Beyond antibodies: using biological principles to guide the development of next-generation protein therapeutics
CURRENT OPINION IN BIOTECHNOLOGY
2013; 24 (6): 1072-1077
Cystine-knot peptides engineered with specificities for alpha(IIb)beta(3) or alpha(IIb)beta(3) and alpha(v)beta(3) integrins are potent inhibitors of platelet aggregation
JOURNAL OF MOLECULAR RECOGNITION
2011; 24 (1): 127-135
Protein-based biologics, which leverage the inherent affinity and specificity of protein-protein interactions, offer an effective strategy for targeting and modulating disease pathways. Despite the broad diversity of the proteome, monoclonal antibodies have been the major focus of such drug discovery efforts. While antibodies have shown great clinical value, the breadth and complexity of human disease highlight the need for alternatives that expand the therapeutic repertoire beyond this single class of proteins. The elucidation of molecular mechanisms underlying human disease has provided new opportunities for protein-based drugs to address challenging clinical problems. Natural ligands and receptors, which inherently modulate complex biological processes, have emerged as promising candidates for protein-based drug discovery efforts. Protein engineering strategies, guided by biological principles, are allowing ligands and receptors to be developed as next-generation therapeutics with improved safety and efficacy.
View details for DOI 10.1016/j.copbio.2013.03.017
View details for Web of Science ID 000328806200017
A truncated form of the Agouti-related protein (AgRP), a member of the cystine-knot family, has shown promise as a scaffold for engineering novel peptides with new molecular recognition properties. In this study, we replaced a constrained six amino acid loop in AgRP with a nine amino acid loop containing an Arg-Gly-Asp integrin recognition motif, and randomized the neighboring residues to create a library of approximately 20 million AgRP variants. We displayed the AgRP mutants as fusions on the surface of yeast and used high-throughput fluorescence-activated cell sorting (FACS) to isolate peptides that bound specifically to the platelet integrin ?(IIb)?(3), a clinically important target for the prevention and treatment of thrombosis. These AgRP peptides had equilibrium dissociation (K(D)) constants for ?(IIb)?(3) integrin ranging from 60 to 90?nM, and did not bind to ?(v)?(3), ?(v)?(5), or ?(5)?(1) integrins. Using an alternate library screening strategy, we identified AgRP peptides that bound to both ?(IIb)?(3) and ?(v)?(3) integrins with K(D) values ranging from 40 to 70?nM and 20 to 30?nM, respectively, and did not bind to ?(v)?(5) or ?(5)?(1) integrins. Unique consensus sequences were identified within both series of AgRP peptides suggesting alternative molecular recognition events that dictate different integrin binding specificities. In addition, the engineered AgRP peptides prevented platelet aggregation as well as or slightly better than the FDA-approved cyclic peptide eptifibatide. Collectively, these data demonstrate that cystine-knot peptides can be generated with high affinity and specificity to closely-related integrins, and provide insights into molecular interactions between small, structured peptide ligands and their receptors.
View details for DOI 10.1002/jmr.1036
View details for Web of Science ID 000289781900013
View details for PubMedID 21194123