RESUMEN
Advances in the treatment of pediatric AML have been modest over the past four decades. Despite maximally intensive therapy, approximately 40% of patients will relapse. Novel targeted therapies are needed to improve outcomes. We identified mesothelin (MSLN), a well-validated target overexpressed in some adult malignancies, to be highly expressed on the leukemic cell surface in a subset of pediatric AML patients. The lack of expression on normal bone marrow cells makes MSLN a viable target for immunotherapies such as T-cell engaging bispecific antibodies (BsAbs) that combine two distinct antibody-variable regions into a single molecule targeting a cancer-specific antigen and the T-cell co-receptor CD3. Using antibody single-chain variable region (scFv) sequences derived from amatuximab-recognizing MSLN, and from either blinatumomab or AMG330 targeting CD3, we engineered and expressed two MSLN/CD3-targeting BsAbs: MSLNAMA-CD3L2K and MSLNAMA-CD3AMG, respectively. Both BsAbs promoted T-cell activation and reduced leukemic burden in MV4;11:MSLN xenografted mice, but not in those transplanted with MSLN-negative parental MV4;11 cells. MSLNAMA-CD3AMG induced complete remission in NTPL-146 and DF-5 patient-derived xenograft models. These data validate the in vivo efficacy and specificity of MSLN-targeting BsAbs. Because prior MSLN-directed therapies appeared safe in humans, MSLN-targeting BsAbs could be ideal immunotherapies for MSLN-positive pediatric AML patients.
RESUMEN
On-target, off-tissue toxicity limits the systemic use of drugs that would otherwise reduce symptoms or reverse the damage of arthritic diseases, leaving millions of patients in pain and with limited physical mobility. We identified cystine-dense peptides (CDPs) that rapidly accumulate in cartilage of the knees, ankles, hips, shoulders, and intervertebral discs after systemic administration. These CDPs could be used to concentrate arthritis drugs in joints. A cartilage-accumulating peptide, CDP-11R, reached peak concentration in cartilage within 30 min after administration and remained detectable for more than 4 days. Structural analysis of the peptides by crystallography revealed that the distribution of positive charge may be a distinguishing feature of joint-accumulating CDPs. In addition, quantitative whole-body autoradiography showed that the disulfide-bonded tertiary structure is critical for cartilage accumulation and retention. CDP-11R distributed to joints while carrying a fluorophore imaging agent or one of two different steroid payloads, dexamethasone (dex) and triamcinolone acetonide (TAA). Of the two payloads, the dex conjugate did not advance because the free drug released into circulation was sufficient to cause on-target toxicity. In contrast, the CDP-11R-TAA conjugate alleviated joint inflammation in the rat collagen-induced model of rheumatoid arthritis while avoiding toxicities that occurred with nontargeted steroid treatment at the same molar dose. This conjugate shows promise for clinical development and establishes proof of concept for multijoint targeting of disease-modifying therapeutic payloads.
Asunto(s)
Artritis Experimental , Corticoesteroides , Animales , Artritis Experimental/tratamiento farmacológico , Cartílago , Humanos , Péptidos , Ratas , EsteroidesAsunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Antígenos CD28/metabolismo , Complejo CD3/metabolismo , Receptores Coestimuladores e Inhibidores de Linfocitos T , Inmunoterapia Adoptiva/métodos , Activación de Linfocitos/inmunología , Anticuerpos Biespecíficos/farmacología , Línea Celular , Resistencia a Medicamentos , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Linfocitos T/inmunologíaRESUMEN
Peptides folded through interwoven disulfides display extreme biochemical properties and unique medicinal potential. However, their exploitation has been hampered by the limited amounts isolatable from natural sources and the expense of chemical synthesis. We developed reliable biological methods for high-throughput expression, screening and large-scale production of these peptides: 46 were successfully produced in multimilligram quantities, and >600 more were deemed expressible through stringent screening criteria. Many showed extreme resistance to temperature, proteolysis and/or reduction, and all displayed inhibitory activity against at least 1 of 20 ion channels tested, thus confirming their biological functionality. Crystal structures of 12 confirmed proper cystine topology and the utility of crystallography to study these molecules but also highlighted the need for rational classification. Previous categorization attempts have focused on limited subsets featuring distinct motifs. Here we present a global definition, classification and analysis of >700 structures of cystine-dense peptides, providing a unifying framework for these molecules.
Asunto(s)
Cistina/química , Péptidos/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Células HEK293 , Humanos , Canales Iónicos/antagonistas & inhibidores , Modelos Moleculares , Biosíntesis de Péptidos , Péptidos/clasificación , Péptidos/farmacologíaRESUMEN
Interleukin-31 (IL-31) is a member of the four helical-bundle gp130/IL-6 cytokine family. Despite its implicated roles in inflammatory diseases, the biosynthetic processes of IL-31 have been poorly investigated. A detailed understanding of IL-31 biosynthesis and the nature of ligand-receptor interactions can provide insights into effective strategies for the design of therapeutic approaches. By using various heterologous protein expression systems, we demonstrated that murine IL-31 was secreted as inter-molecularly disulfide-bonded covalent aggregates. Covalently aggregated IL-31 appeared while trafficking in the secretory pathway, but was not actively retained in the ER. The aggregate formation was not caused by a dysfunctional ER quality control mechanism or an intrinsic limitation in protein folding capacity. Furthermore, secreted IL-31 aggregates were part of a large complex composed of various pleiotropic secretory factors and immune-stimulators. The extent and the heterogeneous nature of aggregates may imply that IL-31 was erroneously folded, but it was capable of signaling through cognate receptors. Mutagenesis revealed the promiscuity of all five cysteines in inter-molecular disulfide formation with components of the hetero-aggregates, but no cysteine was required for IL-31 secretion itself. Our present study not only illustrated various functions that cysteines perform during IL-31 biosynthesis and secretion, but also highlighted their potential roles in cytokine effector functions.
Asunto(s)
Cisteína/metabolismo , Cuerpos de Inclusión/química , Interleucinas/biosíntesis , Animales , Línea Celular , Proliferación Celular , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Células HEK293 , Humanos , Interleucinas/genética , Ratones , Procesamiento Proteico-Postraduccional , Transducción de Señal/fisiologíaRESUMEN
Stimulation of red cell production through agonism of the erythropoietin receptor (EpoR) has historically been accomplished through administration of erythropoietin (EPO), the native ligand. The short half-life of EPO has led to the development of a variety of other agonists, including antibodies. It is of considerable interest to understand how these agents might activate the EpoR and whether or not it is important to bind in a manner similar to the native ligand. The binding epitopes of a panel of eight agonistic, single-chain antibody (scFv-Fc) constructs were determined through scanning alanine mutagenesis as well as more limited arginine mutagenesis of the receptor. It was found that while some of these constructs bound to receptor epitopes shared by the ligand, others bound in completely unique ways. The use of a panel of agonists and scanning mutagenesis can define the critical binding regions for signaling; in the case of the EpoR, these regions were remarkably broad.
Asunto(s)
Epítopos/metabolismo , Eritropoyetina/agonistas , Receptores de Eritropoyetina/metabolismo , Transducción de Señal , Anticuerpos de Cadena Única/metabolismo , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Epítopos/química , Epítopos/genética , Humanos , Cinética , Conformación Molecular , Unión Proteica , Receptores de Eritropoyetina/química , Receptores de Eritropoyetina/genética , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genéticaRESUMEN
The expression of proteins which do not express well on their own can be enhanced by linking them to human serum albumin (HSA) or antibody crystallizable fragment (Fc). The constructs shown here are designed to secrete the proteins after transient transfection of mammalian cell lines. The fusion partners are appended to the N-terminus of the proteins and contain a linker designed to be proteolytically cleaved. Transient transfection and purification protocols are provided as well as experimental results with five interleukins.
Asunto(s)
Células Eucariotas/fisiología , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Animales , Humanos , MamíferosRESUMEN
The expression levels of five secreted target interleukins (IL-11, 15, 17B, 32, and IL23 p19 subunit) were tested with three different fusion partners in 2936E cells. When fused to the N-terminus, human serum albumin (HSA) was found to enhance the expression of both IL-17B and IL-15, cytokines which did not express at measurable levels on their own. Although the crystallizable fragment of an antibody (Fc) was also an effective fusion partner for IL-17B, Fc did not increase expression of IL-15. Fc was superior to HSA for the expression of the p19 subunit of IL-23, but no partner led to measurable levels of IL-32gamma secretion. Glutathione S-transferase (GST) did not enhance the expression of any target and suppressed the production of IL-11, a cytokine which expressed robustly both on its own and when fused to HSA or Fc. Cleavage of the fusion partner was not always possible. The use of HSA or Fc as N-terminal fusions can be an effective technique to express difficult proteins, especially for applications in which the fusion partner need not be removed.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/metabolismo , Interleucinas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Albúmina Sérica/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Interleucinas/química , Interleucinas/genética , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Albúmina Sérica/genética , TransfecciónRESUMEN
The Structural Genomics of Pathogenic Protozoa (SGPP) Consortium aimed to determine crystal structures of proteins from trypanosomatid and malaria parasites in a high throughput manner. The pipeline of target selection, protein production, crystallization, and structure determination, is sketched. Special emphasis is given to a number of technology developments including domain prediction, the use of "co-crystallants," and capillary crystallization. "Fragment cocktail crystallography" for medical structural genomics is also described.
Asunto(s)
Genómica/métodos , Plasmodium/genética , Proteínas Protozoarias/química , Trypanosomatina/genética , Animales , Cristalización , Cristalografía por Rayos X/métodosRESUMEN
The 1.8 A resolution de novo structure of nucleoside 2-deoxyribosyltransferase (EC 2.4.2.6) from Trypanosoma brucei (TbNDRT) has been determined by SADa phasing in an unliganded state and several ligand-bound states. This enzyme is important in the salvage pathway of nucleoside recycling. To identify novel lead compounds, we exploited "fragment cocktail soaks". Out of 304 compounds tried in 31 cocktails, four compounds could be identified crystallographically in the active site. In addition, we demonstrated that very short soaks of approximately 10 s are sufficient even for rather hydrophobic ligands to bind in the active site groove, which is promising for the application of similar soaking experiments to less robust crystals of other proteins.
Asunto(s)
Pentosiltransferasa/antagonistas & inhibidores , Pentosiltransferasa/química , Tripanocidas/química , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Animales , Alcoholes Bencílicos/química , Alcoholes Bencílicos/farmacología , Sitios de Unión , Cristalografía por Rayos X , Indoles/química , Indoles/farmacología , Isoquinolinas/química , Isoquinolinas/farmacología , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Quinolinas/química , Quinolinas/farmacología , Relación Estructura-Actividad , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacosRESUMEN
The structure of ribose 5-phosphate isomerase from Plasmodium falciparum, PFE0730c, has been determined by molecular replacement at 2.09 angstroms resolution. The enzyme, which catalyzes the isomerization reaction that interconverts ribose 5-phosphate and ribulose 5-phosphate, is a member of the pentose phosphate pathway. The P. falciparum enzyme belongs to the ribose 5-phosphate isomerase A family, Pfam family PF06562 (DUF1124), and is structurally similar to other members of the family.
Asunto(s)
Isomerasas Aldosa-Cetosa/química , Plasmodium falciparum/enzimología , Secuencia de Aminoácidos , Animales , Cristalización , Cristalografía por Rayos X , Dimerización , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
As part of a structural genomics initiative, 1000 open reading frames from Plasmodium falciparum, the causative agent of the most deadly form of malaria, were tested in an E. coli protein expression system. Three hundred and thirty-seven of these targets were observed to express, although typically the protein was insoluble. Sixty-three of the targets provided soluble protein in yields ranging from 0.9 to 406.6 mg from one liter of rich media. Higher molecular weight, greater protein disorder (segmental analysis, SEG), more basic isoelectric point (pI), and a lack of homology to E. coli proteins were all highly and independently correlated with difficulties in expression. Surprisingly, codon usage and the percentage of adenosines and thymidines (%AT) did not appear to play a significant role. Of those proteins which expressed, high pI and a hypothetical annotation were both strongly and independently correlated with insolubility. The overwhelmingly important role of pI in both expression and solubility appears to be a surprising and fundamental issue in the heterologous expression of P. falciparum proteins in E. coli. Twelve targets which did not express in E. coli from the native gene sequence were codon-optimized through whole gene synthesis, resulting in the (insoluble) expression of three of these proteins. Seventeen targets which were expressed insolubly in E. coli were moved into a baculovirus/Sf-21 system, resulting in the soluble expression of one protein at a high level and six others at a low level. A variety of factors conspire to make the heterologous expression of P. falciparum proteins challenging, and these observations lay the groundwork for a rational approach to prioritizing and, ultimately, eliminating these impediments.
Asunto(s)
Baculoviridae/metabolismo , Escherichia coli/genética , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Animales , Baculoviridae/genética , Clonación Molecular , Escherichia coli/metabolismo , Expresión Génica , Punto Isoeléctrico , Sistemas de Lectura Abierta , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The structure of a conserved hypothetical protein, PlasmoDB sequence MAL13P1.257 from Plasmodium falciparum, Pfam sequence family PF05907, has been determined as part of the structural genomics effort of the Structural Genomics of Pathogenic Protozoa consortium. The structure was determined by multiple-wavelength anomalous dispersion at 2.17 A resolution. The structure is almost entirely beta-sheet; it consists of 15 beta-strands and one short 3(10)-helix and represents a new protein fold. The packing of the two monomers in the asymmetric unit indicates that the biological unit may be a dimer.
Asunto(s)
Plasmodium falciparum/química , Proteínas Protozoarias/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Alineación de SecuenciaRESUMEN
The crystal structure of D-glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH) from the major malaria parasite Plasmodium falciparum is solved at 2.25 A resolution. The structure of PfGAPDH is of interest due to the dependence of the malaria parasite in infected human erythrocytes on the glycolytic pathway for its energy generation. Recent evidence suggests that PfGAPDH may also be required for other critical activities such as apical complex formation. The cofactor NAD(+) is bound to all four subunits of the tetrameric enzyme displaying excellent electron densities. In addition, in all four subunits a completely unexpected large island of extra electron density in the active site is observed, approaching closely the nicotinamide ribose of the NAD(+). This density is most likely the protease inhibitor AEBSF, found in maps from two different crystals. This putative AEBSF molecule is positioned in a crucial location and hence our structure, with expected and unexpected ligands bound, can be of assistance in lead development and design of novel antimalarials.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasas/química , Plasmodium falciparum/enzimología , Proteínas Protozoarias/química , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Cristalografía por Rayos X , Citoplasma/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , NAD/química , NAD/metabolismo , Plasmodium falciparum/genética , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
X-ray crystallography is a technique which is finding increasing utility in the effort to find new antimalarial drugs. This is in spite of the serious difficulties often encountered in obtaining sufficient quantities of protein to crystallize. This review provides an overview of the Plasmodium falciparum proteins which have been crystallized with bound inhibitors and the methodology employed in the heterologous expression of these proteins. Lactate dehydrogenase, plasmepsin II, and triosphosphate isomerase are the most advanced targets of structure-based drug design, but nine other P. falciparum proteins have been crystallized with inhibitors as well, and this is clearly an area which is moving very quickly. Some consideration will also be given to the limitations of structure-based drug discovery with respect to known antimalarial drugs.
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Antimaláricos/química , Plasmodium falciparum/efectos de los fármacos , Animales , Antimaláricos/síntesis química , Cristalografía por Rayos X , Plasmodium falciparum/química , Plasmodium falciparum/enzimología , Proteínas Protozoarias/químicaRESUMEN
Cloning grills are aluminum grids designed to divide an agar plate into segments, thereby multiplying the number of E. coli cultures which can be streaked out on a single plate. The grills are autoclaved and placed in square petri dishes immediately after hot agar is poured. When the agar solidifies, the grill remains embedded in the media, and each of the 12 lanes accommodates the streaking out of a single culture. As the spacing of the grill lanes is the same as that of a 96-well plate, 12 cultures can be streaked at a time using a 12-channel pipette. This allows a plate of 96 cultures to be rapidly and accurately plated for colony isolation on only eight agar plates.
Asunto(s)
Clonación Molecular/métodos , Genómica/instrumentación , Técnicas Bacteriológicas/instrumentación , Escherichia coli/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Freezing of purified protein drops directly in liquid nitrogen is a convenient technique for the long-term storage of protein samples. Although this enhances reproducibility in follow-up crystallization experiments, some protein samples are not amenable to this technique. It has been discovered that plunging PCR tubes containing protein samples into liquid nitrogen results in more rapid freezing of the samples and can safely preserve some proteins that are damaged by drop-freezing. The PCR-tube method can also be adapted to a PCR-plate freezing method with applications for high-throughput and structural genomics projects.
Asunto(s)
Criopreservación/métodos , ARN Nucleotidiltransferasas/química , Proteínas Represoras/químicaRESUMEN
Cell adhesion, a critical early step in the inflammatory process, has increasingly become the target of drug discovery efforts. Described in this unit are techniques for measuring inhibitors of VLA-4-mediated adhesion to either VCAM or the connecting segment (CS-1) of fibronectin.
Asunto(s)
Adhesión Celular/fisiología , Integrina alfa4beta1/antagonistas & inhibidores , Receptores de Antígeno muy Tardío/fisiología , Molécula 1 de Adhesión Celular Vascular/fisiología , Línea Celular Transformada , Línea Celular Tumoral , Cloruros/farmacología , Recolección de Datos , Fibronectinas/metabolismo , Humanos , Compuestos de Manganeso/farmacología , Manejo de Especímenes/métodosRESUMEN
The discovery of a sulphonamide by-product with VLA-4 antagonistic activity led to a series of potent, small molecule VLA-4 antagonists. Synthesis, SAR and in vivo evaluation of the selected compound will be presented.