Bifunctional Chimera That Coordinately Targets Human Immunodeficiency Virus 1 Envelope gp120 and the Host-Cell CCR5 Coreceptor at the Virus-Cell Interface

Article abstract of DOI:10.1021/acs.jmedchem.8b00477

To address the urgent need for new agents to reduce the global occurrence and spread of AIDS, we investigated the underlying hypothesis that antagonists of the HIV-1 envelope (Env) gp120 protein and the host-cell coreceptor (CoR) protein can be covalently joined into bifunctional synergistic combinations with improved antiviral capabilities. A synthetic protocol was established to covalently combine a CCR5 small-molecule antagonist and a gp120 peptide triazole antagonist to form the bifunctional chimera. Importantly, the chimeric inhibitor preserved the specific targeting properties of the two separate chimera components and, at the same time, exhibited low to subnanomolar potencies in inhibiting cell infection by different pseudoviruses, which were substantially greater than those of a noncovalent mixture of the individual components. The results demonstrate that targeting the virus-cell interface with a single molecule can result in improved potencies and also the introduction of new phenotypes to the chimeric inhibitor, such as the irreversible inactivation of HIV-1.

Full text of DOI:10.1021/acs.jmedchem.8b00477

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Article
Bifunctional Chimera That Coordinately Targets Human
Immunodeficiency Virus 1 (HIV-1) Envelope gp120 and
Host Cell CCR5 Co-receptor at the Virus-Cell Interface
Adel A Rashad, Li-Rui Song, Andrew P Holmes, Kriti Acharya, Shiyu Zhang, Zhi-Long Wang,
Ebony Gary, Xin Xie, Vanessa Pirrone, Michele A Kutzler, Ya-Qiu Long, and Irwin Chaiken
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00477 • Publication Date (Web): 16 May 2018
Downloaded from http://pubs.acs.org on May 16, 2018
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Bifunctional Chimera That Coordinately Targets Human Immunodeficiency Virus 1
(HIV-1) Envelope gp120 and Host Cell CCR5 Co-receptor at the Virus-Cell Interface
Adel A. Rashad#1, LiꢀRui Song#2,3,4, Andrew P. Holmes#1, Kriti Acharya1, Shiyu Zhang1,5,
ZhiꢀLong Wang2, Ebony Gary6, Xin Xie2, Vanessa Pirrone7, Michele A. Kutzler6, YaꢀQiu
Long*2,3, and Irwin Chaiken*1
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Department of Biochemistry and Molecular Biology, Drexel University College of
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Medicine, Philadelphia, PA 19102, USA; CAS Key Laboratory of Receptor Research,
Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China
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College of Pharmaceutical Sciences, Soochow University Medical College, Suzhou
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215123, China; University of Chinese Academy of Sciences，No. 19A Yuquan Road,
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Beijing 100049, China; School of Biomedical Engineering, Science and Health Systems,
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Drexel University, Philadelphia, PA 19104; Department of Medicine, Drexel University
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College of Medicine, Philadelphia, PA 19102; and Department of Microbiology and
Immunology, Drexel University College of Medicine, Philadelphia, PA 19102, USA
# These authors contributed equally to this work.
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BSTRACT
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To address the urgent need for new agents to reduce the global occurrence and spread of
AIDS, we investigated the underlying hypothesis that antagonists of HIVꢀ1 Envelope (Env)
gp120 and host cell coꢀreceptor (CoR) proteins can be covalently joined into bifunctional
synergistic combinations that will improve antiviral. A synthetic protocol was established to
covalently combine a CCR5 small molecule antagonist and a gp120 peptide triazole
antagonist to form the bifunctional chimera. Importantly, the chimeric inhibitor preserved the
specific targeting properties of the two separate chimera components, and at the same time
exhibited low to subꢀnanomolar potencies, in inhibiting cell infection by different
pseudoviruses, that were substantially greater than those of a nonꢀcovalent mixture of
individual components. The results demonstrate that targeting the virusꢀcell interface by a
single molecule can result in improved potencies and also introduction of new phenotypes to
the chimeric inhibitor such as irreversible inactivation of HIVꢀ1.
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NTRODUCTION
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Inhibition of initial entry of HIVꢀ1 into host cells remains a compelling yet elusive means to
prevent infection and spread of the virus. HIVꢀ1 cell infection is mediated by concerted
interactions, at the virusꢀhost cell interface, between trimeric envelope glycoprotein (Env)
spikes on the virus membrane surface and two host cell receptors, CD4 and a coꢀreceptor that
is most commonly either CCR5 or CXCR41. Each Env trimer consists of two nonꢀcovalently
associated glycoproteins, a gp41 transmembrane protein and an external gp120 surface
protein. During viral infection, CD4 interaction with the most exposed Env protein gp120
causes conformational rearrangement of the latter, leading to increased affinity coꢀreceptor
interaction at an initially cryptic site in gp120. This cascade exposes structural components in
gp41 necessary to promote virus and cell membrane lipid mixing, fusion, pore formation, and
infection. Inhibitors that can potently block virusꢀcell interactions and cell entry would hold
great promise of inhibiting initial HIVꢀ1 infection.
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Currently approved inhibitors of entry steps are available clinically but have properties that
limit their therapeutic usefulness. Maraviroc is an approved entry inhibitor that binds to the
coꢀreceptor CCR5 and prevents binding to HIV Env gp120 and, as a consequence, prevents
full exposure of gp41 and suppresses virusꢀcell fusion2, 3. However, effectiveness of this drug
requires matching the administered therapeutic to coꢀreceptor use by the viral variants
infecting each patient. In most cases, HIV infects cells after binding to the CCR5 receptor in
the early stages of disease, but as the disease progresses, the virus can switch to another
receptor, CXCR4. So, patients need to be tested for viral tropism before commencing CCR5
inhibition therapy. In addition, maraviroc induces tropism switch to CXCR4ꢀexpressing
cells4. Hence, maraviroc is currently only approved for use in treatmentꢀexperienced
patients5. Enfuvirtide (T20) is a 36ꢀresidue peptide that mimics part of the Cꢀterminal helix in
gp41 that mediates fusion. As such, it blocks binding of the gp41 Nꢀterminal helix to Cꢀ
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terminal helix and formation of the 6ꢀhelix bundle, which is the process that drives fusion
between viral and cellular membranes. Due to its high cost of production, short drug halfꢀlife
and the need for subcutaneous injections, enfuvirtide is also only approved as salvage therapy
in patients who have failed multiple lines of therapy. Moreover, rapid mutations in a 10ꢀ
residue stretch of gp41 Nꢀterminal helix were observed to lead to resistance to this drug6.
Recently, it also has been shown that HIVꢀ1 can develop resistance to fusion inhibitors and
become inhibitorꢀdependent, due to the critical kinetics required for these inhibitors7.
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Identifying new entryꢀtargeting inhibitor approaches, in particular those that could function in
combinations to increase potency and overcome viral resistance, remains an important goal of
HIVꢀ1 drug discovery efforts. To date, delineation of such combinations has been limited
mainly to combinations of coꢀreceptor inhibitors and T208ꢀ11. Such combinations have
demonstrated the potential for strong synergy with both CCR5 (TAKꢀ220, SCHꢀC, and
Aplaviroc) and CXCR4 (AMDꢀ3100) inhibitors in assays of infection of peripheral blood
mononuclear cells (PBMCs) with clinical HIVꢀ1 isolates. Synergy between coꢀreceptor
inhibitors and Env gp41 inhibitor has been proposed to be due to kinetic linkage between the
steps of entry that they antagonize9ꢀ12. This work points to the potential to achieve increased
potency through combinations of inhibitors targeting different components of the virusꢀcell
interface. This could be particularly important as new smallꢀmolecule coꢀreceptor inhibitors
are developed that target CXCR4 in addition to CCR5, as well as new inhibitors of the Env
protein complex, including the most exposed protein component, gp120.
In the current work, we tested the hypothesis that covalent fusion of HIVꢀ1 gp120 and coꢀ
receptor inhibitors could yield an HIVꢀ1 cell infection inhibitor with enhanced potency
compared to the sum of the potencies of the individual components. New classes of coꢀ
receptor inhibitors have been identified that can target either CCR5 or CXCR4 with strong
antiviral effects13ꢀ17. At the same time, a class of gp120ꢀtargeting peptide triazoles (PTs) has
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been identified that blocks both host cell receptor interactions and causes irreversible virus
inactivation by triggering gp120 shedding18, 19. We chose LJC24015 (coꢀreceptor targeting,
see Figure 2 for structure) and UM1520 (gp120ꢀtargeting, see Figure 2 for structure)
inhibitors for the initial prototype chimera components based on the alreadyꢀestablished
antiviral efficacies of these components on their own. The conjugation points of LJC240 to
UM15 were determined by examining targetꢀbinding models as well as structureꢀactivity
relationships that identify structural elements, of the inhibitors, that are tolerant to
modification. The protocol for the chimera synthesis was derived from the alreadyꢀ
established solid phase synthesis of the peptide triazoles themselves21ꢀ24 with followꢀup
sequential additions of linker and the free acid form of LJC240ꢀCOOH (10) before
introducing the triazole by click conjugation. The functional properties of the resulting
prototype chimera LJC240ꢀL4ꢀUM15 (11) were examined by a combination of cellular,
virological and molecular assays. The results demonstrate that the covalent chimera retained
individual component functions and at the same time a synergistically enhanced antiviral
potency compared to a nonꢀcovalent mixture(s) of the 2 component inhibitors. The work
provides strong support for the hypothesis that the bifunctional chimera could engage and
synergistically block the infectionꢀcausing functions of host cell coꢀreceptor and virus
envelope gp120 at the HIVꢀ1/cell interface.
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R
ESULTS
1. Chimera Design and Synthesis
We designed a bifunctional molecule that is able to target both the HIVꢀ1 envelope and the
cellular coꢀreceptor (here, CCR5). When the HIVꢀ1 envelope encounters the cell receptors,
the released gp120 V3 loop docks into the cellular coꢀreceptor binding site. We envisioned
that a bifunctional molecule that has gp120 and CCR5 targeting components would be able to
bridge the envelopeꢀCCR5 encounter complex. This kind of complex has not been resolved at
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high resolution. However, structural information is available for the individual components.25,
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Since PTs target the CD4 binding area of gp120, we measured the distance between the
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CD4 binding (residue Asp368) site and the tip of the released V3 loop (Figure 1, generated
from pdb code 2QAD27). This distance represents the hypothetical minimal linker length
required to tether gp120 and CCR5 inhibitors together, in a way that mimics the encounter
complex.
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Figure 1. Geometry of gp120 protein, showing the distance between the CD4 binding site and the
released V3 loop tip (pdb code 2QAD27).
We also examined the possible positions for chemical ligation through modeling of the
inhibitors in the context of their individual protein targets (Figure 2), in order to avoid any
unexpected reduction in potency. As shown in Figure 2 left, the UM15 Nꢀterminal
(protonated) residue is in a solventꢀexposed moiety. On the other hand, the fluorine atom of
LJC240 (Figure 2 right) is well solvated and actually can displace water of the crystal
structure of the CCR5 receptor. This docking result, combined with prior structureꢀfunction
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relationships in LJC240ꢀderived CCR5 inhibitors15, argues that structural variation replacing
the fluorine atom should be tolerated.
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Figure 2. Interaction maps of UM15 (left) docked within gp120 of the HIVꢀ1 envelope (pdb code:
5FUU28) and LJC24015 (right) docked into the CCR5 structure (pdb code: 4MBS26). The grey circles
represent the solvated (solvent exposed) moieties.
Based on the predicted topology presented above (Figures 1 and 2), we envisioned a linker
of approximately 59 Å would be required to tether the UM15 Nꢀterminal with the fluoroꢀ
phenyl ring. Hydrophilicity built into the linker was predicted to help keep the two
components apart and avoid any nonꢀspecific hydrophobic interactions with either of the
target proteins. We chose 4 units of aminoꢀethoxyꢀethoxy acetic acid as the linker (hereafter
referred to as L4) as it would provide the distance based on bond length measurements.
We employed solid phase synthesis to build the L4ꢀUM15 precursor. We also synthesized a
modified version of LJC240 (compound 10, Figure 3) in which the fluorine atom (Figure 2)
was replaced by a carboxylic group to enable ligation with the amino group of the L4ꢀUM15
Nꢀterminus (Figure 4). The coupling reagents of HBTU/HOBt and DIEA were selected for
the amideꢀcoupling, followed by onꢀresin click reaction to form the triazole component on
the UM15ꢀazidoꢀPro residue (Figure 4).
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Figure 3. Synthesis of coꢀreceptor inhibitor LJC240ꢀCOOH (10). Reagents and conditions: (a)
NH2OH•HCl, NaHCO3, EtOH/H2O, rt; (b) Na, pentanol, reflux; (c) (Boc)2O, TEA, THF, rt; (d) 10%
Pd/C, HCO2NH4, Methanol, reflux; (e) 1ꢀbromoꢀ3ꢀchloropropane, KI, TEA, DMF, rt; (f) 1ꢀ
acetylpiperidineꢀ4ꢀcarboxylic acid, SOCl2, TEA, DCM, rt; (g) 5, KI, K2CO3, MeCN, reflux; (h) HCl,
Methanol, rt; (i) 4ꢀ(chlorosulfonyl)benzoic acid, Na2CO3, dioxane/H2O, rt.
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Figure 4. Solid phase synthesis of chimera 11. Liberty blue microwave synthesizer was used for the
assembly of the L4ꢀUM15 precursor segment. The latter was joined with LJC240 free acid (10)
followed by ferrocenyl triazole formation using click reaction, leading to the final chimera (11).
2. The Bifunctional Chimera Displays Potent Neutralization of HIV-1 Infection
We measured the antiviral potency of the bifunctional chimera in a cell infection inhibition
assay. Pseudotyped viruses bearing the BaL.01, JRꢀFL and YU2 envelope proteins, and
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carrying a luciferase reporter gene were incubated with serially diluted chimera for 30
minutes at 37 ºC and then added to human osteosarcoma (HOS) cells expressing CD4 and
CCR5 (HOS.T4.R5). Following completion of the infection inhibition assay, the cells were
lysed and monitored for luciferase activity. UM15 and LJC240ꢀL4 (with the same L4 linker
used for the chimera) served as controls for the experiment since they best represent the
parental molecules that make up the chimera. LJC240ꢀL4ꢀUM15 (11) displayed subꢀ
nanomolar to low nanomolar inhibition of HIVꢀ1 envelopeꢀpseudotyped viruses (Figure 5).
The chimera had improved potencies that ranged from > 2 fold (YU2) to > 50 fold (BaL.01)
greater than the most potent parental component, LJC240ꢀL4. We also evaluated an
additional control of a 1:1 nonꢀcovalent mixture of UM15 and LJC240ꢀL4 (against the tierꢀ2
JRFL pseudotyped virus) which would mimic the molar ratio between the inhibitors in the
chimera, to determine whether the covalent attachment resulted in a potential synergy and
increased the potency beyond that of the combination of the individual inhibitors. Indeed, the
bifunctional chimera was more potent (4 nM) than the 1:1 mixture (34 nM, Figure 5B), thus
demonstrating the value of the covalent attachment on potency compared to the sum of its
parts.
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(A)
(B)
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IC50 = 4 ± 0.6 nM
IC50 = 0.359 ± 0.05 nM
IC50 = 20 ± 2 nM
IC50 = 378 ± 23 nM
IC50 = 20 ± 2 nM
IC50 = 34 ± 3.6 nM
IC50 = 498 ± 8 nM
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(C)
(D)
IC50 = 4.8 ± 0.08 nM
IC50 = 10 ± 3 nM
IC50 = 500 ± 65 nM
IC50 = 298 ± 60 nM
IC50 = ND
IC50 = 320 ± 30 nM
[Inhibitor] µM
Figure 5. Enhanced neutralization of HIVꢀ1 Envꢀpseudotyped viruses (A) Bal.01; (B) JRFL; (C)
YU2; and (D) HxBc2 as an example of X4 tropic virus by the bifunctional chimera (red), UM15
(black), LJC240ꢀL4 (blue), 1:1 mixture of UM15 and LJC240ꢀL4 (green). ND = not determined.
2.1. Synergistic effect of the covalent chimera
To further define the value of the covalent attachment of UM15 with LJC240ꢀL4 versus the
nonꢀcovalent mixture, we performed a quantitative synergy analysis. Initially, we evaluated
the nonꢀcovalent mixtures against Bal.01 HIVꢀ1 strain (due to ease of virus production and
assay handling) based on their individual activities reported in Figure 5. Interestingly, both
UM15 and LJC240ꢀL4 showed ~ 7ꢀfold improved potency in presence of each other (Figure
6A); UM15 showed IC50 value of 56 nM in the mixture compared to 378 nM alone (Figure
5A), while LJC240ꢀL4 showed IC50 value of 2.9 nM in the mixture (Figure 6A) compared to
20 nM alone (Figure 5A). The IC50 value obtained for the covalent chimera in the same assay
(0.86 nM, Figure 6A) clearly showed a strong increase in potency vs the noncovalent
mixture. These results argue for the value of the covalently joining the two inhibitors
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together, further confirming the difference with the 1:1 nonꢀcovalent mixture (Figure 5B).
Representation of the data by an isobologram 29 (Figure 6B) shows the synergy between the
nonꢀcovalent mixture (1:30 molar ratio of UM15:LJC240ꢀL4) when we plotted the 50%
inhibition of the mixture (red dot in Figure 6B), compared to the individual IC50 values of the
separate inhibitors (connected by the blue line Figure 6B). Interestingly, plotting the 50%
inhibition by the covalent chimera 11 (green dot in Figure 6B) shows the even stronger
synergistic effect of the latter.
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(A)
(B)
Chimera
LJC240-L4 in mixture
UM15 in mixture
IC50 = 0.86 ± 0.3 nM
IC50 = 2.9 ± 0.6 nM
IC50 = 56 ± 2 nM
Figure 6. (A) Synergy assay results between UM15 and LJC240ꢀL4 against HIVꢀBal.01; IC50 values
(blue for LJC240ꢀL4 and black for UM15) are the effective inhibitory concentration of the individual
component in the mixture, the chimera 11 (red) was also included in the experiment. (B) Isobologram
representation of UM15/LJC240ꢀL4 combination; the blue line connects the IC50 values of the two
inhibitors, the red dot is the effective concentration of the nonꢀcovalent mixture (1:30 ratio which
achieved 50% inhibition) whereas the green dot is the effective concentration of the covalent chimera
11.
3. Individual Components of the Chimera Retain Activity
In order to confirm that the covalent attachment of the inhibitors did not interfere with the
ability of each molecule to bind to its respective target in the virusꢀcell interface, we
performed a series of experiments to measure the magnitudes of the peptide triazole and coꢀ
receptor inhibitor functions of the bifunctional chimera.
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3.1. Activity against X4-tropic HIV
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Firstly, we performed an infection inhibition assay with HxBc2 (Figure 5D), an X4ꢀtropic
virus, on HOS cells expressing CD4 and CXCR4 (HOS.T4.X4). By using an X4ꢀtropic virus
on X4ꢀexpressing cells, we ruled out contribution of the coꢀreceptor inhibitor towards the
potency of the chimera. As expected, the bifunctional chimera potency was nearly identical to
the UM15 control (Figure 5D), thereby demonstrating that the UM15 component of the
chimera retained full activity.
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3.2. Chimera 11 retains full gp120 dual antagonism
Surface Plasmon Resonance (SPR) was carried out to further confirm retention of the UM15
part gp120ꢀbinding efficiency. Both soluble CD4 and 17b (a gp120ꢀtargeting antibody that
stabilizes the bridging sheet) were immobilized separately on sensor chip surfaces, and gp120
was flowed across in increasing concentrations of inhibitor to assess binding. The binding of
the bifunctional chimera was again equivalent to the UM15 control (Figure 7).
Figure 7. Efficacy of chimeric inhibitor for gp120 protein ligand binding determined by SPR gp120
competition analyses. (Left) Representative sensorgrams showing dose dependent inhibition of
monomeric YU2 gp120 binding to the receptors CD4 and 17b by LJC240ꢀL4ꢀUM15. (Right) Dose
response curves derived from inhibition of YU2 gp120 binding sensorgrams by LJC240ꢀL4ꢀUM15
and UM15 (n=3).
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These findings confirm previous observations that Nꢀterminal extensions on the peptide do
not interfere with pharmacophore binding,30, 31 and also match the docking model for UM15
(Figure 2).
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3.3. Chimera 11 irreversibly inactivate HIV via gp120 shedding
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Western blot analysis was used to detect gp120 shedding function of the peptide triazole
component. As observed in infection inhibition (Figure 5D) and gp120 binding (Figure 7),
the shedding ability of UM15 was not affected by the covalent attachment to LJCꢀ240, with
the chimera and UM15 both inducing gp120 shedding with similar potencies (Figure 8). This
argues that the unique irreversible virus inactivation capacity is still retained by the chimeric
inhibitor.
Figure 8. Capacity of the bifunctional chimera LJC240ꢀL4ꢀUM15 to induce gp120 shedding similarly
to UM15.
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3.4. Evaluation of Chimera 11 and individual components against CD4-independent HIV
To further validate the bifunctional approach, we evaluated the chimera 11 and the individual
components against an adapted HIVꢀ1 pseudovirus, J1HX, and its CD4 independent mutant
N197S. Removal of the glycosylation site at asparagine 197 in the V1/V2 stem was found to
be sufficient to enable a conformation that is more primed for CCR5 binding in the absence
of CD4, and for HIVꢀ1 entry into CD4ꢀnegative cells expressing CCR5.32 As shown in
Figure 9, the chimera 11 was functionally active and showed synergy against the parent nonꢀ
CD4 independent J1HX WT with IC50 > 16 fold more active than LJC240ꢀL4 (Figure 9A).
With the CD4 independent mutant, the individual components remained active: the potency
of UM15 component didn’t change compared to the WT, arguing that binding of UM15 to
gp120 was not disrupted, while the LJC240ꢀL4 component showed ~ 3 fold enhanced
potency (Figure 9B) compared to the WT. In contrast with the WT J1HX, the chimera 11
was only slightly more active, if at all, than the LJC240ꢀL4 component alone against J1HX
N197S virus (Figure 9B). The reduced synergistic potency of the chimera 11 observed with
J1HX N197S could be due to either [1] masking of the chimera effect by enhanced LJC240ꢀ
L4 potency or [2] altered dual Env/CCR5 encounter by the chimera components due to
rearrangements in the gp120 V1/V2 region. Further investigation will be required to better
define the mechanism causing reduced synergy.
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Chimera
IC50 = 11 ± 1 nM
LJC240-L4 IC50 = 184 ± 21 nM
UM15 IC50 = 307 ± 39 nM
Chimera
LJC240-L4 IC50 = 59 ± 4 nM
UM15
IC50 = 50 ± 8 nM
(A)
(B)
IC50 = 315 ± 46 nM
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Figure 9. Evaluation of the chimera 11 (red) and the individual components UM15 (black) and
LJC240ꢀL4 (blue) against J1HX (A) and the CD4 independent J1HX mutant N197S (B).
3.5. Chimera 11 retains co-receptor binding efficiency
The coꢀreceptor inhibitory function of the chimera was assessed, initially using a calcium
mobilization assay to determine the extent of antagonism of RANTES function (Figure 10,
top panel). A comparison of LJC24015 (Figure 2) and LJC240ꢀL4 was included to determine
whether the linker would negatively impact antagonistic ability. Indeed, the added linker
appeared to interfere with the potency of the molecule, as judged by an activity decrease from
1.59 nM with LJC240 to 27.44 nM with LJC240ꢀL4. No further decrease of function was
observed with LJC240ꢀL4ꢀUM15 (11), as judged by IC50 value of 22.09 nM.
Antagonism of the coꢀreceptor was confirmed using chemotaxis assays with the parent
molecules, the linkerꢀcontaining inhibitor, and the bifunctional chimera. As shown in Figure
10 bottom panel, the compounds containing coꢀreceptor inhibitor component, at the IC50
values determined in the calcium mobilization assay, all prevented chemotaxis of CEM
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Figure 10. Doseꢀdependent CCR5 antagonism by the bifunctional chimera and the parent small
molecule inhibitors. (Top) The extent of inhibition of RANTES (10 nM) stimulation of calcium
mobilization was measured for LJC240ꢀcontaining compounds. Maraviroc was included as a control.
(Bottom) Chemotaxis inhibition of CEMꢀCCR5 cells to RANTES by LJC240ꢀL4ꢀUM15. One million
CEM CCR5 cells were seeded in the apical chamber of 8um transwell inserts and chemoattraction
was induced using RANTES (10 ng/ml) in the presence or absence (control, “No Chemokine”) of the
inhibitors maraviroc (MRVC), LJC240, LJC240ꢀL4, or the inhibitorꢀpeptide triazole chimera,
LJC240ꢀL4ꢀUM15 at the IC50 value for each compound. Data are presented as cells recovered in the
basolateral chamber post incubation, normalized to cells migrating to RANTES in the absence of
inhibitor. Twoꢀway ANOVA was used to compare the inhibitor treated groups to the RANTES only
control group and Student’s Tꢀ test was used to compare individual groups. Error bars represent mean
± SEM. *=pꢀ 0.05, **p ꢀ0.01. These data are representative of 2 independent experiments.
CCR5 cells to RANTES chemokine in a transwell assay. In vitro cell toxicity was measured
post incubation, and the inhibitors did not lead to cell death measured in the apical and
basolateral chambers post treatment (data not shown). These results support the finding that
the bifunctional chimera retains the function of antagonizing the CCR5 coꢀreceptor.
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4. Cytotoxicity and HIV specificity
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To assess cellular safety and HIV specificity, we evaluated the chimera (11) in a cytotoxicity
WST assay as well as the activity against Acute Murine Leukemia Virus (AMLV). As shown
in Figure 11, the chimera 11 did not show any cytotoxicity nor any activity against AMLV at
the concentration range used.
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Figure 11. Cytotoxicity evaluation of chimera 11 (left) and evaluation against AMLV (Right).
D
ISCUSSION
In this work, we engineered a covalent bifunctional chimera capable of inhibiting HIV cell
infection through a combination of effects on both sides of the virusꢀhost cell interface. A
linker designed to span the distance between the Env and the coꢀreceptor was utilized to join
the peptide triazole and the CCR5 antagonist. The first prototype bifunctional chimera
generated, namely LJC240ꢀL4ꢀUM15 (11), not only displayed enhanced inhibitory potency
compared to a 1:1 (mole/mole) nonꢀcovalent mixture of the parent molecules that make up
the chimera, but also showed better synergistic effect compared to the nonꢀcovalent mixtures
in a quantitative synergy analysis. This potency was made possible because each component
of the chimera is able to function coordinately despite the covalent attachment to the other.
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The observed synergistic function of the two inhibitory components of the CoRIꢀPT chimera
argues that both components can function at the same time. This in turn implies a spatial
proximity of the gp120 and coꢀreceptor binding sites upon virusꢀcell encounter. Several
hypotheses can be surmised to explain this synergy based on the mode of actions of the
chimera components. Since PTs inhibit binding to the coꢀreceptor site likely via affecting the
release of the V3 loop33, we envision that as the virus approaches the cell in the presence of
the chimera, the PT part of the chimera can dock onto the virion Env, inhibiting CD4
attachment and altering the and V3 loop/CCR5 epitope release mechanism. However, the
chimera has another exposed scaffold, which is the CCR5 blocker. This CCR5 blocker brings
the Envꢀchimera complex closer to the cell to dock onto the CCR5 protein. The net result of
this mechanism is locking the virusꢀcell complex in an arrested configuration, leading to
blocking the infection and later on causing gp120 shedding by PT domain function. In other
words, the chimera CCR5ꢀL4 inhibitor part can act as a false V3 loop, bringing the virus
closer to the cell, not for initiating infection, rather to be blocked. On the other hand, the
entropic advantage from the tethered hybrid over the separate two components might be one
cause of the synergistic effect.34 When the chimera binds to one site/protein at the cellꢀvirus
interface, much of the entropy cost of binding to the second site/protein has already been
paid. Thus, the free energy of binding for the chimera is not the sum of the free energies of
the two components because of entropy, leading to a synergistic potency. Importantly, the
shedding function of the chimera was demonstrated in this work. Nonetheless, the sequence
of bifunctional chimera binding events at the cellꢀvirus interface remains speculative at
present.
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While other covalent chimeras have been designed that target HIVꢀ1 infection35ꢀ40, the
prototype chimera LJC240ꢀL4ꢀUM15 (11) is unique in the added value of irreversible
inactivation the virus. Previously, chimeric inhibitors have been reported that target [1] two
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sites on the HIVꢀ1 Env protein alone36, 38, 39, [2] CD4 and Env gp4135 and [3] Env gp41 and
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coꢀreceptor.37,
The latter findings have demonstrated the feasibility of bifunctional
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engagement of both virus Env and host cell receptors. Since CCR5 and CXCR4 coꢀreceptors
can heterodimerize with each other41, the CCR5ꢀtargeting CoRIꢀPT chimera reported in the
current work has an additional potential to bind to the CCR5 in the heterodimer and
neutralize X4ꢀtropic virus by Env inactivation through the PT domain (which is active against
both X4ꢀ and R5ꢀviruses) before CXCR4 encounter.
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Bifunctional chimeras of the CoRIꢀPT type have an important advantage in avoiding virus
resistance, by combining components that can function synergistically or separately against
evolving viruses and hence challenging the virus to combine escape to two different
inhibitory functions coordinately. Analogously with most if not all virus protein targeting
antagonists, mutational escape of the Envꢀbinding PT component can occur31. Further, even
though CoRIs target a host cell protein vs a virus protein, HIVꢀ1 resistance mutations to coꢀ
receptor antagonists have been observed to occur by forming binding sites on the virus Env
protein that can bind the antagonistꢀbound form of the coꢀreceptor and in this way infecting
cells in the presence of CoRIs.42 We envision that, since the CCR5 antagonist in the LJC240ꢀ
L4ꢀUM15 chimera (11) is attached to a relatively large peptide and linker moiety, it would be
difficult for virus to bind the chimeraꢀarmed coꢀreceptor due to increased spatial blockade.
But, even if the virus could overcome this blockade, the added challenge of escaping the PT
component of the chimera would improve the ability of the chimera to avoid overall inhibitor
escape.
The results of the current work open up both future chimera design opportunities and
potential for expanded functional utility. The chimeric inhibitor 11 presented in this work
serves as a prototype for advancing more drugꢀlike chimeric inhibitors. The recent discovery
of macrocyclic PTs (cPTs)19, 43, combined with existing44 and newly discovered CXCR4ꢀ
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targeting small molecules45, provide the potential to develop drugꢀlike and proteaseꢀresistant
CoRIꢀcPT variants that will target both CCR5 and CXCR4 cellular infection with improved
bioavailability. Increased understanding about the structural mechanism of action of the
chimeras will be helpful to guide future chimera design. As the latter is progressing, the
relatively small size of such chimeras will make it feasible to investigate whether these could
inhibit not only virusꢀcell infection but also cellꢀtoꢀcell transmission.
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CONCLUSIONS
In summary, we have designed and produced a fully synthetic biꢀfunctional chimeric
inhibitor that can target both sides of the HIVꢀcell interface. One component blocks the viral
envelope and the second component blocks the cellular coꢀreceptor CCR5. The chimera
showed synergistic potency compared to the nonꢀcovalent mixture(s) of the individual
components. This result demonstrates the ability of making potent, fully synthetic HIVꢀ1
inhibitors by simultaneously targeting the virus Env and its cellular chemokine receptors.
E
XPERIMENTAL SECTION
Docking of LJC240 onto the CCR5 receptor
The recently solved crystal structure of CCR5 bound to inhibitor Maraviroc was used (pdb
code 4MBS26. The pdb file was downloaded from the Protein Data Bank and prepared with
the protein preparation wizard in Maestro 9.9 (Schrödinger Suite 2015; Schrödinger, LLC).
Before docking LJC240, The docking procedures were validated where the coꢀcrystallized
Maraviroc was docked onto the receptor structure (using GlideꢀXP docking protocol) and
showed similar pose to the crystallographically solved bound state (heavy atoms RMSD =
0.31 Å, data not shown) and had a calculated receptorꢀMaraviroc interaction energy value of
∼
ꢀ33.2 kcal/mol as calculated using Szybki 1.8.0.2 (Openeye Scientific Software, Santa Fe,
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NM. http://www.eyesopen.com). LJC240 was built and prepared using the builder and
LigPrep tools in Maestro 9.946. The inhibitor LJCꢀ240 was then docked onto the CCR5
receptor using the same GlideꢀXP protocol. This yielded a stable low energy pose (Figure 2)
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with a calculated receptorꢀligand interaction energy of
∼ ꢀ27.1 kcal/mol (calculated using
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Szybki 1.8.0.2, Openeye Scientific Software, Santa Fe, NM. http://www.eyesopen.com). The
docking showed the fluorophenyl moiety of the inhibitor to be the most solvent exposed part,
with the least important interaction. This was in agreement with the SAR studies (Long Lab)
that changes in this part of the molecule can be tolerated.
Docking of UM15 onto the gp120 protein
Peptide UM15 was built and docked onto gp120 chain A of the SOSIP Env trimer (pdb code
4NCO25, structure as previously described47 using the InducedFit docking protocol48. The N
terminal of the UM15 was shown to be well solvated (Figure 2) and can be used as an
extension point for the Chimera synthesis.
Chemical synthesis
General Information
The starting materials were obtained from commercial sources, such as Adamasꢀbeta, Sigmaꢀ
Aldrich, J&K, TCI and ChemꢀImpex, which were used without further purification. Unless
otherwise specified, all reactions were carried out in ovenꢀdried glassware with magnetic
stirring. All reagents were weighed and handled in air at room temperature. Unless otherwise
stated, commercially obtained materials were used without further purification. Column
chromatography was performed on silica gel (200ꢀ300 mesh). The column output was
monitored by TLC on silica gel (100ꢀ200 mesh) precoated on glass plates (15 x 50 mm), and
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spots were visualized by UV light at 254 nm. All new compounds were characterized by H
NMR, 13C NMR and low/high resolution mass spectroscopy. NMR spectra were recorded on
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a Brucker AVANCE III 400 NMR spectrometer. Chemical shifts for proton magnetic
resonance spectra (1H NMR) were quoted in parts per million (ppm) referenced to the signals
of residual chloroform (7.26 ppm) or methanol (3.30 ppm). All 13C NMR spectra are reported
in ppm relative to deuterochloroform (77.23 ppm) or methanol (49.00 ppm). The following
abbreviations were used to describe peak splitting patterns when appropriate: br = broad, s =
singlet, d = doublet, t = triplet, q = quartet, m = multriplet, dd = doublet of doublet. Coupling
constants, J, were reported in hertz unit (Hz). Mass spectra were recorded using an ESI ion
source unless stated otherwise. All melting points were measured using a BÜCHI 510
melting point apparatus. The yields in this paper refer to isolated yields of compounds
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estimated to be ≥
95% pure as determined by 1H NMR. Unless otherwise specified, the purity
of all new compounds was determined by analytical HPLC on Agilent Technologies 1260
Infinity with Aglient ZORBAX 3.5 ꢁM SBꢀphenyl column (4.6 × 75 mm) in one solvent
system (solvent A, 0.02% TFA in water; solvent B, 0.02% TFA in acetonitrile) with gradient
indicated below; flow rate, 1.5 mL/min; UV detector, 210 and/or 254 nm. Method: 5% B to
90% B in 12 min, 90% B for 3 min.
Synthesis of 8-Benzyl-8-aza-bicyclo[3.2.1]octan-3-one oxime (2)49: A mixture of 8ꢀbenzylꢀ
8ꢀazaꢀbicyclo[3.2.1]octanꢀ3ꢀone (5.00g, 23.2mmol), hydroxylamine hydrochloride (1.78g,
25.6 mmol) and NaHCO3 (2.54g, 30.2mmol) was stirred in EtOH/H2O (60 mL, v:v=1:1) for
18 hours. The reaction mixture was filtered and the residue was washed with water, dried to
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afford the compound 2 as a white solid (3.24g, yield 61%), m.p. 118ꢀ120 C. H NMR (400
MHz, CDCl3) 7.43 (d, J = 7.2 Hz, 2H), 7.38 – 7.32 (m, 2H), 7.32 – 7.29 (m, 1H), 3.70 (s,
δ
2H), 3.43 – 3.34 (m, 2H), 3.02 (d, J = 15.5 Hz, 1H), 2.66 – 2.59 (m, 1H), 2.31ꢀ2.23 (m, 1H),
2.18ꢀ2.05 (m, 1H), 2.10 – 2.00 (m, 2H), 1.66 (t, J = 7.9 Hz, 1H), 1.56 (t, J = 7.6 Hz, 1H). 13C
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NMR (151 MHz, CDCl3)
δ
157.0, 139.3, 128.6, 128.3, 127.0, 58.5, 57.8, 55.6, 37.3, 31.3,
27.6, 26.7. HRMS (ESI) Calcd for C14H19N2O [M+H]+: 231.1492, found 231.1489.
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Synthesis of exo-8-Benzyl-8-aza-bicyclo[3.2.1]oct-3-ylamine (3)50: A solution of the
compound 2 (3.00 g, 13.0 mmol) in pentanol (50 mL) was heated under reflux. Sodium (3.59
g, 156.3 mmol) was added portionwise over 2.5 hours. The reaction was then heated under
reflux for a further 2 hours, then cooled in an ice bath. Water was added until no more
hydrogen gas was evolved. The mixture was acidified using 6N aqueous hydrochloric acid
and the phases separated. The organic layer was extracted with 6N aqueous hydrochloric
acid, the combined aqueous extracts were basified to pH 12 with sodium hydroxide pellets
and the aqueous solution extracted with ethyl acetate. The combined organic solutions were
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dried with MgSO4, filtered and evaporated under reduced pressure to afford the compound 3
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as colorless oil (1.91 g, yield 68%). H NMR (400 MHz, CDCl3)
δ 7.45 – 7.36 (m, 2H),
7.30ꢀ7.25 (m, 2H), 7.26 – 7.19 (m, 1H), 3.63 (s, 2H), 3.38 – 3.23 (m, 3H), 2.09 – 1.97 (m,
2H), 1.96 – 1.80 (m, 4H), 1.67 – 1.53 (m, 2H). 13C NMR (126 MHz, CDCl3)
δ
138.2, 128.4,
127.9, 126.7, 57.5, 54.5, 43.6, 35.7, 26.1. HRMS (ESI) Calcd for C14H21N2 [M+H]+:
217.1699, found 217.1704.
Synthesis of exo-(8-Benzyl-8-aza-bicyclo[3.2.1]oct-3-yl)-carbamic acid tert-butyl ester
(4)51: A mixture of compound 3 (3.00 g, 13.9 mmol), Boc2O (3.33g, 15.3 mmol) and TEA
(3.9 mL, 27.7 mmol) was stirred in THF (52 mL) for 18 hours. The reaction mixture was
evaporated under reduced pressure. The residue was purified by recrystallization, affording
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compound 4 as a white solid (4.19 g, yield 95%), m.p. 174ꢀ175 C. H NMR (400 MHz,
CDCl3) 7.38 (d, J = 7.3 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.26ꢀ 7.23 (m, 1H), 3.56 (s, 2H),
δ
3.31ꢀ3.14 (m , 2H), 2.05ꢀ2.00 (m, 2H), 1.84ꢀ1.78 (m, 2H), 1.73ꢀ1.69 (m, 2H), 1.58ꢀ1.50 (m,
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3H), 1.43 (s, 9H). C NMR (126 MHz, CDCl3)
δ 155.3, 140.0, 128.5, 128.2, 126.8, 79.2,
58.7, 56.1, 42.8, 38.7, 28.4, 26.5. HRMS (ESI) Calcd for C19H29N2O2 [M+H]+: 317.2224,
found 317.2224.
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Synthesis of exo-(8-Aza-bicyclo[3.2.1]oct-3-yl)-carbamic acid tert-butyl ester (5)52: A
solution of the compound 4 (2.73 g, 8.6 mmol) in methanol (40 mL) was added 10% Pd/C
(273 mg, ca.50% water) and HCO2NH4 (3.81g, 60.4 mmol), the above mixture was heated
under reflux for 15 min. Cooled to room temperature, the reaction mixture was evaporated
under reduced pressure. Purification by flash chromatography on silica gel (DCM / methanol
= 10:1) afforded compound 5 as a white solid (1.43 g, yield 73%), m.p. 271ꢀ272 oC. 1H NMR
(400 MHz, CDCl3)
δ
4.80ꢀ4.76 (m, 1H), 4.08ꢀ4.02 (m, 2H), 2.29 – 2.20 (m, 2H), 2.05 – 1.93
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(m, 6H), 1.42 (s, 9H). C NMR (126 MHz, CDCl3)
δ 169.0, 79.8, 54.7, 41.1, 35.4, 28.3,
26.3. HRMS (ESI) Calcd for C12H23N2O2 [M+H]+: 227.1754, found 227.1748.
Synthesis of (3-Chloro-4-methyl-phenyl)-(3-chloro-propyl)-amine (7)53: To a mixture of
1ꢀbromoꢀ3ꢀchloropropane (10.5 mL, 105.9 mmol) in DMF was added KI (586 mg, 3.5
mmol), and the mixture was stirred at room temperature for 30 min, then TEA (19.6 mL,
141.2 mmol) and 3ꢀChloroꢀ4ꢀmethylꢀphenylamine (5.00 g, 35.3 mmol) was added and stirred
at room temperature for further 72h. The reaction mixture was filtered, and the filtrate was
concentrated in vacuum. The residue was diluted with EtOAc, washed with water and brine,
dried with MgSO4, filtered, and concentrated in vacuum. Purification by flash
chromatography on silica gel (petroleum ether / ethyl acetate = 25:1) afforded compound 7 as
yellow oil (5.20 g, yield 68%). 1H NMR (400 MHz, CDCl3) δ 7.01 (d, J = 8.2 Hz, 1H), 6.63
(d, J = 2.4 Hz, 1H), 6.44 (dd, J = 8.2, 2.5 Hz, 1H), 3.65 (t, J = 6.2 Hz, 2H), 3.30 (t, J = 6.6
Hz, 2H), 2.25 (s, 3H), 2.05 (p, J = 6.5 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ 147.0, 134.9,
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131.4, 124.4 113.1, 111.7, 42.5, 41.1, 31.8, 18.9. HRMS (ESI) Calcd for C10H14Cl2N
[M+H]+: 218.0498, found 218.0497.
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Synthesis of 1-Acetyl-piperidine-4-carboxylic acid (3-chloro-4-methyl-phenyl)-(3-
chloro-propyl)-amide (8)53: To a solution of 1ꢀacetylpiperidineꢀ4ꢀcarboxylic acid (6.75 g,
39.4 mmol) in SOCl2 (40 mL) was stirred for 2 h at room temperature. The reaction mixture
was diluted with petroleum ether and filtered to afford the acyl chloride as white solid.
Compound 7 (2.87 g, 13.1 mmol) was dissolved in DCM (50 mL), TEA (5.5 mL, 39.4 mmol)
and acyl chloride were added, then the mixture was stirred at room temperature for further
2h. The residue was diluted with DCM, washed with water and brine, dried with MgSO4,
filtered, and concentrated in vacuum. Purification by flash chromatography on silica gel
(petroleum ether / ethyl acetate = 1:1) afforded compound 8 as a white solid (3.74 g, yield
77%), m.p. 101ꢀ103 oC. 1H NMR (400 MHz, CDCl3) δ 7.31 (d, J = 8.0 Hz, 1H), 7.18 (d, J =
2.0 Hz, 1H), 6.97 (dd, J = 8.0, 2.1 Hz, 1H), 4.51 (d, J = 13.3 Hz, 1H), 3.78ꢀ3.75 (m, 2H),
3.53 (t, J = 6.6 Hz, 2H), 2.87ꢀ2.81 (m, 1H), 2.43 (s, 3H), 2.39 – 2.28 (m, 2H), 2.05 (s, 3H),
2.02ꢀ1.96 (m, 2H), 1.67 – 1.55 (m, 5H). 13C NMR (126 MHz, CDCl3) δ 174.2, 168.8, 140.9,
136.7, 135.4, 132.1, 128.4, 126.2, 47.7, 45.5, 42.3, 40.7, 39.3, 30.8, 28.8, 28.3, 21.4, 19.8.
HRMS (ESI) Calcd for C18H25Cl2N2O2 [M+H]+: 371.1288, found 371.1281.
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Synthesis
of
exo-1-Acetyl-piperidine-4-carboxylic
acid
[3-(3-amino-8-aza-
bicyclo[3.2.1]oct -8-yl)-propyl]-(3-chloro-4-methyl-phenyl)-amide (9): To a mixture of
compound 8 (1.70 g, 4.6 mmol), compound 5 (1.04 g, 4.6 mmol) and KI (760 mg, 4.6 mmol)
in acetonitrile was added NaHCO3 (1.15 g, 13.7 mmol), and the mixture was stirred at reflux
for 12h. After cooling to room temperature, the mixture was filtered, and the filtrate was
concentrated in vacuum. The residue was diluted with EtOAc, washed with water and brine,
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dried with MgSO4, filtered, and concentrated in vacuum. Purification by flash
chromatography on silica gel (DCM/methanol = 25:1) afforded the product as a white foam
(1.72 g, yield 67%). 1H NMR (400 MHz, CDCl3) δ 7.31 (d, J = 8.1 Hz, 1H), 7.20 (d, J = 1.4
Hz, 1H), 7.08 (d, J = 7.7 Hz, 1H), 4.65 – 4.46 (m, 2H), 3.88 – 3.60 (m, 4H), 3.49 – 3.38 (m,
2H), 2.82 (t, J = 13.7 Hz, 1H), 2.57ꢀ2.50 (m, 2H), 2.40 (s, 3H), 2.36 – 2.25 (m, 2H), 2.03 (s,
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3H), 2.00 – 1.95 (m, 2H), 1.84ꢀ1.79 (m, 8H), 1.67 – 1.54 (m, 3H), 1.41 (s, 9H). C NMR
(126 MHz, CDCl3) δ 174.3, 168.8, 155.3, 140.7, 136.7, 135.3, 132.1, 128.4, 126.6, 79.5,
59.7, 54.7, 48.9, 47.5, 45.5, 41.8, 40.7, 39.4, 36.8, 28.9, 28.4, 28.3, 25.8, 21.4, 19.8. HRMS
(ESI) Calcd for C30H46ClN4O4 [M+H]+: 561.3202, found 561.3214. Purtiy: 99.8% (tR=6.63).
Synthesis
of
4-8-(3-(1-Acetyl-N-(3-chloro-4-methylphenyl)piperidine-4-
carboxamido)propyl)-8-azabicyclo[3.2.1]octan-3-yl)sulfamoyl)benzoic acid (10): To a
solution of compound 9 (2.62 g, 4.7 mmol) in methanol, HCl (4.8 M, in methanol) was
added, and the mixture was stirred at room temperature for 4h. After concentrating the
reaction mixture in vacuum, the residue (1.57 g, 3.4 mmol) was dissolved in 1,4ꢀdioxane (40
mL), then 4ꢀ(chlorosulfonyl)benzoic acid (1.50 g, 6.8 mmol) and a solution of Na2CO3 (1.08
g, 10.2 mmol) in H2O (20 mL) were added, and the mixture was stirred at room temperature
for 12h. The residue was evaporated in vacuum, washed with mixture of methanol and DCM
(DCM/methanol= 9:1), filtered, and filtrate was concentrated in vacuum. Purification by flash
chromatography on silica gel (DCM / methanol = 10:1) afforded compound 10 as a white
solid (1.22 g, yield 40% for two steps), m.p. 182ꢀ184 oC. 1H NMR (400 MHz, MeOD) δ 8.01
(d, J = 8.5 Hz, 2H), 7.82 (d, J = 8.5 Hz, 2H), 7.35 (dd, J = 8.5, 5.1 Hz, 2H), 7.11 (dd, J = 8.1,
2.1 Hz, 1H), 4.35 – 4.26 (m, 1H), 3.86 – 3.79 (m, 2H), 3.78 – 3.70 (m, 1H), 3.67ꢀ3.60 (m,
2H), 3.56 – 3.46 (m, 1H), 2.95 – 2.87 (m, 2H), 2.78 (t, J = 11.4 Hz, 1H), 2.42ꢀ2.36 (m, 1H),
2.31 (s, 3H), 2.29ꢀ2.24 (m, 1H), 2.16ꢀ2.08 (m, 2H), 1.95 (s, 3H), 1.86 – 1.74 (m, 8H), 1.59 (d,
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J = 15.2 Hz, 3H), 1.45 (d, J = 16.2 Hz, 1H). 13C NMR (126 MHz, MeOD) δ176.0 (2), 169.7,
143.2, 139.6, 139.0, 136.7, 134.7, 131.9, 129.4, 127.7, 125.9, 125.7, 60.8, 45.9, 44.8, 43.1,
40.0, 38.9, 28.0, 27.5, 23.5, 22.3, 19.3, 18.0. HRMS (ESI) Calcd for C32H42ClN4O6S [M+H]+:
645.2508, found 645.2506. Purtiy: 99.5% (tR=5.61).
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Solid Phase Synthesis of LJC240-L4
Rink amide resin (0.1 mmol, 200ꢀ400 mesh) was swollen in 10 mL of DMF: DCM (1:1).
Fmoc deprotection was achieved using a solution of 20% piperidine/ in DMF. The Fmocꢀ
protected linker (116 mg, 0.3 mmol) was dissolved in DMF, DIEA (124 ꢁl, 0.75 mmol) and
HBTU (110 mg, 0.29 mmol) were added and the mixture was vibrated for 30 min at room
temperature then added to the reaction vessel, vibrated at room temperature for 4 h. Four
units of the linker were installed. LJC240ꢀCOOH (10, 193 mg, 0.3 mmol) was dissolved in
DMF, then DIEA (124 ꢁl, 0.75 mmol), HOBt (41 mg, 0.3 mmol) and HBTU (110 mg, 0.29
mmol) were added, and the mixture was vibrated for 30 min then added to the (linker)4ꢀresin
vessel. The reaction vessel was vibrated at room temperature for 12 h. Cleavage was carried
out using a cocktail of trifluoroacetic acid/ethanedithiol/H2O (4.75/0.125/0.125 v/v) for 2h at
room temperature. Cleaved compound solution was concentrated under a gentle N2 stream,
then dissolved in ACN/H2O. Crude product was purified by semiꢀpreparative HPLC on an
EasySepTMꢀ1050 with XBridge™ Prep C18 reversedꢀphase column (19 × 150 mm,
ACN/H2O/0.1% TFA, tR=17.7 min (gradient: 10−90% ACN over 30 min; flow rate of 4
1
mL/min) and finally evaporated in vacuum to yield ideal product. H NMR (400 MHz,
MeOD) δ 8.06 – 7.99 (m, 4H), 7.51 – 7.46 (m, 2H), 7.24 (dd, J = 7.9, 2.0 Hz, 1H), 4.45 (d, J
= 13.9 Hz, 1H), 4.02 (s, 4H), 3.99 (s, 3H), 3.91 – 3.75 (m, 3H), 3.75 – 3.64 (m, 17H), 3.62ꢀ
3.55 (m, 6H), 3.51 – 3.39 (m, 5H), 3.06 – 2.98 (m, 2H), 2.95 – 2.82 (m, 2H), 2.72 (s, 2H),
2.57 – 2.49 (m, 1H), 2.44 (s, 3H), 2.43 – 2.38 (m, 1H), 2.29 – 2.21 (m, 2H), 2.08 (s, 3H), 2.06
(s, 1H), 2.02 – 1.87 (m, 7H), 1.77 – 1.54 (m, 5H), 1.38 – 1.28 (m, 4H). 13C NMR (126 MHz,
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MeOD) δ 176.0, 174.0, 171.0, 169.6, 166.9, 143.8, 139.6, 137.8, 136.7, 134.7, 131.8, 127.7,
127.6, 126.2, 125.9, 70.1 (4), 69.7 – 69.1 (8), 68.9 – 68.5 (4), 61.2, 45.8, 44.8, 43.1, 40.0,
39.2, 38.9, 37.9, 36.4, 33.5, 28.1, 27.5, 23.4, 22.2, 19.3, 18.0. HRMS (ESI) Calcd for
C56H87ClN9O17S [M+H]+: 1224.5624, found 1224.5627. Purtiy: 99.0% (tR=8.47).
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Solid Phase Synthesis of LJC240-L4-UM15 (11)
Solid Phase synthesis of resin-bound linker-PT
Using Fmocꢀbased synthesis strategy, the following sequence was assembled using
microwave assisted coupling: (linker)4ꢀileꢀasnꢀasnꢀileꢀAzidoProꢀtrpꢀresin. Resin = rink amide
resin (100ꢀ200 mesh size), 0.25 mM synthesis scale. Activation scheme (1 mL 0.5 M DIC/0.5
mL 0.5 M Oxyma) was used. Deprotection of Fmoc groups by 5 mL 20% piperidine+0.1 M
HOBt in DMF.
Coupling LJC-240-COOH to the Linker-PT-resin
The (linker)4ꢀpeptideꢀresin was swollen by soaking in 10 mL of DMF:DCM (1:1) . 1.1
equivalent of LJCꢀ240ꢀCOOH (10) + 2.2 equivalent HBTU + 2.2 equivalent HOBt + 4.4
equivalent DIPEA were added to the reaction vessel. The vessel was vibrated for 10 h at
room temperature and reaction monitored by Kaiser test until negative (no blue color) test
observed. The resinꢀbound complex was then filtered; resin was washed thoroughly by DMF,
Methanol and DCM.
Click reaction of the alkyne (ethynylferrocene) with the azide group on Pro residue
The resinꢀbound fusion was mixed with (5 mL ACN, 4 mL H2O, 1.06 mL DIPEA and 0.53
mL pyridine + 5 equivalent ethynylferrocene), and the reaction vessel was vibrated at room
temperature for 12 h. The resinꢀbound product was washed thoroughly by 5% HCl (2 x 50
mL), DMF (2 x 50 mL) and DCM (2 x 50 mL).
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