Global Health Primer

What is Lipid Biosynthesis?

Lipids are important for energy storage, membrane integrity, hormones, signaling, and numerous other biological functions. Lipids come in several forms including fatty acids, triglycerides, phospholipids, and cholesterol.

Overview

Lipids come in several forms including those that are generated by distinct biosynthetic pathways:

  • Fatty acids
  • Triglycerides
  • Phospholipids
  • Sterols

Fatty acids are long chain hydrocarbons synthesized by the iterative addition of malonyl-CoA to an acyl chain on an acyl carrier protein. This process is carried out by the enzymes acetyl-CoA carboxylase (ACCase) and fatty acid synthase (FAS). FAS enzymes come in two forms: (1) FAS I is a multi-enzyme complex primarily found in mammals, fungi, and plants, and (2) FAS II is a series of separate enzymes that recapitulate the enzymatic activities of the FAS I complex and are primarily found in bacteria and several parasitic protozoa. Fatty acids are important building blocks for more complex lipids.

Triglycerides (or triacylglycerols) and phospholipids are the primary fate of fatty acids. Triglycerides are a major form of energy storage while phospholipids are a major component of lipid membranes. A variety of triglycerides and phospholipids with different functions and fates can be produced depending on the fatty acids that are incorporated and the enzymatic pathways utilized.

Cholesterol is a lipid that is synthesized in the liver, transported in the bloodstream, and used in the construction of cell membranes in animals. Ergosterol is the fungal equivalent to cholesterol. These and other sterols use acetyl-CoA as a primary building block. Synthesis of cholesterol and ergesterol involves more than 20 steps and a variety of enzymes including: mevalonate pathway, isoprenoid pathway, condensation of isoprene to squalene, and cyclization of squalene to lanosterol. Lanosterol can then be converted to cholesterol (animals), stigmasterol (plants) or ergosterol (fungi and several parasitic protozoa).

 

Existing Products

There are numerous lipid biosynthesis inhibitors that are approved or in late stage clinical development for a variety of diseases.

Lipid Biosynthetic Pathway Target Name Development Status
Melvonate pathway HMGCoA reductase Multiple Statins, FDA approved for lowering LDL-cholesterol
Lanosterol conversion to ergosterol CYP51 Posoconazole (Merck), FDA approved for invasive fungal disease
Fatty acid synthesis FAS II Isoniazid and ethionamide, FDA approved for tuberculosis
FAS I Pyrazinamide, FDA approved for tuberculosis

Lipid Biosynthesis Inhibitors as Non-Neglected Tropical Disease Therapeutics

There are several diseases that are not related to neglected tropical diseases for which lipid biosynthesis has been targeted, including atherosclerosis/heart disease and fungal infections.

Statins are inhibitors of the enzyme HMGCoA reductase, a key biosynthetic enzyme in the melvonate pathway that is utilized for the production of cholesterol in humans. High levels of low density lipoprotein (LDL)-cholesterol can lead to the formation of fatty plaques in the arteries, also known as atherosclerosis, and eventually heart disease. HMGCoA reductase inhibitors are the cornerstone for the treatment of high LDL -cholesterol and several products have demonstrated the ability to prevent heart attack and stroke as a result of their effects on LDL-cholesterol.

Fungi produce ergesterol as a primary sterol. This is in contrast to humans and other mammals that produce primarily cholesterol. Although fungi that infect humans may salvage some cholesterol from the host, this is not sufficient to replace de novo ergesterol biosynthesis. The enzyme C14alpha-demethylase (CYP51) is involved in both ergesterol and cholesterol biosynthesis. Inhibitors of CYP51 have been used successfully to treat invasive fungal infections in humans.1 Although CYP51 inhibitors may also inhibit some cholesterol biosynthesis in humans, they do not significantly decrease cholesterol levels since the majority of human cholesterol comes from diet rather than de novo synthesis.

Lipid Biosynthesis Inhibitors as Neglected Tropical Disease Therapeutics

Lipid biosynthesis has been targeted for antibacterial development due to differences between bacterial and mammalian fatty acid biosynthesis. Mycolic acids are long chain fatty acids that are unique to the cell wall of a group of bacteria that includes Mycobacterium tuberculosis, the bacterium that causes human tuberculosis. M. tuberculosis is particularly interesting because it uses both FAS I and FAS II pathways to produce mycolic acids.2 Isoniazid (INH) is the cornerstone of first line tuberculosis treatment. INH inhibits an enzyme in the M. tuberculosis FAS II pathway InhA, an enoyl acyl carrier protein reductase.  Inhibition of InhA interferes with the synthesis of the mycobacterial cell wall. Pyrazinamide (PZA) inhibits the M. tuberculosis FAS I pathway and is used as part of combination therapies with INH and rifampicin for the treatment of tuberculosis.

The success of FAS I and II inhibitors for the treatment of tuberculosis suggests exploitation of differences in fatty acid and other lipid biosynthetic pathways between mammalian, bacterial, and parasitic organisms is a viable strategy for future neglected tropical disease drug development.

 

References

  1. de Souza W and Rodrigues JC (2009) “Sterol biosynthesis pathways as target for anti-trypanosomatid drugs.” Interdisciplinary Perspectives on Infectious Diseases 2009: 642502.
  2. Schroeder EK et al. (2002) “Drugs that inhibit mycolic acid biosynthesis in Mycobacterium tuberculosis.” Current Pharmaceutical Biotechnology 3: 197-225.

 

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PIPELINE

Product/Research ProgramDevelopersDiscoveryPre-clinicalPhase IPhase IIPhase III
E1224Barcelona Centre for International Health Research
CONICET
Doctors Without Borders
Drugs for Neglected Diseases Initiative
Eisai Inc.
Federal University of Ouro Preto
Higher University of San Simon
Juan Misael Saracho Autonomous University

 

 

 

 

PosaconazoleMerck & Co., Inc.
Vall d'Hebron University Hospital

 

 

 

 

SudoterbLupin Pharmaceuticals, Inc.

 

 

 

TAK-187Takeda Pharmaceutical Company LTD

 

 

 

InhA inhibitorsGlaxoSmithKline
Global Alliance for TB Drug Development

 

Lanosterol demethylase inhibitorsConsortium for Parasitic Drug Development
University of Washington

 

Malaria drug discovery programEisai Inc.
Kitasato University
Osaka University

 

ND701NeED Pharma

 

Non-azole CYP51 inhibitorsScripps Research Institute, Florida
University of California, San Francisco

 

Pyrazinamide analogsGlobal Alliance for TB Drug Development
Yonsei University

 

FAS 20013FASgen

 

On Hold

ANALYSIS

Inhibitors of biosynthetic pathways have been validated as therapeutic targets for a variety of diseases. As a target class, lipid biosynthetic pathways benefit from an extensive body of existing research into the understanding of their catalytic mechanisms, reaction mechanism, and structures. Additionally, inhibitors of lipid biosynthetic pathways have the potential to benefit from the recycling of chemical compound libraries developed through previous drug development programs.

The relative strengths, weaknesses, opportunities, and risk for lipid biosynthesis inhibitors that are currently in development for neglected tropical diseases are summarized here.

  Strengths Weaknesses Opportunities Risks
Sterol biosynthesis: Downstream of isoprenoid pathway
Relevant neglected tropical diseases:

Chagas (posaconazole, phase II; E1224 and TAK187, phase I)

Leishmaniasis (lanosterol demethylase inhibitors, discovery

Tuberculosis (ND701, discovery)
Posaconazole is already on market as an antifungal

All of the clinical stage products in this category have unique mechanisms of action relative to on market products for the neglected tropical diseases listed here
No clinical trial data for efficacy in parasitic diseases are available yet

One of the targets of ND701 for tuberculosis, CYP51, is non-essential in that

As many of the targets in this pathway have mammalian homologs host toxicity must be carefully evaluated
Potential for application of products discovered through these programs across multiple neglected tropical diseases

Addition of new products to drug combinations

Exploration of new targets from this pathway
Host toxicity may be an issue
Fatty acid biosynthesis: FAS II
Relevant neglected tropical diseases:

Tuberculosis (INH and ETH on market; multiple programs, discovery through phase II)
Based on well-established existing drugs for tuberculosis treatment Drug resistance and liver toxicity are common with INH, so related compounds may not overcome these weaknesses Application of compounds from tuberculosis programs to protozoan neglected tropical diseases that also have FAS II pathways

Addition of new products to drug combinations
High risk for drug resistance based on experience with INH in tuberculosis

Not clear if new compounds overcome liver toxicities associated with INH
Fatty acid biosynthesis: FAS I
Relevant neglected tropical diseases:

Tuberculosis (PZA on market; PZA analogs, discovery)
Based on well-established existing drug for tuberculosis treatment Early stage, so potential improvements over PZA unclear

Mammalian cells also have FAS I raising concerns about host toxicity
Addition of new products to drug combinations Many more advanced programs for tuberculosis drug development that are likely to be approved before this product

Numerous lipid biosynthetic pathway enzymes have been genetically or chemically validated as potential therapeutic targets for neglected tropical diseases. These validated targets represent the best opportunity for immediate screening of existing small molecule libraries from non-neglected tropical disease lipid biosynthesis drug discovery programs.

Neglected Tropical Disease Fatty Acids Triglycerides and Phospholipids Sterols: Mevalonate pathway Sterols: Isoprenoid pathway Sterols: Downstream of isoprenoid
Chagas1,2 Kinetoplast novel pathway (FAS II-like): endoplasmic reticulum-based elongases (ELOs) for de novo synthesis

FAS II in mitochondrion
  HMG-CoA (preliminary evidence using on market statins) Tc Farnysyl pyrophosphate synthase (TcFPPS) CYP51

Squalene synthase (SQS)

Sterol methyl transferase (SMT)
HAT1,2,3 Kinetoplast novel pathway (FAS II-like): endoplasmic reticulum-based elongases (ELOs) for de novo synthesis

FAS II in mitochondrion
    FPPS inhibitors have effects on replication, but target not confirmed

Tb protein farnesyl transferase (TbPFT)
CYP51 (not proven in vivo – can salvage cholesterol)
Leishmaniasis1,2 Kinetoplast novel pathway (FAS II-like): endoplasmic reticulum-based elongases (ELOs) for de novo synthesis

FAS II in mitochondrion
    LmFPPS Lanosterol demethylase
Malaria1,2,3,4,5 FAS II in apicomplast organelle: PfENR, PfKASI/II, PfHAD Phosphatidylcholine synthesis  Unique mevalonate-independent pathway upstream pre-isoprenoid: PfDOXP  FPPS inhibitors have effects on replication, but target not confirmed

PfPFT
 
Tuberculosis6 FAS II (InhA) and FAS I       CYP51 (non-essential) in combination with CYP121

 

References

  1. Goodman CD and McFadden GI (2008) “Fatty acid synthesis in protozoan parasites: unusual pathways and novel drug targets.” Current Pharmaceutical Design 14: 901-916.
  2. Martin MB et al. (2001) “Bisphosphonates Inhibit the Growth of Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum: A Potential Route to Chemotherapy.” Journal of Medicinal Chemistry 44: 909-916.
  3. Buckner FS et al. (2005) “Protein farnesyl transferase inhibitors for the treatment of malaria and African trypanosomiasis.” Current Opinion in Investigational Drugs 6: 791-797.
  4. Wengelnik K et al. (2002) “A Class of Potent Antimalarials and Their Specific Accumulation in Infected Erythrocytes.” Science 295: 1311-1314.
  5. Seeber F (2003) “Biosynthetic pathways of plastid-derived organelles as potential drug targets against parasitic Apicomplexa.” Current Drug Targets – Immune, Endocrine, & Metabolic Disorders 3: 99-109.
  6. Schroeder EK et al. (2002) “Drugs that inhibit mycolic acid biosynthesis in Mycobacterium tuberculosis.” Current Pharmaceutical Biotechnology 3: 197-225.

 

Get Involved

To learn how you can get involved in neglected disease drug, vaccine or diagnostic research and development, or to provide updates, changes, or corrections to the Global Health Primer website, please view our FAQs or contact us at globalhealthprimer@bvgh.org.

Databases/Resources

The Lipidomics Gateway is a free website for researchers interested in lipid pathways and biology run by the LIPID Metabolites and Pathways Strategy (LIPID MAPS) consortium. The website also details protocols for common assays and other analysis common in lipid research

Assays

There are numerous assays available to monitor lipid biosynthesis, including:

  •  (3H) acetic acid labeling of cultured cells to monitor cholesterol biosynthesis. Following the labeling, lipids are extracted with methanol and chloroform, separated using thin layer chromatography and the cholesterol band is measured by liquid-scintillation counting.  More information available here.
  • A high throughput assay that identifies inhibitors simultaneously against multiple targets within the FASII pathway of most bacterial pathogens. Merck & Co. developed an assay that measures the incorporation of (14C) malonyl-CoA into long hydrophobic acyl chains of acyl-ACP using partially purified bacterial enzymes of the FAS II pathway.  A protocol for this assay is available here.
  • Several assays for high-throughput screening of neutral lipid biosynthesis. These assays include biochemical scintillation proximity assays, cell-based lipotoxicity assays, and cell-based in situ fluorescent quantification assays.  For a review of these techniques please refer to the recent review by Siloto and Weselake available here.
  • There is now an HTS assay for CYP51 based on compound interaction with the active site heme group.

Crystal Structures

Crystal structures of T.cruzi CYP51 are deposited in the PDB database. These include co-crystals with azole drugs and non-azole leads.

 

Get Involved

To learn how you can get involved in neglected disease drug, vaccine or diagnostic research and development, or to provide updates, changes, or corrections to the Global Health Primer website, please view our FAQs or contact us at globalhealthprimer@bvgh.org.

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