1. The study aimed to synthesize and characterize a novel thiol-based HDAC inhibitor and measure its binding energetics with zinc using isothermal titration calorimetry (ITC).
2. Preliminary ITC experiments with the inhibitor in DMSO-containing buffer showed weak binding to zinc, likely due to the solvent lowering the acidity of the inhibitor's thiol group.
3. ITC experiments using the related compound L-cysteine in Tris buffer were able to determine thermodynamic binding parameters between cysteine and zinc. Further experiments will optimize conditions to accurately measure inhibitor-zinc binding.
A optimized process for the synthesis of a key starting material for etodolac...IOSR Journals
Abstract An optimized process developed for the synthesis of 7-ethyltryptophol, a key starting material for etodolac, a non steroidal anti- inflammatory drug. Starting from commercially available 2-ethylphenylhydrazine. HCl and dihydro furan with con. H2SO4 as a catalyst in N, N- dimethyl acetamide ( DMAc). H2O (1:1) as a solvent in 75% yield . the method is easy, inexpensive , without purification getting pure solid. The process is very clean, high yielding & high quality and operationally simple.
Keywords: Etodolac, 7-ethyl tryptophol, 2-ethyl phenyl hydrazine hydrochloride, N,N-dimethyl acetamide.
A optimized process for the synthesis of a key starting material for etodolac...IOSR Journals
Abstract An optimized process developed for the synthesis of 7-ethyltryptophol, a key starting material for etodolac, a non steroidal anti- inflammatory drug. Starting from commercially available 2-ethylphenylhydrazine. HCl and dihydro furan with con. H2SO4 as a catalyst in N, N- dimethyl acetamide ( DMAc). H2O (1:1) as a solvent in 75% yield . the method is easy, inexpensive , without purification getting pure solid. The process is very clean, high yielding & high quality and operationally simple.
Keywords: Etodolac, 7-ethyl tryptophol, 2-ethyl phenyl hydrazine hydrochloride, N,N-dimethyl acetamide.
Recent Structure Activity Relationship Studies of 1,4-Benzodiazepinespeertechzpublication
Structure activity relationship studies of 1,4-benzodiazepines have been discussed especially
with their effects as antianxiety and anticonvulsants. The currently available benzodiazepines are
associated with various side effects. Nowadays the purpose of these studies is to minimize side effects
with these drugs. A very little alteration is possible on the benzene ring while the modification can be
done on the diazepine ring. It can adopt the different conformations and in some cases some aromatic
and heterocyclic rings have been fused with this part in order to see the effect of these conformation
blockers on the pharmacological activity. The structure activity studies are also linked to molecular
modeling studies. This is important in adding some information for the interaction of these drugs with
the receptors and how this interaction can be improved.
Stability studies ensuring the maintenance of product quality, safety and efficacy throughout the shelf life are considered as pre-requisite for the acceptance and approval of any pharmaceutical product. Stability testing is a routine procedure performed on drug substances and products and is employed at various stages of the product development.
Digital Story Literacy Development 6706LSchloesser
Story Content:
1. Getting to Know Literacy Learners (Slide #3-5)
II. Selecting Texts (Slide #6-7)
III. Emergent Literacy Learner Lesson (Slide #8-9)
IV. Beginning Literacy Learner Lesson (Slide #10-11)
V. Reflection (Slide #12)
VI. Insight Gained (Slide #13)
VII. Tell your digital story (Slide #14)
VIII. Reference (Slide #15-16)
Recent Structure Activity Relationship Studies of 1,4-Benzodiazepinespeertechzpublication
Structure activity relationship studies of 1,4-benzodiazepines have been discussed especially
with their effects as antianxiety and anticonvulsants. The currently available benzodiazepines are
associated with various side effects. Nowadays the purpose of these studies is to minimize side effects
with these drugs. A very little alteration is possible on the benzene ring while the modification can be
done on the diazepine ring. It can adopt the different conformations and in some cases some aromatic
and heterocyclic rings have been fused with this part in order to see the effect of these conformation
blockers on the pharmacological activity. The structure activity studies are also linked to molecular
modeling studies. This is important in adding some information for the interaction of these drugs with
the receptors and how this interaction can be improved.
Stability studies ensuring the maintenance of product quality, safety and efficacy throughout the shelf life are considered as pre-requisite for the acceptance and approval of any pharmaceutical product. Stability testing is a routine procedure performed on drug substances and products and is employed at various stages of the product development.
Digital Story Literacy Development 6706LSchloesser
Story Content:
1. Getting to Know Literacy Learners (Slide #3-5)
II. Selecting Texts (Slide #6-7)
III. Emergent Literacy Learner Lesson (Slide #8-9)
IV. Beginning Literacy Learner Lesson (Slide #10-11)
V. Reflection (Slide #12)
VI. Insight Gained (Slide #13)
VII. Tell your digital story (Slide #14)
VIII. Reference (Slide #15-16)
Determination of DNA Methylation Using Electrochemiluminescenc.docxkhenry4
Determination of DNA Methylation Using Electrochemiluminescence
with Surface Accumulable Coreactant
Ryoji Kurita,*,† Kumi Arai,† Kohei Nakamoto,†,‡ Dai Kato,† and Osamu Niwa†,‡
†National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki,
Japan 305-8566
‡Graduate School of Pure and Applied Science, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki, Japan 305-8573
ABSTRACT: Cytosine methylation in DNA was determined by
an enzyme linked immunosorbent assay (ELISA) with electro-
chemiluminescence (ECL) detection and employed for the DNA
methylation assay of a long and real genomic sample for the first
time. The developed method employed an antimethyl cytosine
antibody labeled with acetylcholinesterase, which was added to
recognize single methylated cytosine in a DNA oligomer. The
acetylcholinesterase converted acetylthiocholine (substrate) to
thiocholine (product), which was accumulated on a gold electrode
surface via gold−thiol binding. This surface accumulated
preconcentration made it possible to observe bright and distinctive
ECL by applying a potential to the gold electrode in the presence
of a tris(2,2-bipyridyl)ruthenium complex luminophore when the
analyte DNA contained a methylation region. Methyl-cytosine was measured quantitatively in the 1−100 pmol range, which
exhibits sufficiently high sensitivity to achieve real DNA measurements without amplification by a polymerase chain reaction
(PCR). The proposed ECL method also exhibited high selectivity for methyl-cytosine against nonmethylated cytosine, guanine,
thymine, and adenine nucleotides. Finally, original and methylated DNA samples were clearly distinguished with our method
using a real DNA bacteriophage sample (48 502 base pairs).
DNA methylation is a well-known epigenetic modificationmechanism that regulates gene expression and plays
crucial roles in embryonic development.1 Cytosine methylation
in CpG islands has received particular attention because it is
thought to be involved in controlling genetic expression,
including that in cancer,2 genomic imprinting,3 cellular
differentiation, and Alzheimer’s disease.4 5-Methyl-cytosine is
now recognized as the fifth DNA base containing heritable
information. Therefore, highly sensitive, accurate, and quanti-
tative information concerning cytosine methylation in DNA
would be valuable with respect to genetic disease diagnosis.
Two major cytosine methylation assay methods have been
reported. One is hydrolysis and sequencing with a bisulfite
salt,5,6 and the other is a cleavage assay with methyl-cytosine
sensitive (or insensitive) restriction enzymes.7 A bisulfite based
determination method is very widely used to distinguish
between cytosine and methyl-cytosine. Treatment with bisulfite
converts cytosine to uracil, while methyl-cytosine remains
unaffected. Therefore, information about methyl-cytosine in
DNA can be obtained by combining bisulfite treatment and a
polymer.
Exploring the protein stabilizing capability of surfactants against agitation...Merck Life Sciences
Agitation of therapeutic protein solutions during manufacturing, shipping and handling is one of the major initiators for protein aggregation and particle formation during the life history of a protein drug. Adsorption of protein molecules to liquid-air interfaces leads to the formation of highly concentrated protein surface films. The rupture of these protein films due to various mechanical processes can then result in the appearance of protein aggregates and particles in the bulk solution phase.
One technique to stabilize proteins against stress induced by liquid-air interfaces is the use of non-ionic surfactants. About 91% of antibody formulations commercially available in 2021 contained a surfactant. Polysorbate 20 and 80, composed of a hydrophilic polyoxyethylene sorbitan and hydrophobic fatty acid esters, made up the largest part being employed in 87% of said formulations.
Despite their frequent use in parenteral drug products, concerns have been raised for decades about the application of polysorbates as surfactants in biopharmaceutical formulations. Autoxidation of polysorbate, caused by residual peroxides in polysorbates, can damage the proteins and can further drive the oxidative degradation of polysorbate. Chemical and enzymatic hydrolysis of polysorbate may lead to the formation of free fatty acid particles, which may become visible; and both mechanisms eventually lead to the reduction in polysorbate concentration. Therefore, the purpose of the current study was to compare various molecules for their capabilities to reduced agitation-induced protein aggregation and particle formation; and furthermore, investigate their underlying protein stabilizing mechanisms.
Chemistry Central Journal 12 (1) jun 2018
DOI: 10.1186/s13065-018-0443-0
License CC BY 4.0
Purpose: Free radicals are considered as the causative agents of a variety of acute and chronic pathologies. Natural antioxidants have drawn attention of the researchers in recent years for their ability to scavenge free radicals with minimal or even no side effects. This study evaluates the antioxidant capacity of agathisflavone, a naturally occurring biflavonoid by a number of in vitro methods. Methods: Agathisflavone was subjected to DPPH, ABTS, OH and NO radical scavenging assay, reducing potential and inhibition of lipid peroxidation (TBARS) test using trolox as a standard. Results: Agathisflavone showed concentration-dependent antioxidant activity against all types of free radicals used in this study. The antioxidant capacity, reducing potential and inhibition of lipid peroxidation showed by agathisflavone were comparable to that of trolox. Conclusion: Agathisflavone exhibited antioxidant capacity, which suggests considering this biflavonoid for the use in the prevention and/or treatment of diseases precipitated by oxidative stress.
Exploring the protein stabilizing capability of surfactants against agitation...MilliporeSigma
Agitation of therapeutic protein solutions during manufacturing, shipping and handling is one of the major initiators for protein aggregation and particle formation during the life history of a protein drug. Adsorption of protein molecules to liquid-air interfaces leads to the formation of highly concentrated protein surface films. The rupture of these protein films due to various mechanical processes can then result in the appearance of protein aggregates and particles in the bulk solution phase.
One technique to stabilize proteins against stress induced by liquid-air interfaces is the use of non-ionic surfactants. About 91% of antibody formulations commercially available in 2021 contained a surfactant. Polysorbate 20 and 80, composed of a hydrophilic polyoxyethylene sorbitan and hydrophobic fatty acid esters, made up the largest part being employed in 87% of said formulations.
Despite their frequent use in parenteral drug products, concerns have been raised for decades about the application of polysorbates as surfactants in biopharmaceutical formulations. Autoxidation of polysorbate, caused by residual peroxides in polysorbates, can damage the proteins and can further drive the oxidative degradation of polysorbate. Chemical and enzymatic hydrolysis of polysorbate may lead to the formation of free fatty acid particles, which may become visible; and both mechanisms eventually lead to the reduction in polysorbate concentration. Therefore, the purpose of the current study was to compare various molecules for their capabilities to reduced agitation-induced protein aggregation and particle formation; and furthermore, investigate their underlying protein stabilizing mechanisms.
Cancer Epigenetics: Concepts, Challenges and PromisesMrinmoy Pal
The presentation highlights how recent investigations have shown extensive reprogramming of almost every component of the epigenetic machinery in cancer leading to the emergence of the promising field of epigenetic therapy.
1. HDAC6
Synthesis and Characterization of a
Thiol-Based HDAC Inhibitor
Sarah Lopez, Shrasta Tamrakar, Youya Gao, Lihua Jin* and Caitlin E. Karver*
Department of Chemistry, DePaul University, Chicago IL, 60614
DePaul University Department of Chemistry
Acknowledgments
Method
Background
Summary and Conclusions
Abstract
References
Histone deacetylase (HDAC) is an enzyme involved in histone modification resulting in changes in gene
expression. All NAD+-independent HDACs deacetylate lysine residues on histones and contain a catalytic
zinc ion in their active sites. HDAC inhibitors are appealing anticancer agents because they hinder the
formation of tumors, prevent cell proliferation, and induce terminal differentiation of tumor cells. However,
they have been problematic for their off-target effects as well as poor bioavailability. Thiol-based HDAC
inhibitors are potent metabolically stable small molecules which may be able to combat some of these
issues. The thiol moiety of these inhibitors functions by coordinating zinc ions preventing it from initiating
catalysis. In this work 7-mercapto-N-(4-phenyl-2-thiazolyl)hexanamide was synthesized, purified and
characterized. Its binding energetics with zinc were analyzed by isothermal titration calorimetry (ITC).
Preliminary ITC data indicate complications due to sulfhydryl group oxidation in solution which is
mitigated by zinc chelation. Namely, we see much more oxidation occurring in control runs where the
HDAC inhibitor is titrated into a buffer without zinc. Efforts are underway to select solution conditions that
minimize oxidation, thus, increase accuracy of resulting binding parameters. In summary a novel HDAC
inhibitor has been synthesized. Through investigating binding energetics with zinc, information is obtained
regarding the significance of metal chelation on HDAC inhibition, furthering the development of antitumor
agents.
•Histone acetyl transferase (HAT) relaxes
chromatin; histone deacetylases (HDACs), in
contrast, changes chromatin into a closed
phase.
•Cancerous cells have histone
hypoacetylation. Furthermore, HDACs
promote cancerous cells by preventing the
expression of certain genes.
•This work aims to develop the need for a
variety of compounds to target HDAC as
well as measure binding thermodynamics of
a synthesized HDAC inhibitor.
Figure 1. HDACs role in cancer (K. Garber,
Nature Biotechnology, 2004. 22: p. 364.)
Cancer
•11 out of the 18 HDACS in humans are metal
dependent (typically Zn2+).
•Here a focus is on HDAC6.Consequently,
treatment is directed toward a specific disease,
minimizing the side effects and increasing
potency.
Figure 2. HDAC tetrahedral transition state
Synthesis
Results
ITC is commonly used to study the interaction between two molecules or ions. ITC is capable of
providing a complete thermodynamic profile including stoichiometry (n), binding constant (Ka) (thus,
Gibbs free energy change, ΔG°), enthalpy change (ΔH°) and entropy change (ΔS°) values. During an
ITC experiment, one ligand is titrated in small aliquots into a sample cell containing the other ligand
in a controlled fashion with the use of a motor-operated syringe. ITC studies were performed on a
microcalorimeter VP-ITC instrument (MicroCal Inc.). All solutions were prepared gravimetrically in
buffer or a DMSO:buffer mixture. The buffer consisted of 50 mM Tris and 0.10 M NaCl at pH 7.4
and the DMSO:buffer mixture was a 80:20 by volume mixture with buffer being 50 mM MES, 0.10
M NaCl, pH 6.0.
Figure 3. ITC
Results
•To conserve the thiol product, L-cysteine
was used first to determine suitable
solution conditions.
•In addition, control runs for titrant into
buffer were completed.
•Table 1 values reflect an average of three
independent trials.
1. Suzuki, T., A. Kouketsu, A. Matsuura, A. Kohara, S. Ninomiya, K. Kohdaa, and N.
Miyataa, Thiol-based SAHA analogues as potent histone deacetylase inhibitors Bioorg.
Med. Chem. Lett., 2004. 14: p. 3313-3317.
2. Chekmeneva, E., R. Prohens, J. M.Díaz-Cruz, C. Ariño, and M. Esteban,
Thermodynamics of Cd2+ and Zn2+ binding by the phytochelatin(γ-Glu-Cys)4-Gly and its
precursor glutathione Analytical Biochemistry, 2008. 375: p. 82–89.
3. Glozak, M.A., and E. Seto, Histone deacetylases and cancer Oncogene, 2007. 26: p.
5420–5432.
Figure 5. Raw data and binding
isotherm for 10 mM thiol product
titration in 50 mM MES, 0.10 M
NaCl, pH 6.0 (20% by v), DMSO
(80%) into buffer
n(Zn2+/Cys) Ka (M-1) ΔG° (kcal mol-1) Kd (µM) ΔH° (kcal mol-1) TΔS°(kcal mol-1)
0.204 ± 0.008 1.7 ± 0.3 × 105 -7.1 ± 0.1 6 ± 1 -78 ± 1 -48 ± 2
Table 1. Thermodynamic parameters for Zn2+ binding to L-cysteine
Figure 4. Raw data and binding isotherm for 0.6 mM Zn2+ solution
titration into 0.2 mM cysteine in 50 mM Tris, 0.10 M NaCl buffer, pH 7.4
1.We determined the best solution conditions for the thiol product as 80:20 (% by
volume) DMSO:buffer with the buffer being 50 mM MES, 0.10 M NaCl, pH 6.0.
2. A large control heat implies that the majority of heat is associated with dilution in this
solvent mixture. Furthermore, experiments with Zn2+ titration into the thiol product
indicates weak binding affinity.
3. A related–SH containing compound, cys, was more soluble allowing progress for
optimization in Tris buffer.
•Binding curves in the DMSO:buffer mixture for the interaction of Zn2+ and the thiol
product was far from complete and has a small magnitude of heat. Hence, the binding
affinity was too weak to be determined using ITC. This may be explained by the main
solvent in the mixture, i.e., DMSO, being a much weaker base than water, greatly
lowering the acidity of the –SH group thus its ability to coordinate metal ions.
•Success in using 50 mM Tris, 0.10 M NaCl, pH 7.4 to study L-cysteine thermodynamic
parameters.