Pharmacology of lipid related diseases

Pharmacology of lipid related diseases

Rishi R SapkotaNew Mexico State University

Outline• Introduction to lipids and lipidomics• Cardiovascular diseases and therapeutic strategies• Lipid storage diseases and therapeutic strategies• Lipid post-translational modifications and therapeutic strategies

Prenylation Meristoylation Palmitoylation

• Summary

Lipids and lipidomicsLipids: Hydrophobic organic molecules soluble in organic solventsLipidomics: System-level analysis and characterization of lipids and their interacting moieties.

Fahy et al., Journal of Lipid research, 2005, 46, 839-861

Lipids and Lipidomics• Systematic study of lipids started in 2003- NIH funded “Glue Grant”• PI-Professor Edward A. Denis at University of California, San Diego and

12 co-investigators from different institutions. • lipid metabolism and the active role lipids play in diseases in order to

facilitate development of novel therapeutics • The soft ionization techniques- ES-MS, MALDI( Matrix assisted laser

desorption ionization)- Helped to analyze the biological sample directly- Database is generated.

• ‘lipid MAPS’ (http://www.lipidmaps.org/)

http://www.lipidmaps.org

Examples of lipidsCategory abbreviation Examples

Fatty acyls FA 1-dodecanoic acid

Glycerolipids GL 1-hexadecanoyl-2-(9Z-octadecenoyl)-syn-glycerol

Glycerophospholipids

GP 1-Hexadecanoyl-2-(9Zoctadecenoyl)-sn-glycero-3-phosphocholine

Sphingolipids SP N-(tetradecanoyl)-shing-4-enine

Sterol lipids ST Cholest-5-en-3β-ol

Prenol Lipids PR 2E,6E-farnesol

Saccharolipids SL UDP-3-O-(3R-hydroxy-tetradecanoyl)-αD-N-acetylglucosamine

Polyketides PK aflatoxin


Examples


OOH

O

O(c) Glycerophospholipid: 1-hexadecanoyl-2-(9Z-octadecanoyl)-sn-glycero-3-phosphocholine

POO

OH

N

O

OHH

NH

O(d) Sphingolipid: N-(tetradecanoyl)-sphing-4-enine

HO H

HH

HHO

(e) Sterol Lipids: cholest-5-en-3-ol

OH

(f) Prenol Lipids: 2E, 6E-farnesol

O

OO

H

HO

O O

(h) Polyketides: aflatoxin B1

OHOO

O

OH

HNO

OPO

OOH

P OOH

O

O

OHOH

N

NH

O

O

HO

(g)Saccharolipids: UDP-3-O-(3R-hydroxytetradecanoyl)-D-N-acetylglucosamine


0

1000

2000

3000

4000

5000

6000

7000

8000

FA GL GP SP ST PR SL PK

Lipids per category in LMSD (10/21/10)

Total: 22,500

E. Fahy 2010 ©

Fatty acyls FA

Glycerolipids GL

Glycerophospholipids GP

Sphingolipids SP

Sterol lipids ST

Prenol Lipids PR

Saccharolipids SL

Polyketides PK

Markus. R Wenk, Cell, 2010,143, 888-895

Acetyl CoA

Isopentenyl-PP

O OP

HO

O

OHP

HO

O

O

SCoA

FATTY ACIDSR

O

OH

STEROLS

PRENOLS

O

O

H3CO

H3CO

n

H

H

H

HO

H

H

LIPID BIOSYNTHETIC NETWORKS

H

H

H

O

H

HO

R

SPHINGOLIPIDS GLYCEROPHOSPHOLIPIDS GLYCEROLIPIDS

EICOSANOIDS

ONH

OHH

R

O

HX O

HOO O X

P-O

O

O

O

R1

R2

O

O

HO

O

R2

R1 O R3

O

O

OH

OH

O

HO

E. Fahy 2010 ©

Functions of lipids• Source of energy –triacyl glycerol(TAG)• Thermal insulation- TAG• Structure of cell membrane – phospholipids and cholesterol• Regulatory and co-enzyme activity- fat soluble vitamins, eicosanoids, steroid

hormones• Homeostasis- prostaglandin and steroid hormones• Growth regulation – fatty acids• Signal transduction process – ( E-signaling, T-cell and B-cell antigen receptor

signaling, epidermal growth factor receptor signaling and insulin signaling ) – lipid rafts

Eljamic S. Lipid biochemistry for Medical Science, iUniverse, 2015

Imbalance of lipid and lipid signaling pathways

Berger, E.G.; Smesny, S.; Amminger, G. P. International Review of Psychiatry. 2006, 18(2), 85-98 Miranda, J. P.; Defronzo, A. R.; Callif, M. R.; Guyton, R. J. American Heart Journal. 2005, 149 ,33-45

Altered lipid profiles have been associated with a variety of different metabolic diseases and cancers. Cardiovascular disease is one of the most thoroughly studied lipid related disease.

Cardiovascular diseases• Major cause – increased cholesterol (a sterol lipid) – essential

constituents of mammalian cell membrane- precursor of many steroidal compounds including oxysterol and bile acid.

• Dysregulation of cholesterol- cardiovascular disorders, developmental defects, lysosomal storage syndromes, neurodegeneration and atherosclerosis- promoted by LDL

• 3-hydroxy-3-methyl-glutaryl-Coenzyme A reductase (HMG-CoA)-rate limiting enzyme in the cholesterol biosynthesis pathway

List of scientist who won nobel prize and are involved in cholesterol research1 Albert Szent-Gyorgyi, M.D. 1937

2 Carl Cori, M.D. 1947

3 Bernardo Houssay, M.D. 1947

4 Earl Sutherland, M.D

1971

5 Edwin Krebs, M.D. 1982

6 Joseph Goldstein, M.D. 1985

7 Michael Brown, M.D. 1985

8 Robert Furchgott, Ph.D. 1998

9 Peter Agre, M.D. 2003

10 Mario Capecchi, Ph.D. 2007

11 Martin Chalfie, Ph.D 2008

12 Ralph Steinman, M.D. 2011

13 Robert Lefkowitz, M.D. 2012

Treatment approaches • Inhibitor of HMG-CoA reductase- Statin drugs• Block the absorption of cholesterol in the small intestine• HDL appoprotein expression • PCSK9 target

HMG-CoA-Reductase Mevalonate

First Approach

Inhibit the synthesis of cholesterolFDA approved in 1997125 billion over 14 yearsTop 36th best selling drug in 2014

Rey, K. Journal of Lipid Research. 2013, 54, 2031-2033http://www.crainsnewyork.com/article/20111228/HEALTH_CARE/111229902/lipitor-becomes-worlds-top-selling-drug http://www.pmlive.com/top_pharma_list/pharmaceutical_products/lipitorPhan, B. P.; Dayspring, T. D.; Toth, P. P. Vasc Health Risk Manag. 2012, 8, 415–427http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm?fuseaction=Search.Label_ApprovalHistory#apphist

HMG-CoA

Ezetimibe blocks the absorption of cholesterol in the small intestine. The mechanism of action is believed to involve interaction with the Niemann-Pick C1-like 1 (NPC1L1) protein(sterol transporter protein), and results in accumulation in the brush border region of the small intestine.

Second Approach

Rey, K. Journal of Lipid Research. 2013, 54, 2031-2033http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm?fuseaction=Search.Label_ApprovalHistory#apphist

Fenofibrate activates and increases expression of HDL apolipoprotein, and also increases cellularfatty acid uptake β- oxidation pathway FDA approved in 2002

Third Approach

Erin Crosseya, Marcelo J.A. Amarb, Maureen Sampsonb, Julianne Peabodya, John T. Schillerc, Bryce Chackeriana, Alan T. Remaley, Vaccine, 33 ,2015, 5747–5755

Proprotein convertase subtilisin/kexin type 9 (PCSK9) was identified as a target for the treatment of hypercholesterolemia, atherosclerosis and early cardiovascular disease

Fourth Approach

http://www.pharmacodia.com/web/drug/1_963.html

Alirocumab and Evolocumab monoclonal antibody based drugs-FDA approved in July and August 2015 respectivelyExpensive!

More economic VLP vaccine are under development.

Crossey, E.; Amber, M.; Sampson, M.; Peabody, J.; Schiller, J.T.; Chackerian, B.; Remaley, T. A. Vaccine, 2015, 33, 43, 5747-5755http://www.medscape.com/viewarticle/850715

Lipids Storage Diseases• Lipid storage diseases also known as "Lipidoses" are a group of

inherited autosomal recessive metabolic disorders-accumulates lipids into various tissues and organs.

• Gaucher’s disease is one of the most prevalent- 1 in 20,000 in the world (National Gaucher’s Foundation)

• Glucocerebrosidase is responsible for breaking down of excess of lipids is not produced enough.

Treatment

Deegan, P. B.; Cox, T. M. Drug Des. Devel. Ther. 2012, 6, 81–106http://www.gaucherdisease.org/actelion_drug_information.phpa) http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm410585.htmb) Hughes, D. A.; Pastores, G. M. Lancet. 2015, 385, 2328-2330

Enzyme replacement therapy

Algucerase 1987Genzyme

Imiglugucerase 1994Genzyme

Velagucerase 2010Shire

Taliglucerase 2012Protalix

Miglustat

Substrate Reduction Therapy, inhibit the glucoceramide synthaseFDA approved 2003

Deegan, P. B.; Cox, T. M. Drug Des. Devel. Ther. 2012, 6, 81–106http://www.gaucherdisease.org/actelion_drug_information.php http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm410585.htmHughes, D. A.; Pastores, G. M. Lancet. 2015, 385, 2328-2330

Eliglustat

Selective inhibitor of glucosylceramide synthaseFDA approved in 2014- oral drug

Deegan, P. B.; Cox, T. M. Drug Des. Devel. Ther. 2012, 6, 81–106http://www.gaucherdisease.org/actelion_drug_information.php http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm410585.htmHughes, D. A.; Pastores, G. M. Lancet. 2015, 385, 2328-2330

Post-translational lipid modification of protein • The modification of proteins after or during protein synthesis by the

biochemical attachment of different chemical entities is called post-translational modification and it is important for the localization and activity of a variety of enzymes

• Attachment of chemical species such as methyl, glutathione, and phosphate groups as well as small protein ubiquitin, carbohydrates, and lipids takes place during PTMs.

• post-translational modification of proteins with lipids typically occurs in the cytoplasm or on the cytoplasmic face of the membranes, and in the lumen of the secretory pathway

Howard C. Hang; Published in: Maurine E. Linder; Chem. Rev. 2011, 111, 6341-6358.

Isoprenylation• Also called prenylation-first discovered in fungi in 1978, 1988

mammalian Lamin B was identified as farnesylated.

• About 100 human proteins are identified as substrate for prenylation.

• Responsible for correct cellular localization, activity, and protein–

protein interactions of a number of signaling proteins.

ACS Chem. Biol., 2015, 10 (1), pp 51–62

Isoprenylation involves the addition of 15 carbon farnesyl or the 20 carbon geranylgeranyl groups to thiol cysteine amino acid in the protein –irreversible covalent modifications

ACS Chem. Biol., 2015, 10 (1), pp 51–62

Ras oncogenes H-, N- and K-ras, the Rho and Rab families of GTPases, the nuclear lamins, retinal cGMP phosphodiesterase, rhodopsin kinase and the gamma subunit of the heterotrimeric G proteins

ACS Chem. Biol., 2015, 10 (1), pp 51–62

FPP

GGPP

Farnesylation

Geranylgeranylation

C3

C1H H

...............................................

N

N

N

OO

O

O

O

NHPEPTIDE

S

.. ....

..... .

.. .

Zn2+

PO

OOO

POO

O

......

......

......

.....

......

......

.....

......

......

......

..

Lys 266 Lys 311

His219 Tyr222

Isoprene2

isoprene3

isoprene4

isoprene1

Lys 164

ValOH2

His201 IleArg 173

OH2

Gln 167

H2O

H2O

Glu169

His121

Arg266

Giuseppe, N.; Maria R. J. Inherit Metb. Dis. 2012, 35, 917-926

Involvement of FTase and GGTase in diseases• FTase and GGTase are promising therapeutic targets for various Ras

induced cancers and diseases.• Tumor • Progeria• Parasitic diseases• Other Diseases: Hematologic malignancies, glaucoma, neurological

diseases, and cardiovascular disease are also associated with prenyaltion.

Liu, M.et al. Proc. Natl Accd Sci.U.S.A. 2010,107, 6471-6476

Approaches development of a chemical analogue that competes with the FPP

development of a chemical analogue that competes with the target protein containing the peptide unit (peptidomimetric method)

development of a chemical analogue that competes with the FPP and caax motif, and

competition with protein/caax using a non-peptide analogue. (small molecule)

FTI-276

BMS-186511

Duez,S. et al. Bioorg Med. Chem. 2010, 18,543-556

Lonafarnib

Phase III clinical trail as FTI

GGTI-2418

Advanced to clinical trial phase I for Ras mediated oncogens

Recently molecular docking studies carried out by Reddy et al. showed that the major constituent of the turmeric powder, curcumin, has higher binding affinity than that of the existing GGTIs(CHEML 525185). The interesting result of this study can open the door to develop a new class of isoprenylase inhibitors.

Reddy et al., Bioinformation. 2015,11(5): 248-253

Progeria

A young girl with progeria. A healthy cell nucleus and a progeric cell nucleus.

The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T. PLoS Biology Vol. 3/11/2005

Progeria• Progeria, also known as Hutchinson-Gilford progeria syndrome, is a

genetic disorder caused by the mutation of the Nuclear Lamina A (LMNA) gene .

• When the cytosine in normal genes is mutated and replaced by thymine, a protein called progerin is formed with abnormal CAAX motifs. Because of the C-terminal CAAX motif, the progerin is farnesylated and accumulates at the inner lamina of the nuclear membrane. This accumulation results in different disorders and leads to death in children.

http://www.progeriaresearch.org/about_progeria/

Used for the treatment of progeriaLonafarnib

Parasitic Diseases• Farnesyl transferase has been identified in eukaryotic organisms other than humans

including pathogenic protozoans such as plasmodium, trypanosome, leishmanial and toxoplasma.

• Piggy-back approach to drug development for protozoan parasitic diseases is a logical way around the perennial problems associated with meeting the costs of de novo drug design.

• Martin et al. synthesized a series of farnesyl transferase inhibitors 1-4 (Table 1, 5)with IC50

value in the nanomolar ranges

• Research in the field of malaria treatment through inhibition of isoprenylase is ongoing and none of the drugs have yet progressed to clinical trials

Wiesner, J., Kettler, K., Sakowski, J., Ortmann, R., Katzin, A. M., Kimura, E. A., Silber, K., Klebe, G., Jomaa, H. and Schlitzer, M. 2004, Angewandte Chemie International Edition, 43: 251–254



Molecular docking showing the interaction of the benzophenone drug in the active site of the FTase

Palmitoylation• Lipid palmitoylation involves the addition of the saturated 16-carbon

fatty acyl group of palmitic acid to either a thiol or amine group and is designated as S-palmitoylation or N-palmitoylation, respectively, based on the atom to which acyl functionality is attached

• The S-palmitoylation produces a thioester functional group of intermediate stability, therefore, this is referred to as a reversible lipidation. S-palmitoylation occurs in the cytoplasm under the control of the enzyme acyl transferase DHHC (Asp-His-His-Cys) palmitoyl transferase.

• The N-palmitoylation on the other hand is controlled by the enzyme called Hedgehog acetyl transferase (Hhat). The enzyme Hhat belongs to the MBOAT (membrane bound O-acyl transferase) family of proteins. The N-palmitoylation is associated with the protein trafficking and sub cellular localization.

N-Palmitoylation

Disease• schizophrenia , Huntington’s , Osteoporosis, alopecia and lung cancer• 2- bromopalmitate (2-BP)-suicide inhibitor• Another nonspecific inhibitor of DHHC PAT is RU-SKI 41-did not

proceed to the clinical trial because of no specificity.

2-bromopalmitate RU-SKI-41

Myristoylation• Myristic acid is a 14-carbon saturated fatty acid and ~0.8% of

eukaryotic proteins are modified by this post-translational modification. Myristoylation takes place in the N-terminal of the gylcine rich protein motif.

• Catalyzed by -N-myristoyltransferase 1 and 2 (NMT1, NMT2)

Disease• HIV, malaria, sleeping sickness leishmaniasis • a series of Leishmaniasis-selective NMT inhibitors have been

synthesized and tested against a panel of NMT's from different organisms.

Pf-03402623

Summary• Lipids are important biomolecules with diverse functions.• Dysfunction in the enzymes maintaining the homeostasis of lipid and

PTM of enzymes causes a varieties of diseases.• Understanding of the mechanism provides potential targets for drug

discovery.

If RNA ruled the last decade and DNA dominated the previous one, could the next decade be the one for the lipid?

Cell 143, December 10, 2010, PP 853

Acknowledgement• My mentor- Dr. Jeffrey Arterburn• My Family- Shila Rimal• Lab members- Travis, Rachael and Jared, Dr. Ramesh• Committee members• Faculties, Staffs and friends-Graduate and undergraduate

Questions??

Thank You