Allomed Pharmaceuticals

D-Chiro-Inositol and its Role in Diabetes

and Insulin Resistance

Joseph Larner

The Search for an Insulin Mediator  • Two Enzyme Bioassays

• Initial Mediator Hypothesis and Search• Inositol Glycans Isolated from Rat Liver• Scale up to Beef Liver• Isolate and Determine Novel Structure of Pinitol Galactosamine Pseudodisaccharide Mn++ Chelate

o Bioactivity of Synthetic Pseudodisaccharide• Theoretical Docking and Allosteric Mechanism of Action of Pseudodisaccharide to Activate PP2C• Pathophysiology of Inositol Imbalance and Relation to Insulin Resistance• Epimerization Defect• Repletion with D-Chiro-Inositol• Future New Possible Therapeutic Agents:

o Dibutyryl D-Chiro-Inositolo Pseudodisaccharideo Modified Pseudodisaccharide

PD

H (

% M

axim

um

Act

ivit

y)

TLC chromatogram of 2 purified inositol glycans from rat liver. TLC plate developed in isopropanol:pyridine:acetic acid:H2O (8:8:1:4) sprayed with ninhydrin. Lanes 1 and 4, galactosamine standards; Lane 5, PDH phosphatase stimulator from control rat; Lane 6, PDH phosphatase stimulator from insulin treated rat; Lane 2, cAMP-kinase inhibitor from control rat; Lane 3, cAMP-kinase inhibitor from insulin treated rat.

TLC analysis of silica column fractions from beef liver. Aliquots of fractions 1,2,3 eluted from silica column, stained with ninhydrin together with a galactosamine standard are shown. Solvent used contained pyridine: isopropanol:acetic acid:water (8:8:1:4). Lane 1 – galactosamine standard; lanes 2,3,4 fractions 1,2,3 eluated from the silica column. Note the higher Rf peach staining

spots in lanes 2,3 most prominent in lane 3 separated from the heavier blue ninhydrin staining peptide material in lane 4, fraction 3.

1 2 3 4Allomed Pharmaceuticals

Allomed

Pharmaceuticals 0 15 30 45 60 75 90 105 120 135

40

60

80

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120

Saline (n=6)

-INS-2 (n=4)

-INS-2 (n=6)

***

Time (min)

Pla

sm

a G

luco

se (

% o

f Z

ero

Tim

e)

Allomed

Pharmaceuticals 0 20 40 60 80 100 120 140

0

20

40

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120

140

* +

*

+

oo

*

+

*

*

*

**

***

*

Saline (n=10)

INS-2 2 mg/kg (n=6)

INS-2 8 mg/kg (n=6)INS-2 20 mg/kg (n=5)

o

Time (min)

Pla

sm

a G

luc

os

e (

% o

f Z

ero

Tim

e)

10 -5 10 -4 10 -3 10 -2 10 -1 1 100

10000

20000

30000

40000

50000

* * **

* *

INS2 alone

INS2 with 10nM Insulin

INS-2 Concentration (mM)

[14 C

] G

luc

os

e I

nc

orp

ora

tio

n i

nto

[1

4 C]

Gly

co

ge

n (

CP

M)

Allomed Pharmaceuticals

10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 1 10 1000

10

20

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40

50

60

70

*

**

INS2

Mn++

INS2-Mn++

Basal

INS-2 Mn++ Chelate or Mn++ Concentration(mM)

PD

H P

ho

sp

ha

tas

e R

ea

cti

on

Ve

loc

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(a

rbit

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its

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Allomed Pharmaceuticals

Allosteric site

Catalytic site

Pseudodisaccharide

Phosphorylated peptide substrate

Pseudodisaccharide

Asp 243

ANALYSIS OF INS2 BINDING INTERACTIONS

Using FlexX to find the highest affinity binding conformation, Sybyl was then used to analyze the hydrogen bonding of this conformation of INS2 to PP2C. As we expected, INS2 has strong hydrogen bonding interactions with Asp 243 from two points in the molecule. It was also found that INS2 had a similar interaction with Asp 163. The site mutagenesis data at 163 showing 50% reduced activity upon the removal of this residue helps to support our binding hypothesis for INS2.

Mutations in catalytic site

Mutations in allosteric site

SITE POINT MUTAGENESIS OF PP2C

Pathophysiology of Inositol Imbalance and

Relation to Insulin Resistance

Control Subjects

Type II Diabetic Subjects

Urin

ary

Exc

retio

n of

Ino

sito

ls(µ

mol

/day

± S

EM

)

UVA Pima Indian Subjects

Urin

ary

Inos

itol E

xcre

tion

(nm

ol/d

ay)

Wistar Rats GK rats

Myo-Inositol to Chiro-Inositol

Epimerization Defect

• Conversion of Myo-Inositol to

Chiro-Inositol is defective

in diabetes.

Future New Possible Therapeutic Agents

-13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -30

20

40

60

80

100 Diabetic + Saline

Diabetic + D-chiro-inositol

Euglycemic + Saline

*#

*#*

*

#*

Acetylcholine (log M)

Rel

axat

ion

(%

Ph

e)

-10 -9 -8 -7 -6 -5 -4 -3 -20

20

40

60

80

100

Diabetic + salineDiabetic + D-chiro-inositol

* * * ** *

Normoglycemic

Acetylcholine (- Log M)

Per

fusi

on P

ress

ure

(% P

he)

-17 -16 -15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -50

20

40

60

80

100

Diabetic + saline

Diabetic + D-chiro-inositol

Normoglycemic

*

Sodium Nitroprusside (log M)

Rel

axat

ion

(%Ph

e)

0.1 1 10 100 10000

20

40

60

80

100

Diabetic + saline

Diabetic + D-chiro-inositol

Normoglycemic

*

NO (M)

Rel

axat

ion

(%Ph

e)

-11 -10 -9 -8 -7 -6 -5 -4 -3-100

-75

-50

-25

0

Euglycemic + vehicleDiabetic + vehicleDiabetic + Allo-1

** * *

* * *

Acetylcholine (Log M)

Dec

reas

e in

Per

fusi

on P

ress

ure

(%)

Suppression by D-Chiro-Inositol and Allo-1 of ROSconcentrations in Bovine Aortic Endothelial Cells

Incubated in the presence of 30 mM Glucose

5 m

M G

luco

se

30 m

M G

luco

se

20 µ

M C

hir

o

50 µ

M C

hir

o

100

µM

Ch

iro

20 µ

M A

llo

-1

50 µ

M A

llo

-1

100

µM

All

o-1

0

50

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250

*

* **

* *

**

RO

S (

nm

ole

s/m

L)