Pharmacological reversal of advanced glycation end-product-mediated protein crosslinking
P. Ulrich
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X. Zhang
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The Picower Institute for Medical Research
, Manhasset,
New York, USA
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It is well known that glucose reacts
non-enzymatically with protein amino groups with eventual
formation of covalent protein-protein crosslinks, which are
called advanced glycation end-products (AGEs) [1].
Much work has been done to establish a causative
association between this process and many of the
pathological sequelae observed in diabetes mellitus,
including LDL modification and the vascular changes
which lead to atherosclerosis [2, 3]. AGE crosslinking
also occurs between tissue proteins and
AGE-containing peptide fragments formed from protein
turnover. Such reactive AGE-peptides, now called
glycotoxins, are normally cleared by the kidneys, but they
build up in the serum in conditions such as diabetic
kidney failure, and their random covalent attachment
to proteins may cause widespread tissue damage.
In the early 1980s we suspected that reactive
carbonyls were involved in the mechanism of
crosslinking and tissue damage by AGEs, and so we
developed the carbonyl-blocking agent, aminoguanidine,
as a new pharmaceutical for inhibition of the
development of diabetic complications [4].
Aminoguanidine is now in Phase 2/Phase 3 clinical trials under
the generic name pimagedine. However,
development of improved agents for preventing or reversing
this process in vivo has been hampered by a lack of
detailed knowledge of the chemical nature of the
principal crosslinking structures. The crosslinking
structures identified to date are thought on the basis
of chemical considerations and spectroscopic data to
constitute only a small fraction of the AGE
crosslinking which occurs in vivo, with the major
crosslinking structures still unidentified [5].
We have now found that certain substituted
thiazolium salts can actually break covalent AGE
crosslinks and return the affected proteins to the
uncrosslinked state both in vitro and in vivo [6]. In addition
to demonstrating the unexpected new possibility of
pharmacological reversal of clinically relevant AGE
crosslinking, this cleavage reaction provides new
chemical information about the nature of the major
AGE crosslinks by virtue of the unusual chemistry
of which thiazolium salts are capable. In
collaboration with workers at Alteon Inc., (Ramsey, N. J.,
USA) a number of thiazolium derivatives have been
prepared and studied both for the inhibition and the
reversal of AGE-crosslinking. One compound of
particularly high activity, phenacyl thiazolium bromide
(PTB), has been studied in detail. PTB rapidly
cleaves a model diketone, phenylpropanedione, with
release of benzoic acid.
In an in vitro assay, AGE-BSA was allowed to
crosslink to collagen which had been coated onto
microtitre wells, and unreacted AGE-BSA was washed
away. PTB released BSA that had been crosslinked
to the collagen in a dose- and time-dependent
manner. Aminoguanidine is not able to release
crosslinked BSA in this assay.
Normal rat tail tendon collagen yields
electrophoresible fragments on digestion with cyanogen
bromide. However, tail collagen from diabetic rats
releases almost no such fragments on CNBr treatment.
This is because of extensive additional crosslinking
due to AGE formation. This effect is almost
completely reversed on pre-treatment of the isolated
collagen with PTB prior to cyanogen bromide digestion.
Such a reversal is not seen on pre-treatment with
aminoguanidine.
In diabetic rats IgG becomes covalently bound to
erythrocyte membrane proteins through AGE
reactions. However, in diabetic rats which received PTB
at 10 mg/kg four times per day for up to 4 weeks,
release of erythrocyte-bound IgG occurred, reaching
a level of 6070 % cleavage. In vitro, treatment of
diabetic rat erythrocytes with PTB causes IgG binding
to revert to control levels.
There is evidence that AGE crosslinking may
contribute to amyloid nucleation and increased amyloid
deposition in Alzheimers disease. In vitro, glucose
treatment of -amyloid peptide causes AGE
formation and enhances aggregation. Treatment of
aggregated fibrillar AGE -amyloid with PTB results in
significant disaggregation of the amyloid, as
determined by gel electrophoresis of radiolabelled
material, and electron microscopy.
Our proposed mechanism for AGE crosslink
cleavage by thiazolium salts involves dicarbonyl
containing crosslinks derived from the early glycation
adduct, the Amadori product, a fructosamine derivative
which forms by rearrangement of the initially formed
Schiff-base adduct of glucose with protein amino
groups. The Amadori product can undergo a number
of subsequent rearrangements which lead to the
formation of -dicarbonyl compounds. We have become
interested in the little studied amino dicarbonyl
adducts, the Amadori dione [7, 8] and the Amadori
ene-dione [9] (Fig. 1). These potentially important
AGE intermediates have been neglected due to their
instability and difficulty of synthesis. They were very
interesting to us because they cont (...truncated)