RESUMEN
Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 â increase in IgG1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG1 of -33.0 kcal/mol and -32.7 kcal/mol, and -T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.
Asunto(s)
Inteínas , Staphylococcus aureus , Humanos , Inteínas/genética , Ligandos , Termodinámica , Inmunoglobulina G , Calorimetría/métodos , Unión ProteicaRESUMEN
Cyclic peptides are an emerging class of molecular therapeutics that are increasingly viewed as ideal backbones for modulation of protein-protein interactions. A split-intein based method, termed SICLOPPS, enables the rapid generation of genetically encoded cyclic peptide libraries of around a hundred million members. Here we review recent approaches using SICLOPPS for the discovery of bioactive compounds.
Asunto(s)
Péptidos Cíclicos/química , Descubrimiento de Drogas , Biblioteca de Péptidos , Péptidos Cíclicos/genética , Péptidos Cíclicos/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismoRESUMEN
The phenomenon of protein misfolding and aggregation has been widely associated with numerous human diseases, such as Alzheimer's disease, systemic amyloidosis and type 2 diabetes, the vast majority of which remain incurable. To advance early stage drug discovery against these diseases, investigation of molecular libraries with expanded diversities and ultrahigh-throughput screening methodologies that allow deeper investigation of chemical space are urgently required. Toward this, we describe how Escherichia coli can be engineered so as to enable (1) the production of expanded combinatorial libraries of short, drug-like, head-to-tail cyclic peptides and (2) their simultaneous functional screening for identifying effective inhibitors of protein misfolding and aggregation using a genetic assay that links protein folding and misfolding to cell fluorescence. In this manner, cyclic peptides with the ability to inhibit pathogenic protein misfolding and/or aggregation can be readily selected by flow cytometric cell sorting in an ultrahigh-throughput fashion. This biotechnological approach accelerates significantly the identification of hit/lead molecules with potentially therapeutic properties against devastating diseases.
Asunto(s)
Pliegue de Proteína , Diabetes Mellitus Tipo 2 , Escherichia coli/genética , Humanos , Péptidos , Péptidos Cíclicos , Proteínas , TecnologíaRESUMEN
Backbone macrocyclic structures are often found in diverse bioactive peptides and contribute to greater conformational rigidity, peptidase resistance, and potential membrane permeability compared to their linear counterparts. Therefore, such peptide scaffolds are an attractive platform for drug-discovery endeavors. Recent advances in synthetic methods for backbone macrocyclic peptides have enabled the discovery of novel peptide drug candidates against diverse targets. Here, we overview recent technical advancements in the synthetic methods including 1) enzymatic synthesis, 2) chemical synthesis, 3) split-intein circular ligation of peptides and proteins (SICLOPPS), and 4) in vitro translation system combined with genetic code reprogramming. We also discuss screening methodologies compatible with those synthetic methodologies, such as one-beads one-compound (OBOC) screening compatible with the synthetic method 2, cell-based assay compatible with 3, limiting-dilution PCR and mRNA display compatible with 4.
RESUMEN
Cyclic peptide libraries have successfully been employed for the identification of inhibitors of highly challenging targets. While several methodologies exist for the generation of cyclic peptide libraries, genetically encoded libraries hold several advantages over purely in vitro methods of library generation, including the ability to conduct cell-based functional screens and straightforward hit deconvolution. Here we detail the use of split-intein circular ligation of peptides and proteins (SICLOPPS) for the identification and optimization of several first-in-class and best-in-class inhibitors. We describe the current advances in the identification of SICLOPPS-derived inhibitors, as well as the optimization of library generation through the use of new inteins. Finally, we discuss the production of more diverse libraries as a way of enhancing the hit rate against difficult protein-protein interactions.
Asunto(s)
Inteínas/genética , Biblioteca de Péptidos , Péptidos Cíclicos/genética , Proteínas/antagonistas & inhibidores , Biocatálisis , Química Farmacéutica , Descubrimiento de Drogas/métodos , Genes Reporteros , Ensayos Analíticos de Alto Rendimiento , Humanos , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/uso terapéutico , Empalme de Proteína , Proteínas/metabolismo , Selección Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
With the increasing utilization of high-throughput screening for lead identification in drug discovery, the need for easily constructed and diverse libraries which cover significant chemical space is greater than ever. Cyclic peptides address this need; they combine the advantageous properties of peptides (ease of production, high diversity, high potential specificity) with increased resistance to proteolysis and often increased biological activity (due to conformational locking). There are a number of techniques for the generation and screening of cyclic peptide libraries. As drug discovery moves toward tackling challenging targets, such as protein-protein interactions, cyclic peptide libraries are expected to continue producing hits where small molecule libraries may be stymied. However, it is important to design robust systems for the generation and screening of these large libraries, and to be able to make sense of structure-activity relationships in these highly variable scaffolds. There are a plethora of possible modifications that can be made to cyclic peptides, which is both a weakness and a strength of these scaffolds; high variability will allow more precise tuning of leads to targets, but exploring the whole range of modifications may become an overwhelming challenge.
Asunto(s)
Descubrimiento de Drogas/métodos , Biblioteca de Péptidos , Péptidos Cíclicos/genética , Péptidos Cíclicos/farmacología , Animales , Evaluación Preclínica de Medicamentos/métodos , Humanos , Péptidos Cíclicos/química , Mapas de Interacción de Proteínas/efectos de los fármacos , ARN Mensajero/genética , Relación Estructura-ActividadRESUMEN
The cellular response to hypoxia is orchestrated by HIF-1, a heterodimeric transcription factor composed of an α and a ß subunit that enables cell survival under low oxygen conditions by altering the transcription of over 300 genes. There is significant evidence that inhibition of HIF-1 would be beneficial for cancer therapy. We recently reported a cyclic hexapeptide that inhibits the HIF-1α/HIF-1ß protein-protein interaction in vitro and prevents HIF-1-mediated hypoxia-response signaling in cells. This cyclic peptide was identified from a library of 3.2 × 106 members generated using SICLOPPS split-intein mediated protein splicing. With a view to demonstrating the potential for encoding the production of a therapeutic agent in response to a disease marker, we have engineered human cells with an additional chromosomal control circuit that conditionally encodes the production of our cyclic peptide HIF-1 inhibitor. We demonstrate the conditional production of our HIF-1 inhibitor in response to hypoxia, and its inhibitory effect on HIF-1 dimerization and downstream hypoxia-response signaling. These engineered cells are used to illustrate the synthetic lethality of inhibiting HIF-1 dimerization and glycolysis in hypoxic cells. Our approach not only eliminates the need for the chemical synthesis and targeted delivery of our HIF-1 inhibitor to cells, it also demonstrates the wider possibility that the production machinery of other bioactive compounds may be incorporated onto the chromosome of human cells. This work demonstrates the potential of sentinel circuits that produce molecular modulators of cellular pathways in response to environmental or cellular disease stimuli.
Asunto(s)
Reprogramación Celular/efectos de los fármacos , Cromosomas/efectos de los fármacos , Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Hipoxia/tratamiento farmacológico , Péptidos Cíclicos/farmacología , Transcripción Genética/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Reprogramación Celular/genética , Cromosomas/genética , Células HEK293 , Humanos , Hipoxia/genética , Factor 1 Inducible por Hipoxia/genética , Mapas de Interacción de Proteínas/efectos de los fármacos , Mapas de Interacción de Proteínas/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción/genética , Transcripción Genética/genéticaRESUMEN
Cyclic peptides are an important class of molecules that are increasingly viewed as an ideal scaffold for inhibition of protein-protein interactions (PPI). Here we detail an approach that enables the intracellular synthesis of cyclic peptide libraries of around 108 members. The method utilizes split intein mediated circular ligation of peptides and proteins (SICLOPPS), taking advantage of split intein splicing to cyclize a library of peptide sequences. SICLOPPS allows the ring size, set residues and number of random residues within a library to be predetermined by the user. SICLOPPS libraries have been combined with a variety of cell-based screens to identify cyclic peptide inhibitors of a variety of enzymes and protein-protein interactions.