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1.
Nat Biotechnol ; 40(12): 1845-1854, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35864170

RESUMO

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19).


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Cricetinae , Humanos , SARS-CoV-2/genética , Proteínas de Repetição de Anquirina Projetadas , Microscopia Crioeletrônica , Anticorpos Monoclonais/uso terapêutico , Terapia Combinada de Anticorpos , Anticorpos Neutralizantes
2.
BioDrugs ; 34(4): 423-433, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32583318

RESUMO

The DARPin® drug platform was established with a vision to expand the medical use of biologics beyond what was possible with monoclonal antibodies. It is based on naturally occurring ankyrin repeat domains that are typically building blocks of multifunctional human proteins. The platform allows for the generation of diverse, well-behaved, multifunctional drug candidates. Recent clinical data illustrate the favorable safety profile of the first DARPin® molecules tested in patients. With the positive phase III results of the most advanced DARPin® drug candidate, abicipar, the DARPin® drug platform is potentially about to achieve its first marketing approval. This review highlights some of the key milestones and decisions encountered when transforming the DARPin® platform from an academic concept to a biotech drug pipeline engine.


Assuntos
Repetição de Anquirina , Anticorpos Monoclonais/química , Preparações Farmacêuticas , Anticorpos Monoclonais/imunologia , Humanos
3.
Protein Eng Des Sel ; 30(9): 583-591, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29088432

RESUMO

A long systemic half-life is key for therapeutic proteins. To that end we have generated serum albumin-binding designed ankyrin repeat domains. These domains bind serum albumin of different species with nanomolar affinities, and have significantly improved pharmacokinetic properties both in mouse and cynomolgus monkey compared to non-serum albumin-binding DARPin® domains. In addition, they exhibit high thermal stability and long storage stability, which is an essential feature for their use in drug development. Covalently linking a serum albumin-binding DARPin® domain to domains with other target specificities results in improvements of multiple orders of magnitude in exposure and terminal half-life, both in mouse and cynomolgus monkey. Pharmacokinetic assessment of such constructs revealed terminal half-life values ranging from 27 h to 80 h in mouse, and from 2.6 days to 20 days in cynomolgus monkey. Extrapolation by allometric scaling on these findings suggests terminal half-life values of 5-50 days in human, indicating that pharmacokinetic properties in the range of monoclonal antibodies can be achieved with DARPin® drug candidates. Such serum albumin-binding DARPin® domains are thus valuable tools for the generation of multi-functional drugs with an extended in vivo half-life.


Assuntos
Repetição de Anquirina , Vetores Genéticos/química , Proteínas Recombinantes de Fusão/farmacocinética , Albumina Sérica/genética , Animais , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/metabolismo , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Macaca fascicularis , Camundongos , Ligação Proteica , Estabilidade Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Albumina Sérica/metabolismo
4.
MAbs ; 9(8): 1262-1269, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29035637

RESUMO

MP0250 is a multi-domain drug candidate currently being tested in clinical trials for the treatment of cancer. It comprises one anti-vascular endothelial growth factor-A (VEGF-A), one anti-hepatocyte growth factor (HGF), and two anti-human serum albumin (HSA) DARPin® domains within a single polypeptide chain. While there is first clinical validation of a single-domain DARPin® drug candidate, little is known about DARPin® drug candidates comprising multiple domains. Here, we show that MP0250 can be expressed at 15 g/L in soluble form in E. coli high cell-density fermentation, it is stable in soluble/frozen formulation for 2 years as assessed by reverse phase HPLC, it has picomolar potency in inhibiting VEGF-A and HGF in ELISA and cellular assays, and its domains are simultaneously active as shown by surface plasmon resonance. The inclusion of HSA-binding DARPin® domains leads to a favorable pharmacokinetic profile in mouse and cynomolgus monkey, with terminal half-lives of ∼ 30 hours in mouse and ∼ 5 days in cynomolgus monkey. MP0250 is thus a highly potent drug candidate that could be particularly useful in oncology. Beyond MP0250, the properties of MP0250 indicate that multi-domain DARPin® proteins can be valuable next-generation drug candidates.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antineoplásicos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Administração Intravenosa , Animais , Repetição de Anquirina/genética , Repetição de Anquirina/imunologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Desenho de Fármacos , Feminino , Meia-Vida , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/imunologia , Humanos , Infusões Intravenosas , Macaca fascicularis , Masculino , Camundongos Endogâmicos BALB C , Ligação Proteica/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacocinética , Albumina Sérica Humana/genética , Albumina Sérica Humana/imunologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologia
5.
Angiogenesis ; 16(1): 101-11, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22983424

RESUMO

The next-generation ophthalmic anti-VEGF therapeutics must aim at being superior to the currently available agents with regard to potency and improved drug delivery, while still being stable and safe to use at elevated concentrations. We show here the generation of a set of highly potent VEGF-A antagonistic DARPins (designed ankyrin repeat proteins) delivering these properties. DARPins with single-digit picomolar affinity to human VEGF-A were generated using ribosome display selections. Specific and potent human VEGF-A binding was confirmed by ELISA and endothelial cell sprouting assays. Cross-reactivity with VEGF-A of several species was confirmed by ELISA. Intravitreally injected DARPin penetrated into the retina and reduced fluorescein extravasation in a rabbit model of vascular leakage. In addition, topical DARPin application was found to diminish corneal neovascularization in a rabbit suture model, and to suppress laser-induced neovascularization in a rat model. Even at elevated doses, DARPins were safe to use. The fact that several DARPins are highly active in various assays illustrates the favorable class behavior of the selected binders. Anti-VEGF-A DARPins thus represent a novel class of highly potent and specific drug candidates for the treatment of neovascular eye diseases in both the posterior and the anterior eye chamber.


Assuntos
Inibidores da Angiogênese/farmacologia , Repetição de Anquirina , Proteínas Recombinantes/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Administração Tópica , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/uso terapêutico , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/crescimento & desenvolvimento , Neovascularização de Coroide/tratamento farmacológico , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Olho/irrigação sanguínea , Olho/efeitos dos fármacos , Olho/patologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Injeções Intravítreas , Camundongos , Soluções Oftálmicas/farmacologia , Soluções Oftálmicas/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Coelhos , Ratos , Ratos Endogâmicos BN , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Mol Cell Biol ; 32(19): 3802-13, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22801374

RESUMO

Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory "designed ankyrin repeat proteins" (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies.


Assuntos
Sítio Alostérico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Animais , Expressão Gênica , Humanos , Estrutura Terciária de Proteína , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
7.
Cancer Res ; 70(4): 1595-605, 2010 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-20124480

RESUMO

Slow-clearing, tumor-targeting proteins such as monoclonal antibodies typically exhibit high tumor accumulation but low tissue contrast, whereas intermediate-sized proteins such as scFvs show faster clearance but only moderate tumor accumulation. For both, tumor targeting does not seem to improve further above an optimal affinity. We show here that with very small high-affinity proteins such as designed ankyrin repeat proteins (DARPins), these limits can be overcome. We have systematically investigated the influence of molecular mass and affinity on tumor accumulation with DARPins with specificity for HER2 in SK-OV-3.ip nude mouse xenografts. DARPins with a mass of 14.5 kDa and affinities between 270 nmol/L and 90 pmol/L showed a strong correlation of tumor accumulation with affinity to HER2, with the highest affinity DARPin reaching 8% ID/g after 24 hours and 6.5% ID/g after 48 hours (tumor-to-blood ratio >60). Tumor autoradiographs showed good penetration throughout the tumor mass. Genetic fusion of two DARPins (30 kDa) resulted in significantly lower tumor accumulation, similar to values observed for scFvs, whereas valency had no influence on accumulation. PEGylation of the DARPins increased the circulation half-life, leading to higher tumor accumulation (13.4% ID/g after 24 hours) but lower tumor-to-blood ratios. Affinity was less important for tumor uptake of the PEGylated constructs. We conclude that two regimes exist for delivering high levels of drug to a tumor: small proteins with very high affinity, such as unmodified DARPins, and large proteins with extended half-life, such as PEGylated DARPins, in which the importance of affinity is less pronounced.


Assuntos
Repetição de Anquirina , Sistemas de Liberação de Medicamentos/métodos , Neoplasias/tratamento farmacológico , Proteínas/administração & dosagem , Proteínas/síntese química , Animais , Repetição de Anquirina/fisiologia , Afinidade de Anticorpos , Antineoplásicos/administração & dosagem , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Desenho de Fármacos , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Camundongos , Camundongos Nus , Peso Molecular , Engenharia de Proteínas/métodos , Proteínas/química , Proteínas/uso terapêutico , Especificidade por Substrato/fisiologia , Resultado do Tratamento , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Drug Discov Today ; 13(15-16): 695-701, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18621567

RESUMO

DARPins (designed ankyrin repeat proteins) are a novel class of binding molecules with the potential to overcome limitations of monoclonal antibodies, hence allowing novel therapeutic approaches. DARPins are small, single domain proteins (14 kDa) which can be selected to bind any given target protein with high affinity and specificity. These characteristics make them ideal agonistic, antagonistic or inhibitory drug candidates. Furthermore, DARPins can be engineered to carry various effector functions or combine multiple binding specificities, enabling completely new drug formats. Taken together, DARPins are a prominent member of the next generation of protein therapeutics with the potential to surpass existing antibody drugs.


Assuntos
Anquirinas/farmacologia , Animais , Repetição de Anquirina , Anquirinas/uso terapêutico , Descoberta de Drogas , Humanos , Modelos Moleculares , Ligação Proteica , Engenharia de Proteínas
9.
Acta Crystallogr D Biol Crystallogr ; 64(Pt 4): 339-53, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18391401

RESUMO

As a key regulator of mitosis, the Ser/Thr protein polo-like kinase-1 (Plk-1) is a well validated drug target in cancer therapy. In order to enable structure-guided drug design, determination of the crystal structure of the kinase domain of Plk-1 was attempted. Using a multi-parallel cloning and expression approach, a set of length variants were identified which could be expressed in large amounts from insect cells and which could be purified to high purity. However, all attempts to crystallize these constructs failed. Crystals were ultimately obtained by generating designed ankyrin-repeat proteins (DARPins) selective for Plk-1 and using them for cocrystallization. Here, the first crystal structure of the kinase domain of wild-type apo Plk-1, in complex with DARPin 3H10, is presented, underlining the power of selective DARPins as crystallization tools. The structure was refined to 2.3 A resolution and shows the active conformation of Plk-1. It broadens the basis for modelling and cocrystallization studies for drug design. The binding epitope of 3H10 is rich in arginine, glutamine and lysine residues, suggesting that the DARPin enabled crystallization by masking a surface patch which is unfavourable for crystal contact formation. Based on the packing observed in the crystal, a truncated DARPin variant was designed which showed improved binding characteristics.


Assuntos
Anquirinas/química , Proteínas de Ciclo Celular/química , Proteínas Serina-Treonina Quinases/química , Proteínas Proto-Oncogênicas/química , Sequência de Aminoácidos , Calorimetria , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/isolamento & purificação , Clonagem Molecular , Cristalização , Coleta de Dados , Humanos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Recombinantes/química , Quinase 1 Polo-Like
10.
J Mol Biol ; 376(1): 241-57, 2008 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-18164721

RESUMO

Full-consensus designed ankyrin repeat proteins were designed with one to six identical repeats flanked by capping repeats. These proteins express well in Escherichia coli as soluble monomers. Compared to our previously described designed ankyrin repeat protein library, randomized positions have now been fixed according to sequence statistics and structural considerations. Their stability increases with length and is even higher than that of library members, and those with more than three internal repeats are resistant to denaturation by boiling or guanidine hydrochloride. Full denaturation requires their heating in 5 M guanidine hydrochloride. The folding and unfolding kinetics of the proteins with up to three internal repeats were analyzed, as the other proteins could not be denatured. Folding is monophasic, with a rate that is nearly identical for all proteins ( approximately 400-800 s(-1)), indicating that essentially the same transition state must be crossed, possibly the folding of a single repeat. In contrast, the unfolding rate decreases by a factor of about 10(4) with increasing repeat number, directly reflecting thermodynamic stability in these extraordinarily slow denaturation rates. The number of unfolding phases also increases with repeat number. We analyzed the folding thermodynamics and kinetics both by classical two-state and three-state cooperative models and by an Ising-like model, where repeats are considered as two-state folding units that can be stabilized by interacting with their folded nearest neighbors. This Ising model globally describes both equilibrium and kinetic data very well and allows for a detailed explanation of the ankyrin repeat protein folding mechanism.


Assuntos
Repetição de Anquirina , Anquirinas/química , Anquirinas/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Dicroísmo Circular , Escherichia coli/genética , Expressão Gênica , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Espectrometria de Fluorescência
11.
Structure ; 15(5): 625-36, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17502107

RESUMO

Specific and potent caspase inhibitors are indispensable for the dissection of the intricate pathways leading to apoptosis. We selected a designed ankyrin repeat protein (DARPin) from a combinatorial library that inhibits caspase-2 in vitro with a subnanomolar inhibition constant and, in contrast to the peptidic caspase inhibitors, with very high specificity for this particular caspase. The crystal structure of this inhibitor (AR_F8) in complex with caspase-2 reveals the molecular basis for the specificity and, together with kinetic analyses, the allosteric mechanism of inhibition. The structure also shows a conformation of the active site that can be exploited for the design of inhibitory compounds. AR_F8 is a specific inhibitor of an initiator caspase and has the potential to help identify the function of caspase-2 in the complex biological apoptotic signaling network.


Assuntos
Repetição de Anquirina/fisiologia , Caspase 2/química , Inibidores de Caspase , Cisteína Endopeptidases/química , Engenharia de Proteínas , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular
12.
Proteins ; 65(2): 285-95, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16948156

RESUMO

Two designed ankyrin repeat (AR) proteins (E3_5 and E3_19) are high homologous (with about 87% sequence identity) and their crystal structures have a Calpha atom-positional root-mean-square difference of about 0.14 nm. However, it was found that E3_5 is considerably more stable than E3_19 in guanidinium hydrochloride and thermal denaturation experiments. With the goal of providing insights into the various factors contributing to the stabilities of the designed AR proteins and suggesting possible mutations to enhance their stabilities, homology modeling and molecular dynamics (MD) simulations with explicit solvent have been performed. Because the crystal structure of E3_19 was solved later than that of E3_5, a homology model of E3_19 based on the crystal structure of E3_5 was also used in the simulations. E3_5 shows a very stable trajectory in both crystal and solution simulations. In contrast, the C-terminal repeat of E3_19 unfolds in the simulations starting from either the modeled structure or the crystal structure, although it has a sequence identical to that of E3_5. A continuum electrostatic model was used to estimate the effect of single mutations on protein stability and to study the interaction between the internal ARs and the C-terminal capping AR. Mutations involving charged residues were found to have large effects on stability. Due to the difference in charge distribution in the internal ARs of E3_19 and E3_5, their interaction with the C-terminal capping AR is less favorable in E3_19. The simulation trajectories suggest that the stability of the designed AR proteins can be increased by optimizing the electrostatic interactions within and between the different repeats.


Assuntos
Proteínas/química , Proteínas/metabolismo , Repetição de Anquirina , Biologia Computacional , Simulação por Computador , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Mutação/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/genética
13.
Protein Eng Des Sel ; 19(5): 219-29, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16551653

RESUMO

We describe here the rapid selection of specific MAP-kinase binders from a combinatorial library of designed ankyrin repeat proteins (DARPins). A combined in vitro/in vivo selection approach, based on ribosome display and the protein fragment complementation assay (PCA), yielded a large number of different binders that are fully functional in the cellular cytoplasm. Ribosome-display selection pools of four successive selection rounds were examined to monitor the enrichment of JNK2-specific DARPins. Surprisingly, only one round of ribosome display with subsequent PCA selection of this pool was necessary to isolate a first specific binder with micromolar affinity. After only two rounds of ribosome-display selection followed by PCA, virtually all DARPins showed JNK2-specific binding, with affinities in the low nanomolar range. The enrichment factor of ribosome display thus approaches 10(5) per round. In a second set of experiments, similar results were obtained with the kinases JNK1 and p38 as targets. Again, almost all investigated DARPins obtained after two rounds of ribosome display showed specific binding to the targets used, JNK1 or p38. In all three selection experiments the identified DARPins possess very high specificity for the target kinase. Taken together, the combination of ribosome display and PCA selections allowed the identification of large pools of binders at unparalleled speed. Furthermore, DARPins are applicable in intracellular selections and immunoprecipitations from the extract of eukaryotic cells.


Assuntos
Repetição de Anquirina/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Biblioteca de Peptídeos , Engenharia de Proteínas , Animais , Repetição de Anquirina/genética , Sítios de Ligação , Linhagem Celular , Técnicas de Química Combinatória , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Camundongos , Proteína Quinase 9 Ativada por Mitógeno/química , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/química , Ligação Proteica
14.
Proteins ; 65(2): 280-4, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16493627

RESUMO

Consensus-designed ankyrin repeat (AR) proteins are thermodynamically very stable. The structural analysis of the designed AR protein E3_5 revealed that this stability is due to a regular fold with highly conserved structural motifs and H-bonding networks. However, the designed AR protein E3_19 exhibits a significantly lower stability than E3_5 (9.6 vs. 14.8 kcal/mol), despite 88% sequence identity. To investigate the structural correlations of this stability difference between E3_5 and E3_19, we determined the crystal structure of E3_19 at 1.9 A resolution. E3_19 as well has a regular AR domain fold with the characteristic H-bonding patterns. All structural features of the E3_5 and E3_19 molecules appear to be virtually identical (RMSD(Calpha) approximately 0.7 A). However, clear differences are observed in the surface charge distribution of the two AR proteins. E3_19 features clusters of charged residues and more exposed hydrophobic residues than E3_5. The atomic coordinates of E3_19 have been deposited in the Protein Data Bank. PDB ID: 2BKG.


Assuntos
Proteínas/química , Proteínas/metabolismo , Sequência de Aminoácidos , Repetição de Anquirina , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína
15.
Nat Biotechnol ; 23(10): 1257-68, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16211069

RESUMO

Not all adaptive immune systems use the immunoglobulin fold as the basis for specific recognition molecules: sea lampreys, for example, have evolved an adaptive immune system that is based on leucine-rich repeat proteins. Additionally, many other proteins, not necessarily involved in adaptive immunity, mediate specific high-affinity interactions. Such alternatives to immunoglobulins represent attractive starting points for the design of novel binding molecules for research and clinical applications. Indeed, through progress and increased experience in library design and selection technologies, gained not least from working with synthetic antibody libraries, researchers have now exploited many of these novel scaffolds as tailor-made affinity reagents. Significant progress has been made not only in the basic science of generating specific binding molecules, but also in applications of the selected binders in laboratory procedures, proteomics, diagnostics and therapy. Challenges ahead include identifying applications where these novel proteins can not only be an alternative, but can enable approaches so far deemed technically impossible, and delineate those therapeutic applications commensurate with the molecular properties of the respective proteins.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Desenho de Fármacos , Biblioteca de Peptídeos , Engenharia de Proteínas/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Proteínas/metabolismo , Sítios de Ligação , Simulação por Computador , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Modelos Químicos , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/genética
16.
Structure ; 13(8): 1131-41, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16084385

RESUMO

Aminoglycoside phosphotransferase (3')-IIIa (APH) is a bacterial kinase that confers antibiotic resistance to many pathogenic bacteria and shares structural homology with eukaryotic protein kinases. We report here the crystal structure of APH, trapped in an inactive conformation by a tailor-made inhibitory ankyrin repeat (AR) protein, at 2.15 A resolution. The inhibitor was selected from a combinatorial library of designed AR proteins. The AR protein binds the C-terminal lobe of APH and thereby stabilizes three alpha helices, which are necessary for substrate binding, in a significantly displaced conformation. BIAcore analysis and kinetic enzyme inhibition experiments are consistent with the proposed allosteric inhibition mechanism. In contrast to most small-molecule kinase inhibitors, the AR proteins are not restricted to active site binding, allowing for higher specificity. Inactive conformations of pharmaceutically relevant enzymes, as can be elucidated with the approach presented here, represent powerful starting points for rational drug design.


Assuntos
Repetição de Anquirina/fisiologia , Resistência a Medicamentos/fisiologia , Canamicina Quinase/química , Regulação Alostérica/fisiologia , Sequência de Aminoácidos , Enterococcus/enzimologia , Canamicina Quinase/antagonistas & inibidores , Canamicina Quinase/metabolismo , Dados de Sequência Molecular , Engenharia de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Staphylococcus/enzimologia , Homologia Estrutural de Proteína
17.
Curr Opin Biotechnol ; 16(4): 459-69, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16005204

RESUMO

Over the past 30 years, monoclonal antibodies have become the standard binding proteins and currently find applications in research, diagnostics and therapy. Yet, monoclonal antibodies now face strong competition from synthetic antibody libraries in combination with powerful library selection technologies. More recently, an increased understanding of other natural binding proteins together with advances in protein engineering, selection and evolution technologies has also triggered the exploration of numerous other protein architectures for the generation of designed binding molecules. Valuable protein-binding scaffolds have been obtained and represent promising alternatives to antibodies for biotechnological and, potentially, clinical applications.


Assuntos
Engenharia de Proteínas , Biblioteca de Peptídeos , Ligação Proteica , Engenharia de Proteínas/métodos , Engenharia de Proteínas/tendências
18.
J Biol Chem ; 280(26): 24715-22, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15851475

RESUMO

The specific intracellular inhibition of protein activity at the protein level allows the determination of protein function in the cellular context. We demonstrate here the use of designed ankyrin repeat proteins as tailor-made intracellular kinase inhibitors. The target was aminoglycoside phosphotransferase (3')-IIIa (APH), which mediates resistance to aminoglycoside antibiotics in pathogenic bacteria and shares structural homology with eukaryotic protein kinases. Combining a selection and screening approach, we isolated 198 potential APH inhibitors from highly diverse combinatorial libraries of designed ankyrin repeat proteins. A detailed analysis of several inhibitors revealed that they bind APH with high specificity and with affinities down to the subnanomolar range. In vitro, the most potent inhibitors showed complete enzyme inhibition, and in vivo, a phenotype comparable with the gene knockout was observed, fully restoring antibiotic sensitivity in resistant bacteria. These results underline the great potential of designed ankyrin repeat proteins for modulation of intracellular protein function.


Assuntos
Anquirinas/química , Amicacina/química , Aminoglicosídeos/química , Antibacterianos/farmacologia , Biotinilação , Domínio Catalítico , Cromatografia , Clonagem Molecular , Farmacorresistência Bacteriana , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Biblioteca Gênica , Canamicina/química , Canamicina Quinase/química , Cinética , Modelos Químicos , Modelos Moleculares , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribossomos/química , Sensibilidade e Especificidade , Ressonância de Plasmônio de Superfície
19.
Protein Sci ; 13(11): 2864-70, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15498935

RESUMO

Ankyrin repeats (AR) are 33-residue motifs containing a beta-turn, followed by two alpha-helices connected by a loop. AR occur in tandem arrangements and stack side-by-side to form elongated domains involved in very different cellular tasks. Recently, consensus libraries of AR repeats were constructed. Protein E1_5 represents a member of the shortest library, and consists of only a single consensus repeat flanked by designed N- and C-terminal capping repeats. Here we present a biophysical characterization of this AR domain. The protein is compactly folded, as judged from the heat capacity of the native state and from the specific unfolding enthalpy and entropy. From spectroscopic data, thermal and urea-induced unfolding can be modeled by a two-state transition. However, scanning calorimetry experiments reveal a deviation from the two-state behavior at elevated temperatures. Folding and unfolding at 5 degrees C both follow monoexponential kinetics with k(folding) = 28 sec(-1) and k(unfolding) = 0.9 sec(-1). Kinetic and equilibrium unfolding parameters at 5 degrees C agree very well. We conclude that E1_5 folds in a simple two-state manner at low temperatures while equilibrium intermediates become populated at higher temperatures. A chevron-plot analysis indicates that the protein traverses a very compact transition state along the folding/unfolding pathway. This work demonstrates that a designed minimal ankyrin repeat protein has the thermodynamic and kinetic properties of a compactly folded protein, and explains the favorable properties of the consensus framework.


Assuntos
Repetição de Anquirina , Dobramento de Proteína , Desenho de Fármacos , Modelos Moleculares , Biblioteca de Peptídeos , Desnaturação Proteica , Renaturação Proteica , Temperatura , Ureia
20.
Nat Biotechnol ; 22(5): 575-82, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15097997

RESUMO

We report here the evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding. Using a consensus design strategy, we generated combinatorial libraries of AR proteins of varying repeat numbers with diversified binding surfaces. Libraries of two and three repeats, flanked by 'capping repeats,' were used in ribosome-display selections against maltose binding protein (MBP) and two eukaryotic kinases. We rapidly enriched target-specific binders with affinities in the low nanomolar range and determined the crystal structure of one of the selected AR proteins in complex with MBP at 2.3 A resolution. The interaction relies on the randomized positions of the designed AR protein and is comparable to natural, heterodimeric protein-protein interactions. Thus, our AR protein libraries are valuable sources for binding molecules and, because of the very favorable biophysical properties of the designed AR proteins, an attractive alternative to antibody libraries.


Assuntos
Repetição de Anquirina , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática , Dados de Sequência Molecular , Conformação Proteica
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