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1.
Nucleic Acids Res ; 52(12): 7305-7320, 2024 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-38842936

RESUMEN

The SorC family of transcriptional regulators plays a crucial role in controlling the carbohydrate metabolism and quorum sensing. We employed an integrative approach combining X-ray crystallography and cryo-electron microscopy to investigate architecture and functional mechanism of two prototypical representatives of two sub-classes of the SorC family: DeoR and CggR from Bacillus subtilis. Despite possessing distinct DNA-binding domains, both proteins form similar tetrameric assemblies when bound to their respective DNA operators. Structural analysis elucidates the process by which the CggR-regulated gapA operon is derepressed through the action of two effectors: fructose-1,6-bisphosphate and newly confirmed dihydroxyacetone phosphate. Our findings provide the first comprehensive understanding of the DNA binding mechanism of the SorC-family proteins, shedding new light on their functional characteristics.


Asunto(s)
Bacillus subtilis , Proteínas Bacterianas , Microscopía por Crioelectrón , Modelos Moleculares , Proteínas Represoras , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Unión Proteica , Multimerización de Proteína , ADN/química , ADN/metabolismo , Sitios de Unión , Regulación Bacteriana de la Expresión Génica , ADN Bacteriano/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Operón/genética , Fructosadifosfatos
2.
Nucleic Acids Res ; 51(18): 10011-10025, 2023 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-37615563

RESUMEN

Eukaryotic transcription is dependent on specific histone modifications. Their recognition by chromatin readers triggers complex processes relying on the coordinated association of transcription regulatory factors. Although various modification states of a particular histone residue often lead to differential outcomes, it is not entirely clear how they are discriminated. Moreover, the contribution of intrinsically disordered regions outside of the specialized reader domains to nucleosome binding remains unexplored. Here, we report the structures of a PWWP domain from transcriptional coactivator LEDGF in complex with the H3K36 di- and trimethylated nucleosome, indicating that both methylation marks are recognized by PWWP in a highly conserved manner. We identify a unique secondary interaction site for the PWWP domain at the interface between the acidic patch and nucleosomal DNA that might contribute to an H3K36-methylation independent role of LEDGF. We reveal DNA interacting motifs in the intrinsically disordered region of LEDGF that discriminate between the intra- or extranucleosomal DNA but remain dynamic in the context of dinucleosomes. The interplay between the LEDGF H3K36-methylation reader and protein binding module mediated by multivalent interactions of the intrinsically disordered linker with chromatin might help direct the elongation machinery to the vicinity of RNA polymerase II, thereby facilitating productive elongation.

3.
Int J Mol Sci ; 25(4)2024 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-38396918

RESUMEN

The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin (IrThy), which is secreted in the saliva of Ixodes ricinus ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure-function characterization of IrThy-the first investigated thyropin of parasite origin-and suggests its potential role in host-parasite interactions at the tick bite site.


Asunto(s)
Ixodes , Saliva , Animales , Humanos , Saliva/metabolismo , Cisteína , Glicosaminoglicanos , Catepsinas/metabolismo , Ixodes/metabolismo , Espectroscopía de Resonancia Magnética
4.
Chembiochem ; 22(18): 2741-2761, 2021 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-33939874

RESUMEN

This review describes recent progress in the design and development of inhibitors of human carbonic anhydrase IX (CA IX) based on space-filling carborane and cobalt bis(dicarbollide) clusters. CA IX enzyme is known to play a crucial role in cancer cell proliferation and metastases. The new class of potent and selective CA IX inhibitors combines the structural motif of a bulky inorganic cluster with an alkylsulfamido or alkylsulfonamido anchor group for Zn2+ ion in the enzyme active site. Detailed structure-activity relationship (SAR) studies of a large series containing 50 compounds uncovered structural features of the cluster-containing inhibitors that are important for efficient and selective inhibition of CA IX activity. Preclinical evaluation of selected compounds revealed low toxicity, favorable pharmacokinetics and ability to reduce tumor growth. Cluster-containing inhibitors of CA IX can thus be considered as promising candidates for drug development and/or for combination therapy in boron neutron capture therapy (BNCT).


Asunto(s)
Compuestos de Boro/química , Anhidrasa Carbónica IX/antagonistas & inhibidores , Inhibidores de Anhidrasa Carbónica/química , Sitios de Unión , Compuestos de Boro/metabolismo , Compuestos de Boro/uso terapéutico , Anhidrasa Carbónica IX/metabolismo , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/metabolismo , Inhibidores de Anhidrasa Carbónica/uso terapéutico , Humanos , Simulación de Dinámica Molecular , Neoplasias/tratamiento farmacológico , Compuestos Organometálicos/química , Relación Estructura-Actividad , Sulfonamidas/química
5.
Proc Natl Acad Sci U S A ; 115(30): E7053-E7062, 2018 07 24.
Artículo en Inglés | MEDLINE | ID: mdl-29997176

RESUMEN

Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos , Línea Celular Tumoral , VIH/enzimología , VIH/genética , Integrasa de VIH/genética , Integrasa de VIH/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Subunidad 1 del Complejo Mediador/genética , Subunidad 1 del Complejo Mediador/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Fosforilación/genética , Factores de Transcripción/genética
6.
J Biol Chem ; 294(46): 17371-17382, 2019 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-31558604

RESUMEN

Information on how insulin and insulin-like growth factors 1 and 2 (IGF-1 and -2) activate insulin receptors (IR-A and -B) and the IGF-1 receptor (IGF-1R) is crucial for understanding the difference in the biological activities of these peptide hormones. Cryo-EM studies have revealed that insulin uses its binding sites 1 and 2 to interact with IR-A and have identified several critical residues in binding site 2. However, mutagenesis studies suggest that Ile-A10, Ser-A12, Leu-A13, and Glu-A17 also belong to insulin's site 2. Here, to resolve this discrepancy, we mutated these insulin residues and the equivalent residues in IGFs. Our findings revealed that equivalent mutations in the hormones can result in differential biological effects and that these effects can be receptor-specific. We noted that the insulin positions A10 and A17 are important for its binding to IR-A and IR-B and IGF-1R and that A13 is important only for IR-A and IR-B binding. The IGF-1/IGF-2 positions 51/50 and 54/53 did not appear to play critical roles in receptor binding, but mutations at IGF-1 position 58 and IGF-2 position 57 affected the binding. We propose that IGF-1 Glu-58 interacts with IGF-1R Arg-704 and belongs to IGF-1 site 1, a finding supported by the NMR structure of the less active Asp-58-IGF-1 variant. Computational analyses indicated that the aforementioned mutations can affect internal insulin dynamics and inhibit adoption of a receptor-bound conformation, important for binding to receptor site 1. We provide a molecular model and alternative hypotheses for how the mutated insulin residues affect activity.


Asunto(s)
Factor I del Crecimiento Similar a la Insulina/química , Insulina/química , Receptor IGF Tipo 1/química , Receptor de Insulina/química , Anomalías Múltiples/genética , Secuencia de Aminoácidos/genética , Sitios de Unión/genética , Trastornos del Crecimiento/genética , Humanos , Insulina/análogos & derivados , Insulina/síntesis química , Insulina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/química , Factor II del Crecimiento Similar a la Insulina/genética , Mutación/genética , Resonancia Magnética Nuclear Biomolecular , Unión Proteica/genética , Dominios Proteicos/genética , Receptor IGF Tipo 1/genética , Receptor de Insulina/genética
7.
J Enzyme Inhib Med Chem ; 35(1): 1800-1810, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-32962427

RESUMEN

Human carbonic anhydrase IX (CA IX), a protein specifically expressed on the surface of solid tumour cells, represents a validated target both for anticancer therapy and diagnostics. We recently identified sulfonamide dicarbaboranes as promising inhibitors of CA IX with favourable activities both in vitro and in vivo. To explain their selectivity and potency, we performed detailed X-ray structural analysis of their interactions within the active sites of CA IX and CA II. Series of compounds bearing various aliphatic linkers between the dicarbaborane cluster and sulfonamide group were examined. Preferential binding towards the hydrophobic part of the active site cavity was observed. Selectivity towards CA IX lies in the shape complementarity of the dicarbaborane cluster with a specific CA IX hydrophobic patch containing V131 residue. The bulky side chain of F131 residue in CA II alters the shape of the catalytic cavity, disrupting favourable interactions of the spherical dicarbaborane cluster.


Asunto(s)
Antineoplásicos/química , Compuestos de Boro/química , Anhidrasa Carbónica IX/antagonistas & inhibidores , Inhibidores de Anhidrasa Carbónica/química , Sulfonamidas/química , Secuencia de Aminoácidos , Antígenos de Neoplasias/genética , Antineoplásicos/farmacología , Anhidrasa Carbónica IX/genética , Inhibidores de Anhidrasa Carbónica/farmacología , Dominio Catalítico , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Unión Proteica , Relación Estructura-Actividad , Sulfonamidas/farmacología
8.
Biomacromolecules ; 20(1): 412-421, 2019 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-30485077

RESUMEN

A water-soluble polymer cancerostatic actively targeted against cancer cells expressing a disialoganglioside antigen GD2 was designed, synthesized and characterized. A polymer conjugate of an antitumor drug doxorubicin with a N-(2-hydroxypropyl)methacrylamide-based copolymer was specifically targeted against GD2 antigen-positive tumor cells using a recombinant single chain fragment (scFv) of an anti-GD2 monoclonal antibody. The targeting protein ligand was attached to the polymer-drug conjugate either via a covalent bond between the amino groups of the protein using a traditional nonspecific aminolytic reaction with a reactive polymer precursor or via a noncovalent but highly specific interaction between bungarotoxin covalently linked to the polymer and the recombinant scFv modified with a C-terminal bungarotoxin-binding peptide. The GD2 antigen binding activity and GD2-specific cytotoxicity of the targeted noncovalent polymer-scFv complex proved to be superior to the covalent polymer-scFv conjugate.


Asunto(s)
Antineoplásicos/química , Gangliósidos/inmunología , Nanoconjugados/química , Anticuerpos de Cadena Única/química , Células 3T3 , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Bungarotoxinas/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Doxorrubicina/administración & dosificación , Doxorrubicina/química , Doxorrubicina/farmacología , Ratones , Ácidos Polimetacrílicos/química , Unión Proteica , Anticuerpos de Cadena Única/inmunología
9.
Biochemistry ; 57(16): 2373-2382, 2018 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-29608283

RESUMEN

Insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively) are protein hormones involved not only in normal growth and development but also in life span regulation and cancer. They exert their functions mainly through the IGF-1R or by binding to isoform A of the insulin receptor (IR-A). The development of IGF-1 and IGF-2 antagonists is of great clinical interest. Mutations of A4 and A8 sites of human insulin lead to disproportionate effects on hormone IR binding and activation. Here, we systematically modified IGF-1 sites 45, 46, and 49 and IGF-2 sites 45 and 48, which correspond, or are close, to insulin sites A4 and A8. The IGF-1R and IR-A binding and autophosphorylation potencies of these analogues were characterized. They retained the main IGF-1R-related properties, but the hormones with His49 in IGF-1 and His48 in IGF-2 showed significantly higher affinities for IR-A and for IR-B, being the strongest IGF-1- and IGF-2-like binders of these receptors ever reported. All analogues activated IR-A and IGF-1R without major discrepancies in their binding affinities. This study revealed that IR-A and IGF-1R contain specific sites, likely parts of their so-called sites 2', which can interact differently with specifically modified IGF analogues. Moreover, a clear importance of IGF-2 site 44 for effective hormone folding was also observed. These findings may facilitate novel and rational engineering of new hormone analogues for IR-A and IGF-1R studies and for potential medical applications.


Asunto(s)
Factor II del Crecimiento Similar a la Insulina/química , Factor I del Crecimiento Similar a la Insulina/química , Receptor de Insulina/química , Receptores de Somatomedina/genética , Evolución Molecular , Humanos , Insulina/química , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/genética , Ligandos , Mutación , Fosforilación , Unión Proteica , Isoformas de Proteínas , Receptor IGF Tipo 1 , Receptor de Insulina/metabolismo , Receptores de Somatomedina/química , Transducción de Señal
10.
Cytotherapy ; 20(4): 507-520, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29475789

RESUMEN

BACKGROUND AIMS: Clinical-grade chimeric antigenic receptor (CAR)19 T cells are routinely manufactured by lentiviral/retroviral (LV/RV) transduction of an anti-CD3/CD28 activated T cells, which are then propagated in a culture medium supplemented with interleukin (IL)-2. The use of LV/RVs for T-cell modification represents a manufacturing challenge due to the complexity of the transduction approach and the necessity of thorough quality control. METHODS: We present here a significantly improved protocol for CAR19 T-cell manufacture that is based on the electroporation of peripheral blood mononuclear cells with plasmid DNA encoding the piggyBac transposon/transposase vectors and their cultivation in the presence of cytokines IL-4, IL-7 and IL-21. RESULTS: We found that activation of the CAR receptor by either its cognate ligand (i.e., CD19 expressed on the surface of B cells) or anti-CAR antibody, followed by cultivation in the presence of cytokines IL-4 and IL-7, enables strong and highly selective expansion of functional CAR19 T cells, resulting in >90% CAR+ T cells. Addition of cytokine IL-21 to the mixture of IL-4 and IL-7 supported development of immature CAR19 T cells with central memory and stem cell memory phenotypes and expressing very low amounts of inhibitory receptors PD-1, LAG-3 and TIM-3. CONCLUSIONS: Our protocol provides a simple and cost-effective method for engineering high-quality T cells for adoptive therapies.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Elementos Transponibles de ADN/genética , Interleucina-4/farmacología , Interleucina-7/farmacología , Interleucinas/farmacología , Ingeniería de Proteínas/métodos , Receptores Quiméricos de Antígenos/genética , Linfocitos T , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Células Cultivadas , Electroporación , Vectores Genéticos , Células HEK293 , Humanos , Inmunoterapia Adoptiva/métodos , Lentivirus/genética , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Células PC-3 , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transducción Genética/métodos
11.
J Biol Chem ; 291(40): 21234-21245, 2016 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-27510031

RESUMEN

Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions with insulin becoming a key regulator of metabolism, whereas insulin-like growth factors (IGF)-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and insulin-like growth factor type I receptor (IGF-1R). Identification of structural determinants in IGFs and insulin that trigger their specific signaling pathways is of increasing importance in designing receptor-specific analogs with potential therapeutic applications. Here, we developed a straightforward protocol for production of recombinant IGF-II and prepared six IGF-II analogs with IGF-I-like mutations. All modified molecules exhibit significantly reduced affinity toward IR-A, particularly the analogs with a Pro-Gln insertion in the C-domain. Moreover, one of the analogs has enhanced binding affinity for IGF-1R due to a synergistic effect of the Pro-Gln insertion and S29N point mutation. Consequently, this analog has almost a 10-fold higher IGF-1R/IR-A binding specificity in comparison with native IGF-II. The established IGF-II purification protocol allowed for cost-effective isotope labeling required for a detailed NMR structural characterization of IGF-II analogs that revealed a link between the altered binding behavior of selected analogs and conformational rearrangement of their C-domains.


Asunto(s)
Antígenos CD/química , Factor II del Crecimiento Similar a la Insulina/química , Receptor IGF Tipo 1/química , Receptor de Insulina/química , Sustitución de Aminoácidos , Antígenos CD/genética , Antígenos CD/metabolismo , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , Mutación Missense , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
BMC Biol ; 14(1): 91, 2016 10 19.
Artículo en Inglés | MEDLINE | ID: mdl-27756303

RESUMEN

BACKGROUND: Relapsed acute lymphoblastic leukemia (ALL) is one of the main causes of mortality in childhood malignancies. Previous genetic studies demonstrated that chemoresistant ALL is driven by activating mutations in NT5C2, the gene encoding cytosolic 5´-nucleotidase (cN-II). However, molecular mechanisms underlying this hyperactivation are still unknown. Here, we present kinetic and structural properties of cN-II variants that represent 75 % of mutated alleles in patients who experience relapsed ALL (R367Q, R238W and L375F). RESULTS: Enzyme kinetics measurements revealed that the mutants are consitutively active without need for allosteric activators. This shows that hyperactivity is not caused by a direct catalytic effect but rather by misregulation of cN-II. X-ray crystallography combined with mass spectrometry-based techniques demonstrated that this misregulation is driven by structural modulation of the oligomeric interface within the cN-II homotetrameric assembly. These specific conformational changes are shared between the studied variants, despite the relatively random spatial distribution of the mutations. CONCLUSIONS: These findings define a common molecular mechanism for cN-II hyperactivity, which provides a solid basis for targeted therapy of leukemia. Our study highlights the cN-II oligomerization interface as an attractive pharmacological target.


Asunto(s)
5'-Nucleotidasa/genética , Regulación Leucémica de la Expresión Génica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , 5'-Nucleotidasa/metabolismo , Alelos , Clonación Molecular , Cristalografía por Rayos X , Humanos , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Conformación Proteica , Recurrencia
13.
J Struct Biol ; 191(2): 214-23, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26066970

RESUMEN

The hyaluronate receptor CD44 plays role in cell adhesion and migration and is involved in tumor metastasis. The extracellular domain of CD44 comprises the hyaluronate-binding domain (HABD) and the membrane-proximal stem region; the short intracellular portion interacts with adaptor proteins and triggers signaling pathways. Binding of hyaluronate to CD44 HABD induces an allosteric conformational change, which results in CD44 shedding. A poorly characterized epitope in human CD44 HABD is recognized by the murine monoclonal antibody MEM-85, which cross-blocks hyaluronate binding to CD44 and also induces CD44 shedding. MEM-85 is of therapeutic interest, as it inhibits growth of lung cancer cells in murine models. In this work, we employed a combination of biophysical methods to determine the MEM-85 binding epitope in CD44 HABD and to provide detailed insight into the mechanism of MEM-85 action. In particular, we constructed a single-chain variable fragment (scFv) of MEM-85 as a tool for detailed characterization of the CD44 HABD-antibody complex and identified residues within CD44 HABD involved in the interaction with scFv MEM-85 by NMR spectroscopy and mutational analysis. In addition, we built a rigid body model of the CD44 HABD-scFv MEM-85 complex using a low-resolution structure obtained by small-angle X-ray scattering. The MEM-85 epitope is situated in the C-terminal part of CD44 HABD, rather than the hyaluronate-binding groove, and the binding of MEM-85 induces a structural reorganization similar to that induced by hyaluronate. Therefore, the mechanism of MEM-85 cross-blocking of hyaluronate binding is likely of an allosteric, relay-like nature.


Asunto(s)
Anticuerpos Monoclonales/química , Receptores de Hialuranos/química , Sitios de Unión , Mapeo Epitopo , Humanos , Ácido Hialurónico/química , Células Jurkat , Modelos Moleculares , Mutación , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína
14.
J Enzyme Inhib Med Chem ; 30(1): 63-8, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24506201

RESUMEN

Human mitochondrial 5'(3')-deoxyribonucleotidase (mdN) catalyzes dephosphorylation of nucleoside monophosphates, and thus helps maintain homeostasis of deoxynucleosides required for mitochondrial DNA synthesis. Mature mdN is a 23-kDa dimeric protein with highest expression levels in the heart, brain and skeletal muscle. We have identified an alternative splice variant of the mdN gene containing an 18-nucleotide insertion encoding 6 amino acids (GKWPAT) at the 3'-end of the penultimate exon 4. We recombinantly expressed this enzyme variant and characterized its biochemical and kinetic properties as well as its three-dimensional structure. Our high-resolution (1.27 Å) crystal structure revealed that the insertion forms a loop located in the vicinity of the active site pocket and affects enzyme kinetic parameters as well as protein thermal stability.


Asunto(s)
5'-Nucleotidasa/química , Empalme Alternativo , Proteínas Mitocondriales/química , 5'-Nucleotidasa/genética , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Exones , Expresión Génica , Humanos , Isoenzimas/química , Isoenzimas/genética , Cinética , Proteínas Mitocondriales/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Insercional , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
15.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 2): 461-70, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24531480

RESUMEN

The human 5'(3')-deoxyribonucleotidases catalyze the dephosphorylation of deoxyribonucleoside monophosphates to the corresponding deoxyribonucleosides and thus help to maintain the balance between pools of nucleosides and nucleotides. Here, the structures of human cytosolic deoxyribonucleotidase (cdN) at atomic resolution (1.08 Å) and mitochondrial deoxyribonucleotidase (mdN) at near-atomic resolution (1.4 Å) are reported. The attainment of an atomic resolution structure allowed interatomic distances to be used to assess the probable protonation state of the phosphate anion and the side chains in the enzyme active site. A detailed comparison of the cdN and mdN active sites allowed the design of a cdN-specific inhibitor.


Asunto(s)
Desoxirribonucleótidos/química , Inhibidores Enzimáticos/química , Isoenzimas/química , Nucleotidasas/química , Organofosfonatos/química , Fosfatos/química , Dominio Catalítico , Cristalografía por Rayos X , Citosol/química , Citosol/enzimología , Diseño de Fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Células Eucariotas/química , Células Eucariotas/enzimología , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Mitocondrias/química , Mitocondrias/enzimología , Modelos Moleculares , Simulación del Acoplamiento Molecular , Nucleotidasas/antagonistas & inhibidores , Nucleotidasas/genética , Especificidad de Órganos , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relación Estructura-Actividad
16.
Org Biomol Chem ; 12(40): 7971-82, 2014 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-25178098

RESUMEN

This work describes novel in vitro inhibitors of human mitochondrial (mdN) and cytosolic (cdN) 5'(3')-deoxynucleotidases. We designed a series of derivatives of the lead compound (S)-1-[2-deoxy-3,5-O-(phosphonobenzylidene)-ß-d-threo-pentofuranosyl]thymine bearing various substituents in the para position of the benzylidene moiety. Detailed kinetic study revealed that certain para substituents increase the inhibitory potency (iodo derivative; K = 2.71 µM) and some induce a shift in selectivity toward cdN (carboxy derivative, K = 11.60 µM; iodoxy derivative, K = 6.60 µM). Crystal structures of mdN in complex with three of these compounds revealed that various para substituents lead to two alternative inhibitor binding modes within the enzyme active site. Two binding modes were also identified for cdN complexes by heteronuclear NMR spectroscopy.


Asunto(s)
5'-Nucleotidasa/antagonistas & inhibidores , Citosol/enzimología , Inhibidores Enzimáticos/farmacología , Mitocondrias/enzimología , Organofosfonatos/farmacología , 5'-Nucleotidasa/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Conformación Molecular , Organofosfonatos/síntesis química , Organofosfonatos/química , Relación Estructura-Actividad
17.
Biomacromolecules ; 14(3): 881-9, 2013 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-23373696

RESUMEN

The specificity of polymer conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) bearing cytostatic drugs for cancer cells could be significantly increased by the incorporation of a suitable targeting ligand, such as a monoclonal antibody (mAb). However, direct binding of the protein to the polymer carrier could cause considerable problems, such as decreasing the binding capacity of mAb to its target. Here, we introduce a novel strategy of joining a targeting moiety to a polymeric conjugate with cytostatic drug. The scFv of B1 mAb (specific for BCL1 leukemia cells) was tagged with peptide K ((VAALKEK)4). Peptide E ((VAALEKE)4), which forms a stable coiled coil structure heterodimer with peptide K, was assembled with the HPMA copolymers bearing doxorubicin. Such targeted polymeric conjugates possess very selective and high binding activity toward BCL1 cells. Similarly, targeted polymeric conjugates exert approximately 100 times higher cytostatic activity toward BCL1 cells in comparison to nontargeted conjugates in vitro. At the same time, the conjugates have comparable and rather low cytostatic activity for 38C13 cells, which are used as a negative control, in vitro.


Asunto(s)
Acrilamidas/farmacología , Materiales Biocompatibles/farmacología , Citostáticos/farmacología , Leucemia/tratamiento farmacológico , Polímeros/farmacología , Acrilamidas/síntesis química , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Anticuerpos Monoclonales/química , Materiales Biocompatibles/síntesis química , Línea Celular Tumoral , Proliferación Celular , Citostáticos/química , Doxorrubicina/química , Doxorrubicina/farmacología , Portadores de Fármacos/química , Metacrilatos/química , Ratones , Ratones Endogámicos BALB C , Polímeros/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
18.
Angew Chem Int Ed Engl ; 52(51): 13760-3, 2013 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-24307504

RESUMEN

CA inhibitors: Human carbonic anhydrases (CAs) are diagnostic and therapeutic targets. Various carborane cages are shown to act as active-site-directed inhibitors, and substitution with a sulfamide group and other substituents leads to compounds with high selectivity towards the cancer-specific isozyme IX. Crystal structures of the carboranes in the active site provide information that can be applied to the structure-based design of specific inhibitors.


Asunto(s)
Inhibidores de Anhidrasa Carbónica/farmacología , Diseño de Fármacos , Descubrimiento de Drogas , Humanos , Relación Estructura-Actividad
19.
Int J Pharm ; 648: 123619, 2023 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-37979631

RESUMEN

Antibody-mediated targeting is an efficient strategy to enhance the specificity and selectivity of polymer nanomedicines towards the target site, typically a tumor. However, direct covalent coupling of an antibody with a polymer usually results in a partial damage of the antibody binding site accompanied with a compromised biological activity. Here, an original solution based on well-defined non-covalent interactions between tris-nitrilotriacetic acid (trisNTA) and hexahistidine (His-tag) groups, purposefully introduced to the structure of each macromolecule, is described. Specifically, trisNTA groups were attached along the chains of a hydrophilic statistical copolymer based on N-(2-hydroxypropyl)methacrylamide (HPMA), and at the end or along the chains of thermo-responsive di-block copolymers based on N-isopropylmethacrylamide (NIPMAM) and HPMA; His-tag was incorporated to the structure of a recombinant single chain fragment of an anti-GD2 monoclonal antibody (scFv-GD2). Static and dynamic light scattering analyses confirmed that mixing of polymer with scFv-GD2 led to the formation of polymer/scFv-GD2 complexes; those prepared from thermo-responsive polymers formed stable micelles at 37 °C. Flow cytometry and fluorescence microscopy clearly demonstrated antigen-specific binding of the prepared complexes to GD2 positive murine T-cell lymphoma cells EL-4 and human neuroblastoma cells UKF-NB3, while no interaction with GD2 negative murine fibroblast cells NIH-3T3 was observed. These non-covalent polymer protein complexes represent a new generation of highly specific actively targeted polymer therapeutics or diagnostics.


Asunto(s)
Neoplasias , Polímeros , Ratones , Humanos , Animales , Polímeros/química , Ácido Nitrilotriacético , Sistemas de Liberación de Medicamentos/métodos , Proteínas Recombinantes
20.
J Med Chem ; 66(10): 6652-6681, 2023 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-37134237

RESUMEN

Purine nucleoside phosphorylase (PNP) is a well-known molecular target with potential therapeutic applications in the treatment of T-cell malignancies and/or bacterial/parasitic infections. Here, we report the design, development of synthetic methodology, and biological evaluation of a series of 30 novel PNP inhibitors based on acyclic nucleoside phosphonates bearing a 9-deazahypoxanthine nucleobase. The strongest inhibitors exhibited IC50 values as low as 19 nM (human PNP) and 4 nM (Mycobacterium tuberculosis (Mt) PNP) and highly selective cytotoxicity toward various T-lymphoblastic cell lines with CC50 values as low as 9 nM. No cytotoxic effect was observed on other cancer cell lines (HeLa S3, HL60, HepG2) or primary PBMCs for up to 10 µM. We report the first example of the PNP inhibitor exhibiting over 60-fold selectivity for the pathogenic enzyme (MtPNP) over hPNP. The results are supported by a crystallographic study of eight enzyme-inhibitor complexes and by ADMET profiling in vitro and in vivo.


Asunto(s)
Inhibidores Enzimáticos , Purina-Nucleósido Fosforilasa , Humanos , Purina-Nucleósido Fosforilasa/metabolismo , Inhibidores Enzimáticos/química , Cristalografía
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