Papers by Ankit Srivastava
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neuro... more Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons. Unfortunately,
effective therapeutics against this disease is still not available. Almost 20% of familial ALS (fALS) is suggested to be
associated with pathological deposition of superoxide dismutase (SOD1). Evidences suggest that SOD1-containing
pathological inclusions in ALS exhibit amyloid like properties. An effective strategy to combat ALSmay be to inhibit
amyloid formation of SOD1 using small molecules. In the present study, we observed the fibrillation of one of the
premature forms of SOD1 (SOD1 with reduced disulfide) in the presence of curcumin. Using ThT binding assay,
AFM, TEMimages and FTIR,we demonstrate that curcumin inhibits the DTT-induced fibrillation of SOD1 and favors
the formation of smaller and disordered aggregates of SOD1. The enhancement in curcumin fluorescence on the
addition of oligomers and pre-fibrillar aggregates of SOD1 suggests binding of these species to curcumin. Docking
studies indicate that putative binding site of curcumin may be the amyloidogenic regions of SOD1. Further, there
is a significant increase in SOD1 mediated toxicity in the regime of pre-fibrillar and fibrillar aggregates which is
not evident in curcumin containing samples. All these data suggest that curcumin reduces toxicity by binding to
the amyloidogenic regions of the species on the aggregation pathway and blocking the formation of the toxic
species. Nanoparticles of curcumin with higher aqueous solubility show similar aggregation control as that of
curcumin bulk. This suggests a potential role for curcumin in the treatment of ALS.
The pathogenesis of protein misfolding diseases
is attributed to the cytotoxicity caused by amylo... more The pathogenesis of protein misfolding diseases
is attributed to the cytotoxicity caused by amyloidogenic
prefibrillar aggregates, rather than mature fibrils. The presence
of one or more amyloidogenic stretches in different proteins
has been proven critical for initiating fibril formation. In the
present study, we show that two natural compounds, curcumin
and emetine, bind tightly (Kd < 1.6 μM) to the core
amyloidogenic stretch (182−192) of gelsolin (AGel). Binding
happens in different structural orientations, distinctly modulating the amyloidogenic pathway of AGel. While AGel alone undergoes sigmoidal transition to thioflavin T (ThT)-
responsive fibrillar aggregates with clear lag phase, the
presence of curcumin or emetine abolishes the lag phase and produces starkly different, noncytotoxic end products. Atomic
force microscopy revealed that while curcumin augments fibril formation, emetine arrests it at an intermediate aggregated stage with no fibrillar morphology. FTIR spectroscopy, dynamic light scattering, and ANS fluorescence experiments also suggest that these two species are distinct. Curcumin and emetine also differentially affect the preformed amyloids with the former thickening the fibrils and the latter releasing reclusive oligomers. MD simulations further provided mechanistic insights of differential
interaction by the two compounds modulating amyloid formation. The results were also confirmed on the disease-associated amyloidogenic fragment of gelsolin ( fAGel). Thus, our findings suggest that targeting amyloidogenic stretches in proteins could be useful in designing novel molecules against protein misfolding diseases.
Covalent linkers bridging the domains of multidomain
proteins are considered to be crucial for a... more Covalent linkers bridging the domains of multidomain
proteins are considered to be crucial for assembly and
function. In this report, an exception in which the linker of a
two-domain dimeric l-asparaginase from Pyrococcus furiosus
(PfA) was found to be dispensable is presented. Domains of
this enzyme assembled without the linker into a conjoined
tetrameric form that exhibited higher activity than the parent
enzyme. The global shape and quaternary structure of the
conjoined PfA were also similar to the wild-type PfA, as
observed by their solution scattering profiles and X-ray
crystallographic data. Comparison of the crystal structures of
substrate-bound and unbound enzymes revealed an altogether
new active-site composition and mechanism of action. Thus,
conjoined PfA is presented as a unique enzyme obtained
through noncovalent, linker-less assembly of constituent
domains that is stable enough to function efficiently at
elevated temperatures.
Thermophilic L-asparaginases display high
stability and activity at elevated temperatures. Howev... more Thermophilic L-asparaginases display high
stability and activity at elevated temperatures. However,
they are of limited use in leukemia therapy
because of their low substrate affinity and reduced
activity under physiological conditions. In an attempt to
combine stability with activity at physiological conditions,
3 active-site mutants of Pyrococcus furiosus L-asparaginase
(PfA) were developed. The mutants, specifically
K274E, showed improved enzymatic properties at
physiological conditions as compared to the wild type.
All variants were thermodynamically stable and resistant
to proteolytic digestion. None of the enzymes
displayed glutaminase activity, a highly desirable therapeutic
property. All variants showed higher and significant
killing of human cell lines HL60, MCF7, and K562
as compared to the Escherichia coli L-asparaginase. Our
study revealed that increased substrate accessibility
through the active site loop plays a major role in
determining activity. A new mechanistic insight has
been proposed based on molecular dynamics simulated
structures, where dynamic flipping of a critical Tyr
residue is responsible for the activity of thermophilic
L-asparaginases. Our study not only resulted in development
of PfA mutants with combination of desirable
properties but also gave a mechanistic insight about
their activity.
Natural coumarinolignoids isolated from the
seeds of Cleome viscosa consist of a racemic mixture
... more Natural coumarinolignoids isolated from the
seeds of Cleome viscosa consist of a racemic mixture
of cleomiscosins A, B and C. To screen out
potential lead, anti-inflammatory activity of the
isolated compounds was evaluated through
molecular docking and QSAR studies by using
reported in vivo activity of Swiss albino mice.
Based on docking binding affinity, a possible
mechanism of action has been hypothesized
which constitute toll-like receptors (TLR-4), cluster
of differentiation molecules (CDs), iNOS, COX-
2 and STAT-6 proteins. It was very interesting to
find that the 3D topology of the active site of
COX-2 from the docking was in good agreement
with QSAR model and in silico ADME⁄T parameters.
A forward feed multiple linear regression
model was developed with r2 = 0.92 and
rCV2 = 0.87. This study showed that chemical descriptors,
for example dipole vector-X, dipole vector-
Y, steric energy, LUMO energy, size of
smallest ring, size of largest ring and carboxyl
group count, correlate reasonably well with
experimental in vivo activity (logLD50). QSAR
study indicates that dipole vector-Y and carboxyl
group count have negative correlation with activity.
Cleomiscosins also showed compliance with
95% of in silico ADME⁄ T properties of available
drugs, e.g. serum protein binding, blood–brain
barrier, CNS activity, HERG K+ channel activity,
apparent Caco-2 permeability, apparent MDCK
permeability, skin permeability and human oral
absorption in GI. Besides, toxicity screening study
suggests that cleomiscosin molecules possess no
toxicity risk parameters. This study offer useful
references for understanding and molecular
design of inhibitors with improved anti-inflammatory
activity.
Immunomodulation is the process of alteration in immune response due to foreign
intrusion of mole... more Immunomodulation is the process of alteration in immune response due to foreign
intrusion of molecules inside the body. Along with the available drugs, a large number of herbal
drugs are promoted in traditional Indian treatments, for their immunomodulating activity. Natural
coumarinolignoids isolated from the seeds of Cleome viscose have been recognized as having
hepatoprotective action and have recently been tested preclinically for their immunomodulatory
activity affecting both cell-mediated and humoral immune response. To explore the immunomodulatory
compound from derivatives of coumarinolignoids, a quantitative structure activity
relationship (QSAR) and molecular docking studies were performed. Theoretical results are in
accord with the in vivo experimental data studied on Swiss albino mice. Immunostimulatory
activity was predicted through QSAR model, developed by forward feed multiple linear regression
method with leave-one-out approach. Relationship correlating measure of QSAR model
was 99% (R2 = 0.99) and predictive accuracy was 96% (RCV2 = 0.96). QSAR studies indicate
that dipole moment, steric energy, amide group count, lambda max (UV-visible), and molar
refractivity correlates well with biological activity, while decrease in dipole moment, steric
energy, and molar refractivity has negative correlation. Docking studies also showed strong
binding affinity to immunomodulatory receptors.
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Papers by Ankit Srivastava
effective therapeutics against this disease is still not available. Almost 20% of familial ALS (fALS) is suggested to be
associated with pathological deposition of superoxide dismutase (SOD1). Evidences suggest that SOD1-containing
pathological inclusions in ALS exhibit amyloid like properties. An effective strategy to combat ALSmay be to inhibit
amyloid formation of SOD1 using small molecules. In the present study, we observed the fibrillation of one of the
premature forms of SOD1 (SOD1 with reduced disulfide) in the presence of curcumin. Using ThT binding assay,
AFM, TEMimages and FTIR,we demonstrate that curcumin inhibits the DTT-induced fibrillation of SOD1 and favors
the formation of smaller and disordered aggregates of SOD1. The enhancement in curcumin fluorescence on the
addition of oligomers and pre-fibrillar aggregates of SOD1 suggests binding of these species to curcumin. Docking
studies indicate that putative binding site of curcumin may be the amyloidogenic regions of SOD1. Further, there
is a significant increase in SOD1 mediated toxicity in the regime of pre-fibrillar and fibrillar aggregates which is
not evident in curcumin containing samples. All these data suggest that curcumin reduces toxicity by binding to
the amyloidogenic regions of the species on the aggregation pathway and blocking the formation of the toxic
species. Nanoparticles of curcumin with higher aqueous solubility show similar aggregation control as that of
curcumin bulk. This suggests a potential role for curcumin in the treatment of ALS.
is attributed to the cytotoxicity caused by amyloidogenic
prefibrillar aggregates, rather than mature fibrils. The presence
of one or more amyloidogenic stretches in different proteins
has been proven critical for initiating fibril formation. In the
present study, we show that two natural compounds, curcumin
and emetine, bind tightly (Kd < 1.6 μM) to the core
amyloidogenic stretch (182−192) of gelsolin (AGel). Binding
happens in different structural orientations, distinctly modulating the amyloidogenic pathway of AGel. While AGel alone undergoes sigmoidal transition to thioflavin T (ThT)-
responsive fibrillar aggregates with clear lag phase, the
presence of curcumin or emetine abolishes the lag phase and produces starkly different, noncytotoxic end products. Atomic
force microscopy revealed that while curcumin augments fibril formation, emetine arrests it at an intermediate aggregated stage with no fibrillar morphology. FTIR spectroscopy, dynamic light scattering, and ANS fluorescence experiments also suggest that these two species are distinct. Curcumin and emetine also differentially affect the preformed amyloids with the former thickening the fibrils and the latter releasing reclusive oligomers. MD simulations further provided mechanistic insights of differential
interaction by the two compounds modulating amyloid formation. The results were also confirmed on the disease-associated amyloidogenic fragment of gelsolin ( fAGel). Thus, our findings suggest that targeting amyloidogenic stretches in proteins could be useful in designing novel molecules against protein misfolding diseases.
proteins are considered to be crucial for assembly and
function. In this report, an exception in which the linker of a
two-domain dimeric l-asparaginase from Pyrococcus furiosus
(PfA) was found to be dispensable is presented. Domains of
this enzyme assembled without the linker into a conjoined
tetrameric form that exhibited higher activity than the parent
enzyme. The global shape and quaternary structure of the
conjoined PfA were also similar to the wild-type PfA, as
observed by their solution scattering profiles and X-ray
crystallographic data. Comparison of the crystal structures of
substrate-bound and unbound enzymes revealed an altogether
new active-site composition and mechanism of action. Thus,
conjoined PfA is presented as a unique enzyme obtained
through noncovalent, linker-less assembly of constituent
domains that is stable enough to function efficiently at
elevated temperatures.
stability and activity at elevated temperatures. However,
they are of limited use in leukemia therapy
because of their low substrate affinity and reduced
activity under physiological conditions. In an attempt to
combine stability with activity at physiological conditions,
3 active-site mutants of Pyrococcus furiosus L-asparaginase
(PfA) were developed. The mutants, specifically
K274E, showed improved enzymatic properties at
physiological conditions as compared to the wild type.
All variants were thermodynamically stable and resistant
to proteolytic digestion. None of the enzymes
displayed glutaminase activity, a highly desirable therapeutic
property. All variants showed higher and significant
killing of human cell lines HL60, MCF7, and K562
as compared to the Escherichia coli L-asparaginase. Our
study revealed that increased substrate accessibility
through the active site loop plays a major role in
determining activity. A new mechanistic insight has
been proposed based on molecular dynamics simulated
structures, where dynamic flipping of a critical Tyr
residue is responsible for the activity of thermophilic
L-asparaginases. Our study not only resulted in development
of PfA mutants with combination of desirable
properties but also gave a mechanistic insight about
their activity.
seeds of Cleome viscosa consist of a racemic mixture
of cleomiscosins A, B and C. To screen out
potential lead, anti-inflammatory activity of the
isolated compounds was evaluated through
molecular docking and QSAR studies by using
reported in vivo activity of Swiss albino mice.
Based on docking binding affinity, a possible
mechanism of action has been hypothesized
which constitute toll-like receptors (TLR-4), cluster
of differentiation molecules (CDs), iNOS, COX-
2 and STAT-6 proteins. It was very interesting to
find that the 3D topology of the active site of
COX-2 from the docking was in good agreement
with QSAR model and in silico ADME⁄T parameters.
A forward feed multiple linear regression
model was developed with r2 = 0.92 and
rCV2 = 0.87. This study showed that chemical descriptors,
for example dipole vector-X, dipole vector-
Y, steric energy, LUMO energy, size of
smallest ring, size of largest ring and carboxyl
group count, correlate reasonably well with
experimental in vivo activity (logLD50). QSAR
study indicates that dipole vector-Y and carboxyl
group count have negative correlation with activity.
Cleomiscosins also showed compliance with
95% of in silico ADME⁄ T properties of available
drugs, e.g. serum protein binding, blood–brain
barrier, CNS activity, HERG K+ channel activity,
apparent Caco-2 permeability, apparent MDCK
permeability, skin permeability and human oral
absorption in GI. Besides, toxicity screening study
suggests that cleomiscosin molecules possess no
toxicity risk parameters. This study offer useful
references for understanding and molecular
design of inhibitors with improved anti-inflammatory
activity.
intrusion of molecules inside the body. Along with the available drugs, a large number of herbal
drugs are promoted in traditional Indian treatments, for their immunomodulating activity. Natural
coumarinolignoids isolated from the seeds of Cleome viscose have been recognized as having
hepatoprotective action and have recently been tested preclinically for their immunomodulatory
activity affecting both cell-mediated and humoral immune response. To explore the immunomodulatory
compound from derivatives of coumarinolignoids, a quantitative structure activity
relationship (QSAR) and molecular docking studies were performed. Theoretical results are in
accord with the in vivo experimental data studied on Swiss albino mice. Immunostimulatory
activity was predicted through QSAR model, developed by forward feed multiple linear regression
method with leave-one-out approach. Relationship correlating measure of QSAR model
was 99% (R2 = 0.99) and predictive accuracy was 96% (RCV2 = 0.96). QSAR studies indicate
that dipole moment, steric energy, amide group count, lambda max (UV-visible), and molar
refractivity correlates well with biological activity, while decrease in dipole moment, steric
energy, and molar refractivity has negative correlation. Docking studies also showed strong
binding affinity to immunomodulatory receptors.
effective therapeutics against this disease is still not available. Almost 20% of familial ALS (fALS) is suggested to be
associated with pathological deposition of superoxide dismutase (SOD1). Evidences suggest that SOD1-containing
pathological inclusions in ALS exhibit amyloid like properties. An effective strategy to combat ALSmay be to inhibit
amyloid formation of SOD1 using small molecules. In the present study, we observed the fibrillation of one of the
premature forms of SOD1 (SOD1 with reduced disulfide) in the presence of curcumin. Using ThT binding assay,
AFM, TEMimages and FTIR,we demonstrate that curcumin inhibits the DTT-induced fibrillation of SOD1 and favors
the formation of smaller and disordered aggregates of SOD1. The enhancement in curcumin fluorescence on the
addition of oligomers and pre-fibrillar aggregates of SOD1 suggests binding of these species to curcumin. Docking
studies indicate that putative binding site of curcumin may be the amyloidogenic regions of SOD1. Further, there
is a significant increase in SOD1 mediated toxicity in the regime of pre-fibrillar and fibrillar aggregates which is
not evident in curcumin containing samples. All these data suggest that curcumin reduces toxicity by binding to
the amyloidogenic regions of the species on the aggregation pathway and blocking the formation of the toxic
species. Nanoparticles of curcumin with higher aqueous solubility show similar aggregation control as that of
curcumin bulk. This suggests a potential role for curcumin in the treatment of ALS.
is attributed to the cytotoxicity caused by amyloidogenic
prefibrillar aggregates, rather than mature fibrils. The presence
of one or more amyloidogenic stretches in different proteins
has been proven critical for initiating fibril formation. In the
present study, we show that two natural compounds, curcumin
and emetine, bind tightly (Kd < 1.6 μM) to the core
amyloidogenic stretch (182−192) of gelsolin (AGel). Binding
happens in different structural orientations, distinctly modulating the amyloidogenic pathway of AGel. While AGel alone undergoes sigmoidal transition to thioflavin T (ThT)-
responsive fibrillar aggregates with clear lag phase, the
presence of curcumin or emetine abolishes the lag phase and produces starkly different, noncytotoxic end products. Atomic
force microscopy revealed that while curcumin augments fibril formation, emetine arrests it at an intermediate aggregated stage with no fibrillar morphology. FTIR spectroscopy, dynamic light scattering, and ANS fluorescence experiments also suggest that these two species are distinct. Curcumin and emetine also differentially affect the preformed amyloids with the former thickening the fibrils and the latter releasing reclusive oligomers. MD simulations further provided mechanistic insights of differential
interaction by the two compounds modulating amyloid formation. The results were also confirmed on the disease-associated amyloidogenic fragment of gelsolin ( fAGel). Thus, our findings suggest that targeting amyloidogenic stretches in proteins could be useful in designing novel molecules against protein misfolding diseases.
proteins are considered to be crucial for assembly and
function. In this report, an exception in which the linker of a
two-domain dimeric l-asparaginase from Pyrococcus furiosus
(PfA) was found to be dispensable is presented. Domains of
this enzyme assembled without the linker into a conjoined
tetrameric form that exhibited higher activity than the parent
enzyme. The global shape and quaternary structure of the
conjoined PfA were also similar to the wild-type PfA, as
observed by their solution scattering profiles and X-ray
crystallographic data. Comparison of the crystal structures of
substrate-bound and unbound enzymes revealed an altogether
new active-site composition and mechanism of action. Thus,
conjoined PfA is presented as a unique enzyme obtained
through noncovalent, linker-less assembly of constituent
domains that is stable enough to function efficiently at
elevated temperatures.
stability and activity at elevated temperatures. However,
they are of limited use in leukemia therapy
because of their low substrate affinity and reduced
activity under physiological conditions. In an attempt to
combine stability with activity at physiological conditions,
3 active-site mutants of Pyrococcus furiosus L-asparaginase
(PfA) were developed. The mutants, specifically
K274E, showed improved enzymatic properties at
physiological conditions as compared to the wild type.
All variants were thermodynamically stable and resistant
to proteolytic digestion. None of the enzymes
displayed glutaminase activity, a highly desirable therapeutic
property. All variants showed higher and significant
killing of human cell lines HL60, MCF7, and K562
as compared to the Escherichia coli L-asparaginase. Our
study revealed that increased substrate accessibility
through the active site loop plays a major role in
determining activity. A new mechanistic insight has
been proposed based on molecular dynamics simulated
structures, where dynamic flipping of a critical Tyr
residue is responsible for the activity of thermophilic
L-asparaginases. Our study not only resulted in development
of PfA mutants with combination of desirable
properties but also gave a mechanistic insight about
their activity.
seeds of Cleome viscosa consist of a racemic mixture
of cleomiscosins A, B and C. To screen out
potential lead, anti-inflammatory activity of the
isolated compounds was evaluated through
molecular docking and QSAR studies by using
reported in vivo activity of Swiss albino mice.
Based on docking binding affinity, a possible
mechanism of action has been hypothesized
which constitute toll-like receptors (TLR-4), cluster
of differentiation molecules (CDs), iNOS, COX-
2 and STAT-6 proteins. It was very interesting to
find that the 3D topology of the active site of
COX-2 from the docking was in good agreement
with QSAR model and in silico ADME⁄T parameters.
A forward feed multiple linear regression
model was developed with r2 = 0.92 and
rCV2 = 0.87. This study showed that chemical descriptors,
for example dipole vector-X, dipole vector-
Y, steric energy, LUMO energy, size of
smallest ring, size of largest ring and carboxyl
group count, correlate reasonably well with
experimental in vivo activity (logLD50). QSAR
study indicates that dipole vector-Y and carboxyl
group count have negative correlation with activity.
Cleomiscosins also showed compliance with
95% of in silico ADME⁄ T properties of available
drugs, e.g. serum protein binding, blood–brain
barrier, CNS activity, HERG K+ channel activity,
apparent Caco-2 permeability, apparent MDCK
permeability, skin permeability and human oral
absorption in GI. Besides, toxicity screening study
suggests that cleomiscosin molecules possess no
toxicity risk parameters. This study offer useful
references for understanding and molecular
design of inhibitors with improved anti-inflammatory
activity.
intrusion of molecules inside the body. Along with the available drugs, a large number of herbal
drugs are promoted in traditional Indian treatments, for their immunomodulating activity. Natural
coumarinolignoids isolated from the seeds of Cleome viscose have been recognized as having
hepatoprotective action and have recently been tested preclinically for their immunomodulatory
activity affecting both cell-mediated and humoral immune response. To explore the immunomodulatory
compound from derivatives of coumarinolignoids, a quantitative structure activity
relationship (QSAR) and molecular docking studies were performed. Theoretical results are in
accord with the in vivo experimental data studied on Swiss albino mice. Immunostimulatory
activity was predicted through QSAR model, developed by forward feed multiple linear regression
method with leave-one-out approach. Relationship correlating measure of QSAR model
was 99% (R2 = 0.99) and predictive accuracy was 96% (RCV2 = 0.96). QSAR studies indicate
that dipole moment, steric energy, amide group count, lambda max (UV-visible), and molar
refractivity correlates well with biological activity, while decrease in dipole moment, steric
energy, and molar refractivity has negative correlation. Docking studies also showed strong
binding affinity to immunomodulatory receptors.