Discovery of boronic acid-based potent activators of tumor pyruvate kinase M2 and development of gastroretentive nanoformulation for oral dosing

Article abstract of DOI:10.1016/j.bmcl.2021.128062

Several studies have established that cancer cells explicitly over-express the less active isoform of pyruvate kinase M2 (PKM2) is critical for tumorigenesis. The activation of PKM2 towards tetramer formation may increase affinity towards phosphoenolpyruvate (PEP) and avoidance of the Warburg effect. Herein, we describe the design, synthesis, and development of boronic acid-based molecules as activators of PKM2. The designed molecules were inspired by existing anticancer scaffolds and several fragments were assembled in the derivatives. 6a-6d were synthesized using a multi-step synthetic strategy in 55–70% yields, starting from cheap and readily available materials. The compounds were selectively cytotoxic to kill the cancerous cells at 80 nM, while they were non-toxic to the normal cells. The kinetic studies established the compounds as novel activators of PKM2 and (E/Z)-(4-(3-(2-((4-chlorophenyl)amino)-4-(dimethylamino)thiazol-5-yl)-2-(ethoxycarbonyl)-3-oxoprop-1-en-1-yl) phenyl)boronic acid (6c) emerged as the most potent derivative. 6c was further evaluated using various in silico tools to understand the molecular mechanism of tetramer formation. Docking studies revealed that 6c binds to the PKM2 dimer at the dimeric interface. Further to ascertain the binding site and mechanism of action, rigorous MD (molecular dynamics) simulations were undertaken, which led to the conclusion that 6c stabilizes the center of the dimeric interface that possibly promotes tetramer formation. We further planned to make a tablet of the developed molecule for oral delivery, but it was seriously impeded owing to poor aqueous solubility of 6c. To improve aqueous solubility and retain 6c at the lower gastrointestinal tract, thiolated chitosan-based nanoparticles (TCNPs) were prepared and further developed as tablet dosage form to retain anticancer potency in the excised goat colon. Our findings may provide a valuable pharmacological mechanism for understanding metabolic underpinnings that may aid in the clinical development of new anticancer agents targeting PKM2.

Full text of DOI:10.1016/j.bmcl.2021.128062

Bioorg. Med. Chem. Lett. 42 (2021) 128062
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of boronic acid-based potent activators of tumor pyruvate kinase
M2 and development of gastroretentive nanoformulation for oral dosing
,
,
Rajkumar Patlea 1, Shital Shindea, Sagarkumar Patela 1, Rahul Maheshwarib, Heena Jariyalc,
Akshay Srivastavac, Neelam Chauhanc, Christoph Globischd, Alok Jainc, Rakesh K. Tekadeb
,
,
*
,
*
Amit Sharda
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research-Ahmedabad, India
b Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research-Ahmedabad, India
c Department of Biotechnology, National Institute of Pharmaceutical Education and Research-Ahmedabad, India
d Department of Chemistry, University of Konstanz, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
Several studies have established that cancer cells explicitly over-express the less active isoform of pyruvate ki-
nase M2 (PKM2) is critical for tumorigenesis. The activation of PKM2 towards tetramer formation may increase
affinity towards phosphoenolpyruvate (PEP) and avoidance of the Warburg effect. Herein, we describe the
design, synthesis, and development of boronic acid-based molecules as activators of PKM2. The designed mol-
ecules were inspired by existing anticancer scaffolds and several fragments were assembled in the derivatives.
6a-6d were synthesized using a multi-step synthetic strategy in 55–70% yields, starting from cheap and readily
available materials. The compounds were selectively cytotoxic to kill the cancerous cells at 80 nM, while they
were non-toxic to the normal cells. The kinetic studies established the compounds as novel activators of PKM2
Cancer
Pyruvate kinase M2
Boronic acid
Nanoformulation
MD simulations
Thiolatedchitosan-based nanoparticles (TCNPs)
and
(E/Z)-(4-(3-(2-((4-chlorophenyl)amino)-4-(dimethylamino)thiazol-5-yl)-2-(ethoxycarbonyl)-3-oxoprop-1-
en-1-yl) phenyl)boronic acid (6c) emerged as the most potent derivative. 6c was further evaluated using various
in silico tools to understand the molecular mechanism of tetramer formation. Docking studies revealed that 6c
binds to the PKM2 dimer at the dimeric interface. Further to ascertain the binding site and mechanism of action,
rigorous MD (molecular dynamics) simulations were undertaken, which led to the conclusion that 6c stabilizes
the center of the dimeric interface that possibly promotes tetramer formation. We further planned to make a
tablet of the developed molecule for oral delivery, but it was seriously impeded owing to poor aqueous solubility
of 6c. To improve aqueous solubility and retain 6c at the lower gastrointestinal tract, thiolated chitosan-based
nanoparticles (TCNPs) were prepared and further developed as tablet dosage form to retain anticancer po-
tency in the excised goat colon. Our findings may provide a valuable pharmacological mechanism for under-
standing metabolic underpinnings that may aid in the clinical development of new anticancer agents targeting
PKM2.
Pyruvate kinase is one of the critical metabolic conduits that cata-
lyzes the bio-energetically enriched terminal step in glycolysis.1 It
converts phosphoenolpyruvate (PEP) to pyruvate and produces ATP for
energy production.2 There are four isoforms of pyruvate kinase (PK)
isoenzyme: PKLR (liver isoform L-PK), R-PK (red blood cell isoform),
M1-PK (muscle isoform), and PKM2 (Pyruvate kinase M2 present in
embryonic and tumor cells) forms.3 Out of all isoforms, PKM2 is deeply
associated with tumor growth, survival, and metastasis.4 PKM2 active
state (tetrameric) has more affinity towards PEP, whereas dimeric PKM2
is inactive and has a very low affinity towards PEP.5 The tetrameric form
drags the glycolysis towards pyruvate synthesis. Fructose 1,6 bisphos-
phate (FBP) and serine are natural allosteric activators of PKM2, which
catalyze the tetramer’s formation.6 However, the dimeric PKM2 in
tumor tissues leads to the accumulation of glycolysis intermediates
resulting in activation of alternate metabolic pathways.7
The present research is focused on developing compounds that
* Corresponding authors.
E-mail addresses: rakeshtekade@niperahm.ac.in (R.K. Tekade), amit@niperahm.ac.in (A. Shard).
1
Authors contributed equally
https://doi.org/10.1016/j.bmcl.2021.128062
Received 10 January 2021; Received in revised form 17 April 2021; Accepted 21 April 2021
Available online 24 April 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

R. Patle et al.
Bioorganic & Medicinal Chemistry Letters 42 (2021) 128062
activate PKM2 and act as potent anticancer agents.8 The much-studied
compounds are micheolide, Ml-265, DASA-58, SAICAR, and its deriva-
tives.9–12 Some promising PKM2 activators having appreciable biolog-
ical potential are shown in Fig. 1. Covalent modifiers like micheliode
onsets covalent modification of target leading to anticancer effects.
However, its applicability has been largely impeded owing to associated
safety concerns.13 Several studies have demonstrated significant syner-
gistic effects of PKM2 modulators in combination with other wide range
of established oncology drugs.14 It is imperative to mention that the
development of kinase activators will be highly useful as it will smartly
bypass the functional consequences associated with kinase inhibitors.15
On the contrary, activator binding may stabilize the binding-competent
state. This may increase the target affinity and often pushes the dynamic
range to lower target concentrations.16
The idea to synthesize boronic acid-based molecules stemmed from
our recent findings where nitrile substituted phenylaminothiazoles
(PAT) were found to act as B-cell lymphoma-2 (Bcl-2) inhibitors.32 The
molecules were found to pan assay interference compounds (PAINS) free
and had adequate ADMET properties to guide the apoptosis in cancerous
cells. Considering PAT moiety (blue highlighted) insignia of anticancer
activity, we started tinkering with nitrile moiety isosteric to carbonyl
moiety (yellow highlighted) (Fig. 3).
Since we were inclined to target PKM2, we found that 1,3 dicarbonyl
moiety (yellow highlighted) existing in curcumin directly modulates
PKM2.33 Upon analyzing the structure, it was further found that Knoe-
venagel condensation of carbonyl substituted phenylboronic acid with
active methylene of 1,3 dicarbonyl moiety yields cinnamate, which is
also a known PKM2 modulator.34 We chose ethyl ester owing to the
toxicity of methyl esters. Further, as explained in the introduction, the
boronic acid moiety involved in the modulation of kinases was incor-
porated. Moreover, it may be metabolized to a phenolic moiety (6c-OH)
by simultaneous generation of non-toxic boric acid.26
Thus, there is a need to develop novel PKM2 activators as anticancer
agents. It has been demonstrated that boronic acid-based molecules are
electrophilic and potent antiproliferative agents.17–21 For instance,
bortezomib is chiefly employed in multiple myelocytic leukemia, and
targets the serine/threonine amino acids of proteasome through its
electrophilic boronic acid core (Fig. 2).22 Boron-containing functional
groups like diazaborines, boronic acids, esters, and benzoxaboroles have
been successfully incorporated into therapeutics. Aryl boronic acids also
provide a viable biomimetic of phenolic group having poor bioavail-
ability profiles.23
Further, to synthesize the molecules, phenyl isothiocyanates (2)
were prepared from aniline (1) derivatives and carbon disulfide via
dithiocarbamate salt intermediate (Scheme 1).35 Tetramethylguanidine
adducts (3) were synthesized by the treatment of isothiocyanate de-
rivatives (2) (1 eq.) with 1,1,3,3 tetramethylguanidine (1 eq.) at room
temperature.36
Boronic acid functional group improves the water solubility of potent
anticancer combretastatin CA-4. The moiety also increases the lip-
ophilicity and affinity of a drug towards the hydrophobic region of the
receptor. It can improve the stability of a drug towards metabolic
degradation.24 At the cell surface, an intense cluster of polysaccharides
is present that forms glycocalyx. The boronic acids can readily form
boronate esters with glycocalyx that may enhance drug selectivity to-
wards tumor cells.25 The widespread utility of boronic acids stems from
its vacant p-orbital of boron. Boron has chameleonic behavior that
switches between electrophilic and nucleophilic states under physio-
logical conditions.26
Tetramethylguanidine adducts (3) underwent cyclization in the
presence of 4-chloroacetoacetate that led to thiazole-based (4) core
structure having a side-chain of 1,3 dicarbonyl moiety.37 Desired com-
pounds (6a-6d) were synthesized via the Knoevenagel condensation
method (Fig. 4).38 (For detailed procedure of synthesis, please refer to
the supplementary information).
PKM2 dimeric form is a hallmark of cancer, while the tetrameric
(active) form is incharge of pyruvate generation. The PKM2 enzyme
activity was ascertained by lactate dehydrogenase (LDH)-coupled
enzyme assay.39 The results indicated that 6a-6d are acting as an acti-
vator with AC50 of 7.15 µM, 6.11 µM, 5.69 µM, and 8.17 µM, respec-
tively. Fig. 5 is showing % relative activity of PKM2 following treatment
with 6a-6d.
Here, we designed and synthesized four novel compounds based on
boronic acids centered around the thiazole core, which is generally
considered as master keys in medicinal chemistry.27–30 We further
describe the enzyme assay of the molecules synthesized, their inhibitory
effects on various cancer cell lines, including triple-negative breast
cancer and mesenchymal stem cells, along with computational studies to
elucidate the mechanism of action of our molecules. This was followed
by the development of a formulation to target colon cancer reported to
have the highest expression of PKM2. Considering the poor aqueous
solubility of 6c, a contemporary nanoparticle-based formulation was
developed. Recently in vitro metabolite profiling of 6c was successfully
done and published.31 We speculated that thiolated chitosan-based
formulation may enhance mucoadhesion to colon cells and can have a
marked impact on gastric retention. Our results strongly support that
rationally designed boronic acid-based PKM2 activators can orchestrate
multifaceted cancer cell elimination responses. Thus, targeting PKM2
with activators may represent a novel anticancer strategy. Modelling
studies suggested that boronic acid scaffold could position itself within
proximity of PKM2 dimers which could enhance the binding kinetics.
To further ascertain the potential of 6a-6d, we screened them against
different cell lines (Table 1). Here, we used a wide range of cancer cell
lines, including drug-resistant MDA-MB and drug-sensitive cell lines like
MCF-7, Bcl-2 Jurkat, and Colo-201.40,41 All the cell lines and normal
fibroblast cells were also treated with varying concentrations of 6a-6d
and assayed for the cell viability by MTT (3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyl tetrazolium) assay.42 It is pertinent to mention that all the
cell lines are known to have sufficient PKM2 expression. Cell growth was
inhibited in all treated cancer cell lines (IC50, 80 nM ꢀ 58.7 μM) in a
concentration-dependent manner (Table 1). It may be noted that the
PKM2 activation at a particular concentration leads to the formation of
tetramer and has reverted the cancerous phenotype leading to
cytotoxicity.
The MCF-7 cell line was found to be more sensitive to 6c, with an
IC50 of 0.08 μM. The activities showed (IC50) in Table 1 established that
6c showed promise against almost all cell lines at various
concentrations.
Fig. 1. Reported PKM2 activators (ML-265, DASA-58, Micheliolide).
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R. Patle et al.
Bioorganic & Medicinal Chemistry Letters 42 (2021) 128062
Fig. 2. Boronic acid-based anticancer molecules.
Kinase modulator
(Inspired from Bortezomib)
Cinnamic acid moitey
(PKM2 modulator)
Generation of
phenolic moitey
Curcumin inspired
1,3 dicarbonyl moitey
(PKM2 modulator)
B(OH)2
OH
O
c
O
c
O
O
O
O
NC
N
N
Isosteric
CN ~ CO
N
-H3BO3
S
b
b
S
N
Oxidation
N
S
b
Plausible
metabolism
N
HN
HN
HN
a
a
R1
a
R1
Previously proven
a
6a-d
6c, R1 = 4-Cl
Cl
6c-OH
Bcl-2 inhibitor scaffold
(Phenyl amino thiazole)
Reference 32
Fig. 3. Design of boronic acid-based moieties and their plausible metabolism.
compound could be a potent activator of PKM2 and showed antitumor
PKM2 is involved in cellular proliferation, regulation, apoptosis, and
metastasis of cancer cells.43 Annexin PI assay revealed that 6c induced
annexin positive population is 8.81% and double-positive is 23.50% at a
much lower concentration of 100 nM of 6c (Fig. 6).44 Results indicate
that 6c induces apoptosis at 100 nM. Thus, PKM2 activator (6c) induced
cytotoxicity at a much lower concentration.
activity in vitro.
6c emerged as one of the most bioactive molecules amongst all, and
its effect on inhibiting proliferation was subsequently demonstrated in a
wide variety of cancer cell lines. It was able to inhibit the growth of
different cell lines, as mentioned in Table 1. The compound also showed
Tetrameric PKM2 is the only form with high pyruvate kinase activ-
ity.45 Cancer cells mainly utilize aerobic glycolysis to meet their pro-
liferative demands.46 PKM2 occurs in dimeric (inactive) and tetrameric
(active) forms inside cells. Cancer cells maintain lower levels of tetra-
meric form to increase the glucose uptake of cells and utilize them for
alternate metabolic pathways.47 Furthermore, lower levels of the
tetrameric form allow the entry of key glycolytic intermediates towards
biosynthesis of essential biomolecules, which is required for cell pro-
liferation.48 Accumulation of glycolytic intermediates in cancer cells can
be prevented by converting dimeric PKM2 to tetrameric PKM2. Tetra-
merization of PKM2 was ascertained by disuccinimidyl suberate (DSS)
cross-linker assay.49 We observed that when MCF-7 cells were treated
with 6c, increased tetramer formation at 250 kDa as compared to con-
trol cells (untreated MCF-7 cell) was observed (Fig. 7). Thus, the 6c
an increase in percent activity of PKM2 with an AC50 of 5.6 μM.
Furthermore, 6c also enhanced the tetramer formation in MCF-7 cells
when cells were treated with IC50 concentration of compound compared
to untreated cells.
The compound 6c was found to be the most potent analog among the
four different boronic acid-based ligands, we tried to evaluate compu-
tationally where it binds. The PKM2 monomer consists of 531 amino
acids and aggregates in PKM2 into a homotetrameric structure (A, B, C,
and D chains). In normal physiological conditions, PKM2 remains in
tetramer conformation. However, post-translational modifications or
mutations hinder the tetramer formation process, and the equilibrium is
shifted towards dimer conformation. Therefore, in the docking study, we
only considered the dimeric conformation. Designed molecule 6c and
FBP were docked at the allosteric (FBP binding) site and the interface
3

R. Patle et al.
Bioorganic & Medicinal Chemistry Letters 42 (2021) 128062
Scheme 1. A generalized scheme for the synthesis of boronic acid-based molecules.
monomeric, dimeric, and tetrameric states (data are shown). However,
the best binding mode was observed at the dimeric state where 6c binds
at the monomeric interface site (between both monomers) as illustrated
in Fig. 8. Similar binding modes for other molecules were also reported
for PKM2.50,51 At the monomeric interface, our molecule interacts with
both the protein chains. This offered extra stability to the monomeric
interface but also stabilizes the dimeric interface and supports the
initiation of the tetramer formation process as discussed in the following
section. We monitored the stability of interactions formed by PKM2
residues with 6c during the course of MD simulations. Residues forming
stable interactions with 6c are displayed in Figure SI2. The binding
pocket consists of hydrophobic and hydrophilic residues. Notably, 6c
formed many new interactions that were not present after the docking in
the crystal structure. Polar groups of Asp354, Lys311, and Tyr391
interact with the polar moiety of 6c while the hydrophobic segment of
6c makes contact with Leu353, Ala388, and Tyr391. Herein, we have
only considered the residues which maintained contacts or H-bonds for a
significant fraction of the simulation time. We also observed some
further interactions, but they formed only transiently and are therefore
not reported here. We expect that combinations of such polar and hy-
drophobic contacts make the ligand–protein (6c-PKM2) binding more
specific and possibly reduce binding to the other PK isoforms.
Fig. 4. The desired compounds (6a-6d) and their substitution patterns.
binding site (interface between two chains) as displayed in Fig. 8.
Binding energies of 6c and FBP in dimer conformation at the allosteric
site were ꢀ 51.5 and ꢀ 97.8 kcal/mol, respectively. The binding energies
corroborated very well with non-covalent interaction patterns between
ligands and protein. FBP formed a higher number of H-bonds, and
participated in salt bridge interaction compared with fewer H-bonds by
our designed molecule 6c at the allosteric sites (Fig. 8).
Similar trends were observed in the binding pattern of FBP at the
interface binding site and 6c at the allosteric site and illustrated in SI1).
Based on the interaction pattern and binding energy of 6c and FBP, we
concluded that the designed molecule 6c has a lower affinity towards
the allosteric binding site than FBP. On the other hand, binding energies
of 6c and FBP at the interface site were ꢀ 64.2 and ꢀ 30.7 Kcal/mol,
respectively. The synthesized molecule 6c showed multiple H-bonding,
Proposed mechanism of tetramer formation: Experimental results
demonstrated that our designed molecule 6c drags the system towards
tetramer formation even in the absence of natural allosteric ligand FBP.
To understand the molecular mechanism of tetramer formation, we
performed multiple simulations in the presence and absence of 6c and
FBP ligands as discussed in the method section. During the MD simu-
lation of PKM2 dimer in the apo (without any ligand/FBP) and complex
form, stability at the dimeric interface was closely monitored. Resultant
root means square deviation (RMSD) time plots are illustrated in Fig. 9.
A central dimeric interface was defined by selecting protein residues
within 1.5 nm of the bound ligand (6c). RMSD plots (upper panel of
Fig. 9) clearly demonstrate that in the presence of 6c the central
tetramer interface (magenta helices) is significantly stabilized compared
with the apo simulation. Although this effect is most prominent in one of
the simulations, other simulations showed transient interface stabiliza-
tion. It is important to mention that we do not have a fully optimized
force field for boronic acid. The Lennard Jones (LJ) parameters used by
ATB for Boron corresponded to carbon and were not optimized sepa-
rately. Therefore, we have also performed the docking and subsequent
simulation of 6c-OH molecule (Fig. SI3), which has similar LJ parame-
ters to boron and is the most likely metabolite of 6c formed after its
π-π stacking, and many unspecific hydrophobic interactions that corre-
late with their binding energies, as illustrated in Fig. 8. Overall, the
affinity of 6c is much higher at the interface binding site compared to the
allosteric site. Therefore, its binding is independent of possible muta-
tions at the FBP binding site that inhibit FBP binding and consequently
hinder the tetramerization process. Furthermore, multiple simulations
with the designed molecule 6c, its variant 6c-OH and FBP were per-
formed and are discussed below to confirm that our designed molecules
(6c and 6c-OH) are capable of inducing the necessary conformational
changes in the protein structure (PKM2) to drag the system towards
tetramer (active) formation.
To identify the possible binding site of our molecule 6c with PKM2,
we evaluated two different binding sites in the dimeric state of the PKM2
using the docking protocol discussed in the method section. Our result
suggested a lower binding affinity of 6c at the allosteric binding site
compared with the interface site. We performed docking in the
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R. Patle et al.
Bioorganic & Medicinal Chemistry Letters 42 (2021) 128062
Fig. 5. % Relative pyruvate kinase activity of synthesized molecules (6a-6d) was determined using LDH-coupled enzyme assay. The reaction mixture contains (50
mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl2, 0.6 mM PEP, 0.9 mM ADP, 0.12 mM β NADH and 4.8 U/ml LDH). Pyruvate kinase activity was measured at 25 ◦C
by changing the absorbance of β NADH at 340 nm from 0 to 20 min.
Table 1
Relative cell growth inhibition of 6a-6d in various cancer cell lines.a
Compound
Code
IC50 Value (µM)
Colo-201
BCL-2
Jurkat
MCF-7
MSCs
MDA-MB-
231
6a
2.81 ±
0.12
6.36 ±
0.15
1.5 ±
0.32
40.6 ±
1.21
38.03 ±
0.33
6b
5.58 ±
0.45
0.09 ±
0.29
2.71 ±
0.37
29.43 ±
5.91
36.6 ±
3.59
6c
6d
3.75 ±
0.47
2.82 ±
0.07
0.08 ±
0.24
23.86 ±
0.43
34.85 ±
1.59
5.50 ±
0.03
0.57 ±
0.16
2.72 ±
0.12
25.83 ±
3.18
58.7 ±
9.02
Doxorubicin
0.80 ±
0.40
3.03 ±
0.18
1.08 ±
0.28
2.37 ±
0.80
1.30 ±
0.60
a
MCF-7, breast cancer cell; COLO-201, colorectal adenocarcinoma cells; BCL-
2Jurkat cells, Human Leukemia cells; MDA-MB-231, Triple-negative breast
cancer cells ; MSCs, Mesenchymal Stem Cells.
hydrolysis (Fig. 3).52 As 6c-OH does not contain the boron atom, its
force field is accurately parameterized using ATP. The PKM2 dimer in
the presence of 6c-OH showed a stabilizing effect at the central dimer
interface, illustrated by the blue color line in Fig. 9 upper panel. In
contrast, the PKM2-FBP complex exhibits a totally different picture. We
were expecting the bound FBP will significantly stabilize the tetramer
interface, an advantage to tetramer formation. However, the PKM2-FBP
complex displayed the highest RMSD value (up to 0.33 nm) at the
central dimeric interface. We have also monitored the RMSD value at the
terminal dimer interface (magenta and yellow color secondary struc-
tures) as depicted in Fig. 9 lower panel to solve this mystery. FBP has two
binding sites, one at each monomer, compared with only one binding
site for our ligands (at the monomer interface). Therefore, we have
monitored the effect at both FBP binding sites separately. We have
Fig. 6. Flow cytometric analysis of 6c in the MCF-7 cell line after 48 h of drug
treatment. PI: Propidium Iodide.
observed significantly lower RMSD values at both sites compared with
the apo form (Fig. 9 lower panel and Fig. SI4). The Apo form displayed
an average RMSD value of ~ 0.3 nm compared with an average of ~
0.21 nm observed in the PKM2-FBP complexes, as illustrated in Fig. 9.
Based on the above-mentioned results, we proposed the following model
for the tetramer formation process. Both 6c, 6c-OH, and FBP drag the
dimeric (PKM2) system towards tetramerization via slightly different
mechanisms. 6c and 6c-OH stabilized the central dimeric interface and
possibly trigger the tetramer formation process from there. It was shown
previously50,51 that this interface contains key residues that are critical
5

R. Patle et al.
Bioorganic & Medicinal Chemistry Letters 42 (2021) 128062
efficacy in the ex vivo model. The idea stemmed from the fact that fecal
tumor PKM2 is a highly sensitive marker for colorectal cancer (CRC)
which is the third most prevalent cancer in the world.53,54 PKM2 also
correlates with more advanced stages of CRC, and its reduction is
associated with successful surgical intervention. 5-Fluoro uracil (5-FU),
oxaliplatin, and irinotecan are chiefly employed for parenteral chemo-
therapy of CRC. In recent years a number of cytotoxic drugs such as
capecitabine, vinorelbine, or topotecan have been developed as oral
agents for the parenteral treatment of colon cancer.55 However, to target
the CRC with an orally bioavailable molecule, appropriate release pro-
file and mucoadhesion are vital parameters for sustained release in the
colon.56 To address the challenge a sustained release formulation was
designed. The physicochemical evaluation of the compound was deter-
mined, wherein the molecule was found to be lipophilic in nature,
making it unfavorable for oral administration. The molecule 6c was
bioactive but lacked suitable pharmaceutical properties. Therefore, the
development of an orally bioavailable form of 6c was undertaken.
Excellent solubility and satisfactory dissolution rate are essential con-
ditions for the clinical applications of candidate drugs.57–59 For this
purpose, we selected thiolated chitosan nanoparticles that show
mucoadhesive properties leading to the release of drug at the colonic
mucosal surface.60
Fig. 7. MCF-7 cells were treated with 100 nM of 6c for 48 h. 6c treated cells are
showing more tetramer compared to untreated cells (control). Disuccinimidyl
suberate (DSS) cross-linker assay ascertains tetramer expression of the PKM2 in
MCF-7 cell line on SDS-PAGE.
to initiate the tetramer formation process.
Therefore, the stability of this interface is crucial for tetramer for-
mation. Contrarily, the mode of action of FBP is very different. The
binding of FBP stabilizes the terminal dimeric interfaces at both sides.
The tetramer formation process possibly initiates from this location. The
different binding sites and distinct mechanisms further suggest that our
molecules are independent of FBP binding. The study suggests that in
mutant proteins where FBP cannot bind to the monomers, our molecule
alone is able to drag the system towards tetramer formation. Such in-
silico mutants and their effect on the tetramer formation process need to
be further analyzed.
Zeta sizer measures the size of the particle-based on its light scat-
tering ability.61 Smaller the particle, less is the scattering and more
scattering for larger particles. After treatment with 6c, PKM2 protein is
Further, gastro-retentive tablets of 6c were prepared to check its
FBP at allosteric site
at
6c interface
site
Fig. 8. Docked protein–ligand complex in the PKM2 dimeric state with 6c at interface binding site (cyan) and with FBP at an allosteric site (orange). Monomeric
chains A and C are colored in red and green, respectively. Ligands were shown in ball and stick representations, while protein residues were displayed in the
wireframe. Carbon, nitrogen, oxygen, sulfur, fluorine, hydrogen, and boron were colored in grey, blue, red, yellow, green, white, and pink colors, respectively.
6

R. Patle et al.
Bioorganic & Medicinal Chemistry Letters 42 (2021) 128062
Fig. 9. RMSD values of the central pocket (upper panel) and FBP pocket (lower panel). Part of the protein that forms the central pocket is shown in magenta/blue
color in the upper left panel. FBP pocket is illustrated in the magenta/yellow color in the lower-left panel. 6c I and 6c II represent the data from two independent
simulations.
expected to tetramerize. Due to which its molecular size will increase
and hence the scattering too. Scattering observed by DLS was doubled
after treatment with the compound 6c (Fig. 10).
particle size and zeta potential of the NPs were monitored by optimizing
the ratio of TPP and thiolated chitosan. Finally, 3:1 (S-Ch: TPP) weight
ratio was used to prepare NPs. The average hydrodynamic particle size
of S-Ch NPs was found to be 130.42 ± 3.2 nm, and the zeta potential was
20.92 ± 2.13 mV (Fig. 11A and 11B). Being polysaccharides in nature
First, thiolated chitosan was synthesized following the protocol
described earlier with appropriate modifications.59 The hydrodynamic
Fig. 10. Dynamic Light Scattering (DLS) of PKM2 before treatment (left) with 6c and after (right) treatment with 6c.
7

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Bioorganic & Medicinal Chemistry Letters 42 (2021) 128062
chitosan-based NPs formed via cross-linking, exhibited minimal energy
in the dispersion when they are in the form of NPs.
NPs were monitored by optimizing the ratio of TPP and thiolated
chitosan. Finally, 3:1 (S-Ch: TPP) weight ratio was used to prepare NPs.
The average hydrodynamic particle size of S-Ch NPs was 130.42 ± 3.2
nm, and the zeta potential was 20.92 ± 2.13 mV (Fig. 11A and B). Being
polysaccharides in nature chitosan-based NPs formed via cross-linking
exhibited minimal energy in the dispersion when they are in the form
of NPs. Further, mucin, S-Ch-NPs, and mucin + S-Ch-NPs were tested for
zeta potential to observe the binding of S-Ch-NPs with mucin. Formu-
lation with the experiment was carried out by treating different for-
mulations with excised goat intestinal segment. As shown in Fig. 11D
zeta potential of mucin was changed after the addition of the S-Ch-NPs
indicating that it possesses an affinity for the mucin present in the mu-
cous layer of the intestine where 6c is destined to act. The entrapment
efficiency (EE) indicates the compound’s can be successfully entrapped
into NPs.62 The EE of the S-Ch NPs + 6c was determined with an indirect
method after isolation of NPs by centrifuging them. The EE of S-Ch-NPs
+ 6c was found to be 73.63 ± 1.21%. This high EE might originate from
the tendency of 6c to enter the core of the S-Ch-NPs. The DL of S-Ch-NPs
+ 6c was found to be 30.21 ± 2.1% which might be due to the hydro-
philic nature of the S-Ch.63
Fig. 12. In vitro drug release profile from S-Ch-NP + 6c and Ch-NP + 6c
formulations.
followed by replacement with phosphate buffer (pH 6.8) for 4 and
subsequent replacement with phosphate buffer (pH 7.4) with a total
duration of 12 h. Results are represented as mean ± SD (n = 3). The zero-
order kinetics indicated an initial rapid release of 6c from the S-Ch-NPs
followed by a slow zero-order and regression value of 0.9975 (Fig. SI6).
The fitting of the first-order release kinetics model led to a regression
value of 0.7145, signifying that the formulation does not follow a first-
order release pattern. The Higuchi matrix model regression was found to
be 0.6775, whereas the Hixson Crowell release shows a regression of
0.6639. By calculating and comparing r2 value for all models, zero-order
release was found to be the best fit model, which suggests that the drug
was released at a particular site in a sustained manner. The zero-order
kinetic model was found suitable for the release of drugs from the S-
Ch-NPs, which might be due to a barrier property of chitosan to the
release of 6c.66,67 Fig. 13A and B indicates that the applied force is
directly proportional to the detachment force and work of adhesion.
When applied force was increased, the required detachment force for
sample disc from intestinal mucosa also increased, indicating the greater
Apart from formulation composition, drug release profile may be
another important factor that must be appropriately considered to
obtain required anticancer activity from the developed formulation.64
The in vitro release profile of the 6c-loaded S-Ch-NPs is shown in Fig. 12.
It shows a controlled release profile, suggesting that the S-Ch-NPs acts
as a barrier against the release of entrapped drugs from the polymeric
matrix into the release medium.
Moreover, the release data, when fitted into different kinetic models
viz zero order, first order, Hixon Rowell and Higuchi value of the co-
efficient of correlation (r2) was calculated by linear regression analysis
using graph pad InStat software to evaluate the accuracy of the fit.65 In
vitro drug release was evaluated in three different media using USP
Dissolution apparatus II. The test started with pH 1.5 HCl buffer
Fig. 11. A. Average size of S-Ch-NPs_6c B. Zeta potential of S-Ch-NPs_6c. C. Surface morphology using scanning electron microscope. D. Change in the zeta potential
of tested formulation to predict binding with mucin. Nanoparticles-Ch-NPs + 6c.
8

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Bioorganic & Medicinal Chemistry Letters 42 (2021) 128062
Fig. 13. A. Effect of applied force on the detachment of sample disc from goat intestinal mucosa. B. Effect of different applied force on the work of adhesion (area
under the curve). C. In vitro dissolution investigation of Ch-NPs + 6c and S-Ch-NPs + 6c tablets determined in a different medium to simulate GIT pH from stomach to
lower intestine (colon).
mucoadhesive potential of the synthesized compound. The same phe-
nomenon was observed with the work of adhesion, as increased applied
force showed maximum work of adhesion. The applied force of 5000 mN
showed maximum detachment force and work of adhesion, i. e. 1707.21
mN and 507.42 mN respectively. The further increased applied force
showed rupture of the intestinal mucosa.
sustained drug release and it was found that 79.12 ± 5.2% of 6c was
released within 12 h. It also explains why the release was slower at this
pH compared to that at pH 1.5. Similarly, the 6c formulation showed
antiproliferative activity as compared to 6c (Fig. SI5). Protein cross-
linking assay indicates that 6c based nanoformulation converts
dimeric PKM2 into the tetrameric form of PKM2 (Fig. 14).
Cancer cells anchorage genetic changes that escalate nutrient uptake
and alter their metabolism to support anabolic processes to activate
alternate metabolic pathways. Interfering with this metabolic program
is a strategy for cancer therapy. Altered glucose metabolism is common
in cancer cells and is mediated in part by the expression of PKM2 that
acts as metabolic bug with distinct regulatory properties. FBP
Characterization of S-Ch-NPs þ 6c gastroretentive tablets
Plain tablets (without thiolated chitosan) and tablets containing 6c
entrapped S-Ch-NPs were prepared successfully employing direct
compression method using microcrystalline cellulose as filler, magne-
sium stearate, and Cab-O-Sil as a lubricant and cellulose acetate
phthalate as enteric coating polymer.68,69 The developed core tablets
had a smooth surface, with hardness ranging from 3 ± 1 kg (500 mg
tablet). Moreover, in the weight variation test, randomly selected 20
tablets were weighed individually, and the average weight was found to
be 500 ± 6.72 mg. Evaluated tablet parameters are shown in Table S16
and comply with standard pharmacopoeial limits.
Dissolution data showed that Ch-NPs + 6c tablets released ~ 90% of
6c in 8 h, while ~ 79% of 6c was found released from S-Ch-NPs + 6c
tablets. As shown in Fig. 13C, the release pattern was identical till 3 h
due to the presence of enteric polymer. Afterward, 6c was released at a
faster rate from Ch-NPs + 6c tablets compared to S-Ch-NPs + 6c tablets
attributed to the presence of thiolated chitosan. Besides, less than 10%
of 6c was released in either case and therefore comply with standard
pharmacopeial limits. Conclusively, the S-Ch-NP + 6c tablet provides
Fig. 14. Tetramer formation of PKM2 in MCF-7 cell line on SDS-PAGE. MCF-7
cells were treated with 100 nM of 6c for 48 h. Disuccinimidyl suberate (DSS)
cross-linker assay ascertains conversion of PKM2 dimeric form to tetra-
meric form.
9

R. Patle et al.
Bioorganic & Medicinal Chemistry Letters 42 (2021) 128062
allosterically activates PKM2. It is found to interact with tyrosine-
phosphorylated proteins, resulting in the release of FBP, leading to
decreased enzyme activity. Hence activation of PKM2 might resist and
interfere with the Warburg effect.
Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of
India. The authors gratefully acknowledge Prof. Kiran Kalia, Director,
NIPER-Ahmedabad, for her motivation and constant support throughout
the project. The communication number for this manuscript is NIPER-A/
344/09/2019.
In accordance with the same line of thought, our data show that high
pyruvate kinase activity caused by boronic acid-based small-molecule
PKM2 activation slows down the ability of cancer cells to divide with
limitless replicative potential. Several recent publications have
described activators and inhibitors of PKM2. These range from judi-
ciously designed drug-like molecules to several polyphenols having poor
developability owing to the propensity of PAINS (Pan assay interference
compounds). Although several of these PKM2 modulators may prevent
tumor cell proliferation, our data suggest that judiciously designed
boronic acid derivatives can activate PKM2 enzyme and cancerous cells
can be efficiently challenged with designed ligands.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128062.
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