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Gold Standard Physiological Measurements and Novel Drug Delivery Methods – Session 2
Sponsored by:
Dr. Robert DoyleProfessor of Chemistry & Biology,Syracuse University
InsideScientific is an online educational environment designed for life science researchers. Our goal is to aid in
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tools and laboratory services.
Synthetic, Structural, and Mechanistic Investigations of Vitamin B12 Conjugates of
the Anorectic Peptide PYY3-36
Professor Robert P. Doyle
Syracuse University & SUNY, Upstate Medical University
November 12th 2015
Obesity
CDC Behavioral Risk Factor Surveillance System, 2012, http://www.cdc.gov/obesity/data/adult.html
PYY and Appetite Regulation
• PYY is a 36 aa intestinal hormone that belongs to the pancreatic polypeptide family1
• Synthesized and released by specialized enteroendocrine cells (L cells)1
• PYY has two main receptors, Y1 (orectic effect) and Y2 (anorectic effect)2
• The active anorectic form of PYY is a truncated form known as PYY3-36
2
1 Ekblad et al. Peptides 2002, 23 (2), 251–261.2 Batterham et al. Nature 2002, 418 (6898), 650−654.
PYY3-36 in Obesity Research
• Peripheral administration of PYY3-36 into rodents1
and primates,2 including humans,3 has resulted in an observed reduction in food intake
• Infusion of PYY3-36 into obese individuals (BMI ≥ 30)4
results in a reduced caloric intake comparable to individuals of lower BMI3
• Oral delivery of PYY3-36 via vitamin B12 has been established by the Doyle group in clinically relevant levels (> 180 pg/mL) in rodents5
1 Batterham et al. Nature 2002, 418 (6898), 650−654.2 Moran et al. Am. J. Physiol.: Regul. Integr. Comp. Physiol. 2005, 288 (2), R384−R388.3 Batterham et al. N. Engl. J. Med. 2003, 349 (10), 941–948.4 http://www.nhlbi.nih.gov/health/health-topics/topics/obe/diagnosis.html5 Doyle et al. J. Med Chem. 2011, 54 (24), 8707-8711.
Vitamin B12 (B12/Cobalamin)
1 Nexø et al. Nat. Rev. Gastroentero. 2012, 9 (6), 345-354.2 Russell-Jones et al. Bioconjugate Chem. 1995, 6 (1), 34-42.3 Russell-Jones et al. Bioconjugate Chem. 1999, 10 (6), 1131-1136.
2 | ADVANCE ONLINE PUBLICATION www.nature.com/ nrgastro
and haptocorrin (also known as the R-protein or trans-
cobalamin I). These proteins share the same overall
structural scaffold and each carries a single B12
molecule
(Figure 2).12,13
Intrinsic factor has a crucial function in transporting
B12
to the intrinsic factor–B12
receptor, cubam, which is
expressed on enterocytes in the ileum and is responsible
for the absorption of the vitamin by means of receptor-
mediated endocytosis. Intrinsic factor is also essential for
the actual uptake process as cubam recognizes only the
intrinsic factor–B12
complex and neither intrinsic factor
nor free B12
alone.14,15 In humans, intrinsic factor is syn-
thesized and secreted by parietal cells of the stomach,
and only small amounts have been detected outside the
gastrointestinal tract.16 This carrier protein is highly
Key points
■ A coherent vitamin B12
(B12
) transport pathway from food to the body’s cells has
now been delineated; the pathway includes an ABC transporter for cellular B12
efflux and a receptor for uptake of B12
-bound transcobalamin
■ More than 15 gene products are involved in B12
transport and/ or processing;
several new genes encoding intracellular proteins (including a potential
lysosomal transporter of B12
) have been identified
■ Gastrointestinal uptake of B12
is via cubam, the complex of cubilin and
amnionless
■ Novel genetic causes of B12
deficiency disease have been clarified; many of the
new proteins have been identified by positional cloning of the genes harbouring
the disease-causing mutations
■ New diagnostic assays for B12
deficiency are being developed; plasma level
of holo-transcobalamin is a promising biomarker in combination with existing
markers
glycosylated, which, as well as its specific amino acid
sequence, is thought to protect it from digestion by
intestinal enzymes.17
Structurally, intrinsic factor features a two-domain
architecture where B12
binds at the interface between
the two domains.12 A similar mode of binding of B12
at the interface of two domains is reiterated not only
for transcobalamin and haptocorrin,13 but also in the
B12
-dependent enzymes methionine synthase18 and
methylmalonyl-CoA mutase.19
Owing to the critical role of intrinsic factor in B12
absorption, deficiency of this protein (caused by auto-
immune attack of the parietal cells or rare inborn errors
of synthesis) leads to severe B12
avitaminosis and classic
pernicious anaemia.20,21 Intrinsic factor was discovered
by Castle as the ‘intrinsic factor’ lacking in patients suf-
fering from pernicious anaemia despite normal supply
of the ‘extrinsic factor’ (that is B12
).22
Circulating transcobalamin has an essential role in
transporting B12
absorbed in the ileum to cells of the
body. The importance of transcobalamin is obvious
in the small number of children with inborn errors of
transcobalamin synthesis. The affected child displays few
symptoms at birth, but within months a severe deficiency
develops and, if left untreated, it leads to lifelong impair-
ments due to neurological damage.23–27 Several different
kinds of mutations leading to a lack of transcobalamin
have been identified, including deletions and mutations
resulting in erroneous RNA editing.23–27
Haptocorrin is heavily glycosylated and is expressed in
many, but not all, mammals.28 In humans, haptocorrin is
bCytosol
Mitochondrion
Folate
H3C
N
N
N
N
CH3
H3C
H3C
NH2
O
NH2
O
CH3
CH3
NH2O
Co
CH3
OHN
H2N
H3C
O
O
H2N
O
H2N
O
H3C
P
–O O
O
CH2OH
O
HO
N
N
CH3
CH3
R
Purines, pyrimidines TH-
Folate5-methyl TH-
Methionine synthase Methylcobalamin
Homocysteine
Methionine
Adenosylcobalamin
Methylmalonyl-CoA
Succinyl-CoA
Methylmalonyl-CoAmutase
a
Figure 1 | B12
structure and coenzyme function. a | B12
structure. The core of B12
consists of a corrin ring that encircles a
central cobalt ion. The latter is linked to four nitrogen atoms from the corrin ring, as well as to a nitrogen atom from a
5,6-dimethylbenzimidazole ribonucleotide moiety positioned below the plane of the corrin ring and a variable group (R)
positioned above the plane of the ring.5–8,10 The variable group can be occupied by several ligands, including a hydroxyl,
cyano, methyl, or 5’-deoxyadenosyl group. The enzymatically active cofactor carries either a methyl or a 5' -deoxadenosyl
group at this position. In this Review, the term B12
refers to all variants of the vitamin, unless otherwise stated.
b | Coenzyme function. B12
serves as a coenzyme in two distinct enzymatic processes: the conversion of homocysteine to
methionine by cytosolic methionine synthase and the conversion of methylmalonyl-CoA to succinyl-CoA by mitochondrial
methylmalonyl-CoA mutase. The former reaction is linked to folate metabolism because the methyl group transferred to
homocysteine is provided by the conversion of 5-methyl tetrahydrofolate to tetrahydrofolate. Tetrahydrofolate is essential
for the production of purines and pyrimidines. Prolonged B12
deficiency results in accumulation of 5-methyl tetrahydrofolate
with impaired DNA synthesis as a result. This scenario is known as the methyl-folate-trap. Abbreviation: TH, tetrahydro-.
REVIEWS
© 2012 Macmillan Publishers Limited. All rights reserved
B12 Dietary Uptake Pathway
1 Nexø et al. Nat. Rev. Gastroentero. 2012, 9 (6), 345-354.2 Alpers et al. Pharm. Biotechnol. 1999, 12, 493-520.3 Banerjee et al. J. Biol. Chem. 2013, 288 (19), 13186-13193.4 Doyle et al. Exp. Opin. Drug. Deliv. 2010, 8 (1), 127-140.
B12$
HC$
B12$
HC$
B12$
B12$
B12$ B12$
B12$
B12$
IF$
IF$
CB$
AM$
B12$
IF$
CB$
AM$
to$ileum$
to$stomach$
Kd$≈$0.01$pM$
$to$duodenum$
$pH$>$5$
!
Dietary$source$of$B12$is$broken$in$mouth$releasing$B12;$bound$by$HC$
ileal$enterocyte$B12$
TCII$
MRP1$
B12$
TCII$
?$
B12$
TCII$
CD320$
v! v!MG$
Kd$≈$1.0$pM$
Kd$≈$0.005$pM$
pH$<$3$
B12$
Average$daily$uptake$of$B12$is$about$1O5$μg3$
DietarysourceofB12isbrokendowninthe
mouth,releasingB12;boundbyHC
Hypothesis
Conjugation of B12 to PYY3-36 will have positive pharmacodynamic and pharmacokinetic effects in vivo upon subcutaneous (sc) administration
Specific Aims
1. Synthesize and characterize B12-PYY3-36
conjugates via a series of B12-alkyne precursors2. Test B12-PYY3-36 conjugates for binding,
selectivity, and agonism of the Y2 (anorectic) and Y1 (orectic) receptors in vitro
3. Perform sc in vivo feeding studies with B12-PYY3-
36 conjugates
Synthesis of B12-Alkyne Precursors
Doyle et al. Synlett. 2012, 23 (16), 2363-2366.
Yield (%) MW (g/mol)
84 1406
79 1420
75 1434 EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimideHOBt: hydroxybenzotriazole
Structure and Modification of PYY3-36
PDB: 2DF0
N term. β-Turn α helix C term.I K P E A P G E D A S P E E L N R Y Y A S L R H Y L N L V T R Q R Y
Pederson et al. J. Pept. Sci. 2009, 15 (11), 753-759.
Synthesis of B12-PYY3-36 Conjugates (1-3)
n
1
2
3
Yield (%) MW (g/mol)
93 5481
95 5495
90 5509
TBTA: tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
Representative Purification (1)
RP-HPLC: C18 analytical column, flow rate 1 mL/min, 25 °C, UV detection at 280 nm.A: 0.1% TFA in H2O, B: MeCN, Method: 10% B to 35% B over 25 minutes.
tR = 23.1 min
5456.008
MALDI-ToF MS: 1:1 sample:matrix ratio, CHCA matrix, 10 mg/mL, 50:50 H2O:MeCN with 0.1% TFA.
Expected m/z: 5481 (parent)5455 (-CN)
0
200
400
600
800
1000
1200
Inte
ns
. [a
.u.]
2000 4000 6000 8000 10000m /z
Aim 2: Binding, selectivity, and agonism of the Y2 (anorectic) and
Y1 (orectic) receptors in vitro
Goals
1. Construct and optimize calcium-induced calcium release (CICR) assay via Y2 and Y1 receptors to test activity of conjugates 1-3 vs. PYY3-36 and PYY1-36
2. Confirm Y2 receptor agonism with synthesis and in vitro characterization of a “null” conjugate
1 Jacoby et al. ChemMedChem 2006, 1 (8), 760-782.2 Herzog et al. PNAS 1992, 89 (13), 5794-5798.
GPCR Signal Transduction
PlasmaMembrane
Gq-coupled Gs-coupled Gi-coupled
αq
*αq
αs *αs αi βββγ γγ
PIP2 IP3+DAG
+
+ PLCβ
+
AdenylateCyclase
ATP
IP3Ca++
Ca+++
PKC
βγ--
cAMP
+PKA
*αi
Transcrip onfactors
PromotersCRE,SRE
GeneexpressionDNABP
nucleus
ER
biologicalresponse
CICR Signaling and Detection
λex: 340 and 380 nmλem: 510 nm
1 https://www.lifetechnologies.com/order/catalog/product/F12012 Herzog et al. PNAS 1992, 89 (13), 5794-5798.
O O
N
O
N O
N
O O
OO
O
O
O
O
COO-
Ca2+
O
N
O
O
O
O
O
O
N
OO
OO
O
O
O
O
O
N
O
OO
O
O O
OO
CytosolSES
Fura-2AM Fura-2boundtoCa2+
Y2 Receptor-Stimulated CICR 1 vs. 2 vs. 3
Beck-Sickinger et al. J. Pept. Sci. 2000, 6 (3), 97-122.
PYY3-36
1
2
3
Compound EC50 (nM)
PYY3-36 16
1 72
2 27
3 127
Y1 Receptor-Stimulated CICR
PYY1-36
PYY3-36
2
Beck-Sickinger et al. J. Pept. Sci. 2000, 6 (3), 97-122.
Compound EC50 (nM)
PYY1-36 10
PYY3-36 620
2 2200
Y1 vs. Y2 Receptor
Nygaard et al. Biochemistry 2006, 45 (27), 8350-8357.
PYY1-36
PYY3-36
PYY1-36
PYY3-36
Synthesis of Null Conjugate B12-PYYC36 (4)
SPDP: 3-(2-pyridylthio)propionic acid N-hydroxysuccinimide ester
Doyle et al. ChemMedChem 2014, 9 (6), 1244-1251.
Y2-Receptor Stimulated CICR PYY3-36 & 2 vs. PYYC36 & 4
1 Beck-Sickinger et al. J. Pept. Sci. 2000, 6 (3), 97-122.2 Pederson et al. J. Pept. Sci. 2009, 15 (11), 753-759.3 Beck-Sickinger et al. Eur. J. Biochem. 1994, 225 (3), 947-958.
PYY3-36
2
PYYC36
4
Compound EC50 (nM)
PYY3-36 16
2 27
PYYC36 762
4 1809
Aim 3: In vivo feeding studies (sc) with PYY3-36, 2, and 4 in rats*
Goals
1. Optimize dosing in lean (Sprague Dawley) male rats
2. Acclimate rats to experimental schedule
3. Pharmacodynamic (PD) analysis
4. Pharmacokinetic (PK) analysis
5. Elucidate mechanism of action
6. Repeat sc studies in obese (Zucker) male rats
*All animal studies performed in collaboration with Dr. Christian Roth and Clinton Elfers at Seattle’s Children’s Research Institute in Seattle, WA
Dose Escalation Study with 2
Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.
Thermal/Solution Stability of 2
*All samples ran at 300 nM
Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.
Implanting Microinfusion Pumps
Dosing Schedule
Baseline
PYY3-36
Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.
Baseline
2
Baseline
PYY3-36
Food Intake Trends
4
2
PYY3-36
4
2
PYY3-36
* P < 0.05
Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.
Food Intake Trends
23.7% reduction in food intake due to treatment with 2 and a 13.2% reduction in food intake due to treatment with PYY3-36
4 2 PYY3-36 4 2 PYY3-36
10 day treatment• 2 (n = 6)• PYY3-36 (n = 4)• 4 (n = 4)
5 day treatment• 2 (n = 9)• PYY3-36 (n = 8)• 4 (n = 5)
* P < 0.05** P < 0.01
*** P < 0.001
1 Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749. 2 Reidelberger et al. Am. J. Physiol.: Regul. Integr. Comp. Physiol. 2006, 290 (2), R298-305. 3 Pittner et al. Int. J. Obes. Relat. Metab. Disord. 2004, 28 (8), 963-971.
* P < 0.05 ** P < 0.01
Body Weight Gain
1 Henry et al. Endocrinology 2015, 156 (5), DOI: en.2014-1825. 2 Reidelberger et al. Am. J. Physiol.: Regul. Integr. Comp. Physiol. 2006, 290 (2), R298-305. 3 Pittner et al. Int. J. Obes. Relat. Metab. Disord. 2004, 28 (8), 963-971.
4 2 PYY3-36
Pulses of Drugs and Time of Action
Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.
PYY3-36 B12-PYY3-36 PYY3-36 B12-PYY3-36
PYY3-36 B12-PYY3-36 PYY3-36 B12-PYY3-36 PYY3-36 2 PYY3-36 2
PYY3-36 2 PYY3-36 2
* P < 0.05
2 PYY3-36
4
In Vivo Uptake Studies
Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749.
* * * **
10 nmol/kg 6 10 nmol/kg PYY3-36
10 nmol/kg 2 (n = 4)
10 nmol/kg PYY3-36 (n = 3)
Drug AUC0-∞(pg/h/ml) Cmax(pg/mL) t1/2(h) VD/F(L/kg) CL/F(mL/min/kg)
PYY3-36 3843±1125 1680±243 0.82±0.16 12.8±1.5 188.6±65.6
2 7130±2050 2520±257 1.34±0.28 15.0±1.5 133±32
Tmax = 1 h
* P < 0.05** P < 0.01
PYY3-36: Mechanisms of Action
BRAIN GUT BLOOD
Vagal nerve carries sensory information from the Y2 receptors in the gut to
solitary tract nucleus (NTS)2
Circumventricular organs3
1 Nonaka et al. J Pharmacol. Exp. Ther. 2003, 306 (3), 948-953.2 Abbott et al. Brain Res. 2005, 1044 (1), 127-131. 3 Koda et al. Endocrinology, 2005, 146 (5), 2369-2375.
CENTRAL PERIPHERAL
PYY3-36 crosses BBB and activates Y2 receptors in
the arcuate nucleus (ARC)1
C-Fos Immunohistochemistry
PYY3-36 2 4 Saline
Y2 Receptor Activation
!!Y2 Receptor Activation
Vagus Nerve
1 Doyle R.P. et al. Endocrinology 2015, 156 (5), 1739-1749. 2 Blevins et al. Peptides 2008, 29 (1), 112-119. 3 Schwartz et al. Nature 2000, 404 (6778), 661-671.
* P < 0.052 (n = 9)
PYY3-36 (n = 8)4 (n = 5)
Design of NOTA-2
Doyle R.P. et al. unpublished data.
64Cu-NOTA-2 PET Scan
Administered Dose recovered in brain for 2 vs. PYY3-36. (2-tailed p=0.08). 15 μCiinjected dose 64Cu-labeled conjugate by iv.3 h PET scan of Sprague Dawley rats (n = 3)
Doyle R.P. et al. unpublished data.
Zucker Rats: FI Trends
Av
era
ge
Fo
od
In
take (
g/d
ay)
Baseline 4d Treatment0
10
20
30
40
B12-PYY3-36
PYY3-36
**
2 PYY3-36
* P < 0.052 (n = 3)
PYY3-36 (n = 5)
Doyle R.P. et al. unpublished data.
Zucker Rats: BW Trends
Bo
dy
We
igh
t (
g)
Day 0 Day 10 Day 20 Day 30750
800
850
900
950
1000B12-PYY3-36
PYY3-36
Baseline Treatment Compensation
6
PYY3-36
2
PYY3-36
* P < 0.05D
Bo
dy W
eig
ht
(g)
4 day 10 day
-30
-20
-10
0
B12-PYY3-36
PYY3-36
*
**p<0.05 compared to pretreatment
2
PYY3-36
Av
era
ge
Fo
od
In
take (
g/d
ay)
Baseline 4d Treatment0
10
20
30
40
B12-PYY3-36
PYY3-36
**
2 PYY3-36
Doyle R.P. et al. unpublished data.
Conclusions and Summary
* * * **
10 nmol/kg 6 10 nmol/kg PYY3-36
10 nmol/kg 2
10 nmol/kg PYY3-36
4 2 PYY3-36
2 PYY3-36
4
Av
era
ge
Fo
od
In
take
(g
/da
y)
Baseline 4d Treatment0
10
20
30
40
B12-PYY3-36
PYY3-36
**
D B
od
y W
eig
ht
(g)
4 day 10 day
-30
-20
-10
0
B12-PYY3-36
PYY3-36
*
**p<0.05 compared to pretreatment
Future Work: SUPER PYY!
GLP1-R agonism
Y2-R biased agonism
Doyle R.P. et al. unpublished data; Patent Filed Sept. 2015
Thank you to our event sponsor
Innovative drug infusion technology for laboratory animals.
Dr. Robert [email protected]
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