Engineering of a Potent, Long-Acting NPY2R Agonist for Combination with a GLP-1R Agonist as a Multi-Hormonal Treatment for Obesity

Sam Lear, Elsa P ﬂ imlin, Zhihong Zhou, David Huang, Sharon Weng, Van Nguyen-Tran,

Article

Engineering of a Potent, Long-Acting NPY2R Agonist for

Combination with a GLP-1R Agonist as a Multi-Hormonal Treatment

for Obesity

Sean B. Joseph, Shane Roller, Scott Peterson, Jing Li, Matthew Tremblay, Peter G. Schultz,

and Weijun Shen *

Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c00740

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sı Supporting Information

ABSTRACT: Bariatric surgery results in increased intestinal

secretion of hormones GLP-1 and anorexigenic PYY, which is

believed to contribute to the clinical eﬃcacy associated with the

procedure. This observation raises the question whether

combination treatment with gut hormone analogs might

recapitulate the eﬃcacy and mitigate the signiﬁcant risks associated

with surgery. Despite PYY demonstrating excellent eﬃcacy and

safety proﬁles with regard to food intake reduction, weight loss,

and glucose control in preclinical animal models, PYY-based

therapeutic development remains challenging given a low serum

stability and half-life for the native peptide. Here, combined

peptide stapling and PEG-fatty acid conjugation aﬀords potent

PYY analogs with >14 h rat half-lives, which are expected to translate into a human half-life suitable for once-weekly dosing.

Excellent eﬃcacy in glucose control, food intake reduction, and weight loss for lead candidate 22 in combination with our previously

reported long-acting GLP-1 analog is demonstrated in a diet-induced obesity mouse model.

,8−16

INTRODUCTION

control.

However such therapies have not been fully

■

realized, and while a limited number of PYY analogs have

The obesity epidemic is a major public health crisis and

17−19

progressed into clinical trials,

no approved PYY-based

signiﬁcant burden on healthcare systems worldwide. Obesity

is major risk factor for cardiovascular disease, musculoskeletal

disorders, and cancers. In 2015, 12% of adults worldwide and

over 30% of US adults were obese, with an estimated 4 million

deaths attributed to high body mass index. Thus, there is a

clear unmet medical need for safe and eﬀective weight loss

drugs currently exist. While there has been some controversy

regarding the anorexigenic and anti-obesity eﬀects observed in

20,21

rodent models following administration of PYY alone,

beneﬁcial eﬀects upon combination treatment with other

peptide hormones have been demonstrated, and it is likely that

this type of approach will be necessary to fully reverse the

therapies. Bariatric surgery is currently the only eﬀective

,22

therapeutic intervention for obesity that is demonstrated to

produce a sustained weight loss eﬀect, with the so-called Roux-

en-Y gastric bypass accounting for the majority of all weight

loss surgeries. Furthermore, it is well established that changes

in the secretion of numerous intestinal hormones, including

peptide tyrosine tyrosine (PYY) and glucagon-like peptide-1

eﬀects of obesity. Indeed, improved eﬃcacy through dosing

of PYY analogs in combination with a GLP-1 receptor (GLP-

R) agonist as a second hormonal therapy has been established

23−25

13,26−31

in animal models

and clinical obesity studies,

and

such synergies likely stem from mimicking of the multi-

hormonal eﬀect of gastric bypass, crucially without the

associated risks of surgical intervention.

(GLP-1), occur following this type of surgery. It is these

changes that are thought to play a signiﬁcant role in the

observed weight loss eﬀect and enhancement of glycemic

control, which in some cases results in complete remission of

Received: May 10, 2020

type 2 diabetes. Despite these highly positive results, bariatric

surgery remains a highly invasive procedure, which is not

without signiﬁcant risk. PYY and GLP-1 when directly

administered have shown excellent eﬃcacy and safety proﬁles

with regards to food intake reduction, weight loss, and glucose

XXXX American Chemical Society

J. Med. Chem. XXXX, XXX, XXX−XXX

Article

47,48

Figure 1. Structure of (A) neuropeptide Y bound to Y receptor (NMR structure, receptor omitted; PDB ID: 2DF0),

with residues that

interact with the receptor shown in red (Y20, L24, Y27, and L28) and those outside the binding pocket shown in blue (R19, S23, H26, and L30).

B) Structures of staples used for initial position/length screen, with (C) key peptide sequences.

(

Table 1. Optimization of Staple Position and Length for Stapled PYY Analogs (Activity Data for the Cellular NPY2R

Activation Assay Shown)

hNPY2R EC₅₀(nM)

sequence

PYY1

cysteine substitutions

no staple

S3 10 atom

other staples

none

1.0 ± 0.1

i, i + 7

2, 9

PYY1

2.9 ± 0.5

1.5 ± 0.3

14 ± 2

740 ± 90

69 ± 7

4.4 ± 0.5

4.8 ± 0.7

4.3 ± 0.7

3 ± 1

10, 17

17, 24

18, 25

19, 26

20, 27

21, 28

22, 29

23, 30

24, 31

i, i + 11

10, 21

13, 24

19, 30

i, i + 15

9, 24

S1 (8 atom)

6 ± 1

S2 (9 atom)

8.1 ± 0.9

5 ± 1

190 ± 70

>10,000

5 ± 1

4 ± 2

14 ± 2

0.7 ± 0.8

160 ± 20

6 ± 2

600 ± 300

PYY1

18 ± 2

100 ± 10

8 ± 4

S5 (13 atom)

22 ± 3

8 ± 6

PYY1

PYY2

23 ± 4

8 ± 2

0.41 ± 0.03

S6 (17 atom)

32 ± 2

15 ± 8

15, 30

none

i, i + 7

10, 17

23, 30

PYY2

0.32 ± 0.03

0.13 ± 0.01

0.57 ± 0.04

S4 (11 atom)

0.18 ± 0.02

S7 (11 atom)

0.50 ± 0.05

EC₅₀determined in a cAMP reporter assay using CHO cells overexpressing human NPY2R in the presence of FBS (10%). Cells were treated with

peptides in 12-point dose−response in culture medium with 10 μM forskolin as a positive control. The assay was carried out in triplicate for 30 min

at 37 °C and 5% CO , and the cAMP detection kit from Cisbio was used to quantify cAMP accumulation.

A major challenge for such therapies is the lack of requisite

serum stability and half-life for the native peptides, requiring

high doses and frequent injection for chronic treatment. A

variety of engineering strategies have been adopted in order to

increase the potency and/or half-life of PYY, including

backbone N-methylation and β-homo amino acid replace-

J. Med. Chem. XXXX, XXX, XXX−XXX

Article

Figure 2. Structures of staples used for combined stapling and fatty acid conjugation.

2,33

34−37

ment,

other unnatural amino acid substitutions,

decided to use this truncated form for development as it is the

8,39

40,41

PEGylation,

and lipid

or antibody conjugation. An

Y receptor speciﬁcally that mediates the anorectic eﬀects of

additional consideration is the side eﬀect of severe nausea

observed upon clinical dosing of PYY and GLP-1 analogs,

which poses a further challenge for clinical development.

PYY analogs. A set of PYY1 peptides was synthesized

incorporating di-cysteine mutations at selected stapling

positions representing a scan of the entire sequence and

subsequently conjugated to bromoacetyl-functionalized staples

3,24,38

A slow ramp up of exposure should mitigate this eﬀect, which

is facilitated by a peptide with a suitably long half-life. To this

end, we envisioned a PEG-fatty acid conjugated, stapled PYY

analog with a human half-life suitable for once-weekly dosing.

Furthermore, a synergistic enhancement in eﬃcacy resulting

from combination therapy with GLP-1 oﬀers the possibility of

a reduced dose of either peptide. Given our previously

reported success in engineering the highly potent long-acting

using the solution-phase chemistry previously described.

A scan of i, i + 7 diCys mutants was ﬁrst carried out to ﬁnd

the best position in the sequence for stapling using the 10-

atom staple S3 (Figure 1), which we have previously

demonstrated to be an appropriate length for eﬀective i, i +

alpha-helix stapling. To assess in vitro activity of the

peptides and stapled analogs, we developed a cell-based

NPY2R activation assay, where intracellular cAMP was

quantiﬁed upon receptor activation in CHO cells over-

expressing human NPY2R (see below and Experimental

Section). Assay data are given in Table 1. Multiple diCys

substitution positions were tolerated for unstapled PYY1 (2-9,

GLP-1R agonist “E6” (peptide 1, see below)

with

demonstrated in vivo eﬃcacy in diabetes models, we explored

an approach involving combination dosing of this peptide with

an engineered NPY2R agonist with comparable pharmacoki-

netic proﬁle. To this end, we combined peptide stapling and

PEG-fatty acid conjugation to generate PYY analogs with high

potency and long in vivo half-life, projected to translate to a

human proﬁle suitable for once-weekly dosing. Combination

treatment with peptide 1 demonstrated superior glucose

control, food intake reduction, and weight loss in a mouse

model of diet-induced obesity and diabetes compared to either

peptide alone.

10-17, 20-27, and 23-30), typically with a loss in potency.

However, stapling at position 23-30 with S3 resulted in

subnanomolar potency, similar to that of the native sequence

(

Figure 1). It was also anticipated that longer staples at i, i + 11

and i, i + 15 positions could potentially enhance the proteolytic

stability of the peptides; however, stapling with the length-

matched staples S5 and S6, respectively, adversely aﬀected

activity. In addition, mutations were incorporated into the

PYY1 sequence to further enhance potency of the native

RESULTS AND DISCUSSION

■

peptide (sequence “PYY2”, Figure 1). Given that both diCys

incorporation and stapling at positions 10-17 and 23-30

resulted in the least overall loss of potency for PYY1, these 2

out of the initial 15 stapling positions were tested with the

more active PYY2 sequence. Those PYY2 analogs stapled at

the 10-17 and 23-30 positions were also found to be NPY2R

agonists of equal, or in some cases enhanced, potency.

Interestingly, staples S4 and S7 (which are slightly longer

than S3) were also tolerated at position 10-17. Given this data,

of the initial set of 36 analogs, two stapling positions (10-17

and 23-30) were taken forward in both PYY1 and PYY2

sequences to investigate fatty acid conjugation and incorpo-

ration of serum binding properties for enhanced half-life.

Fatty Acid Conjugation and Optimization. Next, we

attached a PEG-fatty acid moiety onto the staple to enhance

serum protein binding and extend half-life in vivo. A library of

Staple Position Scanning and Length Optimization.

Previously, we have shown that covalent modiﬁcation of two

engineered cysteine residues with a bis-bromoacetyl staple

results in enhanced helicity and stability of alpha-helical

peptide hormones.

was guided by examination of the structure of the homologous

neuropeptide Y (NPY) bound to human G protein-coupled

42−46

The selection of stapling sites on PYY

47,48

neuropeptide Y receptor Y (NPY2R, Figure 1).

Residues

occurring on the face interacting with the receptor (Y20, L24,

Y27, and L28) were avoided when choosing sites for covalent

modiﬁcation so as to minimize disruption of crucial peptide−

receptor interactions. Rapid cleavage at the N-terminus of PYY

by dipeptidyl peptidase-4 (DPP-4) following secretion results

in the truncated peptide PYY₃_‑₃₆(“PYY1” shown in Figure 1)

being the predominant form in circulation. As PYY1 shows

higher speciﬁcity toward the Y receptor subtype than PYY, we

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

to non-fasted male Sprague Dawley rats (n = 3) via femoral

Article

staples was synthesized incorporating a wide variety of PEG

linker and fatty acid types, thus facilitating rapid screening of

conjugates (Figure 2, synthesis detailed in the Supporting

Information). Attachment of the bromoacetyl-functionalized

staples to PYY1 and PYY2 sequences with Cys residues

incorporated at the 10-17 and 23-30 positions was achieved in

solution as previously described (1:1 ammonium bicarbonate

buﬀer/acetonitrile, pH 8.5). Ten-atom staples S8-10 were

initially tested due to the S3 analogs being the most potent in

the previous screen of unlipidated staples.

Table 3. In Vitro CRE Luciferase Assay Activity Data for

PYY Analogs Demonstrating NPY Receptor Subtype

Speciﬁcity (Experimental Details Given in the Experimental

Section)

EC₅₀(nM)

peptide

NPY1R

NPY2R

NPY4R

NPY5R

2000

>10,000

0.20 ± 0.06

1.8 ± 0.4

0.3 ± 0.1

7000

>10,000

410 ± 90

1200 ± 500

>10,000

In vitro activity of the conjugates was assayed as described

previously, and the results are summarized in Table 2. Activity

native PYY1 sequence at position 23-30 with S9 appeared to

aﬀord the best proﬁle in the in vitro assay, with S9 stapling at

position 10-17 being optimal for PYY2.

The “symmetric” staple S11 (Figure 2) was also introduced

to avoid the formation of regioisomers, which can occur upon

stapling with S10. Activity for S11-stapled peptides was shown

to be comparable to that of the corresponding S10-stapled

analogs (Table 4). A clear serum shift was similarly observed

Table 2. In Vitro NPY2R Activation for Stapled PYY

Analogs (i, i + 7) in the Presence (10%) and Absence (0%)

of FBS in the cAMP Assay as Described Above [the Ratio

between EC₅₀Values in the Presence or Absence of FBS

Was Calculated as an Indicator of Serum Binding for a

Variety of Fatty Acid Types/Chain Lengths (Selected

Shown)]

Table 4. In Vitro NPY2R Activation for PYY Analogs

Stapled with “Symmetric” S11 in the cAMP Assay (as

Described Above)

hNPY2R (cAMP)

cysteine

^E^C⁵0 (nM)

(0% FBS)

ratio

10:0% FBS

peptide sequence substitution(s) staple

hNPY2R (cAMP)

ratio

PYY1

PYY2

none

1.2 ± 0.3

2.7 ± 0.3

2.3 ± 0.3

22 ± 7

0.83

1.8

10, 17

23, 30

none

10, 17

23, 30

S10

cysteine

staple/

lipid

^E^C50 (nM)

(0% FBS)

10:0%

FBS

peptide sequence substitution(s)

PYY1

PYY2

none

23, 30

none

10, 17

23, 30

1.2 ± 0.3

7 ± 1

0.83

44 ± 6

7.0

0.7

4.8

S11

1.0 ± 0.1

1.2 ± 0.1

3.1 ± 0.4

40 ± 5

0.29 ± 0.03

0.18 ± 0.03

0.62 ± 0.09

0.85 ± 0.09

1.2 ± 0.1

0.65 ± 0.06

0.44 ± 0.04

0.91 ± 0.08

3.7 ± 0.7

0.29 ± 0.03

0.65 ± 0.05

5 ± 2

1.4

6.4

S11

1.4

0.72

8.4

0.88

S10

for S11-stapled 22, as indicated by dose−response curves in

the presence and absence of serum (Figure 3), implying

enhanced serum binding and longer half-life in vivo.

In Vivo Half-Life. The pharmacokinetic properties of the

conjugates were assessed in vivo in order to determine the half-

life extension eﬀect of the fatty acid moiety (Table 5). Peptides

in sterile saline were administered as a 1 h intravenous infusion

was ﬁrst determined in the absence of serum to avoid any

interference due to sequestration of the fatty acid-conjugated

peptides by serum proteins such as serum albumin present in

the assay. PYY2 demonstrated superior potency over the native

PYY1 sequence as expected, observed previously for the

unlipidated stapled analogs. In general, a large shift was

observed between activity determined in the presence and

absence of serum for staples S9 and S10. This was attributed to

enhanced serum binding in vitro, an eﬀect previously shown to

be indicative of longer in vivo half-life, observed for other

commercially available lipidated peptide therapeutics such as

semaglutide. The peptide conjugates also showed good

selectivity toward the NPY2R receptor over other receptor

subtypes (Table 3), as expected for PYY analogs. The

majority of lipidated analogs were taken forward for

determination of in vivo half-life (see below), selecting based

on the best balance between absolute potency at 0% FBS and

overall shift between 0% and 10% FBS assays, which was

anticipated to provide the optimum serum binding and ideally

the longest half-life. Based on these criteria, stapling of the

Figure 3. PYY analogs stapled with “symmetric” S11, with cAMP

assay concentration−response curves in the presence and absence of

fetal bovine serum (FBS).

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 5. In Vivo Pharmacokinetic Data for PYY Analogs

hNPY2R (cAMP)

peptide

sequence

cysteine substitution(s)

staple

EC₅₀(nM) (0% FBS)

ratio 10:0% FBS

rat T₁_/₂(h)

PYY1

PYY2

10, 17

23, 30

none

10, 17

23, 30

S10

2.3 ± 0.3

22 ± 7

44 ± 6

7.0

4.8

1.4

8.4

6.4

INS

0.45

5.4

INS

2.4

3.9

1.2

INS

1.9

2.0

4.5

1.2 ± 0.1

3.1 ± 0.4

40 ± 5

0.29 ± 0.03

0.62 ± 0.09

0.85 ± 0.09

1.2 ± 0.1

0.91 ± 0.08

3.7 ± 0.7

5 ± 2

S10

S11

vein cannula at a ﬁnal dose of 0.033 mg/kg. Blood samples

were collected at 0.25, 0.5, 0.75, 1, 1.17, 1.33, 1.5, 2, 4, 6, 8, 24,

0, and 48 h after the start of infusion, and the plasma

concentration of the peptides was analyzed via UPLC-MS/MS,

as detailed in the Experimental Section. Despite the substantial

serum shifts previously demonstrated for PYY1 conjugates,

taken to be indicative of enhanced albumin binding, the

longest in vivo half-lives observed were those for the PYY2

analogs. Furthermore, the predicted prolonged half-life for

analogs stapled at the 10-17 position was not borne out in the

in vivo data for either the PYY1 or PYY2 sequence, potentially

due to stapling at this position not being protective against

proteolytic degradation. While PYY2 stapling at position 10-17

retained the greatest activity in vitro, analogs stapled at position

3-30 had the longest half-lives, of up to 15 h in rat, which

correlated with a large in vitro serum shift. For a given

sequence and staple position, staples S9-11, all incorporating

fatty acid moieties bearing carboxylic acid groups, were found

to aﬀord the most favorable pharmacokinetic properties, with

S10-11, decorated with an “internal” carboxylate on the lysine

linker, being superior (see Figure 2 for staple structures).

Although peptide 19 exhibited excellent in vitro potency and

in vivo half-life, it was not taken forward due to the problem of

regioisomer formation previously discussed for staple S10. A

detailed pharmacokinetic proﬁle for the longest-acting

Figure 4. Rat pharmacokinetic data: peptides in sterile saline were

administered via intravenous (IV) infusion to non-fasted male

Sprague Dawley rats (n = 3) via femoral vein cannula at the ﬁnal

dose shown. Blood samples were collected between 0.25 and 48 h

after the start of infusion, and plasma concentration of the peptides

was analyzed via UPLC-MS/MS, as detailed in the Experimental

Section.

intake reduction to peptide 22 dosed at 0.04 mg/kg, the most

robust food intake reduction eﬀect was observed when 0.01

mg/kg 1 was used in combination with 22 at 0.04 and 0.2 mg/

kg, resulting in a 64% and 90% reduction in cumulative food

intake at 24 h, respectively. NPY2R agonists such as PYY₃_‑₃₆

and GLP-1R agonists such as exenatide and liraglutide are

reported to slow gastric emptying and induce conditional taste

aversion; therefore such eﬀects are potentially responsible for

the food intake reduction observed here for either agonist

“

symmetric” stapled analog 22 is shown in Figure 4. Peptide

2 exhibits a greater than 10-fold increase in half-life, with

greatly reduced clearance when compared with the unstapled

sequence (11). This proﬁle is similar to the fatty acid-

conjugated GLP-1R agonist semaglutide, which is dosed once-

weekly in humans. Given the long half-life observed for 22, it

was decided to take this peptide forward into food intake and

body weight reduction models in mice in order to assess its in

vivo eﬃcacy.

Acute Food Intake Reduction Study in Wild-Type

Mice. Given the well-established anorexigenic eﬀect of PYY

administration, a food intake study was carried out in C57BL/

51,52

administered alone or in combination.

A single combined

dose of peptide 1 and 22 (0.01 and 0.2 mg/kg, respectively)

produced a signiﬁcant body weight loss (∼5%) observed at 48

h post dose, indicating a long-acting eﬀect. With this

preliminary data in hand, it was decided that the eﬃcacy of

both analogs (alone and in combination) should be assessed in

a chronic body weight study.

Chronic Body Weight Reduction Study in Obese

Mice. A 2-week chronic study to investigate the eﬀect of daily

administration of peptide 22 on body weight and glucose

homeostasis, either alone or in combination with peptide 1,

was carried out in a diet-induced obesity (DIO) mouse model

(Figure 7). The PYY analog (22) alone at 0.04 and 0.2 mg/kg

wild-type mice using peptide 22 at 0.04 and 0.2 mg/kg (8

and 40 nmol/kg) by subcutaneous injection (SC) (Figure 6).

Peptide 22 was also tested in combination with a previously

published long-acting GLP-1R agonist, peptide 1 (Figure 5).

All groups show considerable reduction in food intake, with

dosing of peptide 22 exhibiting a dose-dependent reduction in

food consumption in the wild-type model. While peptide 1

dosed at 0.01 mg/kg (2 nmol/kg) shows comparable food

J. Med. Chem. XXXX, XXX, XXX−XXX

Figure 5. Full structures of peptides 1 and 22 .

Article

Figure 6. (A) Cumulative food intake in male C57BL/6 mice (n = 6 per group) over 24 h following a single SC dose of peptide or vehicle control

saline); % food intake inhibition relative to vehicle control at 24 h shown on the right side of the graph. (B) Acute change in body weight; %

(

change from initial after 48 h.

demonstrated a dose-dependent reduction in food intake at

day 1 compared to the vehicle control (Figure 7A), although

this eﬀect appeared to diminish over time (day 5, Figure 7B).

As observed in the acute food intake study, peptide 1 alone was

also eﬃcacious; however, the most signiﬁcant body weight

reduction was demonstrated in both combination groups

greater suppressive eﬀects on food consumption compared to

the GLP-1R agonist 1 alone after day 5 of dosing (Figure 7B).

Blood glucose homeostasis at day 14 was evaluated via the

oral glucose tolerance test (OGTT, Figure 7D−F). Treatment

with PYY analog 22 alone at either dose did not have a

signiﬁcant eﬀect on the OGTT result or fasted blood glucose.

While signiﬁcant improvements were observed in the GLP-1R

(

Figure 7C). A dose of 0.01 mg/kg of peptide 1 in

combination with the high dose (0.2 mg/kg) of peptide 22

resulted in nearly 25% body weight reduction after 13 days.

Furthermore, body weight reduction in the combination

groups substantially exceeded the predicted proﬁle based on

additive eﬀects alone (expected additivity plotted), suggesting

a synergistic enhancement of eﬃcacy upon combination

dosing. Similarly, the combination treatment demonstrated

agonist (1) dosing groups, as expected, the combination

groups yielded slightly superior glucose control. The relatively

modest glucose control eﬀect demonstrated was not

unexpected and is potentially due to the somewhat mild

hyperglycemia observed in the prediabetic DIO model. A more

pronounced eﬀect may be observed upon longer treatment

with the combination dose; however, the clear discrimination

J. Med. Chem. XXXX, XXX, XXX−XXX

Article

Figure 7. (A, B) Food consumption 12 h post dose (dark cycle) on (A) day 1 and (B) day 5 in male C57BL/6 DIO mice (n = 6 per group) with

daily SC dosing of peptide or vehicle control (phosphate-buﬀered saline). (C) % change from initial body weight over 13 days. (D) Oral glucose

tolerance test (OGTT) at day 14. (E) AUC for OGTT. (F) Fasted blood glucose at day 14. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001,

relative to vehicle control [one-way ANOVA, or Student’s t-test for (C)].

between those groups dosed with 1 and those without

indicates that it is the eﬀect of the GLP-1R agonist that is

driving the glucose control eﬀect in this study.

reduced ﬁbril formation compared to the unlipidated sequence,

an eﬀect previously reported for lipidated PYY analogs. This

approach serves as the starting point for the development of a

clinical multi-hormone regimen designed to reverse the eﬀects

of obesity and rescue glycemic control. Furthermore, the

engineering approach described is generally applicable to the

translation of bioactive peptides into long-acting molecules

suitable for pharmaceutical development and has found broad

CONCLUSIONS

■

Bariatric surgery has proven to be the most eﬀective, albeit

risky, therapeutic intervention for obesity, providing immediate

improvement in glucose control and a sustained reduction of

body weight. Multi-hormonal therapies utilizing engineered,

long-acting peptide hormones could pharmacologically reca-

pitulate this eﬀect. The peptide engineering strategy presented

here, involving screening for optimal stapling sites followed by

simultaneous stapling and PEG-fatty acid conjugation, aﬀords

potent long-acting PYY analogs. Peptide 22 in particular

demonstrated a signiﬁcantly extended half-life of 14 h in rat.

Further in vivo studies revealed highly favorable food intake

control and signiﬁcant weight loss eﬀect in a chronic eﬃcacy

study in combination with our previously discovered long-

acting GLP-1R agonist 1. Comparison to the approved peptide

therapeutic semaglutide suggests that the observed rodent half-

life is likely to translate to a projected pharmacokinetic proﬁle

in humans suitable for once-weekly dosing. Formulation

optimization for peptide 22, including stability and solubility

screening, is ongoing. In our hands, 22 exhibits favorable

solubility despite lipidation, which may be explained by

42−46,53

application in a variety of therapeutic areas.

EXPERIMENTAL SECTION

■

No unexpected or unusually serious safety hazards were encountered.

Unless otherwise noted, all reagents were purchased from commercial

suppliers (Sigma Aldrich, Fisher, Oakwood) and used without further

puriﬁcation. Peptides were purchased from Cellmano Biotech Limited

(Hefei), InnoPep (San Diego), Shanghai Apeptide Co. (Shanghai) or

Shanghai Dechi Biosciences Co. (Shanghai). Staple S11 was

purchased from WuXi Apptec Co. (Shanghai). All reactions involving

air- or moisture-sensitive reagents or intermediates were performed

under an inert atmosphere of nitrogen or argon. All solvents used

were of HPLC grade. Reactions were monitored by LC-MS or thin-

layer chromatography (TLC) on Merck 50 × 100 mm silica gel 60

aluminum sheets stained using an aqueous solution of KMnO₄.

Peptides were synthesized by standard solid-phase peptide synthesis

(SPPS) techniques and puriﬁed via HPLC (as described below).

Flash chromatography puriﬁcations were performed on silica gel

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

To a solution of 1,2-ethylenediamine (30 μ L, 0.448 mmol, 1 equiv) in

Article

prepacked columns (40 μm, RediSep Rf from Teledyne Isco) on a

CombiFlash Rf (Teledyne Isco). Puriﬁed ﬁnal compounds were

eluted as single and symmetrical peaks (thereby conﬁrming a purity of

to cap the unreacted trityl chloride sites, dried under vacuum, and

stored in a desiccator.

Deprotection of Fmoc Protecting Group (B). To the resin was

added piperidine in DMF (20%). The mixture was shaken for 5 min

and drained. Fresh 20% piperidine was added, and this time the

mixture was shaken for 15 min. Positive ninhydrin and/or TNBS test

was observed. The resin was then washed with DMF (3x), DCM (3x).

Deprotection of ivDde Protecting Group (C). After washing with

DMF and DCM, the resin was treated with 2% hydrazine in DMF (5

mL, 2 × 15 min). Positive ninhydrin and/or TNBS test was observed.

The resin was then washed with DMF (3x) and DCM (3x).

Peptide Coupling (D). The resin was treated with the carboxylic

acid derivative speciﬁed (3 equiv) using coupling reagent HATU (3.3

eq) and DIEA (3.3 eq) in DMF (5 mL) for 2 h or repeated until a

negative ninhydrin and/or TNBS test was observed. The resin was

then washed with DMF (3x) and DCM (3x).

≥

95%). Semi-preparative chromatography were performed on a

Shimadzu HPLC with a Phenomenex Luna column (C18, 100 Å pore

size, 10 μm particle size, 250 × 10.0 mm, ﬂow: 4 mL/min) or an

Agilent 1200 HPLC with a Phenomenex Luna column (C18, 100 Å

pore size, 5 μm particle size, 150 × 21.2 mm, ﬂow: 20 mL/min). H

and ¹³C NMR spectra were recorded on a Bruker 400 system in

DMSO-d , CDCl , or CD OD. Chemical shifts are given in parts per

million (ppm) with tetramethylsilane as an internal standard.

Abbreviations are used as follows: s = singlet, d = doublet, t =

triplet, q = quartet, p = pentet, m = multiplet, dd = doublet of

doublets, br = broad. Coupling constants (J values) are given in hertz

(

Hz). Low-resolution mass spectra were recorded on a Waters

Acquity UPLC with a Phemomenex Luna Omega C18 column (C18,

00 Å pore size, 1.6 μm particle size, 50 × 2.1 mm, ﬂow: 0.4 mL/

min). Solvents: A, H O + 0.1% formic acid; B, MeCN + 0.1% formic

acid; gradient: 0−1 min 10−90% B, 1−1.6 min 90% B, 1.6−1.7 min

0−10% B, and 1.7−2 min 10% B. High-resolution mass spectra

(HRMS) were recorded on an Agilent 1200 Series Accurate Mass

Time-of-Flight (TOF) with an Aeris Widepore column (XB-C8, 3.6

μm particle size, 150 × 2.1 mm, ﬂow: 0.5 mL/min). Solvents: A, H O

0.1% formic acid; B, MeCN + 0.1% formic acid; gradient: 0−2 min

5% B, 2−12 min 5−60% B, 12−13 min 60−80% B, 13−14 min 80−

20% B, 14−15 min 20−80% B, 15−16 min 80−20% B, 16−17 min

20−95% B, 17−20 min 95% B, and 20−21 min 95−5% B.

General Procedure for Bromoacetyl Peptide Stapling/

Conjugation. Peptides were dissolved at a concentration of 2 mM

with 1.5 equiv of bromoacetyl staple in 1:3 (v/v) MeCN/30 mM

NH HCO buﬀer (pH 8.5). The pH of the reaction mixture was

readjusted with ammonium hydroxide to correct the drop in pH

caused by the peptide TFA counterion. More MeCN was added for

particularly insoluble peptides. The reaction was stirred at RT for 2−4

h before acidiﬁcation to pH 5 via dropwise addition of acetic acid.

The resulting solution was lyophilized and puriﬁed by reversed-phase

HPLC.

DCM (5 mL) at 0 °C were added DIEA (172 μ L, 0.985 mmol, 2.2

equiv) followed by bromoacetic anhydride (233 mg, 0.897 mmol, 2

equiv) dissolved in 1 mL of DCM. The reaction mixture was then

stirred for 30 min at 0 °C, 1.5 h at RT, and the solvent was removed.

Puri ﬁ cation by ﬂ ash column chromatography on silica gel provided S1

as a white solid (43.9 mg, 0.145 mmol, 32%); MS (ES ) m /z 302.55

[M + H] ), 304.54 ([M + H] ); H NMR (400 MHz, methanol-d )

δ 2.49 (s, 4H), 2.06 (s, 4H).

Synthesis of Staples S2-6 and S8-10. Synthesis of staples S2

42,43,45

N,N′-(Azanediylbis(ethane-2,1-diyl))bis(2-bromoacetamide)

and S4-6 and S3 and S8-10

was carried out as previously

(

S7). Intermediate S7a .

reported. Brieﬂy, staples S2-6 were synthesized via coupling of the

corresponding diamine and bromoacetic anhydride in solution and

used directly for stapling without further puriﬁcation. Fatty acid-

containing staples S8-10 were synthesized using commercially

available building blocks as follows: S8, coupling of the fatty acid

and mono-Boc-protected diamino-PEG in solution followed by

sequential Boc deprotection (TFA/DCM) and coupling of Boc-

To a solution of tert -butyl bis(2-aminoethyl)carbamate (167 mg, 0.82

mmol, 1 equiv) in DCM (20 mL) at 0 °C were added DIEA (342 μ L,

1.96 mmol, 2.4 equiv) followed by bromoacetic anhydride (426 mg,

.64 mmol, 2 equiv) dissolved in 1 mL of DCM. The reaction mixture

NH-PEG -OH then Boc-Orn(Boc)-OH followed by ﬁnal Boc

deprotection and bromoacetylation of free ornithine amine groups

using bromoacetic anhydride; the ﬁnal staple was puriﬁed using ﬂash

column chromatography, as described; S9, coupling of Boc-Orn-

was then stirred for 30 min at 0 °C, 1.5 h at RT, and the solvent was

removed. Puri ﬁ cation by ﬂ ash column chromatography on silica gel

a ﬀ orded S7a as a white solid (289 mg, 0.65 mmol, 79%); MS (ES )

m /z 445.71 ([M + H] ), 447.7 ([M + H] ); H NMR (400 MHz,

(

Boc)-OH and amino-PEG -azide in solution followed by reduction

of azide (H , Pd/C), coupling of mono-tBu-protected fatty diacid

methanol-d ) δ 3.85 (s, 4H), 3.39 (s, 9H), 1.50 (s, 10H).

followed by ﬁnal Boc deprotection and bromoacetylation;, the ﬁnal

staple was puriﬁed using ﬂash column chromatography, as described;

S10, sequential solid-phase coupling of Fmoc-Lys(ivDde)-OH and

mono-tBu-protected fatty diacid to chlorotrityl chloride resin

S7.

(

protocols A, B, and D below), ivDde deprotection (protocol C

below), sequential coupling of Fmoc-NH-PEG -OH and Fmoc-

Compound S7a (20 mg) was dissolved in TFA/DCM (1:1, v/v, 2

mL), agitated 30 min at RT, and evaporated (co-evaporation with

hexane) to obtain compound S7 as an oil. The product was directly

Orn(Fmoc)-OH (protocols B and D below), deprotection of

ornithine Fmoc groups (protocol B below), and on-resin

bromoacetylation (protocol E below) and cleavage from the solid

support (protocol F below); the ﬁnal staple was puriﬁed using

reversed-phase HPLC, as described.

General Solid-Phase Synthesis Protocols for Staples S10

and S11. Loading of Chlorotrityl Chloride Resin (A). Fmoc-

Lys(ivDde)-OH (60 mg, 100 μmol) was coupled to 2-chlorotrityl

chloride resin (Novabiochem) (100 mg, 80 μmol) by mixing the

amino acid, resin, and DIEA (70 μL, 400 μmol) in 5 mL of DMF and

stirring for 30 min. The resin was then washed with DMF (3x) and

used in further steps; MS (ES ) m /z 345.2 ([M + H] ).

(

S)-1-Bromo-6-(2-(2-bromoacetamido)ethyl)-36-carboxy-

, 7, 10, 20 , 30, 38 -h exaoxo-14, 17 , 24, 27-tetraoxa-

,6,11,21,31,37-hexaazapentapentacontan-55-oic Acid (S11).

Intermediate S11a .

DCM (3x) and treated with CH OH/DCM/DIEA (8:1:1) for 10 min

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

A solution of Fmoc-OSu (131 g, 388 mmol) in DCM (200 mL) was

Article

added dropwise to a solution of diethylenetriamine (20 g, 194 mmol)

cells per well and cultured for 18 h in DMEM with 10% FBS at 37 °C

and 5% CO . Cells were treated with peptide at 12 diﬀerent

in DCM (200 mL) at −40 °C under N and stirred for 2 h. LC-MS

concentrations in triplicate for 24 h and receptor activation was

reported by luminescence intensities using One-Glo (Promega, WI)

luciferase reagent as per the manufacturer’s instructions. The EC₅₀of

each peptide was determined using GraphPad Prism 6 software

(GraphPad, San Diego, CA). Average EC₅₀and standard deviation

(SD) for each peptide were derived from three independent

experiments.

Animal Studies. All animal care and experimental procedures

were approved by the Intarcia Testing Facility Institutional Animal

Care and Use Committee (IACUC) or the IACUC of the California

Institute for Biomedical Research (Calibr) and strictly followed the

NIH guidelines for humane treatment of animals.

showed that the reaction was complete. The crude product in solution

was not puri ﬁ ed and used for the next step directly; H NMR (400

MHz, DMSO-d ) δ 7.88 (d, J = 7.6 Hz, 4H), 7.68 (d, J = 7.6 Hz, 4H),

.43 −7.24 (m, 10H), 4.30 (d, J = 6.4 Hz, 4H), 4.21 (d, J = 6.4 Hz,

H), 3.06 (d, J = 5.6 Hz, 4H), 2.57 (d, J = 7.6 Hz, 4H); MS (ES ) m /

z 548.2 ([M + H] ).

Intermediate S11b .

In Vivo Pharmacokinetic (PK) Study. Peptides were dissolved in

sterile saline and administered as a 1 h intravenous infusion to non-

fasted male Sprague Dawley rats, age 9−20 weeks (median 14 weeks),

body weight 369−554 g (median 460 g) from Charles River

Laboratories (Raleigh, NC), via femoral vein cannula at a ﬁnal dose

of 0.033 mg/kg (n = 3 per compound). All cannulas were externalized

via a three-port Instech vascular access button (Instech Laboratories,

Inc., Plymouth Meeting, PA) located in the rear midscapular region.

Formulations were administered at a rate of 1.67 mL/kg/h. All

animals were singly housed in DiLab caging with free access to food

and water for the duration of the study. Ambient temperature and

humidity were maintained in the study room. The light cycle

consisted of 12 h of light followed by 12 h of darkness. All animals

appeared healthy prior to the start of dosing. Blood samples

To a solution of compound S11a (106 g, 194 mmol) in DCM (400

mL) was added DMAP (4.74 g, 38.8 mmol) and tetrahydrofuran-2,5-

dione (67.9 g, 678 mmol) and stirred at 25 °C for 14 h. LC-MS

showed that the reaction was complete. To the reaction mixture was

added 1 N HCl until pH = 5 −6 and stirred for 15 min, the organic

phase was separated, then the organic phase was washed with water

and saturated NaCl (500 mL) and the aqueous phase was extracted

with DCM (500 mL) twice. The combined DCM was dried over

anhydrous Na SO and concentrated under vacuum. The crude

product was puri ﬁ ed by column chromatography on silica gel using

DCM/MeOH (80:0 −5:1) as an eluent to give compound S11b (57.6

g, 45% yield) as a white solid powder; H NMR (400 MHz, DMSO-

(approximately 250 μL) were collected for pharmacokinetic analysis

d ₆ ) δ 12.09 (s, 1 H), 7.87 (d, J = 7.5 Hz, 4 H), 7.66 (d, J = 7.0 Hz, 4

via a jugular vein cannula at 0.25, 0.5, 0.75, 1, 1.17, 1.33, 1.5, 2, 4, 6, 8,

24, 30, and 48 h post-start of infusion into microtainer tubes

H), 7.23 −7.48 (m, 10 H), 4.24 −4.33 (m, 4 H), 4.14 −4.22 (m, 2 H),

.27 (s, 4 H), 2.95 −3.19 (m, 4 H), 2.37 −2.44 (m, 4 H); MS (ES )

containing K EDTA as an anticoagulant and 25 μL of a protease

m /z 648.2 ([M + H] ).

inhibitor cocktail. Plasma was prepared by centrifugation and stored

at −80 °C until analysis.

S11 (Figure 2). General protocols A, B, D (octadecanedioic acid

mono-tert-butyl ester), C, D (Fmoc-PEG -propionic acid), B, D

PK Plasma Sample Preparation. An aliquot of each plasma

sample was placed into to a 96-well plate. To each well, Tween-20 was

added to a ﬁnal concentration of 0.05%. Plates were then vortex mixed

before three volumes of 0.1% TFA in 2:1 ethanol:acetonitrile

containing an appropriate internal standard was added to each well.

Plates were vortex mixed again and then centrifuged for 10 min at

(

Fmoc-PEG -propionic acid), B, D (compound S11b), and B, E, F.

The crude was puriﬁed by HPLC to aﬀord the product S11 as a white

solid (5.2 g, 11% yield); MS (ES ) m/z 1188.5 ([M + H] ).

In Vitro Assays. Intracellular cAMP Assay. To measure peptide-

induced NPY2R-mediated inhibition of cAMP production, a cAMP

HTRF (cyclic adenosine monophosphate homogeneous time-

resolved ﬂuorescence) assay was performed according to the

manufacturer’s instructions (cAMP, Gs Dynamic kit, Cisbio). Brieﬂy,

cAMP Hunter CHO cells expressing the NPY2R (DiscoveRx) were

seeded overnight in white 384-well plates at 5000 cells per well in

2844g. Supernatants were placed into a clean 96-well plate and

evaporated under a nitrogen stream at 45 °C. Residues were

reconstituted in 20% acetonitrile (aq) containing 0.1% formic acid.

LC-MS Quantiﬁcation of Peptides in Plasma. All calibration

standards were prepared in control rat plasma containing K EDTA

0 μL of F12 medium at 37 °C and 5% CO . The following day, the

and protease inhibitor cocktail. Samples and standards were analyzed

by TurboIonSpray UPLC-MS/MS using a system consisting of a

CTC HTS PAL auto-injector (Leap, Carrboro, NC), an Agilent

Inﬁnity 1290 system with column oven (Palo Alto, CA), a Valco

switching valve (Houston, TX), and either an AB Sciex API 5600

TripleTOF or Sciex API 4000QTrap mass spectrometer (Framing-

ham, MA). Samples were injected onto a 2.1 × 50 mm reverse phase

C18 analytical column, typically a Waters ACQUITY UPLC HSS T3,

medium was removed and replaced with 20 μL of Opti-MEM (Gibco)

in the presence or absence of 10% FBS. Peptides (prepared as 5X

solution in Opti-MEM) at 12 diﬀerent concentrations in triplicate and

forskolin (a direct activator of adenylate cyclase enzyme, ﬁnal

concentration of 10 μM) were added and incubated for 30 min at 37

C. Detection reagent was added and further incubated for 60 min at

room temperature and read on a compatible HTRF reader

PHERAstar). The EC₅₀of each peptide was determined using

(

1.8 μm (Waters Corporation, Milford, MA) or similar. Chromato-

GraphPad Prism 6 software (GraphPad, San Diego, CA). Average

EC₅₀and standard deviation (SD) for each peptide were derived from

three independent experiments.

graphic separation was achieved with a gradient method using water

containing 0.1% formic acid (A) and acetonitrile containing 0.1%

formic acid (B) as a mobile phase. Initial conditions consisted of 95%

A and 5% B. The organic component was increased to 95% B over a

period of 3−4 min, depending on the peptide. Typical ﬂow rates were

600 μL/min. The column temperature was held constant at 40 or 45

°C. Peptides were quantiﬁed my monitoring one or more product

ions produced from a multiply charged parent ion.

Wild-Type Mouse Food Intake Model. C57BL/6 wild-type

male mice (age 15 weeks from Jackson Labs, Bar Harbor, ME)

maintained on regular chow diet were acclimated in reverse light cycle

and administered a single dose of peptide (5 mL/kg) by subcutaneous

injection (n = 6, group housed 2 mice per cage). Food intake was

monitored at 0 (beginning of the dark cycle), 3, 6, 12, and 24 h and

body weight at 0 and 48 h post dose.

CRE-Luciferase Reporter Assay. HEK293 cells were infected with

lentivirus encoding ﬁreﬂy luciferase gene under the control of cAMP

responsive element (CRE) promoter (Qiagen, Netherlands) and then

were selected using 1 μg/mL puromycin (Life Technologies,

Carlsbad) for 1 week. The surviving cells (referred to as CRE-

HEK293) were expanded and then transfected with a G418 selective

mammalian expression plasmid encoding human NPY2R. The

NPY2R plasmid was transfected into CRE-HEK293 cells using

Lipofectamine 2000 and selected with 400 μg/mL geneticin (Life

Technologies, Carlsbad, CA). A single colony stable cell line

overexpressing both CRE-luciferase and NPY2R (HEK293-NPY2R-

CRE) was then established for the in vitro activity assay. HEK293-

NPY2R-CRE cells were seeded in 384-well plates at a density of 5000

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Complete contact information is available at:

Article

Diet-Induced Obesity (DIO) Mouse Body Weight Model.

C57BL/6 diet-induced obesity (DIO) model male mice (age 18

weeks from Jackson Labs, Bar Harbor, ME) maintained on high fat

diet (D12492, 60% fat diet) for 12 weeks were administered a peptide

by daily subcutaneous injection for up to 13 days (n = 6, group

housed 2 mice per cage, regular light cycle). The average body weight

at the beginning of the experiment was 50 g. Mouse body weight was

measured on days 0, 2, 4, 6, 8, 10, 12, and 13. Mice were fasted

overnight prior to the oral glucose tolerance test (OGTT) on day 14

and then dosed with peptide. After 6 h, 1 g of glucose solution per kg

body weight was administered orally, and mouse tail blood glucose

levels were measured before (0 min) and after glucose challenge for 2

h. The data were compared using the unpaired Student’s t-test. Where

appropriate, data were compared using repeated measures or one-way

analysis of variance followed by the Student−Newman−Keuls post

hoc test.

Peter G. Schultz − The Scripps Research Institute, d/b/a Calibr,

a division of Scripps Research, La Jolla, California 92037,

United States; orcid.org/0000-0003-3188-1202

https://pubs.acs.org/10.1021/acs.jmedchem.0c00740

Author Contributions

S.L., E.P., and Z.Z. contributed equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We would like to thank Huafei Zou and Qiangwei Fu for

technical support and useful discussions.

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00740.

■

ABBREVIATIONS USED

■

ANOVA, analysis of variance; AUC, area under the curve;

cAMP, cyclic adenosine monophosphate; CHO, Chinese

hamster ovary; CL, clearance; C_m_a_x, maximum serum

concentration; CRE, cAMP responsive element; DCM,

dichloromethane; DIEA, N,N-diisopropylethylamine; DIO,

diet-induced obesity; DMAP, 4-dimethylaminopyridine;

DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DPP-

Listing of stapled peptides, HPLC and MS data for

peptides, and chronic DIO study initial body weight data

(PDF )

PDB ID: 2DF0 (PDB)

, dipeptidyl peptidase-4; EC , half maximal eﬀective

AUTHOR INFORMATION

Corresponding Author

Weijun Shen − The Scripps Research Institute, d/b/a Calibr, a

division of Scripps Research, La Jolla, California 92037, United

States; orcid.org/0000-0001-8388-729X; Email: wshen@

scripps.edu

■

concentration; EDTA, ethylenediaminetetraacetic acid; ES,

electrospray; FBS, fetal bovine serum; Fmoc, ﬂuorenylmethy-

loxycarbonyl; GLP-1, glucagon-like peptide-1; GLP-1R, GLP-1

receptor; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-

triazolo[4,5-b]pyridinium 3-oxid hexaﬂuorophosphate;

hNPY2R, human NPY2R; HPLC, high-performance liquid

chromatography; HRMS, high-resolution mass spectra; HTRF,

homogeneous time-resolved ﬂuorescence; IACUC, Institu-

tional Animal Care and Use Committee; INS, insigniﬁcant

Authors

Sam Lear − The Scripps Research Institute, d/b/a Calibr, a

division of Scripps Research, La Jolla, California 92037, United

States

Elsa Pﬂimlin − The Scripps Research Institute, d/b/a Calibr, a

division of Scripps Research, La Jolla, California 92037, United

States

Zhihong Zhou − The Scripps Research Institute, d/b/a Calibr, a

division of Scripps Research, La Jolla, California 92037, United

States

David Huang − The Scripps Research Institute, d/b/a Calibr, a

division of Scripps Research, La Jolla, California 92037, United

States

(

half-life too short to be measured); IV, intravenous; ivDde, 1-

4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-methylbutyl; LC-

MS, liquid chromatography−mass spectrometry; MS, mass

spectrometry; MS/MS, tandem mass spectrometry; NMR,

nuclear magnetic resonance; NPY, neuropeptide Y; NPY2R,

neuropeptide Y receptor Y ; OGTT, oral glucose tolerance

test; Orn, ornithine; OSu, N-hydroxysuccinimide ester; PDB,

Protein Data Bank; PEG, polyethylene glycol; PK, pharmaco-

kinetic; PYY, peptide tyrosine tyrosine; RT, room temperature;

SC, subcutaneous; SPPS, solid-phase peptide synthesis; T₁_/₂

half-life; TFA, triﬂuoroacetic acid; TLC, thin-layer chromatog-

raphy; T_m_a_x, time at C_m_a_x; TNBS, 2,4,6-trinitrobenzenesulfonic

acid; TOF, time-of-ﬂight; UPLC, ultra-performance liquid

chromatography

Sharon Weng − Intarcia Therapeutics, Inc., Durham, North

Carolina 27709, United States

Van Nguyen-Tran − The Scripps Research Institute, d/b/a

Calibr, a division of Scripps Research, La Jolla, California

2037, United States

REFERENCES

Sean B. Joseph − The Scripps Research Institute, d/b/a Calibr,

a division of Scripps Research, La Jolla, California 92037,

United States

Shane Roller − The Scripps Research Institute, d/b/a Calibr, a

division of Scripps Research, La Jolla, California 92037, United

States

Scott Peterson − Intarcia Therapeutics, Inc., Durham, North

Carolina 27709, United States

Jing Li − The Scripps Research Institute, d/b/a Calibr, a division

of Scripps Research, La Jolla, California 92037, United States

Matthew Tremblay − The Scripps Research Institute, d/b/a

Calibr, a division of Scripps Research, La Jolla, California

■

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