ABSTRACT
Chemical protein synthesis is important for dissecting the molecular basis of protein function. Here we advance its scope by demonstrating the significant improvement of the multifaceted pharmaceutical profile of small proteins exclusively via a chemical-based approach. The focus of this work centered on CCL-5 (RANTES) derivatives with potent anti-HIV activity. The overall chemical strategy involved a combination of coded and noncoded amino acid mutagenesis, peptide backbone engineering, and site-specific polymer attachment. The ability to alter specific protein residues, as well as precise control of the position and type of polymer attachment, allows for the exploration of specific molecular designs and resulted in novel CCL-5 analogues with significant differences in their respective biochemical and pharmaceutical properties. Using this approach, the complex-interplay of variables contributing to the noncovalent self-association (aggregation) state, CCR-5 specificity, in vivo elimination half-life, and anti-HIV activity of CCL-5-based protein analogues could be empirically evaluated via total chemical synthesis. This work has led to the identification of potent (sub-nanomolar) anti-HIV proteins with significantly improved pharmaceutical profiles, and illustrates the increasing value of protein chemical synthesis in contemporary therapeutic discovery. These antiviral molecules provide a novel mechanism of action for the development of a new generation of anti-HIV therapeutics which are still desperately needed.
Subject(s)
Anti-HIV Agents/chemistry , HIV Antibodies/immunology , Human Immunodeficiency Virus Proteins/immunology , Amino Acids/chemistry , Animals , Anti-HIV Agents/pharmacokinetics , Chromatography, Gel , Male , Models, Molecular , Molecular Structure , Polymers/chemistry , Rats , Receptors, G-Protein-Coupled/metabolismABSTRACT
Chemical synthesis in combination with precision polymer modification allows the systematic exploration of the effect of protein properties, such as charge and hydrodynamic radius, on potency using defined, homogeneous conjugates. A series of polymer-modified synthetic erythropoiesis proteins were constructed that had a polypeptide chain similar to the amino acid sequence of human erythropoietin but differed significantly in the number and type of attached polymers. The analogs differed in charge from +5 to -26 at neutral pH and varied in molecular weight from 30 to 54 kDa. All were active in an in vitro cell proliferation assay. However, in vivo potency was found to be strongly dependent on overall charge and size. The trends observed in this study may serve as starting points for the construction of more potent synthetic EPO analogs in the future.
Subject(s)
Erythropoiesis/physiology , Polymers/chemical synthesis , Proteins/chemical synthesis , Amino Acid Sequence , Animals , Binding Sites/drug effects , Binding Sites/physiology , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Erythropoietin/chemical synthesis , Erythropoietin/metabolism , Erythropoietin/physiology , Humans , Macaca fascicularis , Mice , Molecular Sequence Data , Polymers/metabolism , Polymers/pharmacology , Proteins/metabolism , Proteins/physiology , RatsABSTRACT
A synthetic strategy that allows for the site-specific attachment of polymers such as poly(ethylene glycol) (PEG) to protein pharmaceuticals is described. PEG was attached to a 67-amino acid fully synthetic CCL-5 (RANTES) analogue at its GAG binding site both to reduce aggregation and to increase the circulating lifetime. Effective protection of an Aoaa chemoselective linker during peptide assembly, total chemical protein synthesis, and protein folding was achieved with an isopropylidene group. Mild deprotection of the resulting folded synthetic protein and subsequent polymer attachment occur without interference with the native folded structure and activity.
Subject(s)
Chemokine CCL5/analogs & derivatives , Chemokines, CC/chemistry , Oximes/chemistry , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Binding Sites , Chemokine CCL5/chemistry , Chemokine CCL5/pharmacology , Chemokines, CC/pharmacology , Glycine/chemistry , HIV-1/drug effects , Humans , Models, Molecular , Polyethylene Glycols/chemistry , Protein Folding , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.
