RESUMO
Peptides typically have poor biostabilities, and natural sequences cannot easily be converted into drug-like molecules without extensive medicinal chemistry. We have adapted mRNA display to drive the evolution of highly stable cyclic peptides while preserving target affinity. To do this, we incorporated an unnatural amino acid in an mRNA display library that was subjected to proteolysis prior to selection for function. The resulting "SUPR (scanning unnatural protease resistant) peptide" showed ≈500-fold improvement in serum stability (t1/2 =160â h) and up to 3700-fold improvement in protease resistance versus the parent sequence. We extended this approach by carrying out SUPR peptide selections against Her2-positive cells in culture. The resulting SUPR4 peptide showed low-nanomolar affinity toward Her2, excellent specificity, and selective tumor uptake in vivo. These results argue that this is a general method to design potent and stable peptides for in vivo imaging and therapy.
Assuntos
Evolução Molecular Direcionada , Peptídeo Hidrolases/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Biblioteca de Peptídeos , Peptídeos Cíclicos/farmacocinética , Estabilidade Proteica , RNA Mensageiro/genética , Células Tumorais CultivadasAssuntos
Interleucina-6/antagonistas & inibidores , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Anticorpos/imunologia , Linhagem Celular Tumoral , Evolução Molecular Direcionada , Humanos , Separação Imunomagnética , Interleucina-6/genética , Interleucina-6/metabolismo , Ligantes , Microfluídica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ligação ProteicaRESUMO
Peptides constructed with the 20 natural amino acids are generally considered to have little therapeutic potential because they are unstable in the presence of proteases and peptidases. However, proteolysis cleavage can be idiosyncratic, and it is possible that natural analogues of functional sequences exist that are highly resistant to cleavage. Here, we explored this idea in the context of peptides that bind to the signaling protein Gαi1. To do this, we used a two-step in vitro selection process to simultaneously select for protease resistance while retaining function-first by degrading the starting library with protease (chymotrypsin), followed by positive selection for binding via mRNA display. Starting from a pool of functional sequences, these experiments revealed peptides with 100-400 fold increases in protease resistance compared to the parental library. Surprisingly, selection for chymotrypsin resistance also resulted in similarly improved stability in human serum (~100 fold). Mechanistically, the decreases in cleavage results from both a lower rate of cleavage (kcat) and a weaker interaction with the protease (Km). Overall, our results demonstrate that the hydrolytic stability of functional, natural peptide sequences can be improved by two orders of magnitude simply by optimizing the primary sequence.