Journal: Diabetes, obesity & metabolism

Article Title: A Vitamin B12 Conjugate of Exendin-4 Improves Glucose Tolerance Without Associated Nausea or Hypophagia in Rodents

doi: 10.1111/dom.13222

Figure Lengend Snippet: B12-Ex4 potently suppresses blood glucose in an oral glucose tolerance test but has minimal effects on energy balance control. Food intake and body weight change were measured after SC administration of B12-Ex4. Only the highest dose of drug, 20μg/kg, produced any reduction in feeding (A). No changes in 24h body weight gain were observed as a result of B12-Ex4 administration (B). In a separate experiment, SC injection of B12-Ex4 (0 μg/kg indicated by white circles, 5μg/kg by lighter blue circles, 20μg/kg by darker blue circles) reduced blood glucose in an oral glucose tolerance test from 20-40min after injection (C). *, significantly different from vehicle (p<0.05); &, significantly different from 5 μg/kg (p<0.05). Key in (A) also applies to (B). Data are mean ± SEM.

Article Snippet: Rats (n=4/group) were given IP injection of fluorophore-labeled Ex-4 (Flex; 5μg/kg; 0.0001nM; AnaSpec 51 ), Cy5-B12-Ex4 (5μg/kg; 0.03nM), Cy5-B12 (5μg/kg), or Cy5-B12-Ex4 delivered at an equimolar dose to Flex (0.0001nM).

Techniques: Produced, Injection

Journal: Diabetes, obesity & metabolism

Article Title: A Vitamin B12 Conjugate of Exendin-4 Improves Glucose Tolerance Without Associated Nausea or Hypophagia in Rodents

doi: 10.1111/dom.13222

Figure Lengend Snippet: Systemic administration of Ex4 produces a different profile of metabolic effects than B12-Ex4. In contrast to the potent suppression of blood glucose produced by B12-Ex4, SC injection of Ex4 (0μg/kg indicated by white squares, 5μg/kg by lighter red squares, 20μg/kg by darker red squares) produced a robust hyperglycemic response (A). Food intake (B) and body weight gain (C) were suppressed by SC Ex4. To compare the induction of nausea/malaise by Ex4 with that potentially produced by B12-Ex4, rats were tested for expression of a conditioned taste avoidance (CTA) of a flavor paired with IP injection of B12-Ex4 (5μg/kg), Ex4 (5μg/kg), or LiCl as a positive control (0.15M). The percent acceptance of the drug-paired flavor is shown as a box-and-whiskers plot in (D). Both Ex4 (individual responses represented by black circles, overall group response represented in light gray box) and LiCl (individual responses represented by white circles, overall group response represented in white box) produce avoidance of the drug-paired flavor, as indicated by a reduced acceptance of the flavor. These effects are significantly different from acceptance of the drug-paired flavor in B12-Ex4-treated animals (individual responses represented by white squares, overall group response represented in dark gray box). *, significantly different from vehicle (p<0.05); &, significantly different from all other groups (p<0.05). Key under (B) and (C) applies to both panels. Data in (A-C) are mean ± SEM.

Article Snippet: Rats (n=4/group) were given IP injection of fluorophore-labeled Ex-4 (Flex; 5μg/kg; 0.0001nM; AnaSpec 51 ), Cy5-B12-Ex4 (5μg/kg; 0.03nM), Cy5-B12 (5μg/kg), or Cy5-B12-Ex4 delivered at an equimolar dose to Flex (0.0001nM).

Techniques: Produced, Injection, Expressing, Positive Control

Journal: Diabetes, obesity & metabolism

Article Title: A Vitamin B12 Conjugate of Exendin-4 Improves Glucose Tolerance Without Associated Nausea or Hypophagia in Rodents

doi: 10.1111/dom.13222

Figure Lengend Snippet: Systemic administration of B12-Ex4 or Ex4 suppresses blood glucose in mice. In an intraperitoneal glucose tolerance test, Ex4 (5μg/kg) and B12-Ex4 (dose equimolar to Ex4) suppressed blood glucose levels prior to (t=0min) and after (t=20, 40, 60, 120min) IP glucose administration (A); vehicle versus B12-Ex4: ** p<0.01, *** p<0.001; vehicle versus Ex4: ### p<0.001; B12-Ex4 versus Ex4: §§ p<0.01. Area under the curve analyses from 0-120min (i.e., post-glucose load) show that B12-Ex4 (blue) and Ex4 (red) reduce AUC compared to saline vehicle (white) (B); * p<0.05, *** p<0.001. Data are mean ± SEM.

Article Snippet: Rats (n=4/group) were given IP injection of fluorophore-labeled Ex-4 (Flex; 5μg/kg; 0.0001nM; AnaSpec 51 ), Cy5-B12-Ex4 (5μg/kg; 0.03nM), Cy5-B12 (5μg/kg), or Cy5-B12-Ex4 delivered at an equimolar dose to Flex (0.0001nM).

Techniques:

Journal: Diabetes, obesity & metabolism

Article Title: A Vitamin B12 Conjugate of Exendin-4 Improves Glucose Tolerance Without Associated Nausea or Hypophagia in Rodents

doi: 10.1111/dom.13222

Figure Lengend Snippet: Systemically-delivered fluorescently labeled Ex-4 (Flex) highly penetrates within the DVC, whereas Cy5-B12 and Cy5-B12-Ex4 do not. Images were acquired at 10-20× (A,C,E,G) or 63× (with 2-3× optical zoom) (B,D,F,H) magnifications. Brains were processed for immunohistochemistry to label Flex, Equimolar-Flex, Cy5-B12 and Cy5-B12-Ex4 (yellow), astrocytes (GFAP; green) and neurons (NeuN; red). Sections were counterstained using DAPI (blue) to visualize cell nuclei. (B) Flex and (D) equimolar-Flex immunoreactivity is readily visualized in neurons and astrocytes in the DVC. (F) Cy5-B12 and (H) Cy5-B12-Ex4 are not present either in neurons or in astrocytes within the DVC. AP, area postrema; CC, central canal; DVC, dorsal vagal complex; NTS, nucleus tractus solitarius.

Article Snippet: Rats (n=4/group) were given IP injection of fluorophore-labeled Ex-4 (Flex; 5μg/kg; 0.0001nM; AnaSpec 51 ), Cy5-B12-Ex4 (5μg/kg; 0.03nM), Cy5-B12 (5μg/kg), or Cy5-B12-Ex4 delivered at an equimolar dose to Flex (0.0001nM).

Techniques: Labeling, Immunohistochemistry

Journal: Diabetes, obesity & metabolism

Article Title: A Vitamin B12 Conjugate of Exendin-4 Improves Glucose Tolerance Without Associated Nausea or Hypophagia in Rodents

doi: 10.1111/dom.13222

Figure Lengend Snippet: Systemically-delivered fluorescently labeled Ex-4 (Flex) highly penetrates within the PVN, whereas Cy5-B12 and Cy5-B12-Ex4 do not. Images were acquired at 10-20× (A,C,E,G) or 63× (with 2-3× optical zoom) (B,D,F,H) magnifications. Brains were processed for immunohistochemistry to label Flex, Equimolar-Flex, Cy5-B12 and Cy5-B12-Ex4 (yellow), astrocytes (GFAP; green) and neurons (NeuN; red). Sections were counterstained using DAPI (blue) to visualize cell nuclei. (B) Flex and (D) equimolar-Flex immunoreactivity is readily visualized in neurons in the PVN. (F) Cy5-B12 and (H) Cy5-B12-Ex4 are not present either in neurons or in astrocytes within the PVN. 3V, third ventricle; PVN paraventricular hypothalamic nucleus.

Article Snippet: Rats (n=4/group) were given IP injection of fluorophore-labeled Ex-4 (Flex; 5μg/kg; 0.0001nM; AnaSpec 51 ), Cy5-B12-Ex4 (5μg/kg; 0.03nM), Cy5-B12 (5μg/kg), or Cy5-B12-Ex4 delivered at an equimolar dose to Flex (0.0001nM).

Techniques: Labeling, Immunohistochemistry

Journal: Journal of Medicinal Chemistry

Article Title: Synthesis, Optimization, and Biological Evaluation of Corrinated Conjugates of the GLP-1R Agonist Exendin-4

doi: 10.1021/acs.jmedchem.1c00185

Figure Lengend Snippet: Corrination of Ex4 (Cbi-Ex4) does not prevent GLP-1R agonism in the pancreas but does mitigate agonism in the CNS as tracked by emesis and anorexia. Ex4 alone agonizes GLP-1R populations in both the pancreas and CNS.

Article Snippet: Ex4 K12-azido and Ex40 K40-azido were produced by Genscript (Piscataway, New Jersey, United States).

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Synthesis, Optimization, and Biological Evaluation of Corrinated Conjugates of the GLP-1R Agonist Exendin-4

doi: 10.1021/acs.jmedchem.1c00185

Figure Lengend Snippet: (Left) Structure of the dicyanocobinamide (CN 2 Cbi) starting material, a purple solid prepared via microwave chemistry from B12, with the hydroxyl group boxed used as a site of conjugation to linker series. (Right) Library of linkers used in the conjugation of Cbi to Ex4 peptides. The library of linkers chosen in this study included short hydrophobic alkane chains, amphiphilic PEG, and rigid substituted ethynyl phenyl methanamines, which were coupled to the Cbi hydroxyl group via CDT-mediated amide formation, resulting in Cbi compounds ( 4 – 11 ) with an available alkyne group for subsequent reaction with azido-modified Ex4 peptides via copper-mediated alkyne-azide click chemistry ( Scheme ).

Article Snippet: Ex4 K12-azido and Ex40 K40-azido were produced by Genscript (Piscataway, New Jersey, United States).

Techniques: Conjugation Assay, Modification

Journal: Journal of Medicinal Chemistry

Article Title: Synthesis, Optimization, and Biological Evaluation of Corrinated Conjugates of the GLP-1R Agonist Exendin-4

doi: 10.1021/acs.jmedchem.1c00185

Figure Lengend Snippet: Effect of Cbi conjugation on the secondary structure of Ex4 or Ex40. CD spectra were collected with a sample concentration of 40 μM at pH 7 between 200 and 250 nm. % helicity was measured at 222 nm.

Article Snippet: Ex4 K12-azido and Ex40 K40-azido were produced by Genscript (Piscataway, New Jersey, United States).

Techniques: Conjugation Assay, Circular Dichroism, Concentration Assay

Journal: Journal of Medicinal Chemistry

Article Title: Synthesis, Optimization, and Biological Evaluation of Corrinated Conjugates of the GLP-1R Agonist Exendin-4

doi: 10.1021/acs.jmedchem.1c00185

Figure Lengend Snippet: Conjugation of Cbi to Ex4 or Ex40 peptides maintains agonism at the GLP-1R. Nonlinear regression analysis was performed with GraphPad Prism 8. All compounds were assayed at least as triplicate independent runs. Data are shown as mean ± SEM.

Article Snippet: Ex4 K12-azido and Ex40 K40-azido were produced by Genscript (Piscataway, New Jersey, United States).

Techniques: Conjugation Assay

Journal: Journal of Medicinal Chemistry

Article Title: Synthesis, Optimization, and Biological Evaluation of Corrinated Conjugates of the GLP-1R Agonist Exendin-4

doi: 10.1021/acs.jmedchem.1c00185

Figure Lengend Snippet: 22 increases glucose-stimulated insulin secretion in rat islets relative to glucose controls. Insulin secretion rate from static cultures of Sprague–Dawley rat islets incubated in media containing glucose (10 mM) and Ex4 (10 or 50 nM) or 22 (10 or 50 nM). Data was calculated from three independent experiments and analyzed with repeated-measurements two-way ANOVA followed by Tukey’s posthoc test. Results are expressed as mean ± SEM, **** p < 0.0001.

Article Snippet: Ex4 K12-azido and Ex40 K40-azido were produced by Genscript (Piscataway, New Jersey, United States).

Techniques: Incubation

Journal: Journal of Medicinal Chemistry

Article Title: Synthesis, Optimization, and Biological Evaluation of Corrinated Conjugates of the GLP-1R Agonist Exendin-4

doi: 10.1021/acs.jmedchem.1c00185

Figure Lengend Snippet: Cbi-Ex4 enhances glucose clearance without inducing emesis or body weight loss. (A) In an IPGTT, Ex4, 1 , and 22 (50 nmol/kg, IP) showed similar potency in suppressing BG levels after glucose administration (2 g/kg, IP) compared to saline; vehicle vs 1 : *** P < 0.001; vehicle vs 22 : ### P < 0.001; vehicle vs Ex4: §§§ P < 0.001; 1 vs 22 : ΦΦ P < 0.01 ( n = 12 shrews). (B) Area under the curve (AUC) analysis from 0 (i.e., post-glucose bolus) to 60 min following 1 , 22 , and Ex4. (C) AUC analysis from 0 to 120 min; 22 and Ex4 similarly reduced AUCs compared to vehicle ( P < 0.05). (D) Ex4 and 22 (5 nmol/kg, IP) induced anorexia at 6, 24, and 48 h (and at 72 h for Ex4 only), whereas 1 had no effect on food intake ( n = 10). (E) Ex4-induced anorexia was accompanied by significant body weight loss at 24 h. No significant changes in body weight occurred after 1 and 22 administration compared to controls. (F) The number of single emetic episodes following Ex4, 1 , 22 , or saline systemic administration was recorded for 120 min. The number of animals exhibiting emesis, expressed as a fraction of the total number of animals tested, is indicated above each treatment group. Ex4 induced robust emetic responses that were not observed after 1 or saline injections. 22 induced emesis in two of the animals tested; however, the number of emetic episodes was significantly lower than that of Ex4 and did not differ from animals treated with vehicle or 1 ( n = 10). (G) Heatmaps showing latency, number, and intensity of emesis following Ex4, 1 , and 22 dosing for each individual animal across time. All data expressed as mean ± SEM ( n = 10). Data in panels (A), (D), and (E) were analyzed with repeated-measurements two-way ANOVA followed by Tukey’s posthoc test. Data in panels (B), (C), and (F) were analyzed with repeated-measurements one-way ANOVA followed by Tukey’s posthoc test. Means with different letters are significantly different ( P < 0.05).

Article Snippet: Ex4 K12-azido and Ex40 K40-azido were produced by Genscript (Piscataway, New Jersey, United States).

Techniques: Saline

Journal: Journal of Medicinal Chemistry

Article Title: Synthesis, Optimization, and Biological Evaluation of Corrinated Conjugates of the GLP-1R Agonist Exendin-4

doi: 10.1021/acs.jmedchem.1c00185

Figure Lengend Snippet: EC 50 and IC 50 Values with Hill Slopes and % Helicity of Cbi Conjugates 12 – 27

Article Snippet: Ex4 K12-azido and Ex40 K40-azido were produced by Genscript (Piscataway, New Jersey, United States).

Techniques: