Molecules with: O -acetyl group protect protein glycation by acetylating lysine residues

Article abstract of DOI:10.1039/c6ra11313c

Pharmaceutical intervention for reduction of advanced glycation end products (AGEs) is considered as a therapeutic strategy to attenuate the pathogenesis of diabetes. Many molecules have been reported to possess antiglycation activity, one such example is acetylsalicylic acid (aspirin). It protects proteins from glycation by acetylating the lysine residues. Therefore, in this study we have synthesized and screened molecules containing free N-acetyl, O-acetyl and acetophenone groups. All the selected molecules in this study showed glycation inhibition but interestingly, only molecules with O-acetyl but not N-acetyl and acetophenone groups were capable of acetylating lysine residue. Furthermore, we have demonstrated that pre-acetylation or aspirin treatment prior to the induction of diabetes helps in reducing HbA1c and AGE formation in the streptozotocin induced diabetic mice. Hence pre-acetylation may have an additional therapeutic efficacy of reducing AGE levels in vivo. Incorporation of O-acetyl group into anti-diabetic molecules could be a useful strategy, as it may have an additive effect in reducing AGEs. Identification of such novel acetylating agents represents a new area in the drug discovery process.

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Molecules with O-acetyl group protect protein glycation by
acetylating lysine residues
Garikapati Vannuruswamy# 1, Mashanipalya G. Jagadeeshaprasad#1, 2, K. Kashinath#2, 3 , Suresh K
Kesavan1, Shweta Bhat1, Arvind M. Korwar1, Ashok D. Chougale1, Ramanamurthy Boppana4, D.
Srinivasa Reddy*2, 3, and Mahesh J. Kulkarni*1, 2
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Pharmaceutical intervention for reduction of advanced glycation products (AGEs) is considered as a therapeutic strategy to
attenuate the pathogenesis of diabetes. Many molecules have been reported to possess antiglycation activity, one such
example is acetylsalicylic acid (Aspirin). It protects proteins from glycation by acetylating the lysine residues. Therefore, in
this study we have synthesized and screened molecules containing free N- , O-acetyl and acetophenone group. All the
selected molecules in this study showed glycation inhibition but interestingly, only molecules with O-acetyl but not N-
acetyl and acetophenone group were capable of acetylating lysine residue . Furthermore, we have demonstrated that pre-
acetylation or aspirin treatment prior to the induction of diabetes helps in reducing HbA1c and AGE formation in the
streptozotocin induced diabetic mice. Hence pre-acetylation may have an additional therapeutic efficacy of reducing AGE
levels in-vivo. Incorporation of O-acetyl group into anti-diabetic molecules could be an useful strategy, as it may have an
additive effect in reducing AGEs. Identification of such novel acetylating agents represents a new area in the drug
discovery process.
makes it a promising target for therapeutics. Hence, reducing
AGE levels by both chemical and cellular approaches has been
proposed as an intervention strategy to treat glycation
Introduction
Hyperglycemic condition in diabetes promotes formation of
advanced glycation end products (AGEs), which are implicated
in pathogenesis of diabetic complications including vascular
stiffening1, atherosclerosis2, cataracts3, and age associated
disorders including Parkinson’s disease4, Alzheimer's disease5.
Mounting evidence of involvement of AGEs in various diseases
associated diseases6. Glycation can be intervened at various
strategies viz.(i) Schiff's base formation (ii) deglycation (iii)
transglycation (iv) Amadori product inhibition (v) crosslink
inhibitors (vi) crosslink breakers (vii) RAGE (Receptor for AGEs)
blockers etc. Apart from these strategies, formation of AGEs
can also be prevented by blocking glycation sites, which can be
achieved by modification of amino acid side chains resulting in
such as acetylation, hydroxylation, methylation, formylation
etc. Some of the preexisting FDA approved drugs possess anti
glycation activity including aspirin, which is largely used for its
anti-inflammatory, antipyretic, analgesic and anti-thrombotic
effects. Aspirin in vitro readily acetylates crystallin, a lens
protein, and protects it from glycation by galactose,
glucosamine and glucose7, 8, 9. In vivo studies with aspirin fed
diabetic rats also showed decrease in crystallin glycation and
HbA1c9. Further, lysine acetylation contributes to protect the
biological function of crystallin even in presence of glycation10.
In another in vivo study aspirin prevented inactivation of heme
pathway enzymes δ-aminolevulinic dehydratase and
porphobilinogen deaminase by glycation that would aid in
preventing diabetic complications11. Therefore, implication of
lysine acetylation in various biological processes and complex
association with cellular metabolism offers lot of clinical
relevance. Recently, acetylation and glycation prone sites on
HSA and other plasma proteins were characterized and
quantified for better understanding of their interplay and
1 Mass-spectrometry and proteomics facility, Division of Biochemical Sciences,
CSIR-National Chemical Laboratory, Pune-411008, India.
2Academy of Scientific and Innovative Research (AcSIR), New Delhi, India.
3Organic Chemistry Division, CSIR-National Chemical Laboratory, Pune-411008,
India.
4National Center for Cell Science, Pune-411007, India.
#Equally contributed
*Corresponding authors D. Srinivasa Reddy, PhD
Division of Organic Chemistry
CSIR-National Chemical Laboratory,
Pune-411008, India.
Tel: +91 20 2590 2445
E-mail: ds.reddy@ncl.res.in
Mahesh J. Kulkarni, PhD
Mass spectrometry and Proteomics Facility, Division of Biochemical Sciences,
CSIR-National Chemical Laboratory,
Pune-411008, India.
Tel: +91 20 25902541, Fax: +91 20 25902648.
E-Mail: mj.kulkarni@ncl.res.in.
† Footnotes relaꢀng to the ꢀtle and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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reciprocal competition12, 13. Aspirin mediated acetylation and significant decrease in glycation extent upon incubation with
protein glycation influence each other through a dynamic ASP, BIS and NDS-458 (Figure 2B and Supplementary Figure 5).
equilibrium between acetylation and glycation13. Therefore,
A
acetylation plays a key role in regulation of protein glycation
and molecules capable of acetylating could be useful in
protection against glycation. Hence, in this study, we report
the screening of acetyl group containing molecules for anti-
glycation activity both in vitro and in vivo. Interestingly
molecules having O-acetyl group (such as aspirin, bisacodyl
and NDS-458) are capable to acetylate the protein thereby
decreasing the glycation than N-acetyl and acetophenone
group. In addition, we have shown both in-vitro and in-vivo
that pre-acetylation protects proteins from glycation.
Results and discussion
B
Screening and evaluation of acetyl group molecules for
glycation inhibition
AGE-fluorescence assay
Molecules with acetyl group (Figure 1) were evaluated for their
anti-glycation activity by using BSA-AGE-fluorescence assay
(Excitation 370/Emission 440 nm)14. The results indicate that
Figure 2: (A) Anti-glycation activity by various molecules
containing acetyl group as analyzed by BSA-AGE-fluorescence
all the molecules showed decrease in the relative fluorescence
assay (Excitation 370/Emission 440 nm) (n=3±SE). (B) Western
intensity in comparison with glycated BSA (Figure 2A).
blot analysis utilizing anti-AGE and anti-CML antibodies of 1)
However, aspirin and NDS458 showed strong glycation
Control BSA, 2) Glycated BSA, 3) Glycated BSA with ASP, 4)
inhibition (> 60% with p<0.05).
Glycated BSA with BIS and 5) Glycated BSA with NDS-458.
Insulin glycation assay
Insulin-MALDI based assay to screen the molecules for
glycation inhibition was performed as described earlier14.
Insulin (5808 m/z) upon glycation with glucose (162 m/z) form
glycated Insulin (5970 m/z). The intensity of glycated insulin
was monitored in presence of various acetyl group containing
molecules [ASP, BIS, NDS-458, PAR, ACZ, ACH and NAC] to
study the glycation inhibition activity. The results indicate that
all the molecules showed decrease in the relative intensity of
Figure 1: The molecular structure of acetyl group containing
glycated insulin in comparison with control (Figure 3A). ASP,
BIS and NDS-458 showed strong glycation inhibition (> 70%
with p<0.05).
molecules
Western blot analysis
A qualitative and quantitative result of glycation inhibition by
acetyl group containing molecules was also confirmed by
western blot analysis utilizing anti-AGE and anti-CML
antibodies. The analysis was performed to determine extent of
BSA glycation both with and without incubation with acetyl
group containing molecules. The results showed a clearly
visible difference optical density of glycated BSA reflecting
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12,9,11,15,15,13,14 respectively as shown in Figure 3B, details
of the modified peptides are listed in (Supplementary Table 1).
A representative MS/MS annotation of Amadori modified
peptide of BSA is depicted in (Supplementary Figure 3). Taken
together all the results (qualitative, quantitative and western
blot) strongly support the glycation inhibition of acetyl group
containing molecules.
A
Protein acetylation with acetyl group containing molecules
Protein acetylation assay
Insulin [m/z 5808] was used as a model protein to monitor the
protein acetylation with acetyl group containing molecules.
Formation of acetylated insulin [m/z 5850], which displays a
characteristic mass shift of 42 Da corresponding to acetylation
was monitored. Only three molecules ASP, BIS and NDS-458
were found to be acetylating the insulin as observed by
formation of acetylated insulin peak, while remaining
molecules could not cause acetylation of insulin (Figure 4A).
This was also reflected in BSA acetylation as analyzed by
western blotting with anti-acetylation antibody (Fig 4B and
Supplementary Figure 6). The results showed clearly that, ASP,
BIS and NDS-458 induced BSA lysine acetylation, amongst
these three molecules, NDS-458 showed high intense band
suggesting it strong acetylating capabilities (Figure 4B).
B
A
Figure 3: (A) Insulin-MALDI based assay to screen the
molecules for glycation inhibition; the relative intensity of
glycated insulin was monitored in presence of various acetyl
group containing molecules. (B) Bar graph depicting the
number of glycated modified peptides of in vitro glycated BSA,
glycated BSA in presence of various acetyl group containing
molecules.
LC MS/MS analysis of glycated BSA
Qualitative analysis of glycated BSA was complemented by
quantitative LC-MS/MS analysis. The extent of BSA glycation
inhibition by acetyl group containing molecules was measured
as increase or decrease of glycation modified sites/peptides.
This approach facilitated identification of the 21 Amadori
modified peptides in glycated BSA. Whereas pre-glycated BSA
incubated with acetyl group containing molecules showed
decrease in the glycation. ASP, BIS, NDS-458, PAR, ACZ, ACH
and NAC showed decrease in glycated peptides by
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B
A
Figure 4: A) MALDI-TOF-MS based insulin acetylation inhibition
assay with insulin in presence of various acetyl group
containing molecules. B) Western blot analysis utilizing anti-
Acetyl lysine antibody 1) BSA, 2) BSA with ASP,3) BSA with BIS,
4) BSA with NDS-458, 5) BSA with Paracetamol, 6) BSA with
Acetazolamide, 7) BSA with Acetohexamide and 8) BSA with N-
Acetyl Cysteine.
LC MS/MS analysis of acetylated BSA
BSA acetylation was also studied by LC-MS/MS analysis. The
site specific protein acetylation was monitored by using Q-
Exactive MS. The extent of BSA acetylation by acetyl group
containing molecules was measured as potential acetylating
compound. ASP, BIS, and NDS-458 showed 43, 43, and 58
acetyl modified peptides of BSA respectively as listed in
(Supplementary Table 2, Supplementary Figure 7). Whereas
PAR, ACZ, ACH and NAC did not showed any acetyl modified
BSA peptides.
A representative MS/MS annotation of
acetylated BSA peptide is shown in Supplementary Figure 4.
NDS-458 molecule was synthesized by acetylating the hydroxyl
group present in paracetamol. Taken together all the results
(qualitative, quantitative and western blot) strongly support
that O-acetyl group containing molecules (ASP, BIS, NDS-458)
acetylates the lysine residues of protein rather than N-acetyl
(PAR, ACZ, NAC) or acetophenone molecules (ACH).
Figure 5: (A) In vitro MALDI-TOF-MS based glycation assay in
presence of pre-acetylation insulin with glucose. (B) SDS-PAGE
analysis depicting resistance of pre-acetylated BSA to glycation
of 1) Control BSA, 2) Glycated BSA, 3) Aspirin treated BSA, 4)
Glucose treated with aspirin to BSA and 5) Pre-aspirin treated
BSA then Glucose.
Pre-acetylation protein assay
Pre-acetylated insulin and pre-acetylated BSA were used as
model proteins to examine their ability to resist glycation. Pre-
acetylated insulin showed little glycation as assessed by
MALDI-MS for the presence glycated peak (m/z 5970)
corresponding to increase in mass shift 162 Da. However,
incorporation of aspirin during glycation showed lesser
resistance to glycation as there was formation of glycated
insulin (Fig 5A). Such results were also observed with pre-
acetylated BSA, which showed more resistance to glycation as
observed by lesser formation for glycated BSA products on
SDS-PAGE (Fig 5B). These results suggest that pre-acetylation
is a better strategy in prevention of glycation.
In-vivo protective effect of pre-acetylation against glycation
STZ induced diabetic mice models were utilized to
demonstrate the protective effect of pre-acetylation against
protein glycation. Pre-acetylation of proteins was attained by
oral feeding of aspirin for a month prior to STZ injection. Mice
models chosen for experiments were divided into five groups:
Group (i) healthy control; (ii) STZ induced diabetic; (iii) healthy
control pre-treated with aspirin; (iv) STZ induced diabetic and
aspirin treated and (v) aspirin pre-treated STZ induced
diabetic. Acetylation was monitored by western-blotting with
anti-acetylation antibody. Increased acetylation was observed
in mice of both aspirin pre-treatment and post treatment of
diabetes induction. This was accompanied by reduced AGE
modified serum albumin as measured by western blotting with
anti-CML antibody (Figure 6A, Supplementary Figure 8). This
result was also reflected in HbA1c levels. (Figure 6B).
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mL). The organic layer was dried over NaSO4, concentrated
under reduced pressure. The residue was purified by column
chromatography (silica gel 100-200 mesh, MeOH-CH2Cl2 5:95)
A
to afford 4-acetamidophenylacetate (1.23 g, 97%) as
1
a
colorless solid. NDS 458 was characterized by H NMR and 13C
NMR.
1H NMR (400 MHz, CDCl3) (Supplementary Figure.1 (S1)): δ
7.71 (brs, 1H), 7.53 - 7.41 (m, J = 8.8 Hz, 2H), 7.09 - 6.91 (m, J =
9.0 Hz, 2H), 2.28 (s, 3H), 2.12 (s, 3H); 13C NMR (100 MHz,
CDCl3) (Supplementary Figure.2 (S2)): δ 169.9, 168.6, 146.7,
135.7, 121.9, 120.9, 24.4, 21.1. NMR values (1H &13C) were
compared with reported values and found to be identical18.
B
In vitro glycation of bovine serum albumin (BSA) and AGE
fluorescence assay
The acetyl group containing molecules including aspirin (ASP),
bisacodyl (BIS), NDS-458, paracetamol (PAR), acetazolamide
(ACZ), N-acetyl cysteine (NAC) and acetohexamide (ACH) were
selected for the screening of their in vitro glycation inhibition
studies. 100 mM stock solutions of above mentioned
molecules were prepared in 10% DMSO of 0.1M Phosphate
buffer solution (PBS) (pH 7.4). 10% DMSO in 0.1M PBS was
used as a solvent control.
Figure 6: (A) Western blot analysis utilizing anti-Acetyl lysine
and anti-CML antibodies in in-vivo of plasma proteins B) Bar
graph depicting the HbA1c (n=6±SE) of 1) Control mice, 2)
Diabetic mice,3) Aspirin treated control mice, 4) Diabetic mice
treated with aspirin and 5) Pre-aspirin treated then STZ
induced diabetic mice.
In-vitro glycation of BSA was performed as described earlier19.
Briefly, 450 µl of BSA (50 mg mL-1) in 0.1 M phosphate buffer
(pH 7.4) containing 225µl of D-glucose (2M), 5 mM sodium
azide with or without 10mM of all the above mentioned
molecules were incubated at 37 °C for 15 days. The exact
concentration of the Bisacodyl in the final reaction was
relatively lesser than other drugs (< 10mM) because of its
solubility issue. BSA glycation was monitored by fluorescence
Experimental
Chemicals
All the chemicals were procured from Sigma-Aldrich (Sigma- spectroscopy, excitation at 370 and emission at 440 nm by
Aldrich. MO, USA) unless otherwise are mentioned. Anti-AGE using Varioskan Flash Multimode reader (Thermo Fisher
antibody, protein A-HRP conjugate were purchased from Scientific, Germany).
Merck Millipore (Merck Millipore.MA, USA). Anti-CML
antibody was procured from Merck Abcam (Abcam.
Western blot analysis by using anti-AGE, anti-CML and anti-
Cambridge, UK). Anti-Acetylated Lysine was procured from
acetylated lysine antibodies
Calbiochem (Calbiochem. CA, USA). MS grade water,
acetonitrile (ACN), methanol (MeoH) and were procured from
J.T. Baker (J.T. Baker.PA, USA).
5 µg of Control BSA, glycated BSA and glycated BSA incubated
with 10mM concentration acetyl group containing molecules
[ASP, BIS, NDS-458, PAR, ACZ, ACH and NAC] for 15 days at 37
Synthesis and characterization of 4-Acetamidophenyl acetate
°C were resolved onto 12% SDS-PAGE and transferred onto the
(NDS-458)
PVDF membrane. The membranes were blocked with 5%
skimmed milk. The proteins were probed by anti-AGE, anti-
To a solution of N-(4-hydroxyphenyl) acetamide (1.0 g, 6.6 CML and anti-acetylated lysine antibodies followed by
mmol) and Et3N (1.1 mL, 7.9 mmol) in anhydrous CH2Cl2 (20 incubation with protein A-HRP conjugate. Immunoreactive
mL), acetyl chloride (0.62 mL, 7.9 mmol) was added drop wise bands were visualized by using Western Bright ECL HRP
at 0° C and stirred for 2 h at the same temperature. The substrate (Advansta. CA, USA) and documented by using gel
reaction mixture was diluted with CH2Cl2 (10 mL), washed with doc (Syngene Cambridge, UK).
saturated NaHCO3 solution (5 mL), water (5 mL) and brine (5
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Insulin glycation inhibition assay
MS acquisition: All samples were analyzed on Q-Exactive
Orbitrap MS (Thermo Fisher Scientific, Germany). The
instrument was tuned and calibrated for better performance.
The tune parameters include: spray voltage 4200V, capillary
temperature 320 °C, heater temperature 200 °C, S-lens RF
value 55, sheath and auxiliary gases pressure were 30 and 8 psi
respectively. The samples were acquired in positive ionization
(HESI) mode in data-dependent manner using a top-5 method
with scan range from 350 -1800 m/z. MS spectra were
acquired at a resolution of 70,000 with maximum injection
time (IT) of 120 ms and automatic gain control (AGC) value of 1
× e6 ions and MS/MS spectra were acquired at 17,500
resolution with maximum IT of 120ms and AGC value of 1 × e5
ions. Precursor’s selectivity was performed at isolation width
of 2 m/z, under fill ratio of 0.3% and dynamic exclusion time of
15 sec. The peptide fragmentation was performed using
normalized high energy collision induced dissociation (30 eV)
The in vitro insulin glycation inhibition efficiency of acetyl
group containing molecules [ASP, BIS, NDS-458, PAR, ACZ, ACH
and NAC] was performed by using MALDI based insulin
glycation inhibition assay. Briefly, 50µl of insulin (1.8 mg mL-1)
in 200µl of 0.1 M phosphate buffer (pH 7.4) containing 50µl of
glucose (250mM), 5 mM sodium azide with and without 10mM
concentration of all above mentioned acetyl group containing
drugs were incubated for 7 days at 37 °C. The reaction mixture
was mixed with sinapinic acid and analyzed on MALDI TOF/TOF
5800 (AB SCIEX, MA, USA) in linear mode as described14.
Insulin acetylation assay
21
Insulin (50µl) (1.8 mg mL-1) was incubated with 10 mM
concentration acetyl group containing molecules [ASP, BIS,
NDS-458, PAR, ACZ, ACH and NAC] in 0.1 M phosphate buffer
(pH 7.4) at 37 °C for 7 days. The reaction mixture was mixed
with α-Cyano-4-hydroxycinnamic acid and analyzed on MALDI
TOF/TOF 5800 (AB SCIEX, MA, USA) in linear mode as
described20.
.
Database search and PTM analysis
The MS data set was processed using Proteome discoverer 1.4
(Version 1.4.0.288) (Thermo Fisher Scientific, Germany).
SEQUEST HT search engine was used for peptide identification.
The data was searched against UniProt BSA (P02769) sequence
database. Carbamidomethylation of cysteine (C) and oxidation
of methionine (M) was considered as fixed and variable
Acetylation of BSA
The in vitro acetylation of BSA was performed by incubating modification respectively. Additional glycation modifications at
450 µl of BSA (50 mg/ml) in 0.1 M phosphate buffer (pH 7.4) lysine position Amadori (+162.05 Da), carboxymethyllysine
containing 5 mM sodium azide with or without 10 mM (CML) (+58.005 Da), carboxyethyllysine (CEL) (+72.021 Da) and
concentration acetyl group containing molecules [ASP, BIS, acetylation modification (+42.02 Da) were considered as
NDS-458, PAR, ACZ, ACH and NAC] at 37 °C for 3 days14.
variable modifications. The search was performed using the
following parameters; peptide and fragment mass tolerance-
10ppm and 0.5Da respectively, missed cleavages-2 and false
discovery rate 1%. The glycated peptides were identified as
Tryptic digestion
described earlier19, 22
.
100µg of protein was dissolved in 0.1% RapiGest and reduced
with 100 mM dithiotreitol (DTT) at 60 °C for 15 min followed
by alkylation with 200mM iodoacetamide in dark at room
temperature for 30 min. Then, proteins were subjected to
Animal Experiments
tryptic digestion at 37 °C overnight. The digestion reaction was The animal experiments were approved by Institutional Animal
stopped by adding concentrated HCL and incubated for 10 min Ethics committee of National Centre for Cell Sciences, India.
at 37 °C before vertex and centrifugation and stored at -80 °C The experimental protocols were carried out in accordance
till further use
.
with the guidelines of CPCSEA (Committee for the Purpose of
Control and Supervision of Experiments on Animals), India.
Male BALB/c mice were injected with 50 mg/kg body weight of
streptozotocin (STZ) in 50 mM citrate buffer pH 4.5 for five
consecutive days to induce hyperglycemia, whereas control
mice were injected with 50 mM citrate buffer. The induction of
diabetes was confirmed after 30 days by measuring the blood
glucose levels with glucometer (Bayer. Germany) and HbA1c
level by using HbA1c kit (Bayer. Germany). For each treatment
six animals were grouped into control, diabetic, aspirin treated
control, aspirin pre-treated diabetic and diabetic treated with
aspirin (200 mg/L) for 30 days after induction of diabetes.
LC-HR/AM Q-Exactive Orbitrap Analysis (Full MS/dd-MS2)
Chromatographic Separation: Peptides digest (1.5µg) was
separated on Hypersil Gold C18-RP HPLC column (150X2.1mm,
1.9µm) with 98% of mobile phase A (100% water, 0.1% FA) and
2% of mobile phase B (100% ACN, 0.1% FA) at 350 µl/min flow
rate with a 45 min linear gradient of 2% to 40% mobile phase
B.
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Aspirin was made available through drinking water. Glucose
and HbA1c levels were monitored on 30th, 45th, and 60th day
(0th, 15th and 30th day after initiation of STZ injection).
Animals were euthanized at the end of study after 30 days of
drug administration. Blood samples were collected; blood
glucose and HbA1c were analyzed immediately. Plasma was
obtained by EDTA treatment, which was then centrifuged at
1500 g for 5 min, and the supernatant was stored at -80oC until
further use23. Protein concentration was determined by using
BioRad protein assay kit (BioRad. CA, USA).
This work financially supported by the grants
from Council of Scientific and Industrial research
(CSIR), New Delhi, India (CSC0111). MGJ thank
Indian Council of Medical research (ICMR), India
for research fellowship. KK thank CSIR, New Delhi
for fellowship.Notes and references
1
2
3
4
5
T. J. Sims, L. M. Rasmussen, H. Oxlund and A. J. Bailey,
Diabetologia, 1996, 39, 946-951.
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N. Sasaki, R. Fukatsu, K. Tsuzuki, Y. Hayashi, T. Yoshida, N.
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Amano and Z. Makita, American Journal of Pathology, 1998,
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Statistical Analysis
All experiments were performed in triplicates. Data are
expressed as means ± SD. A p-value < 0.05 was considered
statistically significant.
6
7
8
9
M. J. Kulkarni, A. M. Korwar, S. Mary, H. S. Bhonsle and A. P.
Giri, PROTEOMICS – Clinical Applications, 2013, 7,155-170.
Conclusions
D. Hawkins, R. N. Pinckard and R. S. Farr, Science, 1968, 160
780-781.
,
Aspirin is being used in the treatment of cardiovascular
diseases due to its antithrombotic effects through platelet-
independent mechanisms15. Previous studies have shown that
aspirin has the ability to acetylate proteins16. Therefore, in
this study we investigated the protective effect of acetylation
against glycation by synthesizing acetylating molecules and
screened for their ability to protect glycation. Our results
suggest that molecules with only O-acetyl but not N-acetyl
group or acetophenone compounds were capable of
acetylating lysine residue and protecting against glycation. The
plausible mechanism for this observation could be amine
reacts with O-acetyl carbonyl and then, O to N-acetyl transfer
takes place with loss of corresponding phenolic derivative.
(Supplemntary Figure 9). However, in the case of N-acetyl
compounds (ex:Paracetamol) and acetophenone related
compounds (acetohexamide), corresponding carbonyl groups
are not reactive enough towards attack by the amines under
normal physiological conditions, hence there is no acetyl
migration was observed24-26. Therefore, we propose that
incorporation of O-acetyl group into anti-diabetic molecules
could be a useful strategy, as it may have an additive effect in
reducing AGEs. Identification of such novel acetylating agents
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,
represents
a new area in the drug discovery process.
Furthermore, pre-acetylation or aspirin treatment prior to the
induction of diabetes helps in reducing HbA1c and AGE
formation in the streptozotocin induced diabetic mice. As
accumulation of AGEs is also implicated in development of
insulin resistance17. However, further studies are required to
investigate the usefulness of pre-acetylation in prediabetic or
insulin resistance condition to protect proteins from glycation.
.
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