StSt uctu eructure-Functionunction elationship Relationship

Structure-Function RelationshipSt uctu e unction elationship

Bhimu PatilDirector, Vegetable and Fruit Improvement Center, g p

Professor, Department of Horticultural SciencesTexas A&M UniversityTexas A&M University

O liOutline• Definition• Definition• Descriptors• Limonoids Antifeedant Activity• Limonoids-Antifeedant Activity• Phase II Enzymes inducers

Limonoids– Limonoids– Curcumin

• Quorum Sensing• Quorum Sensing– Limonoids– FlavonoidsFlavonoids

Structure-Activity Relationship (SAR) is a means by which the effect of a drug or bioactive moleculeby which the effect of a drug or bioactive molecule on animal, plant, or the environment can be related

to its molecular structureto its molecular structureor

The relationship between the chemical structure ofThe relationship between the chemical structure of a compound and its biological or pharmacological

activityy




S A R l hSturcture Activity Relationship

• What are factors affecting SAR?• Why Structure matters?

F i fl i SARFactors influencing SAR

• What are the properties that describe a molecule?molecule?

• Molecular structureSh• Shape

• Size• Stereochemical arrangements

Wh SWhy Structure matter

• What are some examples of structure activity relationship?activity relationship?

S iStereoisomers

Isomers which have their atoms connected in the same sequence but differ in the way the atoms are oriented in space - i.e. the difference between two stereoisomer lies only inbetween two stereoisomer lies only in the three dimensional arrangement of atoms. Stereoisomers may beof atoms. Stereoisomers may be classed as cis-trans isomers or optical isomers

How the taste of any material is sensed?

Conventional knowledge

Recent scientific understanding taste ecent scientific unde standing taste

Chandrashekar et al., 2006. Nature:444:288-294

Stereochemistry

+L‐aspartic acid has a flat taste L‐phenylalanine tastes bitter

L‐Aspartame L‐Aspartame

I am

I am bitter!

D‐Aspartame

sweet!

L‐Aspartame :Taste‐200 times more sweeter than sucroseD‐Aspartame: taste bitter

Th f hThe reason for change in taste

L-aspartameSWEET D-aspartame

BITTERBITTER




Li id Vit CLimonoids Vit. C Flavonoids

CarotenoidsPotassiumotass u

PectinFiber

F li A idCoumarins Folic AcidCoumarins

C L dCitrus Limonoids

• 54 known limonoids (Triterpenoids)O22

– Present as

O

O

3 12 1718 20

21 23

• Aglycones • Glucosides

O

OO

O

O

A

A'

B

CD

1

2

5 78

910

11 1314

15

161924

GlucosidesO

Limonin

4

25a25b

5

6

7

Structure of Limonin

1 Li i 19 6 Keto 7β deacet lnomilol

LIMONOIDS AGLYCONES1. Limonin

2. Nomilin

3. Obacunone

4 Deacetylnomilin

19. 6-Keto-7β-deacetylnomilol

20. 6-Keto-7 β-Nominol

21. Isocylocalamin

22. 1-(10-19)Abeo-7 α-acetoxy-10 β-4. Deacetylnomilin

5. Ichangin

6. Deoxylimonin

7 Deoxylimonol

( ) y βhydroxyisoobacunoic acid 3,10-lactone

23. 1-(10-19)Abeo-obacun-9(11)-en-7 α0yl acetate

24. Deacetylnomilinic acid7. Deoxylimonol

8. Limonol

9. Limonyl acetate

10 7α-Obacunyl acetate

24. Deacetylnomilinic acid

25. Nomilinic acid

26. Iso-Obacunoic acid

27. Deoxylimonic acid10. 7α-Obacunyl acetate

11. 7α-Obacunol

12. Ichangensin

13. Citusin

28. Isolimonic acid

29. Epi-iso-obacunoic acid

30. 17-Dehydrolimonoic acid A ring lactone

31 Calaminic acid13. Citusin

14. Calamin

15. Retrocalamin

16. Cyclocalamin

31. Calaminic acid

32. Retrocalaminic acid

33. Cyclocalaminic acid

34. Iso-Obacunoic acid disophenol16. Cyclocalamin

17. Methyl-iso-obacunoate disosphenol

18. Methyl deacetylnomilinate

p

35. Obacunoic acid

36. 19-Hydroxydeacetylnomilinic acid

37. Limonexic acid

Monocarboxylic acids

17-β-D-Glucopyranosides of:

Dicarboxylic acids

17-β-D-Glucopyranosides of:

1. Limonin

2 N ili

10. Nomilinic acid

11 D t l ili i id2. Nomilin

3. Obacunone

4. Ichangensin

11. Deacetylnomilinic acid

12. Obacunoic acid

13. Trans-Obacunoic acidg

5. Deacetylnomilin

6. Ichangin

14. Iso-Obacunoic acid

15. Epi-iso-obacunoic acid

7. Calamin

8. Methyl deacetyl nomilinate

16. Isolimonic acid

17. 19-Hydroxydeacetyl nomilinic acid

9. 6-Keto-7β-deacetylnomilol

Major limonoid found in citrusMajor limonoid found in citrus

Hb = the total maximal number of hydrogen bonds possible either as a H-donor / acceptor.Clogp = calculated octanol/water partition coefficient on the logarithmic scale.

Antifeedant activity of limonoidsLimonin and ten structurally modified limonins were evaluated as antifeedants against 4th instar larvae of the Colorado potato beetle, Leptinotarsa decemlineata Say

Li iLimonin

O

OO

O

O

O

O

O

OH

OO

O

Epilimonol

O

O

OO

O

O

O

O

OH

OO

O

O

Results: The epoxide and furan groups were shown to be essential structural requirements for high antifeedant activity

TetrahydrolimoninDeoxylimoninDeoxylimonol

requirements for high antifeedant activity

Bentley et al., 1988. Entomologia Experimentalis et Applicata. 49: 189-193.

ColorectalBreast Esophageal

SkinProstate

Brain (neuroblastoma) Prostate( )

Mechanism




Phase I Enzymes

C

Pro

CYP’s DNA DAMAGE

ProCarcinogens

Ultimate Phase II Enzyme Carcinogen

GST, QR, UDPGTEXCRETION

+GSH

, Q ,

Conjugated product

H

Conjugated product

(adapted and modified from Wilkinson & Clapper 1997)

Wh h II ?What are phase –II enzymes?

• A group of xenobiotic metabolizing enzymes that are mainly involved in the inactivation and

i f i d h iexcretion of carcinogens and other toxic chemical substances.Hence compounds known to induce phase IIHence compounds known to induce phase-II enzymes are considered to be potential cancer preventive.

Ex: GST( Glutathione S-transferase), QR (Quinone Reductase), UDP-glucuronosyltransferase (UGT), and sulfotransferase (ST)sulfotransferase (ST)

Structure of purified and modified citrus limonoidscitrus limonoids

Perez et al J Agric Food Chem 2009 57 5279 5286Perez et al., J. Agric. Food Chem. 2009, 57, 5279-5286.

GST Activity against 1-Chloro-2,4-dinitrobenzene

Sample stomach intestine liver lungcontrol 0.88 ± 0.07 0.86 ± 0.09 1.78 ± 0.02 0.36 ± 0.08

GST Activity against 1 Chloro 2,4 dinitrobenzene

limonin 0.83 ± 0.13 0.63 ± 0.01 1.63 ± 0.27 0.34 ± 0.05limonin-7-methoxime 0.89 ± 0.18 1.34 ± 0.47 2.36 ± 0.05** 0.36 ± 0.03

d f li i 0 86 0 12 0 79 0 09 1 45 0 54 0 34 0 08defuran limonin 0.86 ± 0.12 0.79 ± 0.09 1.45 ± 0.54 0.34 ± 0.08LG 0.86 ± 0.13 0.75 ± 0.06 1.41 ± 0.08* 0.44 ± 0.09DNAG 1.15 ± 0.38 0.70 ± 0.13 1.35 ± 0.20* 0.60 ± 0.12**

Specific activity (units/mg protein). The results are the means ± SD (n = 4). *indicates statistically significant (p < 0.05) decrease of activity using Student’s t-test. ** indicates statistically significant (p < 0.05) induction of activity using Student’s t-test

Perez et al., J. Agric. Food Chem. 2009, 57, 5279-5286.

LimoninLimonin 7- methoxime

GST activity in different organ homogenates against 4-nitroquinoline 1-oxide, a potent xenobiotic tumorigenic to the lung, esophagus, forestomach, glandular stomach, skin, and other organs. g , , g

The bars indicate the mean ± SD (n = 4). * indicates statistically significant (p < 0.05) decrease in activity. ** indicates statistically significant (p < 0.05) induction using Student’s t-test.


Quinone Reductase ActivityQuinone Reductase ActivitySAMPLE STOMACH INTESTINE LIVER LUNG

control 8.30 ± 1.73 2.33 ± 0.27 0.52 ± 0.04 0.26 ± 0.05control 8.30 ± 1.73 2.33 ± 0.27 0.52 ± 0.04 0.26 ± 0.05

limonin 8.56 ± 1.58 2.47 ± 0.72 0.38 ± 0.10 0.29 ± 0.04

limonin-7-methoxime

10.45 ± 1.12 4.01 ± 1.15 0.85 ± 0.11** 0.34 ± 0.01**

defuran limonin 8.98 ± 0.79 2.00 ± 0.24 0.41 ± 0.02 0.38 ± 0.05**

LG 8.12 ± 2.38 2.45 ± 0.14 0.47 ± 0.06 0.31 ± 0.05

DNAG 9.58 ± 4.07 2.29 ± 0.50 0.23 ± 0.02* 0.28 ± 0.01

Specific activity (units/mg protein). The results are the means ± SD (n = 4). * indicates p y ( g p ) ( )statistically significant (p < 0.05) decrease of activity using Student’s t-test. ** indicates statistically significant (p < 0.05) induction of activity using Student’s t-test.


CurcuminPolyphenolic compound

Chemically DiferuloylmethaneC e ca y e u oy et a e

Rhizomes of Turmeric (Curcuma longa) perennial plant of the ginger familyplant of the ginger family

History of Turmeric• Ayurvedia: Ancient system of health care that is native to

the Indian subcontinent (3000 BC)

• “I have found a plant that has all the qualities of Saffron• I have found a plant that has all the qualities of Saffron, but it is a root” (Marco Polo on Turmeric, 1280 AD)

• Used in India at least since 6000 years

• Europe rediscovered it 700 years ago via Marco Polo and it is used in traditional Brazilian medicine

RECENT CLINICAL STUDIESDose (g/day) Subjects Results References

0.5 – 12g daily,up to 6 months

Patients with pre-neoplasia (25)

Delay of progression – full blown malignancy

Cheng, Anticancer Res. 2001, 21, 2895 –2900.(Taiwan)

2 daily, 6 months/ sulfasalazine

Patients - ulcerative colitis (43)

Prevent relapse Holt, et al., Digest Dis. Sci. 2005, 50, 2191

0.45– 3.6g. daily7 days

Patients with colorectal cancer (23)

Reduction in Serum levels of prostaglandin E-2

Garcea, G. Cancer Epidemiol. BiomarkersPrev. 2005, 14, 120–125.

0.5-12 g / single Healthy volunteers (24) No –toxicity Lao et al., BMC, Complement. Alternate. Med. 2006, 6, 10.

1.1 – 1.65 daily, - Patients -ulcerative Delay in disease Hanai, H., Clin. G t t l H t l

y,3 months colitis/ Crohn’s

disease(10)

yprogression

Gastroenterol. Hepatol. 2006, 4, 1502 –1506.

Structure Function relationship of Curcumin to induce Phase-II enzyme

The study was conduced to understand the S i iStructural requirement to induce Phases-II enzyme (Quinone Reductase) using in murine hepatoma cells.

Structures of curcuminoids tested in this study and their inducer potencies (CD values) in the QR assay in Hepa1c1c7 murine

hepatoma cells

Dinkova-Kostova, A. T. et al. Carcinogenesis 1999 20:911-914; 1

Wh h d l d ?What this study concludes?The presence of two structural elements was found to

be required for high inducer potency:• (i) hydroxyl groups at ortho-position on the

aromatic rings andaromatic rings and• (ii) The β-diketone

LOW TEMP / PH

Diketo form Keto-enol form




103103

1012

Q i

104-106

Quorum sensing

QUORUM SENSINGHow

much is youryour

count?

Q i i d fi d d i d dQuorum sensing is defined as density dependent regulation of gene expression at population l l Th h i i lllevel. The phenomenon was originally identified in marine bacterium Vibrio fisheri.

Diggle et al 2007 Current Biology 17:R907

QUORUM SENSINGQUORUM SENSING

• Cell-cell communication• Multi cellular behavior• Multi-cellular behavior• Signaling molecules

Si li M l lSignaling Molecules

• N-Acylhomoserine Lactones (AHL)• Furanosyl borate dieaster (Autoinducer-2)• Small peptides• Autoinducer-3 (structure unknown)

B

OHHO

O O

HO

OHO

Furanosyl borate diester

LuxN LuxQ

LuxP

LuxU

LuxO

LuxR No Lightg

LuxN LuxQ

LuxP

LuxU

LuxO

LuxR Light




Modified Analogues of Limonoids

O

OOO

OHOO

O

tura

l

d

O

OO

O

O

O

A B

C DA'

O

O

O

OO

O

O

O

OO

O

O GluNa

Mod

ified

O LimoninB

D-furan Limonin Limonin Glucoside

O OModified

O

O

O

OO

OO

O

O

OO

O

O O

NOH

Limonin-7-Oxime

NO CH3

Limonin-7-Methoxime

Antagonistic activity towards cell-cell g ysignaling by Limonin-7-Oxime

Inhibition of AI-1 mediated Cell Signaling by Limonin and Limonin-7-Oxime

506070

on

LimoninLimonin-7-Oxime

Inhibition of AI-2 mediated Cell Signaling by Limonin and Limonin-7-Oxime

60

80

on

01020304050

6.25 12.5 25 50 100

Concentration (μg/ml)

% In

hibi

tio

0

20

40

6.25 12.5 25 50 100Concentration (μg/ml)

% In

hibi

tio

Limonin Limonin-7-Oxime(μg )

Inhibition of Biofilm formation by Limonin and Limonin-7-Oxime

60LimoninLimonin-7-Oxime O

O

O

01020304050

6.25 12.5 25 50 100

% In

hibi

tion

Limonin 7 OximeO

O

O

OO

NOH

Concentration (μg/ml)OH

Limonin-7-Oxime

Antagonistic activity towards cell-cellAntagonistic activity towards cell cell signaling by Limonin-7-Methoxime

Inhibition of AI-1 mediated Cell Signaling by Limonin and Limonin-7-Methoxime

40506070

hibi

tion

LimoninLimonin-7-Methoxime

Inhibition of AI-2 mediated Cell Signaling by Limonin and Limonin-7-Methoxime

40

60

80

ibiti

on

LimoninLimonin-7-Methoxime

0102030

6.25 12.5 25 50 100


% In

h

0

20

40

6.25 12.5 25 50 100


% In

h

Inhibition of Biofilm formation by Limonin and Limonin-7-Methoxime

5060

n

LimoninLimonin-7-Methoxime OO

O

O

01020304050

6.25 12.5 25 50 100

% In

hibi

tion

OOO

NO CH3

Li i 7 M th iConcentration (μg/ml) Limonin-7-Methoxime

Antagonistic activity towards cell-cell g ysignaling by D-furan limonin

Inhibition of AI-1 mediated Cell Signaling by Limonin and D-Furan Limonin

6070

n

LimoninD-Furan Limonin

Inhibition of AI-2 mediated Cell Signaling by Limonin and D-Furan Limonin

60

80

n

01020304050

6.25 12.5 25 50 100

C t ti ( / l)

% In

hibi

tion D Furan Limonin

0

20

40

60

6.25 12.5 25 50 100

C t ti ( / l)

% In

hibi

tion

LimoninConcentration (μg/ml) Concentration (μg/ml)D-Furan Limonin

Inhibition of Biofilm formation by Limonin and D-Furan Limonin

40LimoninD-Furan Limonin OO

OOHO

0

10

20

30

6.25 12.5 25 50 100

% In

hibi

tion

OOO

O

D f Li iConcentration (μg/ml)

D-furan Limonin

Moderate difference in antagonistic activity ff g ytowards cell-cell signaling by Limonin Glucoside

Inhibition of AI-1 mediated Cell Signaling by Limonin and Limonin Glucoside

40506070

ibiti

on

LimoninLimonin Glucoside

Inhibition of AI-2 mediated Cell Signaling by Limonin and Limonin Glucoside

60

80

100

bitio

n

0102030

6.25 12.5 25 50 100


% In

hi

0

20

40

6.25 12.5 25 50 100


% In

hi

LimoninLimonin Glucoside

Inhibition of Biofilm formation by Limonin and Limonin Glucoside

40 LimoninLimonin Glucoside

O

O

0

10

20

30

6.25 12.5 25 50 100

% In

hibi

tion

Limonin GlucosideO

O

OO

O

O Glu

Concentration (μg/ml)Limonin Glucoside

• Modification of 7th position reduces the potency in bioluminescence assays

• However, the same modifications enhanced the potency in biofilm assayspotency in biofilm assays

• Addition of glucose molecule at 17th position reduces g pthe moderate potency in bioluminescence assays as well as in biofilm assays

• Furan ring and 7th position are critical for antagonistic activity against cell-cellfor antagonistic activity against cell cell signaling

• However• However…– Seventh position is important for AI-1 activity

inhibition– Furan ring is probably critical for AI-2 activity

inhibition

B f lBiofilm

• Polysaccharide matrix• Different bacterial cell physiology• Different bacterial cell physiology• Cause recurrence of infection• Resistant/tolerant• Notorious

Adh iAdhesins

• Antigen 43 (Ag43)

• Pilli

• Curli

Effect of Limonin -7-methoxime on ff fdifferent bacterial adhesins

40

50

10

20

30

hibi

tion

-10

0

10

6.25 12.5 25 50 100

% In

h

-20

-10

Concentration (μg/ml)(μg )Pilli Inducing Ag43 Inducing Curli Inducing Curli Non-inducing

Effect of Limonin -7-oxime on different bacterial adhesins

40

2030

40

on

010

6 25 12 5 25 50 100

Inhi

bitio

-30

-20-10 6.25 12.5 25 50 100%

30


Pilli Inducing Ag43 Inducing Curli Inducing Curli Non-inducingPilli Inducing Ag43 Inducing Curli Inducing Curli Non-inducing

C l iConclusion

• Modified limonoids Limonin 7-oxime and limonin 7-methoxime inhibit biofilm by alimonin 7 methoxime inhibit biofilm by a different mechanism.




B f fl idBase structure of flavonoids

3'

O

B18 1'

2' 4'

5'O

A C2

3

7 5'

6'

3

45

6

Presence of double bond between 2-3 fposition (Apigenin)

OH

OHOOH

OH O

OHO

Naringenin

OH O

A i iApigenin

Reduction in antagonistic activity towards cell-g ycell signaling and biofilm formation.

Inhibition of AI-2 mediated cell-cell signaling by Naringenin and Apigenin

100.00

0.00

20.00

40.00

60.00

80.00

% In

hibi

tion

6.25 12.5 25 50 100

Concentration (μg/ml)Naringenin ApigeninInhibition of biofilm formation by Naringenin and

Apigenin

75

on

0

25

50

6 25 12 5 25 50 100

% In

hibi

tio

6.25 12.5 25 50 100

Concentraton (μg/ml)Naringenin Apigenin

Addition of hydroxyl group at positionAddition of hydroxyl group at position 3 (Kaempferol)

OH

OHOHO

OHOOH O

Apigenin

OH O

OHApigenin

Kaempferol

Increase in antagonistic activity towards cell-cell signaling and biofilm formation.

Inhibition of AI-2 mediated cell-cell signaling by Apigenein and Kaempferol

80

100

0

20

40

60

80

% In

hibi

tion

6.25 12.5 25 50 100Concentration (μg/ml)Apigenin Kaempferol

Inhibition of Biofilm formation by Apigenin and Kaempferol

40.00n

0.00

10.00

20.00

30.00

% In

hibi

tion

6.25 12.5 25 50 100

Concentration (μg/ml)Apigenin Kaempferol

Addition of hydroxyl group at position 5’ (Quercetin)

OHO

OH

OHO

OHOH

OH O

OH

Kaempferol

OHOOH

Kaempferol

OH O

OH

Quercetin

I i t i ti ti it t d llIncrease in antagonistic activity towards cell-cell signaling and biofilm formation.

Inhibition of AI-2 mediated cell-cell signaling by Kaempferol and Quercetin

95

100

n

80

85

90

95

% In

hibi

tion

6.25 12.5 25 50 100

Concentration (μg/ml)Kaempferol QuercetinInhibition of Biofilm formation by Kaempferol and

Quercetin

80.00

20.00

40.00

60.00

80.00%

Inhi

bitio

n

0.006.25 12.5 25 50 100

Concentration (μg/ml)Kaempferol Quercetin

Addition of rutinoside at position 3 ddition of utinoside at position 3(Rutin)

OHOH

OHOOH

OHO

OH

OH

OH O

O

OO

OH

OH O

Quercetin

OH

O

OH

OHOHOHOH

Rutin

Reduction in antagonistic activity towards cell-g ycell signaling and biofilm formation.

Inhibition of AI-2 mediated cell-cell signaling by Quercetin and Rutin

80.00

100.00

n

0.00

20.00

40.00

60.00

80.00

% In

hibi

tion

6.25 12.5 25 50 10

Concentration ( μg/ml)Quercetin Rutin Inhibition of Biofilm formation by Quercetin and Rutin

7080

0102030405060

% In

hibi

tion

06.25 12.5 25 50 100

Concentration (μg/ml)Quercetin Rutin

C l iConclusion

Presence of double bond between position 2 and 3 is detrimentalAddition of hydroxyl groups increases the potencyPosition 3 is important for the activity against AI-2 and biofilm 3'against AI 2 and biofilm

O

A

B

C

127

8 1'

2' 4'

5'

6'

3

45

6

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StSt uctu eructure-Functionunction elationship Relationship

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