Synthesis and Characterization of Urofuranoic Acids: In Vivo

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Synthesis and Characterization of Urofuranoic Acids: In Vivo Metabolism of 2-(2-carboxyethyl)-4-methyl-5-propylfuran-3carboxylic acid (CMPF) and Effects on In Vitro Insulin Secretion. Edith Nagy, Ying Liu, Kacey J. Prentice, Kyle W. Sloop, Phillip E. Sanders, Battsetseg Batchuluun, Craig D. Hammond, Michael B. Wheeler, and Timothy B. Durham J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01668 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 8, 2017

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Journal of Medicinal Chemistry

Synthesis and Characterization of Urofuranoic Acids: In Vivo Metabolism of 2-(2-carboxyethyl)4-methyl-5-propylfuran-3-carboxylic acid (CMPF) and Effects on In Vitro Insulin Secretion. Edith Nagy1, Ying Liu2, Kacey J. Prentice2, Kyle W. Sloop1, Phillip E. Sanders1, Battsetseg Batchuluun2, Craig D. Hammond1, Michael B. Wheeler2, and Timothy B. Durham1.* 1

Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, United States.

2

Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada.

KEYWORDS. CMPF, Type 2 Diabetes, Gestational Diabetes, Furanoic Acids, and Urofuranoic Acids, Islets, Beta-cells, Insulin Secretion.

ABSTRACT. CMPF (2-(2-carboxyethyl)-4-methyl-5-propylfuran-3-carboxylic acid) is a metabolite which circulates at high concentrations in Type 2 and gestational diabetes patients.

ACS Paragon Plus Environment

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Further, human clinical studies suggest it might have a causal role in these diseases. CMPF inhibits insulin secretion in mouse and human islets in vitro and in vivo in rodents. However, the metabolic fate of CMPF and the relationship of structure to effects on insulin secretion have not been significantly studied. The synthesis of CMPF and analogues are described. These include isotopically labeled molecules. Study of these materials in vivo has led to the first observation of a metabolite of CMPF. In addition, a wide range of CMPF analogues have been prepared and characterized in insulin secretion assays using both mouse and human islets. Several molecules that influence insulin secretion in vitro were identified. The molecules described should serve as interesting probes to further study the biology of CMPF.

Introduction Diabetes is a significant health crisis.1 In 2012, the US Centers for Disease Control reported 9.3% of the US population as being afflicted, with roughly 40% of those cases being undiagnosed. Diabetes leads to a number of debilitating co-morbidities that have significant impact on the health care system. These morbidities include cardiovascular disease, stroke, neuropathy, blindness, and amputation. Diabetes presents in multiple forms. Type 1 diabetes mellitus (T1DM) is immune system driven and typically occurs in the young. Type 2 diabetes mellitus (T2DM) can occur at any stage of life. Gestational diabetes (GD) occurs during pregnancy. In the US, approximately 9.2% of pregnant women experience GD.2 Typically, GD resolves after delivery of the baby, but in some cases patients progress to T2DM. GD can cause a number of health problems for the mother, including preeclampsia. In some GD cases, the fetus grows larger which leads to


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difficulties during birth. In addition, high birth weight children have a higher risk of obesity, which in turn raises the child’s risk of developing T2DM. Recently, the occurrence of GD and T2DM was shown to correlate with plasma levels of the metabolite 2-(2-carboxyethyl)-4-methyl-5-propylfuran-3-carboxylic acid (1, CMPF, Figure 1).3 Independent studies also showed a strong correlation between elevated levels of CMPF and beta cell dysfunction in women with GD, though others have found less or no correlation of CMPF to impaired glucose tolerance in other human populations.4 CMPF was found at levels around 150 µM in plasma in both pregnant women with GD and T2DM patients.3 Additionally, CMPF was found to dramatically increase in pre-diabetic patients compared to patients with normal glucose tolerance (NGT).3a These observation have led to the proposal that CMPF may have a causative role in GD and T2DM. In the laboratory, treatment of lean,3b DIO,3a or ob/ob3a mice with CMPF leads to impaired glucose clearance during intraperitoneal glucose tolerance test (ipGTT) experiments. Mouse and human islets treated with CMPF show decreased glucose stimulated insulin secretion (GSIS).3b Additionally, mice administered CMPF via intraperitoneal injection had higher numbers of immature insulin granules compared to vehicle treated animals.3a


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O OH OH O

O 1 O OH OH O

O

2 O OH OH O

O

O 3 O OH

OH O OH OH

O

4

Figure 1. Urofuranoic Acids. CMPF enters cells via active transport by organic anion transporters.3b When mouse islets were treated with CMPF, reactive oxygen species (ROS) increased suggesting that CMPF increases oxidative stress in the cell.3b In support of this hypothesis, cells treated with N-acetyl cysteine were protected from the effects of CMPF.3b Ex vivo study of islets from CMPF treated mice shows they have reduced glycolysis and at the same time increased glucose uptake and glycated proteins.3a These ex vivo studies suggest that CMPF alters the islet’s energy production pathways, diminishing cells’ ability to utilize glucose for energy and shifting energy generation to fatty acid oxidation.3a


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CMPF is a urofuranoic acid.5 In humans, CMPF is produced by metabolism of F-acids (Scheme 1).6 F-acids are lipid metabolites produced in plants and believed to enter mammals through the food chain.7 Notably, mammalian metabolism of F-acids appears to differ across species since rats and cows do not metabolize F-acids through oxidation of the C3 methyl. Other urofuranoic acids have also been identified in plasma (ie. Figure 1, compounds 2 and 3).7a,

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CMPF has a very high binding affinity to plasma proteins. For this reason, CMPF has been classified as a uremic toxin.9 Uremic toxins have been linked to chronic kidney disease (CKD).9a, 9b

CKD is a common co-morbidity of diabetes.

O OH

OH O n O m F-Acids m = 2-4; n = 8-12

OH m

O

O

Urofuranoic acids

Scheme 1. Human Metabolism of F-acids. CMPF has been shown to have distinctly different reactivity to oxidants than the parent F-acids.8, 10 For instance, hypochlorite oxidation of CMPF produces dihydroxy furan 4 (Figure 1) which, unlike the typical highly reactive oxidation products of F-acids, can be isolated.11 CMPF has also been shown to react with tert-butyl peroxide to produce a radical species which can be observed by electron paramagnetic resonance (EPR).9c It has been suggested that CMPF radicals could promote reactions in human kidney cells leading to cell damage. Unfortunately, the structure of these species was not able to be determined by EPR.9c Given the data suggesting a potentially causative linkage of CMPF to diabetes, as well as its longstanding connection to CKD, the details of the biology mediated by CMPF are of


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significant interest. The exact mechanism(s) by which CMPF affects glucose metabolism is as of yet unknown. Further, other structurally related urofuranoic acids have been identified, but their potential linkage to diabetes or effects on insulin secretion have not been investigated. Our goal was to use medicinal chemistry approaches to probe CMPF metabolism and explore the effect of structural analogues on islets in order to identify potential tools to support future studies in mechanistic biology. To that end, we report the synthesis of several CMPF analogues and have profiled their effects on insulin secretion in mouse and human islets in vitro. These analogues include a series of putative metabolites of CMPF along with other naturally occurring urofuranoic acid derivatives. Finally, we also made isotopically labeled CMPF analogues to explore the fate of CMPF in vivo. These studies have provided the first observation of a CMPF metabolite in the plasma of a mammal. Chemistry CMPF has been synthesized previously.5a, 12 Both approaches used a Friedel-Crafts acylation to install the C5 propyl chain. To access CMPF and its analogues, we chose to exploit an alternate approach based on the work of Hanson et al13 which was used by Spiteller5b to prepare the naturally occurring urofuranoic acid 2 (Figure 1). In this approach, a β-keto-ester is condensed with an acyloin under Lewis acid promoted conditions to give the tetra-substituted furan. The requisite starting materials are easily prepared by a variety of means. For instance, Meldrum’s acid 6 was acylated with commercially available succinyl chloride 5 and the resulting product refluxed in methanol to provide β-keto-ester 7 (Scheme 2). The necessary acyloins (11) were obtained from commercial sources or were readily prepared by addition of lithiated ethyl vinyl ether to the corresponding aldehyde followed by acid hydrolysis of the enol ether (scheme 2). Refluxing the acyloin (11a-h) with the appropriate β-keto ester (7, 12, or 13) in the presence


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of zinc chloride in methanol provided the urofuranoic acid esters 14-23 in good yield (scheme 3). Hydrolysis of both ester groups was then achieved using forcing conditions. Notably, using this synthetic route we were able to easily make gram quantities of CMPF to support subsequent chemistry and in vivo studies.

Scheme 2.


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Compound

R1

R2

R3

1

nPr

Me

-(CH2)2CO2H

2

nPentyl

Me

-(CH2)2CO2H

14

nPr

Me

-(CH2)2CO2Me

15

nPentyl

Me

-(CH2)2CO2Me

16

Me

Me

-(CH2)2CO2Me

17

Et

Et

-(CH2)2CO2Me

18

nPr

nPr

-(CH2)2CO2Me

19 20

-CH2(CH2)2CH2-CH2CH2OBn

-(CH2)2CO2Me

Me

-(CH2)2CO2Me

21

nPr

Me

-(CH2)3OBn

22

nPr

Me

-(CH2)3OH

23

nPr

Me

nPr

24

Me

Me

-(CH2)2CO2H

25

Et

Et

-(CH2)2CO2H

26

nPr

nPr

-(CH2)2CO2H

27

nPr

Me

-(CH2)3OH

28

-CH2CH2OBn

Me

-(CH2)2CO2H

29

nPr

Me

nPr

Scheme 3.

Additional CMPF analogues were prepared using alternative synthetic approaches (Schemes 4-8). Diketone analogue 31 was prepared from CMPF in three steps via the Weinreb amide intermediate 30 (Scheme 4). CMPF was also decarboxylated using copper/quinolone to give analogue 32 (Scheme 4).


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O

OH

O

O OH

O N

O O

CH3

O

c

a, b N O

O

O 30

1

CH3 O 31

O d

OH O 32

Conditions: a: oxalyl chloride, CH2Cl2; b: MeNOMe-HCl, Et3N, ClCH2CH2Cl, 50% over two steps; c: MeMgBr, THF, 0-45 °C, 50%; d: quinoline, copper, 180 °C, 3h, 45%.

Scheme 4. Naturally occurring keto-analogue 3 was prepared according to the approach described by Pfordt et al (Scheme 5).5a In our hands, Friedel-Crafts acylation of furan 33 proceeded best when using the acid chloride rather than the anhydride. Hydrolysis of ester 34 provided carboxylic acid 3.


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Scheme 5. Furanoic acid 36 was prepared by exploiting the intrinsic chemoselectivty of commercially available dibromide 35 (Scheme 6). The C5 propyl group was installed using a Negishi reaction at room temperature. The resulting bromide was then reacted in a second Negishi reaction at 100 °C. The ester product was then hydrolyzed to furnish acid 36.

Scheme 6. Racemic tetrahydrofuran analogue 42 was prepared using a Lewis acid promoted [3+2] addition reaction. Rhodium promoted cyclopropanantion of 1-pentene with dimethyldiazomalnoate (37) gave diester 38. Tin (IV) chloride promoted [3+2] cycloaddition14 with aldehyde 39 provided the tetrahydrofuran 40 as a 4:1 mixture of diastereomers. Decarboxylation of diester 40 was accomplished using sodium cyanide14 under refluxing conditions to furnish ester 41. Hydrolysis then gave tetrahdyrofuran di-acid 42.


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Scheme 7.

Oxidation products 43 and 4 were synthesized using sodium hypochlorite under buffered conditions (Scheme 8).11 Notably, the oxidation products were stable to silica chromatography. To guard against decomposition, the products were dissolved in DMSO and frozen until needed for testing.

O

O OR2

O OR2

O

NaOCl Acetone pH = 7.4 Buffer

O

OR

OH

OR O

HO 43: R = Me 4: R = H

14: R = Me 1: R = H

Scheme 8. 13

C labeled CMPF analogues were also prepared. We targeted three isotope labeling

patterns to allow suitable opportunity to observe CMPF metabolites in vivo. Analogue 46,


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containing one 13C atom in the propyl chain was prepared from isotopically enriched propionic acid (Scheme 9). Friedel-Crafts acylation of furan 33 followed by hydrolysis gave CMPF analogue 45. Ketone reduction using triethylsilane promoted by BF3-OEt2 provided compound 46 in moderate yield. O C OH

13

a-c

+

33

44 O OH

d

13

C O

OH

O O

45

O OH 13

C

OH

O 46

O

Conditions: a: oxalyl chloride, CH2Cl2; b: AlCl3, ClCH2CH2Cl, 42% over two steps; c: NaOH, MeOH, MW 130 °C, 1 h, 90%; d: Et3SiH, BF3-OEt2, CH2Cl2, reflux, 52%.

Scheme 9. Double labeled analogue 50 was prepared from commercially available 13C succinic acid 47 (Scheme 10). Dehydrative cyclization using acetic anhydride followed by solvolysis gave the succinate mono-ester. Reaction with oxalyl chloride yielded the isotopically enriched acid chloride which was further elaborated by the same approach used in Schemes 2 and 3 for the synthesis of CMPF to provide analogue 50.


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Scheme 10. The final isotopically labeled CMPF analogue 54 was prepared from 13C diethyl malonate (51) (scheme 11). Conversion of the malonate to the mixed β-keto-ester 52 was accomplished in two steps. Keto-ester 52 was then subjected to our standard set of conditions to convert it to labeled CMPF analogue 54.


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Scheme 11. Results and Discussion Our investigations began with studies to explore the potential for metabolism of CMPF in vivo. Plasma concentrations of CMPF in healthy humans range from 20-40 µM. CMPF is excreted as parent in urine in small amounts (~1-5 mg/day). No CMPF conjugates in urine samples from healthy humans have been found.15 We wondered if CMPF might be metabolized through other pathways. In particular, we were interested in seeing if CMPF was being processed through lipid metabolism pathways or alternatively being oxidized in liver.


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To characterize the pharmacokinetics of CMPF, we conducted studies in mice (Figure 2). Because CMPF is believed to be generated via metabolism of F-acids in humans (vide supra), we chose to use IV administration for PK studies rather than oral dosing. IV doses of CMPF produced robust exposures with low volumes of distribution, consistent with a compound that is highly bound to albumin (Table 1). The 3 mg/kg dose produced a C0 of ~130 µM, similar to the plasma levels observed in GD and T2DM patients. CMPF is cleared at a rate well-below hepatic blood flow. Based on these data, we opted to conduct our in vivo metabolism studies using 6 mg/kg doses since we anticipated this would provide a C0 of 200-250 µM, replicating concentrations we intended to use for in vitro work (vide infra).

Figure 2. Plasma Exposure of CMPF in Mice Following IV Administration. Table 1. Pharmacokinetics of CMPF in Mice.

Dose (mg/kg) 1 3

AUC (nM*h) 89300 210000

C0 (nM) 53300 130000

T1/2 (h) 4.5 3.27

Cl (mL/min/kg) 0.66 0.85

Vd (L/kg) 0.2 0.2


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To explore the fate of CMPF in vivo, we chose to use isotopically labeled CMPF. We decided to prepare three CMPF analogues with different label patterns so that we could have the highest likelihood of not only detecting CMPF metabolites, but also determining where modifications were being made. Because we wanted to prepare several analogues and our studies were going to require samples to be shipped between several locations, use of non-radioactive labels was practical. For these studies, CMPF was dosed at 6 mg/kg intraperitoneally for three days. On the fourth and fifth days, labeled molecules 46, 50, and 54 were dosed as 1:1 mixtures of labeled/unlabeled CMPF. On day five, the mice were euthanized 1 h post-dose and plasma, adipose, muscle, and liver samples were collected. Samples were then analyzed by HPLC-MS. Because 1:1 mixtures of labeled/unlabeled CMPF were dosed, any metabolites arising from CMPF would produce distinctive isotope patterns in the MS allowing them to be readily identified. It is important to note that CMPF has a specific fragmentation pattern in MS. The most significant ions observed in negative ESMS of the labeled analogues 46, 50, and 54 are shown in Table 2. These data show that CMPF undergoes preferential loss of the C2 carboxyl during fragmentation. Table 2. Significant Ions Observed in Negative ESMS.

Compound

Ions (m/z)

46 50 54

240, 196, 152 241, 197, 152 241, 196, 152


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In both plasma and tissue samples from treated mice, parent CMPF was the most abundant material observed. However, in plasma samples of treated mice we were able to identify low levels of a glucuronide conjugate of CMPF. MS/MS spectra for the glucuronide product ions derived from the labeled materials are shown in Figure 3. Distinctive peaks at 416/417 m/z from the parent conjugate along with fragmentation peaks arising from CMPF (240/241and 196/197 m/z) and glucuronic acid (193 and 175 m/z) support the assignment of the material as the glucuronide conjugate. No other peaks were observed between 416/417 and 240/241 m/z. Based on the fragmentation pattern, it is not possible to assign which carboxyl group is conjugated to glucuronic acid. To our knowledge, this is the first observation of a metabolite derived from CMPF. Analysis of human samples to determine if the CMPFglucuronide is formed are planned and will be reported as appropriate. Interestingly, no metabolites arising from incorporation of CMPF into lipid biosynthetic pathways (ie, conjugates to glycerol, mono-acylglycerol, or diacylglycerol species) were observed in plasma, muscle, liver or adipose tissues. The study was conducted after several days of dosing to ensure that any metabolic effects caused by refeeding would be observed. In our analysis of the samples, we also did not detect any metabolites derived from oxidative metabolism of CMPF in any compartment. While no other metabolic products were observed besides the CMPF-glucuronide, we cannot exclude that such species are formed given the limit of detection using non-radiolabeled materials.


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Figure 3. MS/MS Spectra of Plasma Glucuronide Molecular Ion. No ions were observed in the regions of the spectra between 250 and 400 m/z. A: Glucuronide derived from compound 50. B: Glucuronide derived from compound 54. C: Glucuronide derived from compound 46. Having explored the fate of CMPF in vivo, we turned our attention to studying the in vitro pharmacology of CMPF. To investigate inhibition of insulin secretion from islets, we prepared several analogues (vide supra). These included other known urofuranoic acids 2 and 3. We also prepared compounds that were designed to allow deconstruction of CMPF (ie


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compounds 27, 29, 31, and 36). We also prepared a series of compounds to explore structural modification at each of the four carbons of the furan. To assess the importance of the furan moiety, we designed tetrahydrofuran 42 as a saturated analogue of CMPF. Molecular modeling of tetrahydrofuran 42 shows that in the low energy conformation all three ring substituents are in pseudo-equatorial orientations, similar to CMPF (Figure 4).

Figure 4. Overlay of Furan 42 and CMPF. Overlay was generated using MOE. Molecules were minimized in a neutral state in the gas phase using a AMBER10 forcefield. Cyan = CMPF. Orange = Compound 42.

With a rich set of analogues in hand, we set out to explore the in vitro properties of our molecules. Our initial profiling approach involved carrying out an assessment of intrinsic cytotoxicity for all of the compounds (Table 3). Here, we chose to use the fold change in caspase 3 and caspase 7 as a measure of intrinsic toxicity. We used two cell lines for our assessment, HEK293 cells and mouse primary hepatocytes. Compounds showing ≥ 2 fold change in caspase 3/7 were considered cytotoxic and were deprioritized for further evaluation. Notably, CMPF (1) showed no change relative to DMSO in either cell line. None of the compounds showed


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cytotoxicity against HEK cells. In mouse hepatocytes, compounds 24, 36, and 42 showed cytotoxicity. Thus we deprioritized these compounds for further follow-up. The difference in toxicity observed could be due to lower levels of expression of transporters in HEK cells.16 Table 3. Cytotoxicity Assay.

Fold Change in Caspase 3/7 Relative to DMSO Mouse Primary Hepatocytes

HEK Cells

Compound Run 1

Run 2

Run 3

Run 1

Run 2

1

0.8

1.1

0.9

2

1.0

1.0

1.0

3

1.1

1.0

1.0

4

1.1

1.0

1.0

15

1.0

1.2

1.1

16

1.0

1.1

1.1

17

0.9

1.0

0.9

18

1.0

1.1

1.1

19

1.1

1.0

1.1

22

1.0

1.1

1.0

24

0.8

1.0

0.9

25

1.0

1.0

1.0

26

1.0

1.0

1.0

27

1.0

1.0

1.0

28

1.1

1.1

NT

29

1.0

2.0

1.5

31

0.9

0.9

0.9

32

1.0

0.9

0.9

34

1.0

1.1

36

1.0

1.2

41

1.0

1.1

NT NT NT

0.8 1.5 1.1 0.9 1.2 0.8 1.0 0.9 0.4 0.6 2.0 1.1 1.8 0.6 1.4 1.4 0.3 0.7 1.5 2.2 1.3

0.7 1.2 1.1 0.9 1.0 0.8 0.9 0.8 0.4 0.6 2.0 1.1 1.6 0.6 NT 1.4 0.3 0.7 NT NT NT


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42

1.0

1.2

NT

43 NT = Not Tested

1.0

0.9

0.9

2.0 0.3

NT 0.3

We next profiled CMPF and a selection of structurally diverse analogues in in vitro assays assessing their microsomal stability to and ability to inhibit cytochrome P450 enzymes (Table 4). CMPF itself did not show detectable levels of degradation in the presence of human or mouse microsomes. Our other analogues had good to modest stability in microsomal preparations, except for primary alcohol 22. CMPF had no detectable inhibition of CYP3A4, CYP2D6, or CYP2C9 at 10 µM. Like CMPF, most of the analogues we evaluated did not show significant inhibition against the CYP enzymes. Table 4. In vitro Metabolism Assessment of CMPF and Select Analogues.

Compound

Human Micorosmal Metabolism (%)

Mouse Micorosmal Metabolism (%)

Human CYP3A4 Inhibition (%)

Human CYP2D6 Inhibition (%)

Human CYP2C9 Inhibition (%)

CMPF (1)

ND

ND

0

0

0

2

5

7

3

3

9

3

ND

ND

3

11

10

15

49

60

59

28

27

16

0

16

22

12

3

19

16

76

48

20

2

22

100

100

0

0

0

25

0

21

0

3

17

26

3

7

0

0

0

27

10

9

2

2

3

29

21

51

13

5

18

31

ND

ND

21

0

0

32

0

20

3

0

9

36

20

0

0

0

0

ND = not determined.


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Compounds were next screened in a mouse islet insulin secretion assay under high glucose conditions to identify which analogues might have effects on islets (Figure 5). Because previous studies have shown CMPF impairs GSIS,3b our initial islet assays were performed in a screening mode using only high glucose conditions (see experimental section for details). The purpose of this assay was to help identify compounds with activity in islets that could be selected for further study. Data are shown as fold change in insulin secretion relative to vehicle treatment for an n=4 experiments. Consistent with previous results, CMPF treated islets showed suppressed insulin secretion. The structurally similar metabolite 2 also showed suppression of insulin secretion (Figure 5). Additionally, analogue 26, in which the C4 substituent has been change to nPr, was also shown to suppress insulin secretion. The two esters 17 and 19 also suppressed insulin secretion. Interestingly, the diacid analogue of 17, compound 25, did not show a meaningful impact on insulin secretion. One possible explanation s is that compound 25 could be inactive in the islet assay due to an inability to penetrate islets. In partial support of this hypothesis, we were unable to measure any passive permeability for compound 25 using a cell permeability assay in MDCK cells (data not shown).


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Figure 5. Insulin Secretion Screening Assay in Mouse Islets. Compounds were tested at 200 µM. The glucose concentration was 20 mM. Y-axis shows fold change relative to vehicle control (dashed line). Values are an average of n = 4 experiments. Error bars indicate standard error of the mean. * = p ≤ 0.05 by the student’s t-test.


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Figure 6. Structures of Compounds Showing Effects in the Mouse Islet Screening Assay. To our surprise, compounds 27, 43, and 4 all showed increased insulin secretion in mouse islets under these assay conditions (Figures 5 and 6). Compounds 27 is a close analogue to CMPF where the C2 carboxyl chain has been reduced to an alcohol. Compounds 43 and 4 are the oxidized CMPF analogues. Based on these results, we chose compounds 4 and 17, which had different responses in the mouse islet screening assay, for more thorough evaluation. Accordingly, we conducted GSIS studies under high and low glucose conditions and KCl stimulated insulin secretion assays in mouse islets (Figure 7). We also measured the intracellular insulin content in islets treated with these compounds. As shown in Figure 7, CMPF (1) displayed suppressed GSIS and KCl


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stimulated insulin secretion but did not change the intracellular insulin content. This is consistent with previous observations from the Wheeler lab which have shown that CMPF treatment of mice increases the amount of immature insulin granules in islets as well as the amount of pro-insulin but had no effect on total insulin content.3a In contrast to what was observed in the screening assay where islets were exposed only to high glucose media (Figure 5), in these assays compound 4 treatment had no significant effect on GSIS, KCl stimulated insulin secretion, or insulin content. Accordingly, we do not know if compounds 27 and 43 will increase insulin secretion in the standard GSIS assay. Compound 17 suppressed GSIS and lowered insulin content in this assay. The observation that compound 17 lowered total insulin content but that CMPF did not suggests the two compounds may act via different mechanisms to reduce GSIS. While the current body of work cannot provide a definitive explanation for this difference, we hypothesize that some of the mechanistic differences may arise from differences in compound reactivity in cells. Like CMPF,9c compound 17 could induce formation of ROS but might do so either to a larger extent or within different microenvironments/compartments of the cell, including the endoplasmic reticulum (ER). Indeed, oxidative and ER stress are have a wide range of effects on protein synthesis and trafficking which are linked to a host of human diseases, including diabetes.17


25


A *

B

Insulin (ng/ g DNA/20 min)

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C

*

Figure 7. Effects of Compounds 1, 4, and 17 in Mouse Islets. All compounds were tested at 200 µM. Error bars indicate standard error of the mean. A: Glucose Stimulated Insulin Secretion assay. Values are the average of a n = 4-5 experiments. * = p

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