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Graph showing the relationship of dPEG® linker length in relation to potency and specificity in extracellular drug conjugates.

Extracellular Drug Conjugates Therapeutically Exploit Protein Proximity

Pharmaceutical company Centrose, founded by James R. Prudent, Ph.D., developed a new class of antibody drug conjugates called extracellular drug conjugates. Nature Publishing Group published the research as a open access paper in its Molecular Therapy journal.1 Apart from the interesting and important development of a new class of antibody drug conjugate (ADC), the research also showed how important linker length 2 is to the potency and specificity of the EDC.

How Extracellular Drug Conjugates Work

Extracellular drug conjugates (EDCs) are designed similarly to ADCs. That is, EDCs consist of a monoclonal antibody (mAb), a linker, and a cytotoxic agent. Antibody drug conjugates require internalization into a diseased cell where the cytotoxic agent can then be released to act on its target.3 The cytotoxic agent may require intracellular modification or degradation to act on its target molecule.

By contrast, extracellular drug conjugates require no internalization. Rather, EDCs target cell surface proteins that are expressed on a target (i.e., cancerous) cell. Moreover, the cytotoxic agent that is linked to the mAb does not target the same protein targeted by the mAb. Rather, the cytotoxic agent kills the targeted cells by affecting a protein or enzyme that is different from the protein or enzyme bound by the mAb but that is closely associated with the target protein or enzyme. Click here to see Centrose's explanation of how EDCs work, including a three-minute video, which you can also view on Centrose's website.

In this particular study, the research team observed that some proteins that are overexpressed on the surface of cancer cells are closely associated with the sodium potassium ATPase1 (NKA, and also known as the sodium potassium pump; see Figure 1). Cardiac glycosides such as digoxin or ouabain inhibit the NKA. Cells affected by these or other cardiac glycosides swell, then undergo necrotic cell death.1,4,5 The team reasoned that by (1) combining antibodies to proteins that are both (a) overexpressed on the cell surface of cancer cells and (b) closely associated with the NKA (2) with cardiac glycosides that strongly inhibit the NKA (3) would create cancer therapeutic antibody drug conjugates that were localized to the extracellular space, hence, Extracellular Drug Conjugates.

 

Creating the Extracellular Drug Conjugates in this Study

Through prior testing (not in this paper) the team found that the cardiac glycoside scillarenin β-L-aminoxyloside (Figure 1) highly inhibited the NKA. This was chosen as the drug for conjugation to the antibody.

Chemical structure of Scillarenin β-L-aminoxyloside, the cardiac glycoside used in this study as the cytotoxic agent for the extracellular drug conjugates.
Figure 1: Scillarenin β-L-aminoxyloside, the cardiac glycoside used in this study as the cytotoxic agent for the extracellular drug conjugates.

The team also developed or acquired nine (9) mAbs. For directly testing the EDCs, the mAbs had to meet one of the following three criteria:

  1. was a marker for metastatic cancer commonly known to associate with the NKA;
  2. was cancer related and thought to associate with the NKA; or
  3. was found by the current study to associate with the NKA and was a current cancer antibody drug target.

As controls, the research team selected mAbs to proteins that were expressed on the cell surface but did not associate with the NKA or that were not expressed on any cell surface.

The researchers also investigated the effect of linker length between the mAb and the drug (abbreviated CG1) using Quanta BioDesign's MAL-dPEG®n-NHS esters. These versatile heterobifunctional linkers come in a variety of specific lengths and are single molecular weight PEG derivatives (i.e., they have no dispersity). The maleimidopropyl group on one end reacts with free sulfhydryl groups forming a thioether linkage, while the NHS ester group on the other end will react with free amines to form a peptide bond. Four lengths of PEG — n = 2, 12, 24, and 36 dPEG® units (27, 56, 105, 144 Angstroms) — were chosen to connect CG1 to the EDC. See Figure 1. Although Figure 1 shows a single CG1-dPEG®n conjugated to the mAb, calculations by Centrose showed that the average EDC had a DAR of four (4). See reference 1, page 5.

 

Chemical structures of the MAL-dPEG®n-NHS-ester linkers used to construct the extracellular drug conjugates used in this study.
Figure 2: MAL-dPEG®n-NHS-ester linkers used to construct the extracellular drug conjugates used in this study.

Extracellular Drug Conjugates Demonstrate In Vitro Efficacy...

The research team examined the efficacy of the extracellular drug conjugates after conjugating CG1 (the cardiac glycoside) to the mAb for dysadherin (a protein marker associated with metastatic cancer). They also measured the efficacy and toxicity of CG1 by itself or conjugated to one of the dPEG® linkers but not conjugated to the EDC.

For the EDC-dPEG®n-CG1 conjugates, the Centrose team first measured antibody binding on the surface of the different cell lines. Then, by monitoring cell viability, they tested the cells' sensitivity to the EDC-dPEG®n-CG1 conjugates at concentrations from 1 to 200,000 pmol/L. The dose-response curve in Figure 3, below, shows that increasing the linker length in EDC-dPEG®n-CG1 conjugates improved target specificity and potency. However, decreasing linker length in dPEG®n-CG1 constructs that were not conjugated to mAb increased toxicity and reduced specificity.

 

Dose-Response Curve and Potency-Specificity Graph for the Extracellular Drug Conjugates Based on Anti-dysadherin. Note that the potency and specificity increase with linker length for the mAb-dPEG®n-CG1 conjugates, but in the dPEG®n-CG1 constructs not conjugated to mAb, the specificity and toxicity increase as the linker length decreases.
Figure 3: Dose-Response Curve and Potency-Specificity Graph for the Extracellular Drug Conjugates Based on Anti-dysadherin. Note that the potency and specificity increase with linker length for the mAb-dPEG®n-CG1 conjugates, but in the dPEG®n-CG1 constructs not conjugated to mAb, the specificity and toxicity increase as the linker length decreases.

Similar dose-response curves were obtained for some of the other tested cell lines. Cell lines expressing a cell surface antigen closely associated with the NKA were particularly sensitive to the EDC-dPEG®n-CG1 conjugates, but control cell lines (those either not expressing a cell surface antigen or expressing a cell surface antigen not associated with the NKA) were relatively insensitive to the EDC-dPEG®n-CG1 conjugates.

 

...And They Work In Vivo Also

The in vitro results also translated to in vivo studies in mice. In xenograft studies in mice bearing human pancreatic cancer tumors, EDC-DYS (EDC specific to dysadherin conjugated to dPEG®-CG1, with a DAR of 4) was compared to the standard dosing regimen of gemcitabine. EDC-DYS outperformed gemcitabine in a dose-dependent manner. Similarly, EDC-CD38 (a marker for various lymphomas and multiple myeloma) beat CHOP, a chemotherapy cocktail used as a standard treatment for Ramos B-cell lymphoma. Likewise, EDC-CD20 (another lymphoma marker) exceeded Rituximab's performance. The control experiments showed that EDC-CONTROL conjugates (mAb targeted to antigens not expressed on the cell surface) did not reduce tumor size in mice.

 

Extracellular Drug Conjugates Offer New Therapeutic Options

EDCs are a new class of antibody drug conjugate, and they offer new, and potentially superior, therapeutic options for patients. Though similar in design and construction to a standard ADC, an EDC is different. The EDC always resides in the extracellular space, and it targets two cell surface proteins. These two features define the EDC. Neither the mAb nor the cytotoxic drug need to be internalized, released, or broken down in order to act. Many cells evolve to evade chemotherapy by rapidly exporting or neutralizing drugs that are released intracellularly. This unique EDC feature impedes cells in evolving resistance to the EDC.

These results show that EDCs are potentially useful in killing cancers that are resistant to multiple drugs, metastatic, and/or aggressive. Thus, in the future, EDCs may offer new therapeutic options for cancers that are otherwise rather difficult to treat.

 

Quanta BioDesign's dPEG® Reagents Were Important to the Success of This Research

Quanta BioDesign's maleimido-dPEG®n-NHS ester products were important to the success of this research on extracellular drug conjugates. Unlike traditional PEG derivatives, our dPEG® derivatives are single molecular weight compounds. We manufacture all of our products entirely in the USA by a patented, proprietary process. Our dPEG®s have no dispersity. Consequently, standard analytical techniques suffice for analyzing them to determine their purity. With traditional, dispersed PEGs, "purity" becomes a much more elusive term and is more difficult to measure.

Whether you are developing a new ADC or something else, Quanta BioDesign can help. We have reagents for many different types of conjugation chemistry, and we are open to custom syntheses. We manufacture products on scales from milligrams to multiple kilograms. Our responsive customer service works hard to get you what you need when you need it. If you want to learn more about us, visit our website, or contact us directly. You will be glad that you did.

 

References/Endnotes

  1. David J. Marshall, Scott C. Harried, John L. Murphy, et al. Extracellular Antibody Drug Conjugates Exploiting the Proximity of Two Proteins, Molecular Therapy advance online publication 19 July 2016; doi: 10.1038/mt.2016.119
  1. A linker is the component of the antibody drug conjugate that joins a monoclonal antibody (mAb) to a cytotoxic drug. An ideal linker is stable in circulation but should release the cytotoxic drug when the mAb reaches the target. "Linker length" refers to the distance between the mAb and the cytotoxic drug. Distance may be expressed in number of atoms or in units of Angstroms.
  1. For more information on how monoclonal antibodies and antibody drug conjugates work in cancer therapy go here and/or here.
  1. Menger, L, Vacchelli, E, Adjemian, S, Martins, I, Ma, Y, Shen, S et al. (2012). Cardiac glycosides exert anticancer effects by inducing immunogenic cell death. Sci Transl Med 4: 143ra99.

Do you have questions or comments about this post? Please leave a comment below. Also, be sure and check out our list of related products below.

 

About the Author

Robert H. Woodman, Ph.D. is a Senior Product Development Scientist and the QC Manager for Quanta BioDesign, Ltd. He is on LinkedIn at https://www.linkedin.com/in/roberthwoodman, on Twitter at @RobertHWoodman and @QuantaBioDesign. Feel free to contact him via social media.

 

Product Pages for the Quanta BioDesign Products Used in This Research

PN10266, MAL-dPEG®2-NHS ester

PN10284, MAL-dPEG®12-NHS ester

PN10314, MAL-dPEG®24-NHS ester

PN10904, MAL-dPEG®36-NHS ester (contact us for information and pricing)

 

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PN10549, MAL-dPEG®2-TFP ester

PN10553, MAL-dPEG®12-TFP ester

PN10554, MAL-dPEG®24-TFP ester

PN10555, MAL-dPEG®36-TFP ester

 

TFP esters are superior to NHS esters. Click here to learn why.

To see all of our crosslinking reagents, click here.

 

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Amphotericin B conjugated to a discrete PEG (dPEG®) linker with a free amine have higher water solubility and lower toxicity than the parent compound.

Amphotericin B+dPEG®: Water-Soluble, Less Toxic, Potent

Amphotericin B conjugated to a discrete PEG (dPEG®) linker with a free amine have higher water solubility and lower toxicity than the parent compound.
Amphotericin B conjugated to a discrete PEG (dPEG®) linker with a free amine has higher water solubility and lower toxicity than the parent compound. Image used by permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.

 

About Amphotericin B

Structure of Amphotericin B. Used with permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.
Figure 1: Structure of Amphotericin B. Image used by permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.

Amphotericin B (Figure 1) is the "gold standard" treatment for systemic fungal infections and diseases caused by the parasite Leishmania. Sometimes it is the only effective treatment because drug resistance renders other treatments useless. Systemic fungal infections are an increasingly serious, widespread problem in medicine. Patients with weakened or suppressed immune systems (caused by HIV/AIDS, diabetes, organ transplants, some cancer treatments) are especially at risk. An estimated 1.5-2 million people die each year from systemic fungal infections (1). Despite its "gold standard" label, Amphotericin B has several well-known difficulties.

A polyene macrolide antifungal drug, Amphotericin B was first discovered in 1953 from the fermentation culture of the bacterium Streptomyces nodosus. It entered the pharmaceutical market in 1958 (2)(3)(4). Nearly insoluble in water, the drug is not absorbed from the human gastrointestinal tract and must be administered intravenously. Amphotericin B was first formulated as a suspension in sodium deoxycholate solution called Fungizone-Squibb (2)(7). Novel, less toxic lipid formulations have been developed recently (5)(6)(7), but these formulations cost more and require high doses to be effective, thus limiting their use (4)(7).

This drug is also toxic. Its side effects are well known, serious, and sometimes fatal (7)(8). Common side effects from taking this drug include high fever coupled with shaking chills ("shake and bake"), headache, kidney problems, low blood pressure, nausea, vomiting, unusual bruising or bleeding, and irregular heartbeat. Anaphylactic shock sometimes occurs with this drug. A 1997 report from the Center for Drug Evaluation and Research, a division of the United States Food and Drug Administration, stated that the toxicity of Amphotericin B was 1.5 mg/Kg in rats. Other reports reach similar conclusions (4)(5)(6)(7)(8).

Since its discovery, numerous researchers have attempted to modify the properties of Amphotericin B to make it more water-soluble, less toxic, or both. PEGylation is one obvious strategy for improving the water solubility of a water-insoluble drug. Greenwald, et al. (9), and Sedlak, et al. (10)(11) created PEGylated prodrug versions of the drug, but these constructs used large, dispersed, polymeric PEGs that are heterogeneous in size and, hence, difficult to characterize.

 

A Different Approach to Water-Soluble Amphotericin B

In a paper published in January 2016 in the Journal of Medicinal Chemistry, Assaf Halperin, Yana Shadkchan, Evgeni Pisarevsky, Alex Szpilman, Hani Sandovsky, Nir Osherov, and Itai Benhar, from Tel-Aviv University and the Israel Institute of Technology chose to work with short PEG chains of defined length and molecular weight. Specifically, they chose to create a stable amide bond on the mycosamine ring and expose a free amine at the opposite end of the linker. After testing PEG linkers with 4, 8, and 44 ethylene oxide units, they settled on the PEG8 linker, because it offered significant improvement in water solubility with little loss in efficacy. By contrast, the PEG4 linker improved water solubility, but less well than the PEG8 and PEG44 linkers. The PEG44 linker improved water solubility greatly, but the derivative's efficacy dropped thirty-fold compared to the parent drug.

 

Synthetic scheme used by Halperin, et al., to create the two PEGylated derivatives of Amphotericin B. Image used with permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.
Figure 2: Synthetic scheme used by Halperin, et al., to create the two PEGylated derivatives of Amphotericin B. Image used by permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.

For the construction of two different PEGylated derivatives, the research group used Quanta BioDesign's Fmoc-N-amido-dPEG®8-NHS ester, product number 10995, purchased from our German distributor, Iris Biotech, GmbH. (Click here for a complete list of our distributors.) The authors stated the reason for their choice in this way:

This approach creates a well-defined medium-molecular-weight product that can be easily separated and characterized by NMR and other common techniques .... The short and specific synthesis and well-defined structural nature of the antifungal conjugate is expected to be an advantage in clinical approval as well. (4, page 1201).

After the dPEG® was conjugated to Amphotericin B, the Fmoc protecting group was removed, exposing the free amine on the dPEG® linker. The synthetic scheme used by the authors is shown in Figure 2.

 

Amphotericin B-dPEG®8 Derivatives Show Promise as Clinical Replacements for the Parent Compound

In testing, both dPEG®ylated derivatives (AB1, the free acid; AM2, the methyl ester; see Figure 3) increased dramatically in water solubility, decreased considerably in toxicity, and maintained (with only a moderate decrease) efficacy.

The solubility of Amphotericin B in water is less than 1 µg/mL (<0.001 mg/mL). The AM2 derivative's water solubility increased to 700 µg/mL (0.7 mg/mL), while AB1 increased to 5,500 µg/mL (5.5 mg/mL). Previous efforts to make useful PEGylated derivatives of Amphotericin B used carbamate linkages between the drug and the PEG. These linkages hydrolyzed readily in phosphate-buffered saline (PBS). By contrast, neither AB1 nor AM2 showed any hydrolysis in PBS after 4 hours, and both hydrolyzed less than 1% after 24 hours in PBS, demonstrating that they are stable. Furthermore, pharmacokinetic studies of AM2 in mice showed no free Amphotericin B after 24 hours, indicating that the derivatives are stable.

Structures of the dPEG®8-amine derivatives of Amphotericin B. Image used by permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.
Figure 3: Structures of the dPEG®8-amine derivatives of Amphotericin B. Image used by permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.

The authors tested the toxicity of AB1 and AM2 against the parent drug in three ways. First, they evaluated nonspecific toxicity of all three compounds by suspending fresh human red blood cells (hRBCs) in PBS and measuring lysis of the cells (hemolysis) in the presence of Amphotericin B or the derivatives. Second, they assayed the viability of mouse embryonic fibroblast cells using an MTT assay (for a detailed, technical discussion of the MTT assay plus procedures for conducting the assay, click here). Third, they measured the LD50 of the three compounds by intravenous injection in mice.

In the first test, Amphotericin B was highly toxic as expected. AB1 was less toxic to hRBCs than the parent compound, while AM2 caused very little hemolysis and thus demonstrated little non-specific toxicity.

MTT Assay comparing Amphotericin B to the AB1 and AM2 derivatives.
Figure 4: MTT Assay comparing Amphotericin B to the AB1 and AM2 derivatives. A color version of this image is at the beginning of this post. AM1 (should be AB1) and AM2 are the derivatives shown in Figure 3. AMB is Amphotericin B (see Figure 1 for structure). Image used by permission from J. Med. Chem. (2016), 59, 1197-1206, copyright 2016, American Chemical Society.

The MTT assay (see Figure 4) showed dramatic differences between Amphotericin B and the derivatives. The MTT assay measures the half-maximal inhibitory concentration, also known as the IC50 (see also here), of a compound against cells in culture. In the assay, AB1 was forty times less toxic to the mouse embryonic fibroblasts than Amphotericin B, while AM2 was at least 600 times less toxic.

The in vivo toxicity testing showed dramatically different results among Amphotericin B, AB1, and AM2. The LD50 of Amphotericin B was 1.1 mg/Kg. Mice injected with twice the LD50 died instantly. By contrast, AB1 was not toxic to mice at the maximum injected dose of 22 mg/Kg, and AM2 was not toxic to mice at the maximum injected dose of 42 mg/Kg.

Efficacy (a measure of the potency of antifungal activity) was assessed both in vitro and in vivo. Both AB1 and AM2 retained activity against a broad spectrum of fungi but were 2-16 times less potent than Amphotericin B. When the length of time needed to kill all fungal cells was measured, AM2 eliminated all fungi in vitro within 2 hours, similar to Amphotericin B. In in vivo testing, Amphotericin B at 1 mg/Kg (near the LD50) cured 10 of 12 mice of fungal infection, compared to AM2, which achieved the same result using 3.5 mg/Kg. AM2, though, cured all mice of fungal infections at a dose of 7 mg/Kg, something that non-PEGylated parent drug could not do, because it was too toxic.

Interestingly, AM2 had a shorter serum half-life than Amphotericin B. This result seems odd at first glance because PEGylation is one of the primary means by which the serum half-life of therapeutic agents is extended. The reason for the difference is that Amphotericin B binds serum proteins, which lengthens the drug's serum half-life. Testing revealed that AM2 does not bind strongly to serum proteins. This allows it to be removed much more quickly from the body.

AB1 and AM2 both show promise as clinical antifungal agents. The increased water solubility and lower toxicity compared to Amphotericin B are important, but it is also important that the efficacy of the derivatives is not greatly diminished from the parent compound. The discrete PEGylation (dPEG®) reagent simplifies analysis of the products, and the specific dPEG® reagent chosen, PN10995, is ideal for conjugating the Amphotericin B derivatives to other molecules after removal of the Fmoc group protecting the terminal amine.

This work also highlights the remarkable power of a relatively small dPEG® linker. Many research groups tend to think that large, dispersed PEGs are the solution to their water solubility problems. This research shows that a dPEG®8 linker imparts significant water solubility and reduces toxicity, with only a modest impact on efficacy. This is truly important for understanding the power and ability of discrete PEGylation.

 

References

  1. Denning, D. W.; Bromley, M. J. Infectious disease: how to bolster the antifungal pipeline. Science (27 March 2015), 347(6229). 1414-1416.
  1. Dutcher, James D. The discovery and development of Amphotericin B. Dis. Chest (October 1968), 54(supplement 1), 40-42.
  1. Gallis, Harry A.; Drew, Richard H.; and Pickard, William W. Amphotericin B: 30 years of clinical experience. Clin. Infect. Dis. (1990), 12(2), 308-329.
  1. Halperin, Assaf; Shadkchan, Yana; Pisarevsky, Evgeni; Szpilman, Alex M.; Sandovsky, Hani; Osherov, Nir; and Benhar, Itai. Novel water-soluble Amphotericin B-PEG conjugates with low toxicity and potent in vivo efficacy. J. Med. Chem. (2016), 59, 1197-1206.
  1. Larabi, M.; Pages, N.; Pons, F.; Appel, M.; Gulik, A.; Schlatter, J.; Bouvet, S.; and Barrat, G. Study of the toxicity of a new lipid complex formulation of amphotericin B. J. Antimicrob. Chemother. (2004), 53, 81-88.
  1. Souza, L. C.; Campa, A. Pharmacological parameters of intravenously administered amphotericin B in rats: comparison of the conventional formulation with amphotericin B associated with a triglyceride-rich emulsion. J. Antimicrob. Chemother. (July 1999) 44(1): 77-84.
  1. Hamill, Richard J. Amphotericin B Formulations: A Comparative Review of Efficacy and Toxicity. Drugs (2013), 73: 919-934
  1. Laniado-Laborín, Rafael; and Cabrales-Vargas, Maria Noemí. Amphotericin B: side effects and toxicity. Rev Iberoam Micol. (2009) 26(4):223–227.

Do you have questions or comments about this post? Please leave a comment below. Also, be sure and check out our list of related products below.

 

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Click here to learn why TFP esters are superior to NHS esters.

PN11000, Fmoc-N-amido-dPEG®4-TFP ester

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Figure 1 shows the process of membrane fusion as mediated by the SNARE protein complex.

SNARE-Mediated Membrane Fusion and dPEG®, Part 3

Part 3: Modeling Membrane Fusion with SNARE Protein Analogs

Click the links for Part 1 and Part 2 of this three-part series.

Membrane fusion is a universal process in living organisms and is critical to proper cellular function. Examples of membrane fusion events include exocytosis, fertilization, envelopment of infecting viruses, and transport of proteins through the Golgi stack. Though essential, membrane fusion is not spontaneous because free energy is required to overcome steric hindrances, electrostatic repulsions, and hydration shells between two membranes as they move toward each other prior to fusion.

In cells membrane fusion is facilitated by a protein superfamily known as SNARE (for Soluble N-ethylmaleimide-sensitive-factor Attachment Receptor), of which there are 36 members in humans. The best studied SNARE-mediated membrane fusion process is exocytosis in neural cells. Indeed, the first proteins of the human SNARE family to be identified were synaptobrevin, syntaxin, and synaptosome-associated protein 25 (SNAP-25). Figure 1 shows a SNARE-mediated exocytosis.

 

Figure 1 shows the process of membrane fusion as mediated by the SNARE protein complex.
Figure 1: Membrane Fusion via SNARE Protein Complex Mediation, By Danko Dimchev Georgiev, M.D. [GFDL or CC-BY-SA-3.0], via Wikimedia Commons
 

Because the native SNARE-mediated process is so complex, numerous research groups have worked to create simplified artificial systems that mimic SNARE-mediated membrane fusion. Numerous systems have been developed using both targeted and non-targeted membrane fusion. Pawan Kumar, Samit Guha, and Ulf Diederichsen have written an excellent review of these SNARE mimetic systems, focusing on targeted membrane fusion. The review is listed in the References section below with a link to the paper, which is behind a paywall.

Most of the targeted SNARE mimetic systems discussed in the review consist of a lipophilic anchor (cholesterol, POPE, and DOPE are discussed), a flexible linker, and a recognition sequence. The flexible linker most often used is a PEG linker, typically a PEG12, though PEG3 has also been used. The purpose of the PEG linker is to site the recognition sequence away from the lipid membrane but to maintain flexibility so that the recognition sequence can move freely in space until it pairs with its target sequence. Example systems using Quanta BioDesign's dPEG®12 linkers (most notably PN10283, Fmoc-N-amido-dPEG®12-acid) have been discussed in Part 1 and Part 2 of this series.

 

 How dPEG® Can Help You

Quanta BioDesign's dPEG® linkers provide the traditional advantages of PEG (water solubility, low immunogenicity, reduced aggregation and precipitation of proteins) but with a critical difference. Traditional PEGs are dispersed, but Quanta BioDesign's patented dPEG® constructs are discrete molecules. Constructs made from dispersed PEGs confound analysis because of the intrinsic heterogeneity of the PEG. Each of Quanta BioDesign's dPEG® constructs is a single molecular entity. There is no range of linker sizes or molecular weights. Thus, products made from dPEG® are well-defined, easily characterized, and amenable to standard analytical techniques.

Whether you are studying SNARE protein-mediated membrane fusion, building a new drug delivery platform, or creating a new peptide or protein drug, if you need PEG, then you need dPEG®. Quanta BioDesign invented and patented the dPEG® construct, and we manufacture our products in the United States.

 

References and Additional Reading

(1) Pawan Kumar, Samit Guha, and Ulf Diederichsen. SNARE protein analog-mediated membrane fusion. J Peptide Sci (2015), 21(8), 621-629. DOI: 10.1002/psc.2773

(2) Lando L. G. Schwenen, et al. Resolving single membrane fusion events on planar pore-spanning membranes. Scientific Reports (2015), 5:12006. DOI: 10.1038/srep12006

(3) Jörg Malsam and Thomas H. Söllner. Organization of SNAREs within the Golgi Stack. Cold Spring Harb Perspect Biol (2011), 3:a005249. DOI: 10.1101/cshperspect.a005249

(4) Yu A. Chen and Richard H. Scheller. SNARE-mediated membrane fusion. Nature Reviews Mol. Cell Biol (February 2001), 2, 98-106.

(5) Reinhard Jahn, Thorsten Lang, and Thomas C. Südhof. Membrane Fusion. Cell (February 21, 2003), 112, 519-533. DOI:10.1016/S0092-8674(03)00112-0

(6) Joseph G. Duman and John G. Forte. What is the role of SNARE proteins in membrane fusion? Am J Physiol Cell Physiol 285: C237–C249, 2003; DOI: 10.1152/ajpcell.00091.2003.

(7) Reinhard Jahn and Richard H. Scheller. SNARES — Engines for Membrane Fusion. Nature Reviews Mol Cell Biol (September 2006), 7, 631-643. DOI:10.1038/nrm2002

 

About the Author

Robert H. Woodman, Ph.D. is a Senior Product Development Scientist and the QC Manager for Quanta BioDesign, Ltd. He is on LinkedIn at https://www.linkedin.com/in/roberthwoodman, on Twitter at @RobertHWoodman and @QuantaBioDesign.

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Figure 1: Structure of the DOPE-PEG12-LPE/LPK lipidated oligopeptides used to study liposome fusion.

SNARE-Mediated Membrane Fusion and dPEG®, Part 2

Part 2: Controlling Liposome Fusion Using SNARE Protein Mimics

For Part 1 of this three-part series, click here. For part 3, click here.

Introduction

Continuing earlier work, researchers at the laboratory of Alexander Kros examined the parameters that affect membrane fusion in liposomes composed of the minimal model developed in their earlier paper (1). In this paper (2), the research group demonstrated that, using the model system previously developed, "the rate and extent of fusion and the number of fusion rounds can be manipulated by adjusting the fusogen and liposome concentrations" (quote from the abstract).

The SNARE protein model for membrane fusion developed by the research group contained 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) connected to one of two oligopeptides (LPE or LPK) via a dPEG®12 linker derived from PN10283, Fmoc-N-amido-dPEG®12-acid, where the PEG linker served as a means to provide a flexible spacer between the DOPE and the oligopeptides. See Figure 1, below. The coiled-coil forming oligopeptides each contain three-heptad repeats, and when populations of liposomes containing the LPE and LPK constructs are mixed, they associate and lead cleanly to liposome fusion of the two different liposome populations. As discussed in the earlier post on this subject, the model system developed by Kros and colleagues is an accurate, minimalist model of SNARE-mediated liposome fusion.

 

Figure 1: Structure of the DOPE-PEG12-LPE/LPK lipidated oligopeptides used to study liposome fusion.
Figure 1: Structure of the DOPE-PEG12-LPE/LPK lipidated oligopeptides used to study liposome fusion.

For this research, the authors characterized the influence on liposome fusion of lipopeptide concentration, lipid concentration, and lipids of positive curvature. The authors also described how liposome fusion in the model system varies with liposome collision, docking, and lipid mixing rates.

 

 

How Liposome Fusion Works

The group first studied whether liposome fusion was targeted (that is, molecular recognition between two different fusogens) was required for fusion to occur or non-targeted (no molecular recognition required). This analysis was carried out by mixing populations of liposomes not modified with either LPE or LPK with populations of liposomes carrying either LPE or LPK. In these experiments, no liposome fusion occurred. However, in experiments in which LPE-modified liposomes were mixed with LPK-modified liposomes, liposome fusion occurred. This indicated that molecular recognition between the LPK and LPE peptides was required for liposome fusion to occur; hence, the fusion was targeted.

Next, the researchers examined the effect of concentration of the two fusogens on liposome fusion. By mixing decreasing proportions (mole%) of the lipopeptide fusogens in a fixed amount of total lipids, the scientists were able to determine the lower limit and optimal amount of fusogens required for liposome fusion. By this method, it was determined that 0.05 mole% lipopeptide was the lower limit of fusogens required for at least one round of liposome fusion to occur. The optimal amount of fusion was found to be 0.75 mole% LPE or LPK incorporated into the liposome membranes. Circular dichroism data demonstrated that the lipidated oligopeptides (LPE and LPK) were most likely distributed evenly across the membrane surface at 0.75 mole%.

 

The general trend of increasing membrane fusion rates with increasing proportions of fusogens arises because two liposomes that diffuse into close proximity are more likely to be displaying complementary peptides in the correct orientation for binding in the approaching area, and hence are more likely to dock and undergo fusion. (2) at page 1048.

 

Above 0.5 mole% lipopeptide concentration, the researchers found that completely lipid mixing occurred rapidly. Large particles ("giant liposomes") and clusters of docked and fused particles formed, and the particles sedimented in a matter of hours. Conversely, at 0.25 mole% or less lipopeptide concentration, the particles that form from liposome fusion are initially small, increase in hydrodynamic diameter size, then decrease again over time before plateauing "at the size corresponding to the fusion of two liposomes in which the volume is conserved rather than the outer surface area" (2, p. 1050). This indicates that the SNARE mimetic liposome fusion is "clean," meaning that no loss of content occurs during fusion.

The research group analyzed whether lipid concentration affected membrane fusion, and if so, by how much. Using FRET experiments, the group found that complete lipid mixing was more efficient if there were more fusogens (the DOPE-PEG12-oligopeptides) on fewer liposomes than if there were fewer fusogens on more liposomes. A lower limit of 0.025 mM lipids (0.25 µM lipopeptide) was established as the lower limit of lipid concentration at which liposome fusion occurred efficiently.

Finally, the group examined the effect of changing lipid curvature on liposome fusion. Using the positive curvature lipid lysophosphatidylcholine (LPC) instead of DOPE altered the shape of the liposomal surface and prevented formation of the so-called "stalk intermediate" that is required in SNARE-mediated fusion (the stalk intermediate requires a negative curvature in order to form). With as little as 15 mole% LPC incorporated into liposomes, the researchers found that lipid mixing decreased dramatically; however, docking (the first step in liposome fusion) was not inhibited, but hemifusion and full fusion were prevented from occurring.

The research performed provided detailed understanding of how the reduced SNARE model works to promote liposome fusion. This detailed degree of understanding is important for developing applications of liposome fusion (or membrane fusion) such as targeted delivery of drugs to cells and controlled mixing of reagents at nanoliter volumes, such as would occur in a lab-on-a-chip application.

 

dPEG® Role in This Research

As mentioned in an earlier post, the DOPE-PEG12-LPE/LPK constructs used PN10283, Fmoc-N-amido-dPEG®12-acid to create the flexible extension between the lipid membrane surface and the oligopeptides. If you need to add PEG to your supramolecular construct, why not consider dPEG®? There are numerous advantages to adding PEG to your peptide or protein construct. First, PEG is amphiphilic. Second, PEG reduces aggregation and precipitation of biomolecules. Third, PEG diminishes the immunogenicity of molecules to which it is conjugated. Conventional PEG, though, is polydispersed, meanign that it is composed of numerous PEG molecules. The "weight" of a conventional PEG is expressed as an average molecular weight across the range of sizes. Quanta BioDesign's dPEG® constructs — designed, patented, and manufactured in the United States — are single molecular weight PEG molecules functionalized with various chemical moieties. Whereas conventional PEGs confound analysis by presenting an intractable mixture of different size molecules, dPEG® constructs enhance analysis by being a single, discrete molecule that can be accurately characterized using ordinary analytical chemistry tools.

We encourage you to look at all our products and find what you need for your next research project. You'll be pleasantly surprised at what you can do with dPEG®!

 

References

(1) Hana Robson Marsden, Nina A. Elbers, Paul H. H. Bomans, Nico A. J. M. Sommerdijk, and Alexander Kros. A Reduced SNARE Model for Membrane Fusion. Angew. Chem. Int. Ed (2009), 48, 2330-2333. DOI: 10.1002/anie.200804493

(2) Hana Robson Marsden, Alexander V. Korobko, Tingting Zhen, Jens Voskuhl, and Alexander Kros. Controlled liposome fusion mediated by SNARE protein mimics. Biomaterials Science (2013), 1, 1046-1054. DOI: 10.1039/c3bm60040h

 

About the Author

Robert H. Woodman, Ph.D. is a Senior Product Development Scientist and the QC Manager for Quanta BioDesign, Ltd. He is on LinkedIn at https://www.linkedin.com/in/roberthwoodman, on Twitter at @RobertHWoodman and @QuantaBioDesign.

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Chemical structure of PN10283, Fmoc-N-amido-dPEG®12-acid

SNARE-Mediated Membrane Fusion and dPEG®, Part 1

Part 1: A Reduced SNARE Model for Membrane Fusion

The cells of all living things depend on membrane fusion for intra- and intercellular transport of molecules. In both cellular membrane fusion and intracellular vesicle fusion, the fusion process is controlled and guided by SNARE proteins. SNARE is an acronym for Soluble NSF Attachment Protein Receptor. NSF stands for N-ethylmaleimide-Sensitive Factor. Reviews of SNARE protein structure and function can be found here, here, here, and here. An example of a SNARE protein is synaptobrevin. Click part 2 and part 3 to read the other pieces in this series. Figure 1 shows a diagram of the process of membrane fusion. Figure 2 shows detail of two membranes fusing with SNARE proteins involved in the process. Both images are from Wikipedia and have a creative commons license.

FIgure 1 shows the process of lipid bilayer fusion such as is found in cell membrane fusion and vesicle fusion.
Figure 1: Lipid Bilayer Fusion Process (image from Wikipedia; image is not altered from the original)

Lipid bilayer fusion by MDougM. Licensed under Public Domain via Creative Commons

Figure 2 shows the exocytosis machinery in membrane fusion.
Figure 2: The Exocytosis Machinery Showing How Membrane Fusion Works (image from Wikipedia.com via a Creative Commons License; image is not altered from the original).

Exocytosis-machinery by Danko Dimchev Georgiev, M.D. - http://en.wikipedia.org/wiki/Image:Exocytosis-machinery.jpg. Licensed under CC BY-SA 3.0 via Creative Commons

Membrane Fusion Modeling

It is critical to model membrane fusion in order to understand and control it. A good model of membrane fusion via SNARE proteins helps scientists understand the fusion process and may permit them to conduct membrane fusion under highly controlled conditions to carry out advanced applications in medical diagnostics and therapeutics. To create a good model for membrane fusion, researchers must find the minimal biochemical machinery needed to reproduce membrane fusion fully in a test system.

In 2009, researchers at the lab of Alexander Kros, Leiden Institute of Chemistry, Leiden University, The Netherlands, took a significant step toward that goal(1). In the process, they used Quanta BioDesign's product PN10283, Fmoc-N-amido-dPEG®12-acid as part of the model system(2). Starting with 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), Kros' group coupled PN10283 to DOPE. The DOPE-PEG construct was then coupled to one of two lipidated oligopeptides, designated LPE and LPK. Each peptide contains a three-heptad repeat (designated E and K) that are the shortest known coiled-coil pair to form a stable heterodimer. See Figure 3, below.

Figure 3: (a) Space-filling model of the two lipidated oligopeptides, LPE and LPK. (b) The two lipidated oligopeptides are spontaneously incorporated into lipid bilayers resulting in liposomes with either LPE or LPK at the surface. When a population of liposomes carrying LPE is mixed with a population of liposomes carrying LPK, the liposomes spontaneously fuse. (c) Diagram showing the fusion of liposomes with the minimal SNARE model of the paper (left) and the SNARE-based protein model (right).
Figure 3: (a) Space-filling model of the two lipidated oligopeptides, LPE and LPK. (b) The two lipidated oligopeptides are spontaneously incorporated into lipid bilayers resulting in liposomes with either LPE or LPK at the surface. When a population of liposomes carrying LPE is mixed with a population of liposomes carrying LPK, the liposomes spontaneously fuse. (c) Diagram showing the fusion of liposomes with the minimal SNARE model of the paper (left) and the SNARE-based protein model (right).

The purpose of this design was to mimic the essential functions of a SNARE protein complex but to minimize the number of different elements needed to direct membrane fusion. The DOPE portion of the construct thus spontaneously anchors the LPE and LPK oligopeptides into the membrane. The dPEG® linker provides a short, flexible spacer that extends the oligopeptides away from the membrane. The two peptides themselves provided the driving force to bring the two membranes together, allowing for membrane fusion.

Using circular dichroism and fluorescence resonance energy transfer (also called Förster resonance energy transfer, known popularly by the acronym FRET) assays with liposome constructs, the researchers were able to distinguish and analyze three distinct stages in membrane fusion. Each stage required an energy input in order to proceed. In the first stage, the liposomes were brought into close proximity. A FRET assay using nitrobenzofuran and lissamine rhodamine dyes attached to the lipid bilayer surfaces showed that the LPE/LPK coiled coil complex that forms spontaneously when the liposomes are in close proximity provides sufficient energy to bring the liposomes together and initiate lipid mixing of the outer lipid layers (known as hemifusion). Further FRET experiments showed that the fusion process initiated by the LPE/LPK coiled coil complex provided enough energy to drive the fusion process to the point of mixing both the outer and inner lipid layers. Furthermore, once the lipid layers were mixed, the contents of the liposomes were observed to mix also.

Conclusions

The approach taken in this paper allowed the research group to build a minimal model of membrane fusion. Indeed, the authors concluded,

"The reduced SNARE model presented herein has been shown to meet all of the characteristics of native membrane fusion, and this similarity combined with the ease of use makes the system a true minimal model for SNARE mediated membrane fusion." (1) at page 2333

Quanta BioDesign Helps You Build Complex Constructs

This research shows the power of modeling even for complex systems such as membrane fusion. Moreover, this research demonstrates the flexible utility of dPEG® products, in this case PN10283, Fmoc-N-amido-dPEG®12-acid.

If you need to add PEG to your supramolecular construct, why not consider dPEG®? There are numerous advantages to adding PEG to your peptide or protein construct. First, PEG is amphiphilic, providing enhanced solubility in both water and organic solvents. Second, PEG has been proven to reduce aggregation and precipitation of biomolecules. Third, PEG reduces immunogenicity of molecules to which it is conjugated.

Dispersity is the key advantage of using Quanta BioDesign's dPEG® products instead conventional PEG products. Conventional PEG is dispersed. Quanta BioDesign's dPEG® products are single molecular weight compounds. That is, rather than having a single discrete molecular size and weight, a conventional PEG is composed of numerous PEG molecules. The "weight" of a conventional PEG is expressed as an average molecular weight across the range of sizes. Quanta BioDesign's dPEG® constructs — designed, patented, and manufactured in the United States — are single molecular weight PEG molecules functionalized with various chemical moieties. Whereas conventional PEGs confound analysis by presenting an intractable mixture of different size molecules, dPEG® constructs enhance analysis by being a single, discrete molecule that can be accurately characterized using ordinary analytical chemistry tools.

Check out our latest products online. You'll be pleasantly surprised at what you can do with dPEG®!

References

(1) Hana Robson Marsden, Nina A. Elbers, Paul H. H. Bomans, Nico A. J. M. Sommerdijk, and Alexander Kros. A Reduced SNARE Model for Membrane Fusion. Angew. Chem. Int. Ed (2009), 48, 2330-2333. DOI: 10.1002/anie.200804493

(2) The dPEG® was purchased from Iris Biotech GmbH, one of Quanta BioDesign's highly valued distributors. Iris Biotech is located in Marktredwitz, Germany.

About the Author

Robert H. Woodman, Ph.D. is a Senior Product Development Scientist and the QC Manager for Quanta BioDesign, Ltd. He is on LinkedIn at https://www.linkedin.com/in/roberthwoodman, on Twitter at @RobertHWoodman and @QuantaBioDesign.

Read More
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