RESUMEN
Elucidation of the relationship between targeting molecule binding properties and the adhesive behavior of therapeutic or diagnostic nanocarriers would aid in the design of optimized vectors and lead to improved efficacy. We measured the adhesion of 200-nm-diameter particles under fluid flow that was mediated by a diverse array of molecular interactions, including recombinant single-chain antibodies (scFvs), full antibodies, and the avidin/biotin interaction. Within the panel of scFvs, we used a family of mutants that display a spectrum of binding kinetics, allowing us to compare nanoparticle adhesion to bond chemistry. In addition, we explored the effect of molecular size by inserting a protein linker into the scFv fusion construct and by employing scFvs that are specific for targets with vastly different sizes. Using computational models, we extracted multivalent kinetic rate constants for particle attachment and detachment from the adhesion data and correlated the results to molecular binding properties. Our results indicate that the factors that increase encounter probability, such as adhesion molecule valency and size, directly enhance the rate of nanoparticle attachment. Bond kinetics had no influence on scFv-mediated nanoparticle attachment within the kinetic range tested, however, but did appear to affect antibody/antigen and avidin/biotin mediated adhesion. We attribute this finding to a combination of multivalent binding and differences in bond mechanical strength between recombinant scFvs and the other adhesion molecules. Nanoparticle detachment probability correlated directly with adhesion molecule valency and size, as well as the logarithm of the affinity for all molecules tested. On the basis of this work, scFvs can serve as viable targeting receptors for nanoparticles, but improvements to their bond mechanical strength would likely be required to fully exploit their tunable kinetic properties and maximize the adhesion efficiency of nanoparticles that bear them.
Asunto(s)
Nanopartículas , Ingeniería de Proteínas , Anticuerpos de Cadena Única/metabolismo , Avidina/metabolismo , Biotina/metabolismo , Cinética , Unión Proteica , Anticuerpos de Cadena Única/químicaRESUMEN
Yeast surface display has become an increasingly popular tool for protein engineering and library screening applications. Recent advances have greatly expanded the capability of yeast surface display, and are highlighted by cell-based selections, epitope mapping, cDNA library screening, and cell adhesion engineering. In this review, we discuss the state-of-the-art yeast display methodologies and the rapidly expanding set of applications afforded by this technology.
Asunto(s)
Saccharomyces cerevisiae/genética , Animales , Moléculas de Adhesión Celular/genética , ADN Complementario/biosíntesis , ADN Complementario/genética , Mapeo Epitopo , Humanos , Ingeniería de ProteínasRESUMEN
Activated lymphocyte function-associated antigen-1 (LFA-1, alphaLbeta2 integrin) found on leukocytes facilitates firm adhesion to endothelial cell layers by binding to intercellular adhesion molecule-1 (ICAM-1), which is up-regulated on endothelial cells at sites of inflammation. Recent work has shown that LFA-1 in a pre-activation, low-affinity state may also be involved in the initial tethering and rolling phase of the adhesion cascade. The inserted (I) domain of LFA-1 contains the ligand-binding epitope of the molecule, and a conformational change in this region during activation increases ligand affinity. We have displayed wild-type I domain on the surface of yeast and validated expression using I domain specific antibodies and flow cytometry. Surface display of I domain supports yeast rolling on ICAM-1-coated surfaces under shear flow. Expression of a locked open, high-affinity I domain mutant supports firm adhesion of yeast, while yeast displaying intermediate-affinity I domain mutants exhibit a range of rolling phenotypes. We find that rolling behavior for these mutants fails to correlate with ligand binding affinity. These results indicate that unstressed binding affinity is not the only molecular property that determines adhesive behavior under shear flow.
Asunto(s)
Antígeno CD11a/química , Molécula 1 de Adhesión Intercelular/química , Antígeno-1 Asociado a Función de Linfocito/química , Mutación , Epítopos/química , Humanos , Cinética , Conformación Molecular , Unión Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Adherencias Tisulares , Regulación hacia ArribaRESUMEN
The emergence of drug resistance is a major limitation of current antimalarials. The discovery of new druggable targets and pathways including those that are critical for multiple life cycle stages of the malaria parasite is a major goal for developing next-generation antimalarial drugs. Using an integrated chemogenomics approach that combined drug resistance selection, whole-genome sequencing, and an orthogonal yeast model, we demonstrate that the cytoplasmic prolyl-tRNA (transfer RNA) synthetase (PfcPRS) of the malaria parasite Plasmodium falciparum is a biochemical and functional target of febrifugine and its synthetic derivative halofuginone. Febrifugine is the active principle of a traditional Chinese herbal remedy for malaria. We show that treatment with febrifugine derivatives activated the amino acid starvation response in both P. falciparum and a transgenic yeast strain expressing PfcPRS. We further demonstrate in the Plasmodium berghei mouse model of malaria that halofuginol, a new halofuginone analog that we developed, is active against both liver and asexual blood stages of the malaria parasite. Halofuginol, unlike halofuginone and febrifugine, is well tolerated at efficacious doses and represents a promising lead for the development of dual-stage next-generation antimalarials.
Asunto(s)
Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Antimaláricos/farmacología , Inhibidores Enzimáticos/farmacología , Malaria Falciparum/tratamiento farmacológico , Piperidinas/farmacología , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/antagonistas & inhibidores , Quinazolinas/farmacología , Quinazolinonas/farmacología , Aminoacil-ARNt Sintetasas/metabolismo , Animales , Antimaláricos/química , Antimaláricos/toxicidad , Diseño Asistido por Computadora , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Resistencia a Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/toxicidad , Eritrocitos/parasitología , Hígado/parasitología , Malaria Falciparum/sangre , Malaria Falciparum/parasitología , Ratones , Modelos Moleculares , Estructura Molecular , Terapia Molecular Dirigida , Piperidinas/química , Piperidinas/toxicidad , Plasmodium falciparum/enzimología , Proteínas Protozoarias/metabolismo , Quinazolinas/química , Quinazolinas/toxicidad , Quinazolinonas/química , Quinazolinonas/toxicidad , Relación Estructura-Actividad , Factores de TiempoRESUMEN
The inserted (I) domain of αLß2 integrin (LFA-1) contains the entire binding site of the molecule. It mediates both rolling and firm adhesion of leukocytes at sites of inflammation depending on the activation state of the integrin. The affinity change of the entire integrin can be mimicked by the I domain alone through mutations that affect the conformation of the molecule. High-affinity mutants of the I domain have been discovered previously using both rational design and directed evolution. We have found that binding affinity fails to dictate the behavior of I domain adhesion under shear flow. In order to better understand I domain adhesion, we have developed a novel panning method to separate yeast expressing a library of I domain variants on the surface by adhesion under flow. Using conditions analogous to those experienced by cells interacting with the post-capillary vascular endothelium, we have identified mutations supporting firm adhesion that are not found using typical directed evolution techniques that select for tight binding to soluble ligands. Mutants isolated using this method do not cluster with those found by sorting with soluble ligand. Furthermore, these mutants mediate shear-driven cell rolling dynamics decorrelated from binding affinity, as previously observed for I domains bearing engineered disulfide bridges to stabilize activated conformational states. Characterization of these mutants supports a greater understanding of the structure-function relationship of the αL I domain, and of the relationship between applied force and bioadhesion in a broader context.