellent ability to target poor
druggable targets such as PPIs. Macrocycles possess good pharmaceutical
properties despite their relatively larger molecular weights compared to the
conventional small molecule drugs.19 The evolving cPT inhibitors of HIVꢀ1 Env PPI
interactions with the host cellular receptor proteins are a potentially important class
of inhibitors that could lead to novel antiꢀHIV therapeutics, especially with their
unique modes of action, namely inhibition of Envꢀcellular protein interactions and
triggering gp120 shedding from the virions. The pioneering work of Villar et al.18
helped derive rules, “Villar’s rule of macrocycles”, for designing orally bioavailable
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macrocyclic drugs by controlling macrocyclic physiochemical properties. According
to Villar’s rules,18 a macrocycle should be orally bioavailable if it has 1) molecular
weight of 600ꢀ1200 daltons, 2) clogP value of ꢀ2 to 6, 3) polar surface area of 180ꢀ
320 Å2, 4) number of Hꢀbond donors < 12, 5) number of Hꢀbond acceptors in the
range of 12ꢀ16 and 6) number of rotatable bonds < 15. Interestingly, analysis of our
lead compound 36 discovered in this study (Figure 8) shows that it actually fits these
rules, leading to the question whether these molecules might be orally bioavailable. If
true, the potential for oral bioavailability, proteolytic stability and good aqueous
solubility (reflected by clogD7.4) make this class of cPTs viable candidates for
development as HIV therapeutics.
Conclusions
In summary, we have explored the effect of chemical changes around the cPT
scaffold including the pharmacophore region as well as the cyclization bridge. A
major task going forward will be to further utilize the SAR data derived from this work
at both the “activity tuner” and “conformational tuner” sites for optimizing the scaffold
to increase the potency and further improve the molecular properties of cPTs. This
will
help
identify
lead
cPTs
suitable
for
advanced
ADME
(absorption, distribution, metabolism and excretion) as well as pharmacokinetics
studies.
Experimental
I. Chemistry
I.1. Chemical synthesis of cPTs (3ꢀ38)
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A CEM microwave synthesizer (Liberty Blue) was used for solid phase peptide
synthesis of cPTs as previously described.20 Fmocꢀ4ꢀazidoꢀProline was synthesized
as previously reported.20 All other Fmocꢀ, Bocꢀ protected amino acids, N,N'ꢀ
diisopropylcarbodiimide (DIC), ethyl (hydroxyimino) cyanoacetate (OxymaPure) and
rink amide resin (100ꢀ200 mesh size, 0.53 meq/g substitution) were purchased from
ChemꢀImpex International, INC. Alkynes and CuI catalyst for the click reaction, and
hydrazine, were purchased from Sigma Aldrich. Benzyl bromides were purchased
form Alfa Aesar. HPLC purifications were performed using a Waters® HPLC system
with reverse phase (RP) C18 prep columns. All synthesized cPTs were >95 % pure
as judged by purity checks using an analytical C18 RPꢀHPLC column and
BeckmanCoulter® HPLC system. HPLC grade ACN, MilliporeꢀMilliQ water and 0.1%
TFA were used as solvents for the HPLC purification. Alkynes a10ꢀa16 were
synthesized from the corresponding benzyl bromides a1ꢀa9 following the procedures
previously described45 (see below). Alkynes a10ꢀa16 were used in click reactions
without purification because they are volatile and unstable upon storage. Mass
validation of all cPTs was performed using Thermo Scientific LTQ XL Ion Trap
LC/MS (Supplementary Table S2).
I.1.a General synthesis of alkynes a9-a16
To a solution of ethynyltrimethylsilane (40 mmol) in 20 mL THF at 0 °C, was added iꢀ
PrMgCl (2 M solution in THF) dropwise and the mixture was stirred at 0 °C for 30 min
and allowed to warm up to r.t. for 30 min. CuBr (6 mmol) was added and the mixture
was stirred at r.t. for 1 h. The corresponding benzyl bromide a1-a8 was added and
the mixture was heated under reflux for 4 h. The reaction mixture was then cooled to
r.t. before pouring onto saturated NH4Cl solution (500 mL). The mixture was
29