ABSTRACT
Peptides are highly selective, high-affinity ligands for a diverse array of disease targets, but suitably derivatizing them for application as diagnostic or therapeutic agents often presents a significant challenge. Covalent modification with metal chelates frequently results in decreased binding affinity, so a variety of strategies must be explored to find suitable locations for modification and facile peptide conjugation chemistries that maintain or enhance binding affinity. In this chapter, we present a paradigm for systematically optimizing peptide binding and determining the favorable sites and methods for peptide conjugation. This strategy is illustrated by two case studies of peptide-based targeted gadolinium contrast agents: EP-2104R for diagnosis of thrombosis and EP-3533 for diagnosis of cardiac perfusion and fibrosis. Two different architectures for the peptide-metal complex conjugation were designed: EP-2104R contains a total of four gadolinium (Gd) chelates linked at the N- and C-termini, whereas EP-3533 is derivatized with three Gd chelates, two on the N-terminus and one on a lysine side chain. Detailed protocols are provided for two Gd chelate conjugation methods.
Subject(s)
Contrast Media , Magnetic Resonance Imaging , Peptides , Amino Acid Sequence , Collagen/chemistry , Collagen/metabolism , Contrast Media/chemistry , Fibrin/metabolism , Gadolinium/chemistry , Gadolinium DTPA/chemistry , Heterocyclic Compounds/chemistry , Humans , Molecular Sequence Data , Organometallic Compounds/chemistry , Peptides/chemical synthesis , Peptides/chemistry , Staining and Labeling , Structure-Activity RelationshipABSTRACT
Peptides that bind to fibrin but not to fibrinogen or serum albumin were selected from phage display libraries as targeting moieties for thrombus molecular imaging probes. Three classes of cyclic peptides (cyclized via disulfide bond between two Cys) were identified with consensus sequences XArXCPY(G/D)LCArIX (Ar = aromatic, Tn6), X(2)CXYYGTCLX (Tn7), and NHGCYNSYGVPYCDYS (Tn10). These peptides bound to fibrin at â¼2 sites with K(d) = 4.1 µM, 4.0 µM, and 8.7 µM, respectively, whereas binding to fibrinogen was at least 100-fold weaker. The peptides also bind to the fibrin degradation product DD(E) with similar affinity to that measured for fibrin. The Tn7 and Tn10 peptides bind to the same site on fibrin, while the Tn6 peptides bind to a unique site. Alanine scanning identified the N- and C-terminal ends of the Tn6 and Tn7 peptides as most tolerant to modification. Peptide conjugates with either fluorescein or diethylenetriaminepentaaceto gadolinium(III) (GdDTPA) at the N-terminus were prepared for potential imaging applications, and these retained fibrin binding affinity and specificity in plasma. Relaxivity and binding studies on the GdDTPA derivatives revealed that an N-terminal glycyl linker had a modest effect on fibrin affinity but resulted in lower fibrin-bound relaxivity.
Subject(s)
Bacteriophages/chemistry , Fibrin/chemistry , Peptides/chemistry , Amino Acid Sequence , Molecular Sequence DataABSTRACT
A strategy for preparing high relaxivity, metabolically stable peptide-based MR contrast agents is described.
Subject(s)
Contrast Media/chemistry , Fibrin/chemistry , Magnetic Resonance Imaging/methods , Thrombosis/diagnosis , Amino Acid Sequence , Animals , Disease Models, Animal , Drug Evaluation, Preclinical , Gadolinium/chemistry , Guinea Pigs , Molecular Sequence Data , Molecular Structure , Sensitivity and SpecificityABSTRACT
Thrombus (blood clot) is implicated in a number of life threatening diseases, e.g., heart attack, stroke, pulmonary embolism. EP-2104R is an MRI contrast agent designed to detect thrombus by binding to the protein fibrin, present in all thrombi. EP-2104R comprises an 11 amino acid peptide derivatized with 2 GdDOTA-like moieties at both the C- and N-terminus of the peptide (4 Gd in total). EP-2104R was synthesized by a mixture of solid phase and solution techniques. The La(III) analogue was characterized by and 1D and 2D NMR spectroscopy and was found to have the expected structure. EP-2104R was found to be significantly more inert to Gd(III) loss than commercial contrast agents. At the most extreme conditions tested (pH 3, 60 degrees C, 96 hrs), less than 10% of Gd was removed from EP-2104R by a challenge with a DTPA based ligand, while the commercial contrast agents equilibrated within minutes to hours. EP-2104R binds equally to two sites on human fibrin (Kd = 1.7 +/- 0.5 microM) and has a similar affinity to mouse, rat, rabbit, pig, and dog fibrin. EP-2104R has excellent specificity for fibrin over fibrinogen (over 100-fold) and for fibrin over serum albumin (over 1000-fold). The relaxivity of EP-2104R bound to fibrin at 37 degrees C and 1.4 T was 71.4 mM(-1) s(-1) per molecule of EP-2104R (17.4 per Gd), about 25 times higher than that of GdDOTA measured under the same conditions. Strong fibrin binding, fibrin selectivity, and high molecular relaxivity enable EP-2104R to detect blood clots in vivo.
Subject(s)
Contrast Media/chemistry , Gadolinium/blood , Gadolinium/chemistry , Magnetic Resonance Angiography/methods , Peptides/blood , Peptides/chemistry , Thrombosis/blood , Contrast Media/metabolism , Fibrin/chemistry , Fibrin/metabolism , Fibrinogen/chemistry , Fibrinogen/metabolism , Heterocyclic Compounds/blood , Heterocyclic Compounds/chemistry , Humans , Kinetics , Nuclear Magnetic Resonance, Biomolecular/methods , Organometallic Compounds/blood , Organometallic Compounds/chemistry , Protein Binding , Substrate Specificity , Thermodynamics , Thrombosis/diagnosisABSTRACT
Clinically relevant relaxivity enhancement of a magnetic resonance imaging (MRI) contrast agent has been achieved by using prodrug Gd3+ complexes (see picture, DTPA=diethylenetriaminepentaaceto). Enzymatic cleavage of lysine residues from the prodrug exposes a group that has a high affinity to human serum albumin and promotes enhanced relaxivity, thus enabling the detection of targets at submicromolar concentrations.
