Download Beili B24 25 Insulin

Site Specific Introduction of Unnatural Amino Acids
at Sites Critical to Insulin Receptor Recognition & Biological Activity
B.Quan, D.Smiley, V.Gelfanov, and R.DiMarchi
Department of Chemistry Indiana University, Bloomington Indiana 47405 U.S.A
Abstract
Experimental Design
60
CH2
3.55
50
49
Insulin
43
LEU
LEU
42
GLU
VAL
LYS
41
GLN
ARG
A-CHAIN
40
GLY
GLY
39
VAL
2
ILE
38
GLN
3 VAL
LEU
GLU
4
S
5
ASP 36
S
GLN
CONNECTING
PEPTIDE
CYS
CYS
6
ALA
SER
7
8
1 PHE
VAL
9
ILE
10
S
CYS SER
ASN
LEU TYR GLN
CYS
LEU GLU ASN TYR
20
0.51
11
12
13
14
15
16
17
18
19
20
GLN
4
S
LEU
PRO
6
7
THR
S
GLY
TYR
SER
8
HIS
LEU
9
10
11
VAL
12
ARG
GLU ALA
GLU
LEU TYR LEU VAL CYS GLY
13
14
15
16
17
18
19
20
21
22
GLY
40
0
CH2
1E-3
0.01
0.1
1
10
16.58
100
0.49
1E-3
0.01
0.1
CF3
[Peptide], nM
1
10
100
1000
[Peptide], nM
Fig 3. Binding Affinity of Insulin Analogs with Comparable Amino Acid Substitution at B24 & B25
B24(2-Me)
B24(3-Me)
B24(4-Me)
200
B24 IC50 nM
CH2
18.25
CH2
22.54
CH3
N
60
CH2
40
6.78
CH2
0.63
S
Insulin
CH3
20
35
CH2
34
B24(4-pyridyl)
B24(4-thienyl)
180
80
160
140
120
5.25
PHE
PHE
24
1E-3
32
0.01
0.1
1
10
100
80
Results & Conclusion
60
40
Insulin
0
-20
1E-3
CH3
1000
0.01
[Peptide], nM
30
30
0.1
1
10
100
[Peptide], nM
29
28
27
26
Fig 4. Binding Affinity of Insulin Analogs Substituted at Position B24
25
23
Fig 1. Primary Structure of Human Proinsulin
B25 IC50 nM
B25 IC50 nM
140
>1000
Insulin B25 Phe(4-CF3)
Theoretical MW= 5875
0.46 Χ 15cm Vydac C18
1ml/min, 45C, 214nm,
Linear gradient of CH3CN in
0.1% TFA(aq)
Ubiquitin
(M+H)+
average
Ubiquitin
(M+2H)
2+average
CH2
CH2
13.15
B25(4-Me)
B25(4-Br)
B25(4-CH2NH2)
hPhe25
B25(4-COOH)
120
100
CH2
0.67
CH3
80
CH2
60
40
CH2
Insulin
1000
Nineteen different amino acids (17 non-natural) were substituted for the native
phenylalanine residues at positions B24 and B25 in insulin. The analogs were
successfully prepared by SPPS from individual A and B chains, in total yields that varied
between 1 and 10%. The relative binding affinity of this group of insulin analogs for the
insulin receptor was determined to differ by more than thousand-fold from the most
potent to the least potent peptide studied. Consistent with previous observations
predominantly employing native amino acids, we observed that insulin activity was
highly dependent upon aromatic character at these two residues (1). Position B24 was
extremely restrictive to structural modification while B25 was extremely permissive.
In Figure 3 we report the extreme difference between the same substitutions at B24 and
B25. Each of the B25 insulin analogs were of high affinity while only one of the B24
analogs did not exhibit a sizable reduction in binding affinity. The highly homologous
B24 Phe(4-F) analog is a rare example of an l-amino acid with comparable potency to
the native hormone, and represents an opportunity for structural study in the receptor
recognition site with isotopically enriched amino acid. Further study at B24 (Figure 4)
illustrates that even a single methyl group added to the phenyl ring is deactivating and
most notably when ortho to the ring. Phenylalanine heterocycle-based mimetics were
studied. An appreciable difference was observed among two commonly used
derivatives. The 5-membered thiophene ring provided nearly native insulin affinity but
the more alkaline 6-membered pyridine ring was sizably reduced in potency.
Additional study at position B25 (Figure 5) demonstrated that movement of the phenyl
ring closer or further from the peptide backbone had a significant deactivating impact
upon binding affinity (2). Additional modifications that selectively introduced negative
charge (4-COOH) and size (4-Br) relative to the native Phe were largely without effect.
Although, the alkaline amino-methyl substitution (4-CH2NH2) reduced binding affinity to
a significant degree. This observation is consistent with the more subtle reduction noted
for the less alkaline 4-NH2 modification (Figure 3).
3.15
20
CH2NH2
0
0.01
0.1
1
10
100
1000
References
CH2
0.55
[Peptide], nM
Fig 2. Purified Insulin Analog Chromatographic
& Mass Spectral Analysis
0.83
COOH
1E-3
Receptor Binding : The binding affinity of each insulin analog for the insulin receptor
was measured in a competition binding assay utilizing scintillation proximity assay
technology. Serial 3-fold dilutions of the peptides were made in scintillation proximity
assay buffer (0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.1% w/v bovine serum albumin) in
a 96 well plate (Corning Inc., Acton, MA) with 0.05 nM (3-[125I]-iodotyrosyl) TyrA14
Insulin. An aliquot of 1-6 micrograms of plasma membrane fragments prepared from
cells over-expressing the human insulin receptor and 1 mg/well polyethyleneiminetreated wheat germ agglutinin type A scintillation proximity assay beads (Amersham
Biosciences, Piscataway, NJ) were added to each well. The plate was incubated for
twelve hours at room temperature and measurements made with MicroBeta1450 liquid
scintillation counter (Perkin-Elmer, Wellesley, MA). Non-specifically bound (NSB)
radioactivity was measured in the wells with fourfold greater concentration of “cold”
native ligand than the highest concentration in test samples and total bound radioactivity
was detected in the wells with no competitor. Percent specific binding was calculated as
following: % Specific Binding = Bound-NSB / Total bound- NSB x 100. IC50 values were
determined by using Origin software (OriginLab, Northampton, MA).
100
20
0
31
THR
LYS
CYS
IGF-1
20
0.91
0
ARG
HIS
5
60
Insulin
Synthesis of B-chain analogs : B chain analogs with unnatural amino acids at B24
and B25 were successfully synthesized by solid phase methodology using Fmoc
chemistry. Peptides were cleaved from the support in strong anhydrous acid and purified
under acidic conditions by preparative high performance reverse-phase chromatography
in the free sulfhydryl form. Each peptide was characterized by MALDI-MS and HPLC
analysis to be at least 90% pure prior to chain combination.
Chain Combination Reaction : Each B-chain analog was mixed with a molar
equivalent of native A-chain S-sulfonate (gift from Eli Lilly) in a 0.05 M glycine buffer, pH
10.5. A molar equivalent of DTT relative to each S-sulfonate was used to facilitate
disulfide interchange. The reaction was conducted at 4ºC, overnight. The product was
purified by preparative high performance reverse phase chromatography in a slightly
alkaline NH4HCO3 buffer with CH3CN elution. The insulin analogs were obtained in step
yield that varied from 5-30%, and characterized by MALDI-MS and HPLC analysis to be
greater than 95% pure.
33
21
ARG
S
80
F
GLU
ASN
3
GLU
THR
B-CHAIN
2
37
NH2
CH2
48
44
1.03
IGF-1
%Specific Binding
1
55
100
0.72
6.79
100
GLN
64
65
56
51
47
LEU SER GLY
59
PRO GLN
46
LEU
ALA
GLY
ALA
60
PRO
45
LEU
GLY
61
GLU
GLY
GLY
GLY
SER
62
63
57
52
0.67
OH
%Specific Binding
58
53
0.67
80
120
54
CH2
CH2
40
B25(4-CF3)
B25(4-F)
B25(4-NH2)
B25 (4-OH)
120
%Specific Binding
100
B25 IC50 nM
%Specific Binding
Our work has focused on two amino acids in the C-terminus of the Bchain that are central to insulin activity, specifically positions B24 and 25.
The selective introduction of a series of Phe-related derivatives at each
position has yielded an insightful perspective on insulin receptor
recognition. Position 24 has proven highly restrictive to changes that can
be introduced and the results emphasize the importance of backbone
conformation to full potency. In comparison, position B25 is quite
accommodating to sizable changes in side-chain structure but
surprisingly highly demanding in that the phenyl ring must be positioned
at the beta-carbon. These collective observations establish a foundation
for application of unnatural amino acids as a route to insulin
pharmacology that may not be obtainable with natural amino acids
alone.
B24 IC50 nM
B24(4-F)
B24(4-NH2)
B24 (4-OH)
B24(4-CF3)
120
%Specific Binding
Insulin constitutes a hormone of central importance in physiology and a
vital element in glucose management. Its use in diabetes care has been
of seminal significance for nearly a century. The advent of rDNA
biosynthesis provided human insulin in virtually unlimited quantity. More
importantly, it provided a mechanism by which improved biosynthetic
insulin analogs could be synthesized, evaluated and registered as new
medicines. The advent of chemical biotechnology (biosynthesis with
unnatural amino acids) provides a new venue for optimizing insulin
pharmacology through the use of synthetic chemistries that otherwise
would be prohibitively expense for commercialization.
1. Mirmira, R.G., Nakagawa S.H., and Tager H.S. (1991) J. Biol. Chem. 266, 1428-1436
2. Nakagawa, S. H. And Tager, H.S. (1986) J. Biol. Chem. 261, 7332-7341
Br
Fig 5. Binding Affinity of Insulin Analogs Substituted at Position B25
Acknowledgements
Beili Quan is financially supported by an Indiana University Gill Center Fellowship.