Getting Things into Cells, Pt. 1: Peptides
An idea I had for this platform was organizing and formalizing my literature reviews and sharing them here. These will likely be incredibly boring posts for most (sorry), but I do hope they will be helpful to those learning about basic translational science concepts and those without easy access to academic literature.
— SUMMARY —
Why we care: Peptide drugs may be an interesting therapeutic strategy for some indications. Getting things into cells is hard. Cool drug targets are inside cells.
Take home notes: Peptides have a short half-life in the body and are hard to get into cells. This may be addressable. Cell-penetrating-peptides are in the clinic and nearing market approval.
— TABLE OF CONTENTS —
(I) The Basics
An introduction to proteins.
(II) Peptides as Therapeutics
An introduction to the current status of peptides in the clinic and their advantages and disadvantages.
(III) Cell Membranes
A quick introduction to cell membranes and permeability constants of molecules.
(IV) Permeability: Development of Cell Penetrating Peptides
Important historical developments, characteristics, cell entering mechanisms, and the use of cell penetrating peptides in clinic, and an introduction to macrocyclic peptides.
(V) Improving Half Life in vivo
How to improve the half-life of peptide therapies in the body to improve pharmacodynamics of these drugs.
(VI) Applications in Longevity and Aging
Case studies of peptide use in FOXO4 inhibition, cancer therapeutics development, botulinum conjugates, and Alzheimer’s disease.
(VII) References
— (I) THE BASICS —
An amino acid is an organic compound characterized by its amine (-NH2), carboxyl (-COOH), and R group (varying structures). The R group determines the characteristics of the specific amino acid. Peptides are short chains of amino acids connected by peptide bonds. Proteins are large chains of one or multiple polypeptides, generally considered to be larger than 50 amino acids.
Amino acids are coded for by three nucleotides and their combination into peptides and proteins is encapsulated within the human genome. The central dogma of molecular biology is DNA -> RNA -> protein.
Peptides play roles in human physiology as neurotransmitters, growth factors, hormones, and broadly as signaling molecules.
— (II) PEPTIDES AS THERAPEUTICS —
There are over 200 peptide and protein-based therapeutics approved by the FDA. [2] Usmani et al of the CSIR-Institute of Microbial Technology in India have developed an open-access database of these therapies (THPdb: link).
If we assume that this list is representative of most peptide drugs on the market, it illustrates a few key points:
- Peptide drugs, at least some, are mild enough to be used for indications which are not severe and even as a diagnostic tool;
- All are delivered non-orally;
- They largely target membrane-bound receptors — therefore it is highly likely that none of these drugs are able to cross the cell membrane;
- The half life varies from as short as approximately 1 minute (oxytocin) to 15 hours (liraglutide);
- These are largely recombinantly-produced analogs of naturally existing peptides;
Some of these drugs are massively successful: liraglutide (trade name Victoza), leuroprolide (Lupron), glatiramer (Copaxone), and goserelin (Zoladex) are/were blockbusters or pretty close to it! [3]
If we want to develop peptide therapies for intracellular targets, two significant challenges will be cell membrane permeability and half-life. Before I dig further into those issues and how some are tackling it, a quick summary:
Benefits of Peptide Therapies
- Highly selective for their targets
- Broadly applicable to many indications
- Manufacturing protocols are established
- Generally safe and tolerable
- Demonstrated efficacy in some uses
- Theoretically amenable to modification
- Can combine and co-synthesize with other drug types
Drawbacks
- They are unstable both chemically and physically
- Prone to chemical reactions
- Short half life in vivo and quick elimination
- Low membrane permeability
- Not orally bioactive
- Potential for immunogenicity
- Specificity and ability to target them to specific tissues
- Higher cost than small molecules
(the above is modified from a protein drug SWOT diagram found in [2] and [8])
— (III) CELL MEMBRANES —
I cannot resist an excuse to review the cellular membrane. (AKA — let’s see how much basic biology has leaked out of my brain since freshman year!) Most of this section is distilled from [4] unless otherwise noted, which I found to be a great overview of drug permeability.
The eukaryotic cellular membrane is composed of lipids and membrane proteins. Lipids are these funny little molecules which have hydrophobic (water-fearing) and hydrophillic (water-loving) character to them. These lipids form a bilayer with the hydrophilic ends facing outwards and the hydrophobic inside. The lipid bilayer is only 5 nm (0.000000005 meters) thick, yet it is an effective barrier and gate control for the cell. Membrane proteins include those such as the receptors to the peptide drugs in the table above. These membrane proteins play a lot of rolls including pumping in and out critical ions and other molecules including peptides, receiving signals from other cells, and sending signals to other cells.
Selected laboratory methods of assaying if a molecule has crossed the membrane:
- Artificial lipid bilayers
- Cell monolayers (can be modified to up- or down-regulate certain transporters)
- Cellular fractionation to identify residues in the cytosol
- Fluorescence assays to visualize localization
- Biological effect that only occurs if the molecule is endocytosed
The permeability of a specific molecule by passive diffusion is measured by its permeability coefficient in units of cm/s. To give scale to this: a highly permeable molecule, O2, has a permeability constant of 2.3*10¹ cm/s, and molecule which is not very permeable, Na+, has a constant of at 10^-14. (Protein sodium channels in the membrane facilitate entry of these ions which are critical to many cellular processes, including neuronal action potentials). The title “highly permeable” is associated with permeability coefficients of 10^-5 or larger. Generally, large polar molecules and all charged molecules have a hard time getting across the membrane on their own. Non-polar, uncharged, small molecules have the best shot of getting across the membrane. This source states that these values aren’t often calculated for peptide drugs, although it is unclear whether this is because assaying membrane penetrance of peptides is inherently more challenging than of other molecules, or because most peptides to date inherently cannot cross the cell membrane. Molecules that cannot passively diffuse through the membrane can enter by protein receptors in the membrane. Some toxins and viruses have evolved to take advantage of these pathways to inflict their pathology and people use these to get things into cells. An example is the adeno-associated virus, which has had clinical success as a vector for gene therapy in the eye and other indications.
— (IV) PERMEABILITY: DEVELOPMENT OF CELL PENETRATING PEPTIDES —
Cell penetrating peptides have transduction domains which facilitate their crossing of the membrane. These are not yet a honed technology, but there is a lot of potential here for allowing the use of therapeutic peptides and other molecules intracellularly. As far as I know, there are currently no CPP-mediated drugs on the market.
The reference [5] does a fantastic job of outlining the current status and potential of CPPs in translational medicine. Much of below is adapted from this paper.
(IV.a) Important Historical Developments
1988 — the transactivator of transcription (Tat) protein was isolated from HIV and shown to be able to enter cells in vitro by Frankel et al and Green et al
1991 — the homeodomain of Antennapedia, a protein found in Drosophila melanogaster, is shown to be able to enter cells in vitro by Joliot et al.
1994—the 16 AA CPP penetratin is defined, which is derived from Antennapedia, by Derossi et al
(IV.b) Characteristics of Cell Penetrating Peptides
CPPs are generally five to thirty amino acids in length and can cross the cell membrane in a receptor-independent manner. [6] They may be cationic, amphipathic, or hydrophobic:
Cationic: positive net charge. Often rich with arginine and lysine residues. Includes Tat and penetratin. Arginine is thought to be a primary driver of these CPP’s ability to cross the membrane
Amphipathic: contain both polar (hydrophilic, water-loving) and non-polar (hydrophobic, water-fearing) regions. Rich in hydrophobic residues.
Hydrophobic: non-polar residues. High affinity for hydrophobic domains of the membrane. They are thought to be able to cross the membrane in an energy-independent manner, which the former are not capable of.
CPPs can carry a wide variety of cargo which may be attached covalently or non-covalently. Covalent binding, while strong, can change the properties of the cargo and therefore non-covalent binding may be preferable in some instances. [6] Non-covalent interactions for CPPs may also serve to promote a longer half-life by inhibiting enzymatic degradation of the conjugate.
CPPs can also be classified by their origin: natural products, natural products which have been somehow engineered or modified, protein fragments, and full engineered novel peptides. [7]
(IV.c) Endocytosis
CPPs can broadly enter cells either via endocytosis (energy-dependent) or membrane transduction (energy-independent), the former being more common than the latter.
After endocytosis, CPPs and their cargo must escape the endosomes, which use a high pH to degrade things that the cell does not want inside. This is thought to be a major rate-limiting step. Methods of facilitating CPP endosome escape:
- Insertion of a peptide sequence which is sensitive to the high pH characteristic of endosomes, which then function to destabilize the lipid membrane of the endosomes;
- The addition of histidine moieties into the CPP sequence, which accept protons at the aforementioned high pH and increases the osmotic pressure into the cell (makes it more energy-favorable for the peptides to exit the endosome);
- PepFects, a series of peptides which have special residues to facilitate endosomal escape from the Langel group at Stockholm University
(IV.d) Cell penetrating peptides in the clinic
CPPs are already in the clinic, with two candidates by Xigen SA and Sarepta Therapeutics having reached Phase III studies indicated for intraocular pain with inflammation and Duchenne muscular dystrophy respectively.
I plan to expand on this topic significantly in a further post. Understanding the conditions surrounding the successes and failures of CPPs in the clinic is important for assessing the translational capacity of the platform as a whole.
(IV.e) Macrocyclic Peptides
A cyclic peptide is one where the peptide is not completely linear but has cyclic or circular structure due to extra bonds between the amino acids. An amino acid residue in a linear peptide only has up to two bonds (the amide bonds with its two neighbors). An example below is linaclotide, which demonstrates disulfide bonds:
Cyclization of peptides can improve in vivo stability and permeability of the peptide by impairing the ability of an enzyme to recognize and reach its target amino acid sequence where it has catalytic activity.
Some natural cyclic peptides exist which are cell permeable. These have a number of shared characteristics: N-terminus methylation (a post-translational modification), intramolecular hydrogen bonds, and hydrophobic groups which function to reduce polar surface area of the peptide, decreasing the desolvation energy inherent of the molecule. [9] (Remember, water is highly polar and interacts well with highly polar (hydrophilic) molecules, while hydrophobic molecules are non-polar. If you reduce the polar surface area of a molecule, it “wants” to leave water (the extracellular environment) more, so it is more energy favorable to interact with the cell membrane and receptors).
This website provides a good overview of molecular interactions and their impact on drug pharmacodynamics: link
— (V) INCREASING HALF-LIFE IN VIVO—
The half-life of a molecule is the amount of time it takes for the amount of this molecule to drop to half of its original value. The half-life of a drug is a critical factor in its biological activity.
Peptides generally have very short half-lives due to enzymatic activity and renal clearance. This is not ideal from a pharmacodynamic perspective. Short half-life can inhibit the drugs’ bioavailability, and in the clinic could require the patient receive the drug multiple times per day to achieve the therapeutic benefit — something that is unideal in general and makes peptide use in non-critical indications less appealing. Oral bioavailability is favorable as it is minimally invasive, able to be done at home, and generally has increased patient compliance.
An interesting method used to increase half-life indirectly is used by leuprolide, the gonadotrophin-releasing hormone agonist and blockbuster drug. A large amount of the peptide is deposited IM into the patient, from which the peptide releases slowly for over a month, providing long term pharmacological activity.
Proteolysis is the degradation of peptides and proteins by enzymes which break the amide bond between each amino acid. The N-terminus residue of a protein can give a clue to the expected half-life of a specific peptide. (Proteins and peptides have a C-terminus and an N-terminus. The C-terminus is the end of the protein where the free -COOH and the N-terminus is the end with the free -NH2 group). Proteolysis resistance can be conferred by protection of these termini, where many enzymes have their proteolytic activity, by replacing L-amino acids with D-amino acids, and by unnaturally modifying some residues in enzyme binding sites. [7] Amino acids have two isomers (These amino acids are structurally identical, but mirror images of each other. You can visualize this by looking at your hands, which are enantiomers of each other). The L-isomer is used by cells while the D-isomer is not.
Enhanced renal stability has been demonstrated by binding the peptide drug to other proteins which resist degradation and filtering, especially marcomolecules and endogenous plasma proteins. [7]
— (VI) APPLICATIONS IN LONGEVITY AND AGING —
Yes! The exciting bit. How is relevant to therapeutics targeting the mechanisms of aging and longevity? This topic of this post was partially inspired by the announcement of Cleara Therapeutics (link), a new biotechnology company launched by Peter de Keizer and Apollo Ventures (link). Cleara will be developing peptide therapies for FOXO4-associated cellular senescence. I’ve quickly summarized a few aging-related fields where peptide therapies have been contemplated or explored.
(VI.a) FOXO4 inhibition [10]
The inhibition of FOXO4 with a peptide-CPP construct was shown to selectively induce apoptosis in senescent cells and restore mouse fitness.
Senescent cells are cells which have permanently exited the replicative cycle. They are thought to have negative impact on their microenvironment and have been associated with multiple diseases of aging. Senolytics are a novel class of candidate therapeutics which selectively eliminate these cells; these work via a variety of molecular pathways. FOXO4 is elevated in senescent cells and is thought to bind p53 and inhibit p53-mediated-apoptosis. In this paper, the authors used FOXO4-DRI, a peptide, to compete with the FOXO4-p53 interaction. (DRI stands for D-retro-inverso, reflecting the use of D-amino acids instead of L-amino acids. See Increasing Half Life in Vitro for a succinct explanation of L versus D isomers). FOXO-DRI was fused with Tat, the HIV-derived CPP discussed previously, to facilitate cell permeability.
(VI.b) Cancer [11]
Peptides therapies are being explored for use in cancer. These are being explored in a wide variety of pathways.
Antimicrobial peptides form pores on the bacterial membrane via electrostatic interactions and induce apoptosis. These can also target cancer cells, disrupting the cells’ membrane in a similar manner and inducing apoptosis or necrosis. Cell-penetrating peptides have been explored as a delivery mechanism for cytotoxic agents to the cancer cell. Tumor-targeting peptides are peptides which target specific receptors on the cancer cell membrane. These can be used to inhibit specific molecular pathways, or to facilitate tumor-specific peptide-mediated cargo delivery into cancer cells. Pathways of particular interest are the MAPK pathway, which is constitutively activated in many cancers. Therapeutic peptides may also be able to target the cell cycle, specifically the drivers of overactive cell proliferation as is characteristic of cancer cells, and to block inhibition of tumor suppressors. FOXO4-DRI is an example of this, as its blocking of p53-FOXO4 interaction frees p53, a tumor suppressor, from inhibition.
(VI.c) Botulinum conjugates
Ok — this one isn’t strongly age-related, but I found it an interesting use of a CPP. Revance Therapeutics is developing CPP-conjugated botulinum-toxin A (the active ingredient in Botox) for multiple therapeutic and cosmetic indications, including wrinkles. (The CPP is referred to as the TransMTS carrier peptide system by the company) Botox does not target the mechanism of skin aging, it acts as a local paralytic. However, this mechanism has proved useful in non-cosmetic indications such as migraines and overactive bladder.
Revance Therapeutics’ CPP-botulinum toxin A conjugate is in Phase II and entering Phase III clinical trials for cervical dystonia (involuntary and painful neck contraction) and plantar fasciitis (heel pain).
(VI.d) Alzheimer’s disease [12]
Alzheimer’s disease is a notoriously difficult disease to develop drugs for, and is infamous for the many drugs which have failed in Phase III for this indication. With that, I have to emphasize that success in Alzheimers animal models is not a very strong predictor of clinical success. Some peptides have been shown to be able to cross the blood-brain barrier (Things that can cross the blood-brain barrier are impressive! This highly selective membrane between the central nervous system and the body’s circulatory system is notoriously difficult to cross).
The peptide explored in this paper is R8-A-beta(25–35) conjugated to polyethylenimine (PEI), which has been used previously for protein transduction across the membrane, delivered INN to APP/PS1 transgenic mice and in vitro. These mice have elevated beta-amyloid production in neurons. The therapeutic conjugate also contained a segment of polyR, a cell penetrating peptide. The cargo was intended to inhibit A-beta aggregation and propagation by entering the cell and binding A-beta peptides, preventing amyloidosis. In young transgenic mice, daily IN delivery of this conjugate reduced A-beta levels and improved performance on memory assays in the mice after 4 months of treatment. In older mice with established amyloid plaques, the number of plaques remained constant but their size and abundance in the CNS decreased by approximately 20%. It is unclear if these mice showed improvement in memory assays. No immunoreactivity to the peptide was shown.
— — —
I hope this will be helpful to others. Please do not hesitate to ping me if I have missed a crucial fact or misstated something! Also, please do let me know if these style posts are too boring to stand, although I cannot promise that will stop me from writing more of them…
— (VII) REFERENCES —
[1] Fosgerau K and Hoffmann T. Peptide therapeutics: current and future directions. Drug Discovery Today. 2015; 20(1): 122–128.
[2] Usmani et al. THPdb: Database of FDA-approved peptide and protein therapeutics. PLoS One. 2017; 12(7).
[3] Pharmaceutical Technology Editors. Peptides Gain Traction in Drug Development. 02 June 2012. Accessed: 19 March 2018. Available at: http://www.pharmtech.com/peptides-gain-traction-drug-development?id=&pageID=1&sk=&date=
[4] Yang N and Hinner M. Getting Across the Cell Membrane: An Overview for Small Molecules, Peptides, and Proteins. Methods Mol Bio. 2015;1266: 29–53.
[5] Guidotti G, Brambilla L, Rossi D. Cell-Penetrating Peptides: From Basic Research to Clinics. Trends in Pharmacological Sciences. 2017;38(4): 406–424.
[6] Guo Z, Peng H, Kang K, and Sun D. Cell-penetrating peptides: Possible transduction mechanisms and therapeutic applications (Review) Biomedical Reports. 2016;4: 528–534.
[7] Walport L, Obexer R, and Suga H. Strategies for transitioning macrocyclic peptides to cell permeable drug leads. Current Opinion in Biotechnology. 2017;48: 242–250.
[8] Li Di. Strategic Approaches to Optimizing Peptide ADME Properties. The AAPS Journal. 2015;17(1): 134–143.
[9] Qian Z, Dougherty P, and Pei D. Targeting intracellular protein-protein interactions with cell-permeable cyclic peptides. Current Opinion in Chemical Biology. 2017;38: 80–86.
[10] Baar et al. Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging. Cell. 2017;169: 132–147.
[11] Marcus S, Pirogova E, Piva T. Evaluation of the use of therapeutic peptides for cancer treatment. Journal of Biomedical Science. 2017;24: 1–15.
[12] Cheng Y et al. An intranasally delivered peptide drug ameliorates cognitive decline in Alzheimer transgenic mice. EMBO Molecular Medicine. 2017;9(5): 703–715.
