Volume 12

Number 12
December 2021
Pages 1979–2074

RSC
Medicinal Chemistry
rsc.li/medchem

ISSN 2632-8682

RESEARCH ARTICLE
Sanela Martic, Ngong Kodiah Beyeh et al.
Functionalized resorcinarenes effectively disrupt
the aggregation of αA66-80 crystallin peptide related
to cataracts
 RSC
Medicinal Chemistry
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RESEARCH ARTICLE View Journal | View Issue

Functionalized resorcinarenes effectively disrupt

the aggregation of αA66-80 crystallin peptide
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Cite this: RSC Med. Chem., 2021, 12,

2022 related to cataracts†
Kwaku Twum, a Avik Bhattacharjee, b Erving T. Laryea, a
Josephine Esposto, c George Omolloh, b Shaelyn Mortensen,c Maya Jaradi, a

Naomi L. Stock, d Nicholas Schileru,ae Bianca Elias,a Elan Pszenica,a

Theresa M. McCormick, b Sanela Martic *cd and Ngong Kodiah Beyeh *a

Received 2nd September 2021,
Accepted 13th October 2021
DOI: 10.1039/d1md00294e
rsc.li/medchem
Cataracts, an eye lens clouding disease, are debilitating and while operable, remain without a cure. αA66-
80 crystallin peptide abundant in cataracted eye lenses contributes to aggregation of αA-crystallin protein
leading to cataracts. Inspired by the versatility of macrocycles and programmable guest selectivity through
discrete functionalizations, we report on three water-soluble ionic resorcinarene receptors (A, B, and C)
that disrupt the aggregation of αA66-80 crystallin peptide. A and B each possess four anionic sulfonate
groups, while C includes four cationic ammonium groups with four flexible extended benzyl groups.
Through multiple non-covalent attractions, these receptors successfully disrupt and reverse the
aggregation of αA66-80 crystallin peptide, which was studied through spectroscopic, spectrometric,
calorimetric, and imaging techniques. The αA66-80·receptor complexes were also explored using
molecular dynamics simulation, and binding energies were calculated. Even though each of the three
receptors can bind with the peptide, receptor C was characterized by the highest binding energy and
affinity for three different domains of the peptide. In effect, the most efficient inhibitor was a cationic
receptor C via extended aromatic interactions. These results highlight the potential of versatile and tunable
functionalized resorcinarenes as potential therapeutics to reverse the aggregation of α-crystallin dominant
in eye cataracts.

Introduction compromised with aging due to extensive chemical

Cataract disease remains without a cure and is caused by the
clouding of the eye lens.1 The onset of lens clouding is due to
aging, protein post-translational modifications, and damage
by radicals such as reactive oxygen species (ROS) generated by
exogenous and endogenous factors.1–3 The αA-crystallin, a
heat-shock protein, maintains the solubility and stability of
lens proteins and lens clarity by preventing protein
aggregation, which may lead to lens clouding and eventually
cataracts.4–6 The chaperone property of αA-crystallin may be
a
Department of Chemistry, Oakland University, 146 Library Drive, Rochester, MI
48309-4479, USA. E-mail: beyeh@oakland.edu
b
Department of Chemistry, Portland State University, 1710 SW 10th Ave, Portland,
OR 97201, USA
c
Department of Forensic Science and Environmental and Life Sciences Program,
Trent University, ON, K9 L0G2, Canada. E-mail: sanelamartic@trentu.ca
d
Water Quality Centre, Trent University, ON, K9L 0G2, Canada
e
Department of Osteopathic Medicine, Midwestern University, 555 31st St.,
Downers Grove, IL 60515, USA
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1md00294e
modifications and degradation of αA crystallin. The αA-
crystallin protein level in the eye lens decreases significantly
with aging leading to low molecular weight peptide (LMW)
fragments.7 The LMW fragments are present in aggregates of
cataract lenses, pointing to their essential roles in the
disease.8 In particular, the αA66-80 peptide fragment of αA-
crystallin, among others, was observed at a high level in
cataracts eye lenses.5 The αA-crystallin peptide sequence has
also been evaluated for its aggregation and ROS production.9
The αA66-80 crystallin aggregation is a viable therapeutic
target, and several small molecules have been assessed as
aggregation inhibitors to include aspirin, flavonoids, orange
G, among others.9,10 Currently, there are no proven cures for
cataracts other than surgery, which is performed at a later
stage of the disease and continues to be debilitating to the
people affected as they wait for the surgery.11 Lanosterol, an
oxysterol, was recently found to dissolve lenticular opacities;
the non-covalent attraction of the steroid to hydrophobic cores
of aggresomes forced the opacities to dissolve.12 However, it
was later proved that lanosterol's interaction relevant for
binding to crystallin was too weak and non-selective.13

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RSC Medicinal Chemistry
Cavity-containing macrocyclic compounds can be
modified with specific functional groups and used as a target
for biological materials.14,15 Depending on the
complementary features of the macrocycle and the
biomaterials, the macrocycles can also function as
sensors,16,17 glue to connect biopolymers18 or as components
to help disrupt aggregates.19 Understandably, designing
synthetic macrocycles that can target peptides or proteins
offer a suitable avenue for applications in hybrid materials,
diagnostics and potentially in this study as aggregation
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inhibitors. Receptors like molecular tweezers have been used
to target aggregation of amyloidogenic peptides through their
amino acid residues;20–22 however, the crystallin aggregation
has not been previously targeted using this approach. In this
contribution, we hypothesize that receptors that can target
specific amino acid sites on αA crystallin (Fig. 1) can disrupt
the aggregation pattern inherent with its morphological
changes. To this effect, we used three different
resorcinarenes-based ionic macrocyclic receptors (A, B and
C). Receptors A and B, Fig. 1, are functionalized
resorcinarenes with anionic sulfonate groups at the upper
and lower rim respectively. Receptor C is a resorcinarene salt
receptor with an extended cavity with four ammonium
groups at the upper rim and four hydroxyl groups at the
lower rim (Fig. 1). Receptor C has a strong intramolecular
circular hydrogen bond seam (⋯NR′R″H2+⋯Cl−⋯NR′R″
H2+⋯Cl−⋯)2 between the ammonium groups and the
chloride anion referred to as the hydrogen bond analogs of
deep cavity cavitands.23 The anionic receptors A and B have
the potential to interact via electrostatic attractions to the
cationic amino acids, arginine (R), lysine (K) and histidine
(H) on the αA66-80 peptide while receptor C can interact with
various amino acids including aspartic acid (D) and others
through ionic and aromatic interactions. All the receptors
possess hydrophobic internal cavities that can accommodate
Fig. 1 Structure of resorcinarene-based receptors (A, B and C) and
crystallin peptide chain αA66-80.
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neutral side groups based on size complementarity. The
effect of the receptors on the aggregation/de-aggregation of
αA66-80 peptide are investigated at the macroscopic level
through fluorescence aggregation assay, dynamic light
scattering (DLS), transmission electron microscopy (TEM),
and mass spectrometry. Interactions at the molecular level
between the receptors and the individual amino acids are
investigated qualitatively via NMR spectroscopy, and
quantitatively via isothermal titration calorimetry (ITC). The
binding of receptors A, B, and C with αA66-80 peptide chain
was studied using DFT, molecular docking, and molecular
dynamics (MD) simulations. The calculated MMGBSA
binding energies support the experimental observations.
Results & discussion
The receptors used in this study were synthesized according
to reported procedures.24–27 The crystallin αA66-80 peptide
was purchased from GenScript and used without further
purification.
Fluorescence aggregation assay
Resorcinarenes are notably weak fluorescent compounds
which prompted the use of the Proteostat detection kit for
qualitative detection of peptide aggregates. This rotational
proprietary dye specifically intercalates into quaternary
protein structures typical of aggregated β-pleated sheet
peptides and proteins, resulting in fluorescence emission
enhancement.28 Moreover, the dye's free intramolecular
rotation along a single central bond eliminates fluorescent
emissions in the absence of aggregates. As a result, the dye
has been reliably used as an indicator of β-pleated sheet
formation during crystallin aggregation.28–31 As shown in
Fig. 2a, pure crystallin aggregation resulted in formation of
dynamic structural assemblies that produced a high
fluorescence intensity. To assess the level of aggregation of
the peptide with the receptors, the mixtures of receptor:
peptide ratios at 0.2, 0.4, 0.6, 0.8, 1.0 and 5.0 were incubated
at 37 °C in 10 mM Tris buffer (pH 7.4) for seven days.
Compared to the pure crystallin incubated for the same
period, all ratios showed a concentration-dependent decrease
in the extent of peptide aggregation (Fig. 2a and S1–S3†).
From these results, receptors A, B and C all have an
inhibiting effect on the extent of crystallin peptide
aggregation in the seven days incubation period. To rule out
any inconsistent fluorescence contributions from the
Proteostat dye, we also run positive and negative controls
(Fig. S4†) to make sure that, a) each freshly prepared dye
solution is effective, b) there is little to no fluorescence
contributions from free dye solutions and c) the receptors are
not artificially quenching fluorescence from the dye
irrespective of peptide aggregation. In queries a) and b), we
see high and minimal fluorescence intensities from the
proprietary positive and negative controls respectively. The
normalized inhibitory response was also used to derive IC50
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Fig. 2 a) Bar chart showing results of fluorescence aggregation of crystallin peptide (5.4 mM) in different host ratios. Error bars represent
standard deviations of the mean of repetitive measurements. b) Graph showing percent intensities of Z-average sizes in solution of pure crystallin
(red), 1 : 1 αA66-80 crystallin peptide: receptor A (sea blue), 1 : 1 αA66-80 crystallin peptide: receptor B (green), 1 : 1 αA66-80 crystallin peptide:
receptor C (dark blue), pure receptor B (grey), pure receptor A (orange) and pure receptor C (yellow). c) Transmission electron microscopy (TEM)
imaging of one week incubated samples of i) pure crystallin, ii) 1 : 1 αA66-80 crystallin peptide: receptor A, iii) 1 : 1 αA66-80 crystallin peptide:
receptor B, and iv) 1 : 1 αA66-80 crystallin peptide: receptor C.

values of 203, 89, and 418 μM for receptors A, B, and C
respectively (Fig. S5–S7†).
Dynamic light scattering (DLS)
To clarify the effect of the receptors on peptide aggregation
without another chemical dye, we resorted to dynamic light
scattering (DLS) experiments (Fig. 2b). DLS is used for
measuring the size distribution of small particles, peptides,
cells, carbohydrates, nanoparticles, or polymers in solution.32
These measurements were also carried out on increasing
receptor: peptide molar ratios (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 :
1) after seven days of incubation at 37 °C. The size (Z-)
average in d nm of triplicate measurements of the different
concentration ratios reflects a similar inhibitory effect of
receptors A, B, and C against aggregation of crystallin (Fig.
S8–S10†). In the incubations of pure αA66-80 crystallin
peptide without any receptor, the Z-average size is (1.20 ±
0.15) × 104 d nm after seven days of incubation. All the
incubations revealed substantially different size distribution
profiles showing smaller average size intensity profiles
compared to the crystallin peptide (Fig. 2b). The mixture
between receptor A, B and the peptide show some large size
distributions on an average of replicate measurements,
however, the overall sizes show substantially smaller
distributions compared to the pure αA66-80 crystallin
peptide. The average sizes from triplicate measurements of a
1 : 1 concentration ratio of receptor A, B, and C: crystallin are
1918 ± 897, 3739 ± 644, and 202 ± 2.2 d nm respectively.
Receptor C, the deep cavity cavitand, was most effective at

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RSC Medicinal Chemistry Research Article

disrupting the aggregation of the crystallin peptide in the

DLS experiments.

Transmission electron microscopy (TEM)

Transmission electron microscopy (TEM) was used to
virtually characterize αA66-80 crystallin peptide aggregates in
the presence of the inhibitors. TEM analysis of pure αA66-80
crystallin peptide, peptide–receptor A, B and C complexes at
1 : 1 concentration ratio, and pure receptor A, B and C were
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performed after aging the samples for seven days. TEM

images (Fig. 2c, S11 and S12†) are representative from
different sections of the TEM grids. Fig. 2c–i shows
significant aggregates of the crystallin peptide (dark spots).
These are evidentially less upon incubation with the
receptors A, B and C (2ii, 2iii and 2iv respectively). TEM of
pure crystallin reveals large, fibrillar-like peptide aggregates
congruent with the results from fluorescence and DLS
experiments both initially and after seven days. The
macrocycles show significant potential to disrupt the
crystallin aggregation. Additionally, receptor C, decorated
with functional groups on both sides of the receptor, is
superior to receptors A or B at equimolar concentrations
regarding disrupting crystallin αA66-80 aggregation. αA66-80
crystallin peptide: receptor C complex morphology showed
significantly less fibrillar aggregates when compared to αA66-
80 crystallin peptide: receptor A or B complex morphology,
suggesting its superior inhibitory role. TEM imaging however
shows amorphous small protein aggregates in the αA66-80
crystallin peptide: receptor A, B and C complexes with less Fig. 3 Positive mode ESI-MS spectra of a) doubly charged receptor A–
aggregated fibrils. Overall, these results suggest that while all peptide complexes showing [A + peptide–4Na + 6H]2+ and [A +
peptide–3Na + 5H]2+; d) receptor B + peptide complexes showing [B +
receptors A, B, and C inhibit the aggregation of the crystallin
peptide–4Na + 6H]2+ and [B + peptide–3Na + 5H]2+ (experimental: a),
peptide, receptor C acts as a more potent inhibitor to the d); theoretical isotope distribution: b), c), e) and f).
peptide over time.

Mass spectrometry
To further investigate this phenomenon of crystallin de-
aggregation, we turned to gas phase measurements, devoid
of solution interruptions to tease out possible host-αA66-80
crystallin peptide complexes. Electrospray ionization mass
spectrometry (ESI-MS), a soft ionization method, was used to
characterize each receptor and the peptide using freshly
made sample solutions, as well as their respective complexes.
In the positive ion mode, the mass spectrum for the peptide
shows the [M + 3H]3+ peak at m/z 622.6646, associated with
the triply charged peptide (Fig. S11a†). The mass spectra for
the receptor A, B, and C show peaks at m/z 1065.097 [M +
H]+, 1009.0296 [M + H]+, and 599.3110 [M–4HCl + 2H]2+
respectively (Fig. S13b–d†). The MS analysis of freshly
prepared samples, compared to aged samples (72 h at 37 °C),
yielded more prominent receptor + peptide complexes. Fig. 3
shows the observed (Fig. 3a) and theoretical isotope patterns
for doubly charged receptor A + peptide complexes as [A +
peptide–4Na + 6H]2+ and [A + peptide–3Na + 5H]2+ (Fig. 3b, c
and S16†). In addition, the triply charged receptor A–peptide
complexes were observed with increasing sodium loss. Fig. 3
shows the MS spectrum of the observed (Fig. 3d) and
theoretical isotope patterns of the doubly charged complexes
of receptor B as [B + peptide–4Na + 6H]2+ and [B + peptide–
3Na + 5H]2+ (Fig. 3e, f and S18†). The receptor B + peptide
complexes were also observed as triply and doubly charged
ions. The multiply charged ions are expected due to the mass
of the complexes, the multiple basic amino acids present in
the peptide and the chosen ionization method. Equimolar
receptor C and peptide solution were also analyzed, but no
peaks associated with receptor C + peptide complexes were
observed under the same experimental conditions (Fig. S20†).
Receptor C is cationic and structurally very different from the
anionic receptor A and B. However, the receptor C + peptide
complex could not be observed under the ionization
methods.
NMR spectroscopy
To further understand the interactions between the receptors
and the αA66-80 crystallin peptide, we investigated the
binding ability of the receptors towards the individual amino

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Research Article RSC Medicinal Chemistry

acids within the αA66-80 peptide through 1H NMR
experiments. This was done by monitoring the complexation-
induced 1H chemical shift changes of the receptor–amino
acid mixtures and comparing that to the pure receptor and
the amino acids in D2O (Fig. S23–S48†). Guest complexation
in the resorcinarene cavity is typically indicated by shielding
and broadening guests' signals while hydrogen bond
interactions are generally observed as downfield shifts.25
With receptors A and B, both upfield and downfield shifts of
lysine and arginine signals were observed. While the
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under physiological conditions using isothermal calorimetric
titrations (ITC). Individual amino acid experiments were
limited to the arginine, lysine, histidine, and aspartic acid
residues based on established supramolecular attraction to
the receptors inferred from their 1H-NMR chemical shift
changes. Moreover, ITC experiments were also conducted
between the receptors A, B, and C with αA66-80 crystallin
peptide (Table 1). The sodium sulfonate substitution pattern
on receptors A and B does not have a marked difference in
the thermodynamic binding parameters with the amino acids

receptors can encapsulate both amino acid residues, extra
exo-cavity interactions are also observed with the de-shielded
protons. No significant shift changes were also observed for
the other amino acids, including histidine and aspartic acid
(Fig. 4 and S23–S48†). If the association cannot be
established with these individual amino acids, there is no
reason to believe they are viable binding pockets on the
crystallin peptide. Moreover, receptor C showed significant
shift changes for complexation with lysine, arginine,
histidine, and minor shift changes for aspartic acid (Fig.
S41–S48†). These results indicate that these receptors have a
peculiar preference for specific amino acids due to both size
and complementarity of the weak interactions. Summary of
complexation induced chemical shift changes have included
in Table S5.†
Isothermal titration calorimetry
If these interactions can translate into any consequential
effect on the morphological changes in a peptide, we needed
to understand the thermodynamics of those interactions
from our initial experiments. However, the binding affinity
and energy are highest with receptor A where the functional
groups are closest to the macrocyclic cavity. Complexation
was enthalpically favorable with entropic compensations
except for histidine, enthalpically and entropically favorable
in complexation with both receptors. The supramolecular
attraction of the persistent hydrophobic cavity and the
different functional groups on both sides of the receptor C
are highlighted in the nonspecific nature of the ITC curvature
with amino acids (Fig. S52†). The multiple binding processes
between receptor C and arginine, lysine could not be fitted to
one or two sets of sites binding model without significant
errors. However, complexation with histidine was found to be
enthalpically favorable with minor entropic compensations.
Aspartic acid, a negatively charged amino acid, was found to
be both enthalpically and entropically favorable in
complexation with receptor C. Ultimately, the binding event
between C and the αA66-80 crystallin peptide is a one-to-one
association. It is worth mentioning that for the ITC
measurements, the titrant is added, and the associated heat
changes are measured immediately.33 In effect, samples for
these measurements cannot be aged, a limitation of the
technique. Again, we recognize the unpredictable and three-
dimensional folding of the crystallin peptide is the most

Table 1 ITC interaction parameters between receptors A, B, and C and

selected amino acids in water. All the data shown were fitted to one set
of site binding models at 298 K

TΔS°
Ka (× 103) ΔH° kcal kcal ΔG° kcal ΔG° kJ
Complex M−1 mol−1 mol−1 mol−1 mol−1
A·
Arginine 7.27 ± 1.28 −15.70 ± 3.85 −10.43 −5.27 −22.05
Lysine 5.89 ± 0.88 −12.07 ± 2.04 −6.45 −5.62 −23.51
Histidine 2.94 ± 0.39 −2.59 ± 0.16 6.91 −9.50 −39.75
Crystallin 1360 ± 490 −8.28 ± 0.21 0.092 −8.37 −35.02
B·
Arginine 3.64 ± 0.47 −15.15 ± 1.00 −10.25 −4.90 −20.50
Lysine 4.45 ± 0.69 −6.74 ± 0.69 −1.76 −4.98 −20.84
Histidine 3.44 ± 0.51 −1.05 ± 0.07 3.75 −4.80 −20.08
Crystallin 380 ± 33 −3.23 ± 0.027 4.38 −7.61 −31.84
Fig. 4 Sections of the 1H NMR spectra in D2O at 298 K of (a) receptor C·
B, (c) lysine, (e) arginine, (g) histidine, (i) aspartic acid, and equimolar Arginine — — — —
mixtures of (b) receptor B + lysine, (d) receptor B + arginine, (f) Lysine — — — —
receptor B + histidine, and (h) receptor B + aspartic acid. Inset: Histidine 14.90 ± 1.70 −7.53 ± 0.34 −1.84 −5.69 −23.81
Downfield sections of (a), (f), and (g) highlighting the aromatic signals Aspartic 7.30 ± 0.10 −1.00 ± 0.06 2.90 −3.90 −16.32
of histidine. The dashed arrows indicate the signal changes in ppm. acid
Star represents the residual D2O solvent. Crystallin 2.40 ± 0.95 4.89 ± 2.66 9.48 −4.59 −19.20

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RSC Medicinal Chemistry Research Article

predominating factor influencing peptide: receptor
complexation and the subsequent disruption of aggregates.
Computational studies
Lastly, we resorted to computational modeling to back up the
fundamental knowledge obtained from our earlier
spectroscopic, microscopic, mass spectrometry and
calorimetric studies. The receptor structures were optimized
using Gaussian 09 suite of quantum chemistry programs at
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peptide–receptor complexes were solvated in a 10 Å cubic box
containing buffer and TIP3P water molecules. A 150 mM
NaCl solute concentration was added to the matrix.38 Sodium
ions were then added to neutralize the corresponding total
charge on the complex. OPLS2005 force-field was used for the
MD simulation,39 and the system minimized in Desmond.
Production MD was carried out for 100 ns in the NPT
ensemble at 300 K without any particle restraint. A Nose–
Hoover chain with a thermostat relaxation time of 1 ps was
used. In addition, a barostat of Martyna–Tobias–Klein

b3lyp/6-31g** level of theory in PCM water solvation.34 AM1-
BCC charges were added to the hosts to prepare them for the
subsequent computational analyses. αA66-80 peptide, protein
database (pdb) ID: 6t1r was obtained from the pdb
database.35
The protein preparation involved removing extraneous
chains, ions, alternate conformations, and solvent molecules
using UCSF Chimera.36 Missing hydrogen atoms were added,
histidine and aspartic acid residues were visually adjusted for
correct protonation states. The protein was capped and
minimized to correct the unfavorable interactions and atom
clashes. First, molecular docking was performed holding the
receptor and peptide rigid. Then the top three poses were
redocked holding the peptide still but the receptor molecules
flexible using Dock6 software suite.37 The molecular
dynamics (MD) system preparation was done using Maestro
and simulations were performed using Desmond. The
method with 2 ps relaxation time and an isotropic coupling
style were employed. A multiple time step RESPA integrator
with 2 fs for near and 6 fs for far sampling were chosen. A
smooth particle mesh Ewald with cutoff at 9.0 Å and an
Ewald tolerance of 1 × 10−9 were used. Energy and time
evolution recording was done every 1.2 ps and 100 ps
respectively. The binding energy for the MD trajectory
calculations were then carried out using MMGBSA method.40
The small root means square deviation (RMSD) of the
peptide backbone (0.1–0.4 Å) (Fig. 5a) was a clear indication
of the acceptable equilibration protocol employed. The
receptors interact with the side chain of the amino acid
residues of the αA66-80 peptide chain through hydrogen
bonding, hydrophobic, and ionic interactions (Fig. 5b).
Receptor A starts out interacting with the amino acid
residues LYS70, VAL72, and PHE74 using the anionic
sulfonate groups. This interaction lasts for 7.6 ns and then

Fig. 5 Molecular dynamics (MD) simulation of the αA66-80·receptors binding starting from the docking structures. The MD simulations were
performed solvating the complexes in a cubic box with a 10 Å buffer from edges containing water molecules (omitted for clarity), and the system
was studied employing OPLS2005 force-field for 100 ns at 300 K. (a) Backbone root mean square deviation (RMSD) of the peptide backbone for
peptide–receptor complexes throughout the simulation time (peptide·X: A black, B blue, C green), (b) starting configuration of peptide·A, peptide·B,
and peptide·C complexes, respectively. The interaction between the receptors and different amino acid residues of the peptide are shown in
yellow (hydrogen bonding), and blue (pi-stacking) dotted lines, (c) time evolution of the peptide·C complex: 0–34 ns, 35–67 ns, 68–100 ns,
respectively.

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Research Article
the receptor flips and binds with LYS70 using the sulfonate
group, LYS78 using one of the anionic phenoxides through
hydrogen bonding and the interaction between VAL72, and
PHE74 with the hydrophobic portion of the receptor. This
conformation remains stable for the rest of the simulation
time and contributes to the overall binding energy of the
peptide–receptor complex of −21.06 ± 7.29 kcal mol−1
(Table 2, Fig. S53†). Receptor B interacts with LYS70, ASP76,
and LYS78 using the ionic sulfonate and hydroxyl groups
through hydrogen bonding and with PHE74 using the
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aromatic phenyl rings through pi-stacking interactions. This
conformation is retained throughout the simulation time and
the binding energy for this complex is −24.73 ± 7.65 kcal
mol−1 (Table 2, Fig. S53†).
However, receptor C samples three different domains of the
peptide throughout the simulation time and settles for the best
binding domain characterized by the highest binding energy. It
starts out by interacting with PHE74, LEU75, ASP76, VAL77, and
LYS78 using the benzyl groups. After 34 ns it moves to the
second domain of the peptide chain to interact with the amino
acid residues ASP67, ARG68, and PHE71 using the benzyl groups
and remains in that region for about 32 ns. Then it moves to the
hydrophobic domain of the peptide and stays there for the rest
of the simulation time. The interaction in this domain is
facilitated by LYS70, VAL72, PHE74, LEU75, VAL77, and PHE80
amino acid residues. The average binding energy for receptor C
and peptide complex is −29.03 ± 7.16 kcal mol−1 (Table 2,
Fig. 5c). The strongest binding mode for receptor C is mostly
controlled by the hydrophobic interactions. From the detailed
computational studies, we can conclude that all the macrocyclic
receptor molecules reported here bind effectively with the αA66-
80 peptide, disrupting the aggregation mechanism.
However, from the magnitude of binding energies of the
receptor–peptide complexes it is clearly visible that receptor
C presents with the best binding interaction followed by B
and A. These results are in good agreement with the results
obtained from the DLS experiments as receptor C proves to
be the best inhibitor of the peptide aggregation as it has
affinity for three different domains of the peptide, revealed
from the detailed MD simulation.
Table 2 MMGBSA binding energies of the αA66-80 peptide·receptor
complexes showing the amino acid residues involved and the types of
interaction obtained from the MD simulations
Binding energy
Complex (kcal mol−1) Residues (type of interaction)
Peptide·A −21.06 ± 7.29 LYS70,LYS78 (H-bonds)
LYS70, PHE74, VAL72 (hydrophobic)
LYS70,LYS78 (ionic)
Peptide·B −24.73 ± 7.65 SER66, LYS70, ASP76, VAL77, LYS78
(H-bonds)
PHE74, LYS78 (hydrophobic)
LYS70, LYS78 (ionic)
Peptide·C −29.03 ± 7.16 LYS70 (H-bond)
LYS70, VAL72, PHE74, LEU75, VAL77,
PHE80 (hydrophobic)
LYS70 (ionic)
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Conclusions
The present work demonstrates that three resorcinarene-
based receptors can successfully disrupt αA66-80 crystallin
peptide which is abundant in cataracts eye lenses. Two of the
receptors possess anionic sulfonate groups at the upper and
lower rim. The third receptor possesses cationic ammonium
group and flexible benzyl groups. All three receptors possess
internal hydrophobic cavities. Macroscopic experiments such
as fluorescence with a Proteostat dye, dynamic light
scattering, and TEM microscopy show the receptors to
successfully interact and disrupt the aggregate of the αA66-80
crystallin peptide. These macroscopic experiments also show
receptor C with the cationic ammonium groups and benzyl
arms to be the optimum receptor for this process. Qualitative
NMR experiments reveal the receptors prefer specific amino
acids which are all part of the peptide backbone. Isothermal
titration calorimetric experiments provide some
thermodynamic data showing the interaction between the
receptors and the amino acids, or the αA66-80 crystallin
peptide to be spontaneous at the given temperature. The
interactions were mostly enthalpy-driven with some entropic
contributions. The binding constant between the receptors
and the αA66-80 crystallin peptide shows receptor C to
bind weakly, contrary to other techniques which could be
due to the inability to measure aged mixtures, a limitation
in ITC technique. Nonetheless, the detailed computational
studies support that all the macrocyclic receptors reported
here effectively bind with the αA66-80 peptide, disrupting
their aggregation mechanism. However, receptor C presents
with the highest binding energy, hence the best activity
over receptor B, followed by receptor A. MD simulations
have also revealed that receptor C has the affinity toward
three different domains of the peptide structure. It samples
all of them during the simulation time whilst receptor A
has two modes of binding. Receptor B involves only one
part of the molecule to bind with a specific part of the
peptide, which further rationalizes the claim of C being the
best macrocyclic receptor that can inhibit the aggregation
of the αA66-80 peptide. We are currently expanding the
library of these resorcinarene based receptors. We envision
that based on these results, resorcinarenes can be
developed into potential therapeutics for crystallin
disruption.
Funding sources
This research was partly funded by Oakland University
Provost's Graduate Student Research Awards to K. T. and
NSERC to S. M.
Abbreviations
DFT Density functional theory
MMGBSA Molecular mechanics generalized born surface
area
pdb Protein data bank
This journal is © The Royal Society of Chemistry 2021

RSC Medicinal Chemistry
PCM Polarized continuum model
OPLS Optimized potentials for liquid simulations
RESPA Reversible reference system propagation algorithm
Author contributions
The manuscript was written and edited through the
contributions of all authors. NKB, SM: original concept. KT,
EL, MJ, NS, BE: NMR, ITC, fluorescence, DLS. JE: TEM. SM,
NS, & EP: mass spectrometry. AB & GO: computation. TM:
Published on 01 November 2021. Downloaded by York University on 9/12/2022 9:34:30 PM.
supervised the work at Portland, USA. SM and NLS:
supervised the work at Trent, Canada. NKB: supervised the
work at Oakland, USA. All authors have approved the final
version of the manuscript.
Conflicts of interest
The authors declare no competing conflicts of interest.
Acknowledgements
The authors gratefully acknowledge financial support from
Oakland University (NKB, KT, MJ, NS, BE), the Oakland
University Provost Graduate Research Scholarship (KT). SM,
JE, NS, and SM acknowledge support from Trent University,
and Stephanie Gao for performing additional MS experiments
on receptor C and peptides. Calculations were performed on
the high-performance computing cluster, Coeus at Portland
State University.
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