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1.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 7): 320-325, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32627748

RESUMO

Proline/alanine-rich sequence (PAS) polypeptides represent a novel class of biosynthetic polymers comprising repetitive sequences of the small proteinogenic amino acids L-proline, L-alanine and/or L-serine. PAS polymers are strongly hydrophilic and highly soluble in water, where they exhibit a natively disordered conformation without any detectable secondary or tertiary structure, similar to polyethylene glycol (PEG), which constitutes the most widely applied precipitant for protein crystallization to date. To investigate the potential of PAS polymers for structural studies by X-ray crystallography, two proteins that were successfully crystallized using PEG in the past, hen egg-white lysozyme and the Fragaria × ananassa O-methyltransferase, were subjected to crystallization screens with a 200-residue PAS polypeptide. The PAS polymer was applied as a precipitant using a vapor-diffusion setup that allowed individual optimization of the precipitant concentration in the droplet in the reservoir. As a result, crystals of both proteins showing high diffraction quality were obtained using the PAS precipitant. The genetic definition and precise macromolecular composition of PAS polymers, both in sequence and in length, distinguish them from all natural and synthetic polymers that have been utilized for protein crystallization so far, including PEG, and facilitate their adaptation for future applications. Thus, PAS polymers offer potential as novel precipitants for biomolecular crystallography.


Assuntos
Alanina/química , Cristalografia por Raios X/métodos , Peptídeos/química , Polietilenoglicóis/química , Prolina/química , Cristalização/métodos , Interações Hidrofóbicas e Hidrofílicas , Muramidase/química , Proteínas de Plantas/química , Proteína O-Metiltransferase/química , Solubilidade
2.
Annu Rev Pharmacol Toxicol ; 60: 391-415, 2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31914898

RESUMO

The concept of engineering robust protein scaffolds for novel binding functions emerged 20 years ago, one decade after the advent of recombinant antibody technology. Early examples were the Affibody, Monobody (Adnectin), and Anticalin proteins, which were derived from fragments of streptococcal protein A, from the tenth type III domain of human fibronectin, and from natural lipocalin proteins, respectively. Since then, this concept has expanded considerably, including many other protein templates. In fact, engineered protein scaffolds with useful binding specificities, mostly directed against targets of biomedical relevance, constitute an area of active research today, which has yielded versatile reagents as laboratory tools. However, despite strong interest from basic science, only a handful of those protein scaffolds have undergone biopharmaceutical development up to the clinical stage. This includes the abovementioned pioneering examples as well as designed ankyrin repeat proteins (DARPins). Here we review the current state and clinical validation of these next-generation therapeutics.


Assuntos
Descoberta de Drogas/métodos , Engenharia de Proteínas/métodos , Proteínas/administração & dosagem , Animais , Repetição de Anquirina , Humanos , Ligação Proteica , Proteínas/metabolismo , Proteínas/farmacologia
3.
Curr Opin Biotechnol ; 60: 230-241, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31207556

RESUMO

Initially emerging as a highly innovative concept in the late 1990s, the concept of creating novel binding reagents based on stable protein scaffolds from outside the immunoglobulin (Ig) superfamily has become a well-developed area of research and discovery today. Numerous scaffolds based on extracellular, membrane-bound or intracellular proteins (or their domains) have been recruited, yielding versatile research reagents and even biological drug candidates to serve as a viable alternative to antibodies. This minireview discusses both established and novel concepts in this field and summarizes the current state of clinical development of the more advanced protein scaffolds, in particular Affibody, Adnectin, Anticalin and DARPin drug candidates.


Assuntos
Engenharia de Proteínas , Proteínas
4.
Structure ; 26(4): 649-656.e3, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29526433

RESUMO

We describe the comparative X-ray structural analysis of three Anticalin proteins directed against the extra-domain B (ED-B) of oncofetal fibronectin (Fn), a validated marker of tumor neoangiogenesis. The Anticalins were engineered from the human lipocalin 2 (Lcn2) scaffold via targeted randomization of the structurally variable loop region and selection by phage display, resulting in 15-19 exchanged residues. While the four reshaped loops exhibit diverse conformations (with shifts in Cα positions up to 20.4 Å), the ß-barrel core of the lipocalin remains strongly conserved, thus confirming the extraordinary robustness of this scaffold. All three Anticalins bind the cc' hairpin loop of ED-B, the most exposed motif in the context of its neighboring Fn domains, but reveal entirely different binding modes, with orientations differing by up to 180°. Hence, each Anticalin recognizes its molecular target in an individual manner, in line with the distinct epitope specificities previously seen in binding experiments.


Assuntos
Antígenos de Neoplasias/química , Epitopos/química , Fibronectinas/química , Lipocalina-2/química , Peptídeos/química , Sequência de Aminoácidos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Epitopos/genética , Epitopos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Lipocalina-2/genética , Lipocalina-2/metabolismo , Modelos Moleculares , Biblioteca de Peptídeos , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
5.
Bioorg Med Chem ; 26(10): 2882-2887, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29102080

RESUMO

Pharmacokinetic (PK) extension is no longer just a means to create improved second generation biologics (so-called biobetters), but constitutes an accepted strategy in biopharmaceutical drug development today. Although PEGylation has become a widely applied methodology to furnish therapeutic proteins and peptides with prolonged plasma half-life, the immunogenicity and missing biodegradability of this synthetic polymer has prompted an evident need for alternatives. PASylation is based on biological polypeptides made of the small l-amino acids Pro, Ala and/or Ser (PAS), which adopt a random coil structure in aqueous buffers with surprisingly similar biophysical properties as PEG. In contrast, PAS sequences can be conjugated to pharmaceutically active proteins and peptides both via chemical coupling and at the genetic level, as so-called fusion proteins. PASylation has been successfully applied to numerous biologics, including cytokines, growth factors, antibody fragments, enzymes as well as various peptides, and validated in diverse animal models, from mice to monkeys. Here we compare PASylation with other current strategies for half-life extension and we discuss the utility of these approaches for the design of innovative peptide-based therapeutics.


Assuntos
Descoberta de Drogas/métodos , Peptídeos/química , Peptídeos/farmacocinética , Alanina/química , Alanina/farmacocinética , Animais , Meia-Vida , Humanos , Modelos Moleculares , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Prolina/química , Prolina/farmacocinética , Serina/química , Serina/farmacocinética
6.
Protein Eng Des Sel ; 29(7): 263-70, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27261456

RESUMO

ANTIC ALIGN: is an interactive software developed to simultaneously visualize, analyze and modify alignments of DNA and/or protein sequences that arise during combinatorial protein engineering, design and selection. ANTIC ALIGN: combines powerful functions known from currently available sequence analysis tools with unique features for protein engineering, in particular the possibility to display and manipulate nucleotide sequences and their translated amino acid sequences at the same time. ANTIC ALIGN: offers both template-based multiple sequence alignment (MSA), using the unmutated protein as reference, and conventional global alignment, to compare sequences that share an evolutionary relationship. The application of similarity-based clustering algorithms facilitates the identification of duplicates or of conserved sequence features among a set of selected clones. Imported nucleotide sequences from DNA sequence analysis are automatically translated into the corresponding amino acid sequences and displayed, offering numerous options for selecting reading frames, highlighting of sequence features and graphical layout of the MSA. The MSA complexity can be reduced by hiding the conserved nucleotide and/or amino acid residues, thus putting emphasis on the relevant mutated positions. ANTIC ALIGN: is also able to handle suppressed stop codons or even to incorporate non-natural amino acids into a coding sequence. We demonstrate crucial functions of ANTIC ALIGN: in an example of Anticalins selected from a lipocalin random library against the fibronectin extradomain B (ED-B), an established marker of tumor vasculature. Apart from engineered protein scaffolds, ANTIC ALIGN: provides a powerful tool in the area of antibody engineering and for directed enzyme evolution.


Assuntos
Alinhamento de Sequência/métodos , Software , Sequência de Aminoácidos , Modelos Moleculares , Engenharia de Proteínas/métodos , Análise de Sequência de DNA/métodos , Análise de Sequência de Proteína/métodos
7.
Int J Cancer ; 138(5): 1269-80, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26421425

RESUMO

The standard of care for diagnosis and therapy monitoring of gliomas is magnetic resonance imaging (MRI), which however, provides only an indirect and incomplete representation of the tumor mass, offers limited information for patient stratification according to WHO-grades and may insufficiently indicate tumor relapse after antiangiogenic therapy. Anticalins are alternative binding proteins obtained via combinatorial protein design from the human lipocalin scaffold that offer novel diagnostic reagents for histology and imaging applications. Here, the Anticalins N7A, N7E and N9B, which possess exquisite specificity and affinity for oncofetal fibronectin carrying the extra domain B (ED-B), a well-known proangiogenic extracellular matrix protein, were applied for immunohistochemical studies. When investigating ED-B expression in biopsies from 41 patients with confirmed gliomas of WHO grades I to IV, or in non-neoplastic brain samples, we found that Anticalins specifically detect ED-B in primary glioblastoma multiforme (GBM; WHO IV) but not in tumors of lower histopathological grade or in tumor-free brain. In primary GBM samples, ED-B specific Anticalins locate to fibronectin-rich perivascular areas that are associated with angiogenesis. Anticalins specifically detect ED-B both in fixed tumor specimen and on vital cells, as evidenced by cytofluorometry. Beyond that, we labeled an Anticalin with the γ-emitter (123) I and demonstrated specific binding to GBM-tissue samples using in vitro autoradiography. Overall, our data indicate that ED-B specific Anticalins are useful tools for the diagnosis of primary GBM and related angiogenic sites, presenting them as promising tracers for molecular tumor imaging.


Assuntos
Anticorpos/metabolismo , Neoplasias Encefálicas/diagnóstico , Fibronectinas/análise , Glioblastoma/diagnóstico , Lipocalinas/imunologia , Imagem Molecular/métodos , Neoplasias Encefálicas/química , Linhagem Celular Tumoral , Fibronectinas/metabolismo , Glioblastoma/química , Humanos , Imuno-Histoquímica , Lipocalinas/metabolismo , Biblioteca de Peptídeos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Ligação Proteica , Estrutura Terciária de Proteína
8.
J Mol Biol ; 425(4): 780-802, 2013 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-23238252

RESUMO

The oncofetal isoform of the extracellular matrix protein fibronectin (Fn), which carries the extra-domain B (ED-B) and is exclusively expressed in neovasculature, has gained interest for tumor diagnosis and therapy using engineered antibody fragments. We have employed the human lipocalin 2 (Lcn2) as a small and robust non-immunoglobulin scaffold to select ED-B-specific Anticalins from a new advanced random library using bacterial phage display and ELISA screening against appropriately engineered Fn fragments. As a result, we have isolated and biochemically characterized four different Anticalins that all show low nanomolar affinities for ED-B, right in the range between the monomeric and dimeric forms of the single-chain variable antibody fragment L19 that has been widely applied in this area before. All Anticalins can be readily expressed in Escherichia coli as soluble and strictly monomeric proteins, and they show specific staining of ED-B-positive tumor cells in immunofluorescence microscopy while BIAcore affinity analyses indicate recognition of distinct ED-B epitopes. The crystal structure for one Anticalin, N7A, in complex with the Fn7B8 fragment, was solved at 2.6Å resolution and reveals binding to the gfcc' sheet and cc' loop on ED-B. This is the second example of a protein-specific Lcn2-based Anticalin, which illustrates the remarkable plasticity of the calyx-like ligand pocket of lipocalins with their four structurally hypervariable loops supported by a highly conserved ß-barrel. The ED-B-specific Anticalins resulting from this study should provide useful reagents in research and biomedical drug development, both for in vivo imaging and for directed cancer therapy.


Assuntos
Anticorpos/metabolismo , Fibronectinas/metabolismo , Lipocalinas/imunologia , Biblioteca de Peptídeos , Sequência de Aminoácidos , Anticorpos/química , Anticorpos/genética , Sítios de Ligação/genética , Ligação Competitiva , Células CACO-2 , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Fibronectinas/química , Fibronectinas/genética , Humanos , Lipocalinas/genética , Lipocalinas/metabolismo , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície
9.
J Biol Chem ; 287(21): 17578-17588, 2012 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-22442152

RESUMO

The type III extra-domain B (ED-B) is specifically spliced into fibronectin (Fn) during embryogenesis and neoangiogenesis, including many cancers. The x-ray structure of the recombinant four-domain fragment Fn(III)7B89 reveals a tightly associated, extended head-to-tail dimer, which is stabilized via pair-wise shape and charge complementarity. A tendency toward ED-B-dependent dimer formation in solution was supported by size exclusion chromatography and analytical ultracentrifugation. When amending the model with the known three-dimensional structure of the Fn(III)10 domain, its RGD loop as well as the adhesion synergy region in Fn(III)9-10 become displayed on the same face of the dimer; this should allow simultaneous binding of at least two integrins and, thus, receptor clustering on the cell surface and intracellular signaling. Insertion of ED-B appears to stabilize overall head-to-tail dimerization of two separate Fn chains, which, together with alternating homodimer formation via disulfide bridges at the C-terminal Fn tail, should lead to the known macromolecular fibril formation.


Assuntos
Fibronectinas/química , Multimerização Proteica , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
10.
Methods Enzymol ; 503: 157-88, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22230569

RESUMO

Anticalins are a novel class of small, robust proteins with designed ligand-binding properties derived from the natural lipocalin scaffold. Due to their compact molecular architecture, comprising a single polypeptide chain, they provide several benefits as protein therapeutics, such as high target specificity, good tissue penetration, low immunogenicity, tunable plasma half-life, efficient Escherichia coli expression, and suitability for furnishing with additional effector functions via genetic fusion or chemical conjugation. The lipocalins are a widespread family of proteins that naturally serve in many organisms, including humans, for the transport, storage, or sequestration of small biological compounds like vitamins and hormones. Their fold is dominated by an eight-stranded antiparallel ß-barrel, which is open to the solvent at one end. There, four loops connect the ß-strands in a pairwise manner and, altogether, they form the entry to a ligand-binding site. This loop region can be engineered via site-directed random mutagenesis in combination with genetic library selection techniques to yield "Anticalins" with exquisite specificities-and down to picomolar affinities-for prescribed molecular targets of either hapten or antigen type. Several Anticalins directed against medically relevant disease targets have been successfully engineered and can be applied, for example, for the blocking of soluble signaling factors or cell surface receptors or for tissue-specific drug targeting. While natural lipocalins were already subject to clinical studies in the past, a first Anticalin has completed Phase I trials in 2011, thus paving the way for the broad application of Anticalins as a promising novel class of biopharmaceuticals.


Assuntos
Proteínas de Fase Aguda/química , Proteínas de Transporte/química , Sistemas de Liberação de Medicamentos/métodos , Lipocalinas/química , Biblioteca de Peptídeos , Proteínas Proto-Oncogênicas/química , Proteínas de Fase Aguda/síntese química , Proteínas de Fase Aguda/uso terapêutico , Animais , Proteínas de Transporte/síntese química , Proteínas de Transporte/uso terapêutico , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/química , Vetores Genéticos/química , Humanos , Lipocalina-2 , Lipocalinas/síntese química , Lipocalinas/uso terapêutico , Mutagênese Sítio-Dirigida/métodos , Plasmídeos/química , Engenharia de Proteínas/métodos , Proteínas Proto-Oncogênicas/síntese química , Proteínas Proto-Oncogênicas/uso terapêutico , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapêutico , Especificidade por Substrato
11.
Curr Opin Chem Biol ; 13(3): 245-55, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19501012

RESUMO

Antibodies have been the paradigm of binding proteins with desired specificities for more than one century and during the past decade their recombinant or humanized versions have entered clinical application with remarkable success. Meanwhile, a new generation of receptor proteins was born, which is derived from small and robust non-immunoglobulin "scaffolds" that can be equipped with prescribed binding functions using the methods of combinatorial protein design. Their ongoing development does not only provide valuable insights into the principles of molecular recognition and protein structure-function relationships but also yields novel reagents for medical use. This technology goes hand in hand with our expanding knowledge about the molecular pathologies of cancer, immunological, and infectious diseases. Currently, questions regarding the choice of suitable medically relevant targets with regard to a certain protein scaffold, the methodology for engineering high affinity, arming with effector functions, routes of administration, plasma half-life, and immunogenicity are in the focus. While many protein scaffolds have been proposed during the past years, the technology shows a trend toward consolidation with a smaller set of systems that are being applied against multiple targets and in different settings, with emphasis on the development of drug candidates for therapy or in vivo diagnostics: Adnectins, Affibodies, Anticalins, DARPins, and engineered Kunitz-type inhibitors, among others. Only few data from early clinical studies are available yet, but many more are likely to come in the near future, thus providing a growing basis for assessing the therapeutic potential--but possibly also some limitations--of this exciting new class of protein drugs.


Assuntos
Anticorpos/uso terapêutico , Descoberta de Drogas/métodos , Engenharia de Proteínas/métodos , Anticorpos/genética , Anticorpos/farmacologia , Modelos Moleculares , Ligação Proteica , Receptores de Superfície Celular/antagonistas & inibidores
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