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1.
Int J Biol Sci ; 16(6): 981-993, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32140067

RESUMO

Ly6/uPAR/α-neurotoxin domain (LU-domain) is characterized by the presence of 4-5 disulfide bonds and three flexible loops that extend from a core stacked by several conversed disulfide bonds (thus also named three-fingered protein domain). This highly structurally stable protein domain is typically a protein-binder at extracellular space. Most LU proteins contain only single LU-domain as represented by Ly6 proteins in immunology and α-neurotoxins in snake venom. For Ly6 proteins, many are expressed in specific cell lineages and in differentiation stages, and are used as markers. In this study, we report the crystal structures of the two LU-domains of human C4.4A alone and its complex with a Fab fragment of a monoclonal anti-C4.4A antibody. Interestingly, both structures showed that C4.4A forms a very compact globule with two LU-domain packed face to face. This is in contrast to the flexible nature of most LU-domain-containing proteins in mammals. The Fab combining site of C4.4A involves both LU-domains, and appears to be the binding site for AGR2, a reported ligand of C4.4A. This work reports the first structure that contain two LU-domains and provides insights on how LU-domains fold into a compact protein and interacts with ligands.

2.
Exp Mol Med ; 2020 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152451

RESUMO

The function of the fibrinolytic system was first identified to dissolve fibrin to maintain vascular patency. Connections between the fibrinolytic system and many other physiological and pathological processes have been well established. Dysregulation of the fibrinolytic system is closely associated with multiple pathological conditions, including thrombosis, inflammation, cancer progression, and neuropathies. Thus, molecules in the fibrinolytic system are potent therapeutic and diagnostic targets. This review summarizes the currently used agents targeting this system and the development of novel therapeutic strategies in experimental studies. Future directions for the development of modulators of the fibrinolytic system are also discussed.

3.
Life Sci ; 246: 117404, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32035128

RESUMO

AIMS: The study aims to investigate the effect of plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of fibrinolytic process, on blood glucose in type 2 diabetes mellitus (T2DM) and its mechanism. MATERIALS AND METHODS: We developed a highly potent and highly specific PAI-1 inhibitor, named PAItrap3, based on the inactivated urokinase. Meanwhile, a single point mutation of PAItrap3 (i.e., PAItrapNC) was parallelly prepared as negative control. PAItrap3 was intravenously injected into type 2 diabetic (T2D) mice and its effect on metabolic system was evaluated by measuring the levels of blood glucose, PAI-1, and tumor necrosis factor alpha (TNF-α) in T2D mice. KEY FINDINGS: PAItrap3 significantly reduced the high blood glucose level and PAI-1 level in streptozotocin-induced T2D mice. PAItrapNC did not have any hypoglycemic effect at all on T2D mice. Mechanistically, both PAI-1 and TNF-α levels were attenuated by the administration of PAItrap3. In addition, we observed that PAItrap3 reduced the amount of fat droplets in adipocytes. SIGNIFICANCE: These findings provide clear evidence for PAI-1 to participate in inflammation and obesity mediated hyperglycemia, and open up a new prospect for the treatment of T2DM by PAI-1 inhibition.


Assuntos
Glicemia/análise , Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Inibidor 1 de Ativador de Plasminogênio/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/uso terapêutico , Adipócitos/efeitos dos fármacos , Animais , Masculino , Inibidor 1 de Ativador de Plasminogênio/sangue
4.
Biosci Biotechnol Biochem ; : 1-7, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32019421

RESUMO

Peptidic inhibitors of proteases are attracting increasing interest not only as drug candidates but also for studying the function and regulation mechanisms of these enzymes. Previously, we screened out a cyclic peptide inhibitor of human uPA [Formula: see text] and found that Ala substitution of P2 residue turns upain-1 to a substrate. To further investigate the effect of P2 residue on the peptide behavior transformation, we constructed upain-1-W3F, which has Phe replacement in the P2 position. We determined KD and Ki of upain-1-W3F and found that upain-1-W3F might still exist as an inhibitor. Furthermore, the high-resolution crystal structure of upain-1-W3F·uPA reveals that upain-1-W3F indeed stays as an intact inhibitor bind to uPA. We thus propose that the P2 residue plays a nonnegligible role in the conversion of upain-1 to a substrate. These results also proposed a strategy to optimize the pharmacological properties of peptide-based drug candidates by hydrophobicity and steric hindrance.Abbreviations : uPA: urokinase-type plasminogen activator; SPD: serine protease domain; S1 pocket: specific substrate-binding pocket.

5.
Int J Nanomedicine ; 15: 151-167, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32021171

RESUMO

Purpose: Combination therapy for tumors is an important and promising strategy to improve therapeutic efficiency. This study aims at combining tumor targeting, chemo-, and photodynamic therapies to improve the anti-tumor performance. Patients and Methods: Human serum albumin (HSA), as a nontoxic and biodegradable drug carrier, was used to load hydrophobic photosensitizers (mono-substituted ß-4-pyridyloxy phthalocyanine zinc, mPPZ) by a dilution-incubation-purification (DIP) strategy to form molecular complex HSA:mPPZ. This complex was cross-linked as nanoparticles, and then chemotherapy drug doxorubicin (DOX) was adsorbed into the nanoparticles to achieve combined photodynamic therapy and chemotherapy. Next, the surface of the obtained composite was modified by a tumor surface receptor (urokinase receptor) targeting agent (ATF-HSA) using a noncovalent method to obtain the final product (ATF-HSA@HSA:mPPZ:DOX nanoparticles, AHmDN). Results: AHmDN exhibited strong stability, remarkable cytotoxicity and higher uptake to tumor cells. Cell imaging analysis indicated that DOX was separated from AHmDN and uniformly distributed in cell nucleus while mPPZ localized in cytoplasm. The PDT activity of all the samples had been confirmed by the detection of intracellular ROS. In animal experiments, AHmDN was demonstrated to have a prominent tumor-targeting effect using a 3D imaging system. In addition, the enhanced antitumor effect of AHmDN in tumor-bearing mice was also been observed. Importantly, the tumor-targeting effect of such nanoparticles lasted for about 14 days after one injection. Conclusion: These albumin nanoparticles with combined functions of tumor targeting, chemotherapy and photodynamic therapy can highly enhance the anti-tumor effect. This drug delivery system can be applied to package other hydrophobic photosensitizers and chemotherapy drugs for improving therapeutic efficacy to tumors.

6.
ACS Appl Mater Interfaces ; 12(4): 4221-4230, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31909969

RESUMO

Paclitaxel (PTX) is a widely used anticancer drug that works by inhibiting microtubule disassembly. PTX safety was greatly enhanced by embedding it with human albumin. Here, we study the synergistic effects of PTX with photodynamic therapy (PDT) both in vitro and in vivo by constructing photosensitizer-PTX nanotheranostics (PPNTs). PPNTs were fabricated via noncovalent hydrophobic interactions and π-π stacking between an amphipathic photosensitizer and PTX with an average diameter of ∼80 nm, and these showed high stability in biological conditions. In a tumor-bearing mouse model, PPNTs were shown to accumulate at the tumor site based on three-dimensional fluorescence tomographic imaging. Under 680 nm light irradiation, PPNTs exhibited a superior solid tumor ablation effect in a mouse model, with a dose of PTX (0.2 mg/kg) that is 10-fold lower than that typically used. Mechanistically, PPNTs induced a strong apoptotic response in cells under light illumination and showed an increased antitumor efficacy that is 47.2-fold and 57.6-fold higher than that of the photosensitizer nanoparticles (PNTs) and free PTX, respectively. In addition, PPNTs showed enhanced cellular uptake with focused mitochondria and lysosome colocalization compared to that of PNTs and the amount of PTX delivered in PPNTs was sufficient to induce cell cycle arrest in the G2/M phase. These findings indicated that the current combination therapy has advantages over monotherapy in promoting tumor regression and ultimately achieving tumor elimination.

7.
J Mater Chem B ; 8(3): 504-514, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31840729

RESUMO

Inhibition of pro-cancer proteases is a potent anticancer strategy. However, protease inhibitors are mostly developed in the forms of small molecules or peptides, which normally suffer from insufficient metabolic stability. The fast clearance significantly impairs the antitumor effects of these inhibitors. In this study, we report a nanometer-sized inhibitor of a pro-cancer protease, suppressor of tumorigenicity 14 (st14), which has been reported as a potent prognostic marker for multiple cancers. This st14 inhibitor was fabricated by conjugating a recombinant st14 inhibitor (KD1) with carbon quantum dots (CQDs). CQD-KD1 not only demonstrated high potency of inhibiting st14 activity in biochemical experiments, but also remarkably suppressed the invasion of breast cancer cells. In contrast to the original recombinant KD1, CQD-KD1 demonstrated a prolonged retention time in plasma and at the tumor site because of the reduced renal clearance. Consistently, CQD-KD1 demonstrated enhanced efficacies of suppressing tumor growth and cancer metastases in vivo. In addition, CQD-KD1 precisely imaged tumor tissues in cancer-grafted mice by specifically targeting the over-expressed st14 on the tumor cell surface, which indicates CQD-KD1 as a potent probe for the fluorescence guided surgery of tumor resection. In conclusion, this study demonstrates that CQD-KD1 is a highly potent diagnostic and therapeutic agent for cancer treatments.

8.
Bioorg Med Chem Lett ; 29(24): 126753, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31679971

RESUMO

The oral K+-sparing diuretic amiloride shows anti-cancer side-activities in multiple rodent models. These effects appear to arise, at least in part, through moderate inhibition of the urokinase-type plasminogen activator (uPA, Ki = 2.4 µM), a pro-metastatic trypsin-like serine protease that is upregulated in many aggressive solid malignancies. In applying the selective optimization of side-activity (SOSA) approach, a focused library of twenty two 6-substituted amiloride derivatives were prepared, with multiple examples displaying uPA inhibitory potencies in the nM range. X-ray co-crystal structures revealed that the potency increases relative to amiloride arise from increased occupancy of uPA's S1ß subsite by the appended 6-substituents. Leading compounds were shown to have high selectivity over related trypsin-like serine proteases and no diuretic or anti-kaliuretic effects in rats. Compound 15 showed anti-metastatic effects in a xenografted mouse model of late-stage lung metastasis.

9.
Int J Nanomedicine ; 14: 6799-6812, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31692522

RESUMO

Background: Photodynamic therapy (PDT), a clinical anticancer therapeutic modality, has a long history in clinical cancer treatments since the 1970s. However, PDT has not been widely used largely because of metabolic problems and off-target phototoxicities of the current clinical photosensitizers. Purpose: The objective of the study is to develop a high-efficiency and high-specificity carrier to precisely deliver photosensitizers to tumor sites, aiming at addressing metabolic problems, as well as the systemic damages current clinical photosensitizers are known to cause. Methods: We synthesized a polydopamine (PDA)-based carrier with the modification of folic acid (FA), which is to target the overexpressed folate receptors on tumor surfaces. We used this carrier to load a cationic phthalocyanine-type photosensitizer (Pc) and generated a PDA-FA-Pc nanomedicine. We determined the antitumor effects and the specificity to tumor cell lines in vitro. In addition, we established human cancer-xenografted mice models to evaluate the tumor-targeting property and anticancer efficacies in vivo. Results: Our PDA-FA-Pc nanomedicine demonstrated a high stability in normal physiological conditions, however, could specifically release photosensitizers in acidic conditions, eg, tumor microenvironment and lysosomes in cancer cells. Additionally, PDA-FA-Pc nanomedicine demonstrated a much higher cellular uptake and phototoxicity in cancer cell lines than in healthy cell lines. Moreover, the in vivo imaging data indicated excellent tumor-targeting properties of PDA-FA-Pc nanomedicine in human cancer-xenografted mice. Lastly, PDA-FA-Pc nanomedicine was found to significantly suppress tumor growth within two human cancer-xenografted mice models. Conclusion: Our current study not only demonstrates PDA-FA-Pc nanomedicine as a highly potent and specific anticancer agent, but also suggests a strategy to address the metabolic and specificity problems of clinical photosensitizers.


Assuntos
Ácido Fólico/farmacologia , Indóis/farmacologia , Nanopartículas/uso terapêutico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Polímeros/farmacologia , Animais , Linhagem Celular Tumoral , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Estabilidade de Medicamentos , Feminino , Ácido Fólico/química , Células HeLa , Humanos , Indóis/química , Células MCF-7 , Camundongos , Nanomedicina , Nanopartículas/química , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Fármacos Fotossensibilizantes/administração & dosagem , Polímeros/química , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Nat Chem Biol ; 15(12): 1241-1248, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31611704

RESUMO

The Holliday junction (HJ) is a key intermediate during homologous recombination and DNA double-strand break repair. Timely HJ resolution by resolvases is critical for maintaining genome stability. The mechanisms underlying sequence-specific substrate recognition and cleavage by resolvases remain elusive. The monokaryotic chloroplast 1 protein (MOC1) specifically cleaves four-way DNA junctions in a sequence-specific manner. Here, we report the crystal structures of MOC1 from Zea mays, alone or bound to HJ DNA. MOC1 uses a unique ß-hairpin to embrace the DNA junction. A base-recognition motif specifically interacts with the junction center, inducing base flipping and pseudobase-pair formation at the strand-exchanging points. Structures of MOC1 bound to HJ and different metal ions support a two-metal ion catalysis mechanism. Further molecular dynamics simulations and biochemical analyses reveal a communication between specific substrate recognition and metal ion-dependent catalysis. Our study thus provides a mechanism for how a resolvase turns substrate specificity into catalytic efficiency.


Assuntos
Cloroplastos/metabolismo , Resolvases de Junção Holliday/metabolismo , Proteínas de Plantas/metabolismo , Resolvases de Junção Holliday/química , Simulação de Dinâmica Molecular , Conformação Proteica , Especificidade por Substrato
11.
Cancer Metastasis Rev ; 38(3): 507-524, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31471691

RESUMO

Matriptase is a type II transmembrane serine protease, which has been suggested to play critical roles in numerous pathways of biological developments. Matriptase is the activator of several oncogenic proteins, including urokinase-type plasminogen activator (uPA), hepatocyte growth factor (HGF) and protease-activated receptor 2 (PAR-2). The activations of these matriptase substrates subsequently lead to the generation of plasmin, matrix metalloproteases (MMPs), and the triggers for many other signaling pathways related to cancer proliferation and metastasis. Accordingly, matriptase is considered an emerging target for the treatments of cancer. Thus far, inhibitors of matriptase have been developed as potential anti-cancer agents, which include small-molecule inhibitors, peptide-based inhibitors, and monoclonal antibodies. This review covers established literature to summarize the chemical and biochemical aspects, especially the inhibitory mechanisms and structure-activity relationships (SARs) of matriptase inhibitors with the goal of proposing the strategies for their future developments in anti-cancer therapy.


Assuntos
Neoplasias/tratamento farmacológico , Serina Endopeptidases/metabolismo , Inibidores de Serino Proteinase/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular , Metástase Neoplásica , Neoplasias/enzimologia , Neoplasias/patologia , Serina Endopeptidases/química , Inibidores de Serino Proteinase/administração & dosagem , Bibliotecas de Moléculas Pequenas/farmacologia
12.
Res Pract Thromb Haemost ; 3(3): 412-419, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31294329

RESUMO

Background: Blood coagulation factor VIIa (FVIIa) plays its critical physiological role in the initiation of hemostasis. Even so, recombinant FVIIa is successfully used as a bypassing agent for factor VIII or IX in the treatment of bleeds in patients with severe hemophilia with inhibitors. To investigate the utility of more potent FVIIa variants with enhanced intrinsic activity, molecules such as V21D/E154V/M156Q-FVIIa (FVIIaDVQ) were designed. Methods: Surface plasmon resonance was used to characterize the binding of mAb4F5 to FVIIaDVQ and related variants. X-ray crystallography was used to determine the structure of the Fab fragment of mAb4F5 (Fab4F5). Molecular docking and small angle X-ray scattering led to a model of FVIIaDVQ:Fab4F5 complex. Results: The binding experiments, functional effects on FVIIaDVQ and structure of mAb4F5 (originally intended for quantification of FVIIaDVQ in samples containing FVII(a)) pinpointed the epitope (crucial role for residue Asp21) and shed light on the role of the N-terminus of the protease domain in FVIIa allostery. The potential antigen-combining sites are composed of 1 hydrophobic and 1 negatively charged pocket formed by 6 complementarity-determining region (CDR) loops. Structural analysis of Fab4F5 shows that the epitope interacts with the periphery of the hydrophobic pocket and provides insights into the molecular basis of mAb4F5 recognition and tight binding of FVIIaDVQ. Conclusion: The binary complex explains and supports the selectivity and functional consequences of Fab4F5 association with FVIIaDVQ and illustrates the potentially unique antigenicity of this FVIIa variant. This will be useful in the design of less immunogenic variants.

13.
Front Microbiol ; 10: 1232, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31244791

RESUMO

Intramembrane proteases hydrolyze peptide bonds within the cell membrane as the decision-making step of various signaling pathways. Sporulation factor IV B protease (SpoIVB) and C-terminal processing proteases B (CtpB) play central roles in cellular differentiation via regulated intramembrane proteolysis (RIP) process which activates pro-σK processing at the σK checkpoint during spore formation. SpoIVB joins CtpB in belonging to the widespread family of PDZ-proteases, but much remains unclear about the molecular mechanisms and structure of SpoIVB. In this study, we expressed inactive SpoIVB (SpoIVBS378A) fused with maltose binding protein (MBP)-tag and obtained the solution structure of SpoIVBS378A from its small angle X-ray scattering (SAXS) data. The fusion protein is more soluble, stable, and yields higher expression compared to SpoIVB without the tag. MBP-tag not only facilitates modeling of the structure in the SAXS envelope but also evaluates reliability of the model. The solution structure of SpoIVBS378A fits closely with the experimental scattering data (χ2= 1.76). Comparing the conformations of PDZ-proteases indicates that SpoIVB adopts a PDZ-protease pattern similar to the high temperature requirement A proteases (HtrAs) rather than CtpB. We not only propose that SpoIVB uses a more direct and simple way to cleave the substrates than that of CtpB, but also that they work together as signal amplifiers to activate downstream proteins in the RIP pathway.

14.
FEBS Lett ; 593(11): 1236-1247, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31044429

RESUMO

The urokinase-type plasminogen activator receptor (uPAR) is a cell surface receptor that is capable of binding to a range of extracellular proteins and triggering a series of proteolytic and signaling events. Previous structural studies of uPAR with its ligands uPA and vitronectin revealed that its three domains (DI, DII, and DIII) form a large hydrophobic cavity to accommodate uPA. In the present study, the structure of unoccupied murine uPAR (muPAR) is determined. The structure of DII and DIII of muPAR is well defined and forms a compact globular unit, while DI could not be traced. Molecular dynamic simulations further confirm the rigid binding interface between DII and DIII. This study shows overall structural flexibility of uPAR in the absence of uPA.

15.
Biosci Biotechnol Biochem ; 83(10): 1815-1821, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31117893

RESUMO

Human coagulation factor XII, the initiating factor in the intrinsic coagulation pathway, is critical for pathological thrombosis but not for hemostasis. Pharmacologic inhibition of factor XII is an attractive alternative in providing protection from pathologic thrombus formation while minimizing hemorrhagic risk. Large quantity of recombinant active factor XII is required for screening inhibitors and further research. In the present study, we designed and expressed the recombinant serine protease domain of factor XII in Pichia pastoris strain X-33, which is a eukaryotic expression model organism with low cost. The purification protocol was simplified and the protein yield was high (~20 mg/L medium). The purified serine protease domain of factor XII behaved homogeneously as a monomer, exhibited comparable activity with the human ßFXIIa, and accelerated clot formation in human plasma. This study provides the groundwork for factor XII inhibitors screening and further research.


Assuntos
Fator XII/metabolismo , Pichia/genética , Serina Proteases/metabolismo , Amidas/metabolismo , Sequência de Aminoácidos , Fator XII/genética , Fator XII/isolamento & purificação , Vetores Genéticos , Hemostasia , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Serina Proteases/genética , Serina Proteases/isolamento & purificação , Trombose/metabolismo
16.
Biochem Biophys Res Commun ; 510(4): 539-544, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30737033

RESUMO

Streptococcus pyogenes (group A Streptococcus, GAS) has caused a wide variety of human diseases. Its multifunctional surface dehydrogenase (SDH) is crucial for GAS life cycle. Furthermore, GAS infection into human pharyngeal cells has been previously shown to be mediated by the interaction between SDH and host urokinase-type plasminogen activator receptor (uPAR). However, the structural information of SDH remains to be elucidated and there are few detailed studies to characterize its interaction with uPAR. In-depth research on these issues will provide potential targets and strategies for combating GAS. Here, we prepared recombinant SDH tetramer in Escherichia coli BL21 (DE3) cells. After purification and crystallization, we determined its crystal structure at 1.74 Å. The unique characteristics might be potentially explored as drug targets or vaccine immunogen. We subsequently performed gel filtration chromatography, native-polyacrylamide gel electrophoresis (PAGE) and in vitro pull-down analyses. The results showed that their interaction was too weak to form stable complexes and the role of uPAR involved in GAS infection needs further demonstration. Altogether the current work provides the first view of SDH and deepens the knowledge of GAS infection.


Assuntos
Proteínas de Bactérias/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus pyogenes/metabolismo , Proteínas de Bactérias/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Mapas de Interação de Proteínas , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/química
17.
Theranostics ; 9(3): 884-899, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30809315

RESUMO

Cancer cell expresses abundant surface receptors. These receptors are important targets for cancer treatment and imaging applications. Our goal here is to develop nanoparticles with cargo loading and tumor targeting capability. Methods: A peptide targeting at cancer cell surface receptor (urokinase receptor, uPAR) was expressed in fusion with albumin (diameter of ~7 nm), and the fusion protein was assembled into nanoparticles with diameter of 40 nm, either in the presence or absence of cargo molecules, by a novel preparation method. An important feature of this method is that the nanoparticles were stabilized by hydrophobic interaction of the fusion protein and no covalent linking agent was used in the preparation. The stability, the cargo release, in vitro and in vivo properties of such formed nanoparticles were characterized by transmission electron microscopy, dynamic light scattering, gel shift assay, laser scanning confocal microscopy and 3D fluorescent molecular tomography. Results: The nanoparticles were stable for more than two weeks in aqueous buffer, even in the buffer containing 10% fetal bovine serum. Interestingly, in the presence of urokinase receptor, the uPAR-targeting nanoparticle disintegrated into 7.5 nm fragments and released its cargo, but not the non-targeting nanoparticles made from albumin by the same preparation method. Such nanoparticles also showed higher uptake and cytotoxicity to the receptor-expressing cancer cells in vitro and higher tumor accumulation in xenografted tumor-bearing mice in vivo compared to the non-targeting nanoparticles. Conclusion: Our results demonstrate a new function of cell surface receptor as a responsive trigger to disassemble nanoparticles, besides its common use to enrich targeting agents. Such nanoparticles were thus named receptor-responsive nanoparticles (RRNP).


Assuntos
Carcinoma Hepatocelular/terapia , Portadores de Fármacos/administração & dosagem , Terapia de Alvo Molecular/métodos , Nanopartículas/administração & dosagem , Fármacos Fotossensibilizantes/administração & dosagem , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos , Transplante de Neoplasias , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacocinética , Ligação Proteica , Transplante Heterólogo , Resultado do Tratamento
18.
J Med Chem ; 62(4): 2172-2183, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30707839

RESUMO

Urokinase-type plasminogen activator (uPA) is a diagnostic marker for breast and prostate cancers recommended by American Society for Clinical Oncology and German Breast Cancer Society. Inhibition of uPA was proposed as an efficient strategy for cancer treatments. In this study, we report peptide-based uPA inhibitors with high potency and specificity comparable to monoclonal antibodies. We revealed the binding and inhibitory mechanisms by combining crystallography, molecular dynamic simulation, and other biophysical and biochemical approaches. Besides, we showed that our peptides efficiently inhibited the invasion of cancer cells via intervening with the processes of the degradation of extracellular matrices. Furthermore, our peptides significantly suppressed the tumor growth and the cancer metastases in tumor-bearing mice. This study demonstrates that these uPA peptides are highly potent anticancer agents and reveals the mechanistic insights of these uPA inhibitors, which can be useful for developing other serine protease inhibitors.

19.
Anal Chim Acta ; 1053: 98-104, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30712574

RESUMO

Plasminogen activator inhibitor-1 (PAI-1) is a labile molecule that exists in four different forms: active, latent, cleaved and target bound form. Although there have been many methods to measure the total PAI-1, the measurement of active form of PAI-1 antigen is still challenging. Here we developed a novel ELISA to detect the active form of PAI-1 based on a highly specific PAI-1 capturing agent which binds to active PAI-1 with high affinity. We also used a highly stable PAI-1 mutant as an assay calibrator to enhance the method's reproducibility. This ELISA has the advantage of measuring both the antigen level and activity of PAI-1 at the same time. The assay had a sensitivity of 0.167 ng/ml and a working range of 0.195-25 ng/ml. The intra- and inter-assay variations were 6.7% and 11.3% respectively. The mean recovery of spiked standard was 102%. We used this strategy to measure the active PAI-1 level in plasma of healthy donors, and had an interesting observation: the PAI-1 level reduced by half after plasma storage for 6 h at room temperature. This finding represents the first observation of activity loss in plasma PAI-1 samples, and may explain large variations in PAI-1 levels (0-100 ng/ml) observed in human samples using commercial assays.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Inibidor 1 de Ativador de Plasminogênio/análise , Animais , Ácido Cítrico/metabolismo , Humanos , Modelos Moleculares , Inibidor 1 de Ativador de Plasminogênio/sangue , Inibidor 1 de Ativador de Plasminogênio/química , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Conformação Proteica
20.
Thromb Haemost ; 119(1): 77-86, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30597502

RESUMO

Recombinant tissue-type plasminogen activator (r-tPA) was approved by U.S. Food and Drug Administration as a thrombolytic drug. However, a high dose of r-tPA (up to 100 mg/person) is typically used in clinical applications. Such high dosage leads to severe side effects including haemorrhage and neurotoxicity, which can be fatal. To improve the proteolytic properties of tPA to enhance thrombolytic therapy, we designed a series of mutants in tPA serine protease domain (tPA-SPD) based on the crystal structure of tPA-SPD:plasminogen activators inhibitor-1 (PAI-1) complex that we determined recently. We found that the A146Y substitution in tPA-SPD(A146Y) enhanced resistance to PAI-1 inactivation by 30-fold compared with original tPA-SPD. Interestingly, the tPA-SPD(A146Y) variant showed fivefold higher activation for plasminogen compared with tPA-SPD. The variant also demonstrated thrombolytic activity stronger than tPA-SPD in a clot lysis assay. In vivo, we showed tPA-SPD(A146Y) possessed higher thrombolytic efficacy in a pulmonary embolism model compared with original tPA-SPD. Furthermore, a mouse tail bleeding assay showed that tPA-SPD(A146Y) did not increase bleeding risk compared with clinical drug r-tPA. Together, our findings reveal novel functions of A146Y variant, which not only increases the catalytic efficiency of the enzyme, but also enhances resistance to PAI-1 inhibition, and demonstrating that tPA-SPD (A146Y) variant is a much improved agent for thrombolytic therapy.


Assuntos
Inibidor 1 de Ativador de Plasminogênio/metabolismo , Mutação Puntual , Ativador de Plasminogênio Tecidual/genética , Animais , Tempo de Sangramento , Coagulação Sanguínea , Catálise , Esquema de Medicação , Resistência a Medicamentos , Fibrinólise/efeitos dos fármacos , Fibrinolíticos/uso terapêutico , Variação Genética , Hemoglobinas/análise , Hemorragia/tratamento farmacológico , Humanos , Cinética , Masculino , Camundongos , Domínios Proteicos , Embolia Pulmonar/prevenção & controle , Proteínas Recombinantes/metabolismo , Terapia Trombolítica , Trombose/tratamento farmacológico
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