DARPin
Encyclopedia
DARPins are genetically engineered antibody mimetic
proteins typically exhibiting highly specific and high-affinity target protein
binding. They are derived from natural ankyrin
proteins and consist of at least three, usually four or five repeat motifs
of these proteins. Their molecular mass
is about 14 or 18 kDa (kilodaltons) for four- or five-repeat DARPins, respectively.
DARPins are used as investigational tools, and diagnostic and therapeutic applications are being aimed at. In 2010, the first of these molecules, codenamed MP0112, entered clinical trial
s.
, Switzerland
. They are derived from naturally occurring ankyrin proteins, a protein class that is mediating high-affinity protein-protein interactions in nature. Sequence alignment
s of several thousand natural ankyrin repeat motifs (of about 33 amino acid
s each) combined with structure based design and recombinant DNA
methods is used for generation of these proteins. DARPins are composed of repetitive structural units forming a stable protein domain
with a large potential target interaction surface. Typically, DARPins are composed of four or five repeats, corresponding to the average size of natural ankyrin repeat protein domains. Proteins with less than three repeats do not form a tertiary structure. The molecular mass depending on the number of repeats is as follows:
Libraries
of DARPins with randomized potential target interaction residues with diversities of over 1012 variants have been generated at the DNA level. From these libraries, DARPins binding the target of choice with picomolar affinity and specificity can be selected using ribosome display
or signal recognition particle
(SRP) phage display
. DARPins can be designed to act as receptor agonists, antagonists
, inverse agonists, enzyme inhibitors, or simple target protein binders.
of Escherichia coli
at high levels (over 10 g/l in fermentation, 1 g/l in shake flask) in soluble form. The proteins exhibit high thermal and thermodynamic stability (denaturation midpoint
: Tm > 66 °C, equilibrium unfolding
: ∆G > 9.5 kcal/mol), which is increasing with increasing repeat number. DARPins are stable in human blood serum
and do not contain T-cell epitope
s. The high specificity and affinity of binding DARPins has been attributed rigid body
binding mode. Multi-specific or multi-valent constructs made by genetic fusion show similar properties as single domain DARPins. The absence of cysteine
s in the scaffold enables engineering of site-specific cysteines, allowing site-directed coupling of chemicals to the molecule.
based Molecular Partners AG is currently pursuing the development of therapeutic DARPins. MP0112, the first therapeutic DARPin candidate, is a vascular endothelial growth factor
(VEGF) inhibitor and has entered clinical trials for the treatment of wet macular degeneration and diabetic macular edema in early 2010.
Antibody mimetic
Antibody mimetics are organic compounds that, like antibodies, can specifically bind antigens, but that are not structurally related to antibodies. They are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa...
proteins typically exhibiting highly specific and high-affinity target protein
Target protein
Target proteins are functional biomolecules that are addressed and controlled by biologically active compounds. The identification of target proteins, the investigation of signal transduction processes and the understanding of their interaction with ligands are key elements of modern biomedical...
binding. They are derived from natural ankyrin
Ankyrin
Ankyrins are a family of adaptor proteins that mediate the attachment of integral membrane proteins to the spectrin-actin based membrane skeleton. Ankyrins have binding sites for the beta subunit of spectrin and at least 12 families of integral membrane proteins...
proteins and consist of at least three, usually four or five repeat motifs
Ankyrin repeat
The ankyrin repeat is a 33-residue motif in proteins consisting of two alpha helices separated by loops, first discovered in signaling proteins in yeast Cdc10 and Drosophila Notch. Ankyrin repeats mediate protein–protein interactions and are among the most common structural motifs in known proteins...
of these proteins. Their molecular mass
Molecular mass
The molecular mass of a substance is the mass of one molecule of that substance, in unified atomic mass unit u...
is about 14 or 18 kDa (kilodaltons) for four- or five-repeat DARPins, respectively.
DARPins are used as investigational tools, and diagnostic and therapeutic applications are being aimed at. In 2010, the first of these molecules, codenamed MP0112, entered clinical trial
Clinical trial
Clinical trials are a set of procedures in medical research and drug development that are conducted to allow safety and efficacy data to be collected for health interventions...
s.
Origin, structure and generation
DARPins have been developed mainly at the University of ZurichUniversity of Zurich
The University of Zurich , located in the city of Zurich, is the largest university in Switzerland, with over 25,000 students. It was founded in 1833 from the existing colleges of theology, law, medicine and a new faculty of philosophy....
, Switzerland
Switzerland
Switzerland name of one of the Swiss cantons. ; ; ; or ), in its full name the Swiss Confederation , is a federal republic consisting of 26 cantons, with Bern as the seat of the federal authorities. The country is situated in Western Europe,Or Central Europe depending on the definition....
. They are derived from naturally occurring ankyrin proteins, a protein class that is mediating high-affinity protein-protein interactions in nature. Sequence alignment
Sequence alignment
In bioinformatics, a sequence alignment is a way of arranging the sequences of DNA, RNA, or protein to identify regions of similarity that may be a consequence of functional, structural, or evolutionary relationships between the sequences. Aligned sequences of nucleotide or amino acid residues are...
s of several thousand natural ankyrin repeat motifs (of about 33 amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s each) combined with structure based design and recombinant DNA
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
methods is used for generation of these proteins. DARPins are composed of repetitive structural units forming a stable protein domain
Protein domain
A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins consist of several structural...
with a large potential target interaction surface. Typically, DARPins are composed of four or five repeats, corresponding to the average size of natural ankyrin repeat protein domains. Proteins with less than three repeats do not form a tertiary structure. The molecular mass depending on the number of repeats is as follows:
| Repeats | 3 | 4 | 5 | 6 | 7 | ... |
|---|---|---|---|---|---|---|
| Approximate mass (kDa) | 10 | 14 | 18 | 22 | 26 | ... |
Libraries
Library (biology)
In molecular biology, a library is a collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning...
of DARPins with randomized potential target interaction residues with diversities of over 1012 variants have been generated at the DNA level. From these libraries, DARPins binding the target of choice with picomolar affinity and specificity can be selected using ribosome display
Ribosome display
Ribosome display is a technique used to perform in vitro protein evolution to create proteins that can bind to a desired ligand. The process results in translated proteins that are associated with their mRNA progenitor which is used, as a complex, to bind to an immobilized ligand in a selection step...
or signal recognition particle
Signal recognition particle
The signal recognition particle is an abundant, cytosolic, universally conserved ribonucleoprotein that recognizes and targets specific proteins to the endoplasmic reticulum in eukaryotes and the plasma membrane in prokaryotes....
(SRP) phage display
Phage display
Phage display is a method for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. Phage Display was originally invented by George P...
. DARPins can be designed to act as receptor agonists, antagonists
Receptor antagonist
A receptor antagonist is a type of receptor ligand or drug that does not provoke a biological response itself upon binding to a receptor, but blocks or dampens agonist-mediated responses...
, inverse agonists, enzyme inhibitors, or simple target protein binders.
Properties
DARPins are expressed in the cytoplasmCytoplasm
The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...
of Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
at high levels (over 10 g/l in fermentation, 1 g/l in shake flask) in soluble form. The proteins exhibit high thermal and thermodynamic stability (denaturation midpoint
Denaturation midpoint
Assuming two-state protein folding, denaturation midpoint is defined as that temperature or denaturant concentration at which both the folded and unfolded states are equally populated at equilibrium....
: Tm > 66 °C, equilibrium unfolding
Equilibrium unfolding
In biochemistry, equilibrium unfolding is the process of unfolding a protein or RNA molecule by gradually changing its environment, such as by changing the temperature or pressure, adding chemical denaturants, or applying force as with an atomic force microscope tip. Since equilibrium is...
: ∆G > 9.5 kcal/mol), which is increasing with increasing repeat number. DARPins are stable in human blood serum
Blood serum
In blood, the serum is the component that is neither a blood cell nor a clotting factor; it is the blood plasma with the fibrinogens removed...
and do not contain T-cell epitope
Epitope
An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that recognizes the epitope is called a paratope...
s. The high specificity and affinity of binding DARPins has been attributed rigid body
Rigid body
In physics, a rigid body is an idealization of a solid body of finite size in which deformation is neglected. In other words, the distance between any two given points of a rigid body remains constant in time regardless of external forces exerted on it...
binding mode. Multi-specific or multi-valent constructs made by genetic fusion show similar properties as single domain DARPins. The absence of cysteine
Cysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...
s in the scaffold enables engineering of site-specific cysteines, allowing site-directed coupling of chemicals to the molecule.
Applications and clinical development
DARPins have been used as research tools, as diagnostic agents and as therapeutic agents. ZurichZürich
Zurich is the largest city in Switzerland and the capital of the canton of Zurich. It is located in central Switzerland at the northwestern tip of Lake Zurich...
based Molecular Partners AG is currently pursuing the development of therapeutic DARPins. MP0112, the first therapeutic DARPin candidate, is a vascular endothelial growth factor
Vascular endothelial growth factor
Vascular endothelial growth factor is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis. It is part of the system that restores the oxygen supply to tissues when blood circulation is inadequate....
(VEGF) inhibitor and has entered clinical trials for the treatment of wet macular degeneration and diabetic macular edema in early 2010.