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1.
Org Lett ; 22(10): 3739-3743, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32186890

RESUMO

A novel 6/6/5/6 tetracyclic polyketide named chartspiroton (1) was isolated from a medicinal plant endophytic Streptomyces in Dendrobium officinale. The complete structure assignment with absolute stereochemistry was elucidated through spectroscopic data, computational calculations, and single-crystal X-ray diffraction. Chartspiroton features an unprecedented naphthoquinone derivative spiro-fused with a benzofuran lactone moiety. A plausible polyketide biosynthetic pathway for 1 suggested intriguing oxidative rearrangement steps to form the five-membered lactone ring.

2.
ACS Chem Biol ; 15(2): 342-352, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31868341

RESUMO

Hepatitis C virus, causative agent of chronic viral hepatitis, infects 71 million people worldwide and is divided into seven genotypes and multiple subtypes with sequence identities between 68 to 82%. While older generation direct-acting antivirals had varying effectiveness against different genotypes, the newest NS3/4A protease inhibitors including glecaprevir (GLE) have pan-genotypic activity. The structural basis for pan-genotypic inhibition and effects of polymorphisms on inhibitor potency were not well-known due to lack of crystal structures of GLE-bound NS3/4A or genotypes other than 1. In this study, we determined the crystal structures of NS3/4A from genotypes 1a, 3a, 4a, and 5a in complex with GLE. Comparison with the highly similar grazoprevir indicated the mechanism of GLE's drastic improvement in potency. We found that, while GLE is highly potent against wild-type NS3/4A of all genotypes, specific resistance-associated substitutions (RASs) confer orders of magnitude loss in inhibition. Our crystal structures reveal molecular mechanisms behind pan-genotypic activity of GLE, including potency loss due to RASs at D168. Our structures permit for the first time analysis of changes due to polymorphisms among genotypes, providing insights into design principles that can aid future drug development and potentially can be extended to other proteins.

3.
Bioconjug Chem ; 30(12): 3021-3027, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31661952

RESUMO

It is very popular to fuse a protein drug or drug candidate to the Fc domain of immunoglobulin G (IgG) in order to prolong the in vivo half-life. In this study, we have designed, prepared, and tested an Fc-fused thermostable cocaine esterase (CocE) mutant (known as E196-301, with the T172R/G173Q/L196C/I301C substitutions on CocE) expressed in E. coli. As expected, Fc-fusion does not affect the in vitro enzyme activity and thermal stability of the enzyme and that Fc-E196-301 can favorably bind FcRn with Kd = 386 ± 35 nM. However, Fc-fusion does not prolong the in vivo half-life of E196-301 at all; Fc-E196-301 and E196-301 have essentially the same PK profile (t1/2 = 0.4 ± 0.1 h) in rats. This is the first time demonstrating that Fc-fusion does not prolong in vivo half-life of a protein. This finding is consistent with the mechanistic understanding that E196-301 and Fc-E196-301 are all degraded primarily through rapid proteolysis in the body. The Fc fusion cannot protect E196-301 from the proteolysis in the body. Nevertheless, it has been demonstrated that PEGylation can effectively protect E196-301, as the PEGylated E196-301, i.e., PEG-E196-301, has a significantly prolonged in vivo half-life. It has also been demonstrated that both E196-301 and PEG-E196-301 have dose-dependent in vivo half-lives (e.g., 19.9 ± 6.4 h for the elimination t1/2 of 30 mg/kg PEG-E196-301), as the endogenous proteolytic enzymes responsible for proteolysis of E196-301 (PEGylated or not) are nearly saturated by the high plasma concentration produced by a high dose of E196-301 or PEG-E196-301.

4.
Nucleic Acids Res ; 47(14): 7676-7689, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31424549

RESUMO

The potent antiretroviral protein APOBEC3G (A3G) specifically targets and deaminates deoxycytidine nucleotides, generating deoxyuridine, in single stranded DNA (ssDNA) intermediates produced during HIV replication. A non-catalytic domain in A3G binds strongly to RNA, an interaction crucial for recruitment of A3G to the virion; yet, A3G displays no deamination activity for cytidines in viral RNA. Here, we report NMR and molecular dynamics (MD) simulation analysis for interactions between A3Gctd and multiple substrate or non-substrate DNA and RNA, in combination with deamination assays. NMR ssDNA-binding experiments revealed that the interaction with residues in helix1 and loop1 (T201-L220) distinguishes the binding mode of substrate ssDNA from non-substrate. Using 2'-deoxy-2'-fluorine substituted cytidines, we show that a 2'-endo sugar conformation of the target deoxycytidine is favored for substrate binding and deamination. Trajectories of the MD simulation indicate that a ribose 2'-hydroxyl group destabilizes the π-π stacking of the target cytosine and H257, resulting in dislocation of the target cytosine base from the catalytic position. Interestingly, APOBEC3A, which can deaminate ribocytidines, retains the ribocytidine in the catalytic position throughout the MD simulation. Our results indicate that A3Gctd catalytic selectivity against RNA is dictated by both the sugar conformation and 2'-hydroxyl group.


Assuntos
Desaminase APOBEC-3G/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , RNA/metabolismo , Desaminase APOBEC-3G/química , Desaminase APOBEC-3G/genética , Biocatálise , Citidina/química , Citidina/metabolismo , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Desaminação , HIV-1/genética , HIV-1/metabolismo , Humanos , Ligação Proteica , RNA/química , RNA/genética , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Especificidade por Substrato , Vírion/genética , Vírion/metabolismo
5.
Drug Alcohol Depend ; 202: 168-171, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31352306

RESUMO

BACKGROUND: Cocaine is a commonly abused drug and there is no approved medication specifically to treat its addiction or overdose. Bacterial cocaine esterase (CocE)-derived RBP-8000 is currently under clinical development for cocaine overdose treatment. It is proven to be effective for human use to accelerate cocaine metabolism into physiologically inactive products. Besides cocaine, RBP-8000 may hydrolyze the neurotransmitter acetylcholine (ACh), however, no study has reported its cholinesterase activity. The present study aims to examine RBP-8000's cholinesterase activity and substrate selectivity to address the potential concern that this enzyme therapy might produce cholinergic side-effects. METHODS: Both computational modeling and experimental kinetic analysis were carried out to characterize the potential cholinesterase activity of RBP-8000. Substrates interacting with RBP-8000 were modeled for their enzyme-substrate binding complexes. In vitro enzymatic kinetic parameters were measured using Ellman's colorimetric assay and analyzed by Michaelis-Menten kinetics. RESULTS: It is the first demonstration that RBP-8000 catalyzes the hydrolysis of acetylthiocholine (ATC). However, its catalytic efficiency (kcat/KM) against ATC is 1000-fold and 5000-fold lower than it against cocaine at 25 °C and 37 °C, respectively, suggesting RBP-8000 has the desired substrate selectivity for cocaine over ACh. CONCLUSION: Given the fact that clinically relevant dose of RBP-8000 displays insignificant cholinesterase activity relative to endogenous cholinesterases in human, administration of RBP-8000 is unlikely to produce any significant cholinergic side-effects. This study provides supplemental evidences in support of further development of RBP-8000 towards a clinically used pharmacotherapy for cocaine overdose.

6.
SLAS Technol ; 24(4): 420-428, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31225974

RESUMO

Affordable and physiologically relevant three-dimensional (3D) cell-based assays used in high-throughput screening (HTS) are on the rise in early drug discovery. These technologies have been aided by the recent adaptation of novel microplate treatments and spheroid culturing techniques. One such technology involves the use of nanoparticle (NanoShuttle-PL) labeled cells and custom magnetic drives to assist in cell aggregation to ensure rapid 3D structure formation after the cells have been dispensed into microtiter plates. Transitioning this technology from a low-throughput manual benchtop application, as previously published by our lab, into a robotically enabled format achieves orders of magnitude greater throughput but required the development of specialized support hardware. This effort included in-house development, fabrication, and testing of ancillary devices that assist robotic handing and high-precision placement of microtiter plates into an incubator embedded with magnetic drives. Utilizing a "rapid prototyping" approach facilitated by cloud-based computer-aided design software, we built the necessary components using hobby-grade 3D printers with turnaround times that rival those of traditional manufacturing/development practices at a substantially reduced cost. This approach culminated in a first-in-class HTS-compatible 3D system in which we have coupled 3D bioprinting to a fully automated HTS robotic platform utilizing our novel magnetic incubator shelf assemblies.

7.
J Virol ; 93(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30381484

RESUMO

Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting the influenza virus hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza virus receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole-genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure.IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly conserved stem region of the influenza virus hemagglutinin (HA) can neutralize many influenza virus strains. To understand how influenza virus can become resistant or escape such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations by whole-genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function.


Assuntos
Farmacorresistência Viral , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Mutação , Neuraminidase/genética , Animais , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vacinas contra Influenza , Células Madin Darby de Rim Canino , Modelos Moleculares , Neuraminidase/química , Testes de Neutralização , Genética Reversa , Análise de Sequência de RNA , Proteínas Virais/química , Proteínas Virais/genética
8.
J Chem Theory Comput ; 15(1): 637-647, 2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30457868

RESUMO

APOBEC3 (A3) proteins, a family of human cytidine deaminases, protect the host from endogenous retro-elements and exogenous viral infections by introducing hypermutations. However, overexpressed A3s can modify genomic DNA to promote tumorigenesis, especially A3B. Despite their overall similarity, A3 proteins have distinct deamination activity. Recently determined A3 structures have revealed the molecular determinants of nucleotide specificity and DNA binding. However, for A3B, the structural basis for regulation of deamination activity and the role of active site loops in coordinating DNA had remained unknown. Using advanced molecular modeling followed by experimental mutational analysis and dynamics simulations, we investigated the molecular mechanism of DNA binding by A3B-CTD. We modeled fully native A3B-DNA structure, and we identified Arg211 in loop 1 as the gatekeeper coordinating DNA and critical residue for nucleotide specificity. We also identified a unique autoinhibited conformation in A3B-CTD that restricts access and binding of DNA to the active site. Our results reveal the structural basis for DNA binding and relatively lower catalytic activity of A3B and provide opportunities for rational design of specific inhibitors to benefit cancer therapeutics.


Assuntos
Citidina Desaminase/metabolismo , DNA de Cadeia Simples/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Domínio Catalítico , Citidina Desaminase/química , Citidina Desaminase/genética , Humanos , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/genética , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
9.
SLAS Discov ; 23(8): 842-849, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29750582

RESUMO

Glioblastoma (GBM) is the most aggressive primary brain cancer with an average survival time after diagnosis of only 12-14 months, with few (<5%) long-term survivors. A growing body of work suggests that GBMs contain a small population of glioma stem cells (GSCs) that are thought to be major contributors to treatment resistance and disease relapse. Identifying compounds that modulate GSC proliferation would provide highly valuable molecular probes of GSC-directed signaling. However, targeting GSCs pharmacologically has been challenging. Patient-derived GSCs can be cultured as neurospheres, and in vivo these cells functionally recapitulate the heterogeneity of the original tumor. Using patient-derived GSC-enriched cultures, we have developed a 1536-well spheroid-based proliferation assay and completed a pilot screen, testing ~3300 compounds comprising approved drugs. This cytotoxic and automation-friendly assay yielded a signal-to-background (S/B) ratio of 161.3 ± 7.5 and Z' of 0.77 ± 0.02, demonstrating its robustness. Importantly, compounds were identified with anti-GSC activity, demonstrating the applicability of this assay for large-scale high-throughput screening (HTS).


Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Ensaios de Triagem em Larga Escala , Células-Tronco Neoplásicas/metabolismo , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/imunologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Descoberta de Drogas , Glioma/tratamento farmacológico , Glioma/imunologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Bibliotecas de Moléculas Pequenas , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Oncogene ; 37(32): 4372-4384, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29743592

RESUMO

The RAS proteins are the most frequently mutated oncogenes in cancer, with highest frequency found in pancreatic, lung, and colon tumors. Moreover, the activity of RAS is required for the proliferation and/or survival of these tumor cells and thus represents a high-value target for therapeutic development. Direct targeting of RAS has proven challenging for multiple reasons stemming from the biology of the protein, the complexity of downstream effector pathways and upstream regulatory networks. Thus, significant efforts have been directed at identifying downstream targets on which RAS is dependent. These efforts have proven challenging, in part due to confounding factors such as reliance on two-dimensional adherent monolayer cell cultures that inadequately recapitulate the physiologic context to which cells are exposed in vivo. To overcome these issues, we implemented a high-throughput screening (HTS) approach using a spheroid-based 3-dimensional culture format, thought to more closely reflect conditions experienced by cells in vivo. Using isogenic cell pairs, differing in the status of KRAS, we identified Proscillaridin A as a selective inhibitor of cells harboring the oncogenic KRasG12V allele. Significantly, the identification of Proscillaridin A was facilitated by the 3D screening platform and would not have been discovered employing standard 2D culturing methods.


Assuntos
Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Antineoplásicos/farmacologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Fenótipo , Proscilaridina/farmacologia , Transdução de Sinais/genética
11.
Sci Rep ; 8(1): 7511, 2018 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-29760455

RESUMO

The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, secondary structure, and solution pH. We identified the A3A ssDNA binding motif as (T/C)TC(A/G), which correlated with enzymatic activity. We also validated that A3A binds RNA in a sequence specific manner. A3A bound tighter to substrate binding motif within a hairpin loop compared to linear oligonucleotide, suggesting A3A affinity is modulated by substrate structure. Based on these findings and previously published A3A-ssDNA co-crystal structures, we propose a new model with intra-DNA interactions for the molecular mechanism underlying A3A sequence preference. Overall, the sequence and structural preferences identified for A3A leads to a new paradigm for identifying A3A's involvement in mutation of endogenous or exogenous DNA.


Assuntos
Citidina Desaminase/química , Citidina Desaminase/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas/química , Proteínas/metabolismo , RNA/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , DNA de Cadeia Simples/genética , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , RNA/química , Especificidade por Substrato
12.
SLAS Discov ; 23(6): 574-584, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29673279

RESUMO

Traditional high-throughput drug screening in oncology routinely relies on two-dimensional (2D) cell models, which inadequately recapitulate the physiologic context of cancer. Three-dimensional (3D) cell models are thought to better mimic the complexity of in vivo tumors. Numerous methods to culture 3D organoids have been described, but most are nonhomogeneous and expensive, and hence impractical for high-throughput screening (HTS) purposes. Here we describe an HTS-compatible method that enables the consistent production of organoids in standard flat-bottom 384- and 1536-well plates by combining the use of a cell-repellent surface with a bioprinting technology incorporating magnetic force. We validated this homogeneous process by evaluating the effects of well-characterized anticancer agents against four patient-derived pancreatic cancer KRAS mutant-associated primary cells, including cancer-associated fibroblasts. This technology was tested for its compatibility with HTS automation by completing a cytotoxicity pilot screen of ~3300 approved drugs. To highlight the benefits of the 3D format, we performed this pilot screen in parallel in both the 2D and 3D assays. These data indicate that this technique can be readily applied to support large-scale drug screening relying on clinically relevant, ex vivo 3D tumor models directly harvested from patients, an important milestone toward personalized medicine.


Assuntos
Antineoplásicos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Organoides/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Linhagem Celular Tumoral , Células HT29 , Ensaios de Triagem em Larga Escala , Humanos , Medicina de Precisão/métodos
13.
Sci Rep ; 8(1): 5205, 2018 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-29581541

RESUMO

Human mPGES-1 is recognized as a promising target for next generation of anti-inflammatory drugs without the side effects of currently available anti-inflammatory drugs, and various inhibitors have been reported in the literature. However, none of the reported potent inhibitors of human mPGES-1 has shown to be also a potent inhibitor of mouse or rat mPGES-1, which prevents using the well-established mouse/rat models of inflammation-related diseases for preclinical studies. Hence, despite of extensive efforts to design and discover various human mPGES-1 inhibitors, the promise of mPGES-1 as a target for the next generation of anti-inflammatory drugs has never been demonstrated in any wild-type mouse/rat model using an mPGES-1 inhibitor. Here we report discovery of a novel type of selective mPGES-1 inhibitors potent for both human and mouse mPGES-1 enzymes through structure-based rational design. Based on in vivo studies using wild-type mice, the lead compound is indeed non-toxic, orally bioavailable, and more potent in decreasing the PGE2 (an inflammatory marker) levels compared to the currently available drug celecoxib. This is the first demonstration in wild-type mice that mPGES-1 is truly a promising target for the next generation of anti-inflammatory drugs.


Assuntos
Anti-Inflamatórios/química , Inibidores Enzimáticos/química , Inflamação/tratamento farmacológico , Prostaglandina-E Sintases/química , Animais , Anti-Inflamatórios/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Humanos , Inflamação/genética , Inflamação/patologia , Camundongos , Prostaglandina-E Sintases/antagonistas & inibidores , Ratos , Relação Estrutura-Atividade
14.
AAPS J ; 20(3): 53, 2018 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-29556863

RESUMO

Cocaine abuse is a worldwide public health and social problem without a US Food and Drug Administration (FDA)-approved medication. Accelerating cocaine metabolism that produces biologically inactive metabolites by administration of an efficient cocaine hydrolase (CocH) has been recognized as a promising strategy for cocaine abuse treatment. However, the therapeutic effects of CocH are limited by its short biological half-life (e.g., 8 h or shorter in rats). In this study, we designed and prepared a set of Fc-fusion proteins constructed by fusing Fc(M3) with CocH3 at the N-terminus of CocH3. A linker between the two protein domains was optimized to improve both the biological half-life and catalytic activity against cocaine. It has been concluded that Fc(M3)-G6S-CocH3 not only has fully retained the catalytic efficiency of CocH3 against cocaine but also has the longest biological half-life (e.g., ∼ 136 h in rats) among all of the long-acting CocHs identified so far. A single dose (0.2 mg/kg, IV) of Fc(M3)-G6S-CocH3 was able to significantly attenuate 15 mg/kg cocaine-induced hyperactivity for at least 11 days (268 h) after the Fc(M3)-G6S-CocH3 administration.


Assuntos
Hidrolases de Éster Carboxílico/química , Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Fragmentos Fc das Imunoglobulinas/química , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas Recombinantes/química , Animais , Células CHO , Catálise , Cricetulus , Meia-Vida , Masculino , Engenharia de Proteínas , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/farmacocinética
15.
Oncotarget ; 9(19): 14764-14790, 2018 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-29599906

RESUMO

This manuscript follows a single patient with pancreatic adenocarcinoma for a five year period, detailing the clinical record, pathology, the dynamic evolution of molecular and cellular alterations as well as the responses to treatments with chemotherapies, targeted therapies and immunotherapies. DNA and RNA samples from biopsies and blood identified a dynamic set of changes in allelic imbalances and copy number variations in response to therapies. Organoid cultures established from biopsies over time were employed for extensive drug testing to determine if this approach was feasible for treatments. When an unusual drug response was detected, an extensive RNA sequencing analysis was employed to establish novel mechanisms of action of this drug. Organoid cell cultures were employed to identify possible antigens associated with the tumor and the patient's T-cells were expanded against one of these antigens. Similar and identical T-cell receptor sequences were observed in the initial biopsy and the expanded T-cell population. Immunotherapy treatment failed to shrink the tumor, which had undergone an epithelial to mesenchymal transition prior to therapy. A warm autopsy of the metastatic lung tumor permitted an extensive analysis of tumor heterogeneity over five years of treatment and surgery. This detailed analysis of the clinical descriptions, imaging, pathology, molecular and cellular evolution of the tumors, treatments, and responses to chemotherapy, targeted therapies, and immunotherapies, as well as attempts at the development of personalized medical treatments for a single patient should provide a valuable guide to future directions in cancer treatment.

16.
Cell Rep ; 21(11): 3243-3255, 2017 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-29241550

RESUMO

The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with M53 and ending with N58. G6 binding studies demonstrate the remarkable breadth of binding to 51p1 IGHV1-69 Abs with diverse CDR-H3, light chain, and antigen binding specificities. These studies detail the broad expression of the G6 cross-reactive idiotype (CRI) that further define its potential role in precision medicine.


Assuntos
Anticorpos Anti-Idiotípicos/química , Anticorpos Monoclonais Humanizados/química , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Receptores de Antígenos de Linfócitos B/química , Sequência de Aminoácidos , Anticorpos Anti-Idiotípicos/genética , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Modelos Moleculares , Orthomyxoviridae/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
17.
SLAS Discov ; 22(5): 516-524, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28346088

RESUMO

Evaluation of drug cytotoxicity traditionally relies on use of cell monolayers, which are easily miniaturized to the 1536-well plate format. Three-dimensional (3D) cell culture models have recently gained popularity thanks to their ability to better mimic the complexity of in vivo systems. Despite growing interest in these more physiologically relevant and highly predictive cell-based models for compound profiling and drug discovery, 3D assays are currently performed in a medium- to low-throughput format, either in 96-well or 384-well plates. Here, we describe the design and implementation of a novel high-throughput screening (HTS)-compatible 1536-well plate assay that enables the parallel formation, size monitoring and viability assessment of 3D spheroids in a highly consistent manner. Custom-made plates featuring an ultra-low-attachment surface and round-bottom wells were evaluated for their compatibility with HTS requirements through a luminescence-based cytotoxicity pilot screen of ~3300 drugs from approved drug and National Cancer Institute (NCI) collections. As anticipated, results from this screen were significantly different from a parallel screen performed on cell monolayers. With the ability to achieve an average Z' factor greater than 0.5, this automation-friendly assay can be implemented to either profile lead compounds in a more economical plate format or to interrogate large compound libraries by ultra-HTS (uHTS).


Assuntos
Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Citotoxinas/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Triagem em Larga Escala/métodos , Esferoides Celulares/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Células HT29 , Humanos
18.
SLAS Discov ; 22(7): 887-896, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28346094

RESUMO

Primary hyperoxaluria is the underlying cause of oxalosis and is a life-threatening autosomal recessive disease, for which treatment may require dialysis or dual liver-kidney transplantation. The most common primary hyperoxaluria type 1 (PH1) is caused by genetic mutations of a liver-specific enzyme alanine:glyoxylate aminotransferase (AGT), which results in the misrouting of AGT from the peroxisomes to the mitochondria. Pharmacoperones are small molecules with the ability to modify misfolded proteins and route them correctly within the cells, which may present an effective strategy to treat AGT misrouting in PH1 disorders. We miniaturized a cell-based high-content assay into 1536-well plate format and screened ~4200 pharmacologically relevant compounds including Food and Drug Administration, European Union, and Japanese-approved drugs. This assay employs CHO cells stably expressing AGT-170, a mutant that predominantly resides in the mitochondria, where we monitor for its relocation to the peroxisomes through automated image acquisition and analysis. The miniaturized 1536-well assay yielded a Z' averaging 0.70 ± 0.07. Three drugs were identified as potential pharmacoperones from this pilot screen, demonstrating the applicability of this assay for large-scale high-throughput screening.


Assuntos
Hiperoxalúria/tratamento farmacológico , Ionóforos/farmacologia , Nefropatias/tratamento farmacológico , Animais , Células CHO , Cricetulus , Avaliação Pré-Clínica de Medicamentos/métodos , Hiperoxalúria/genética , Hiperoxalúria/metabolismo , Hiperoxalúria Primária/tratamento farmacológico , Hiperoxalúria Primária/genética , Hiperoxalúria Primária/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Transplante de Rim/métodos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação/genética , Peroxissomos/efeitos dos fármacos , Peroxissomos/genética , Peroxissomos/metabolismo , Diálise Renal/métodos , Transaminases/genética , Transaminases/metabolismo
19.
BMC Biotechnol ; 16(1): 72, 2016 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-27756365

RESUMO

BACKGROUND: A recently reported cocaine hydrolase (CocH3) fused with fragment crystallizable (Fc) region of human immunoglobulin G1, denoted as CocH3-Fc, is known as a promising therapeutic candidate for the treatment of cocaine overdose and addiction. A challenge for practical therapeutic use of this enzyme exists in the large-scale protein production and, therefore, it is interesting to identify a low-cost and feasible, sustainable source of CocH3-Fc production. RESULTS: CocH3-Fc was transiently expressed in plant Nicotiana benthamiana leaves. The plant-expressed protein, denoted as pCocH3-Fc, was as active as that expressed in mammalian cells both in vitro and in vivo. However, compared to the mammalian-cell expressed CocH3-Fc protein, pCocH3-Fc had a shorter biological half-life, probably due to the lack of protein sialylation in plant. Nevertheless, the in vivo half-life was significantly extended upon the PEGylation of pCocH3-Fc. The Fc fusion did not prolong the biological half-life of the plant-expressed enzyme pCocH3-Fc, but increased the yield of the enzyme expression in the plant under the same experimental conditions. CONCLUSIONS: It is feasible to express pCocH3-Fc in plants. Further studies on the pCocH3-Fc production in plants should focus on the development of vectors with additional genes/promoters for the complete protein sialylation and for a better yield.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/uso terapêutico , Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/metabolismo , Tabaco/metabolismo , Animais , Hidrolases de Éster Carboxílico/genética , Transtornos Relacionados ao Uso de Cocaína/prevenção & controle , Relação Dose-Resposta a Droga , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Dose Letal Mediana , Masculino , Camundongos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tabaco/genética
20.
Proc Natl Acad Sci U S A ; 113(2): 422-7, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26712009

RESUMO

Cocaine abuse is a world-wide public health and social problem without a US Food and Drug Administration-approved medication. An ideal anticocaine medication would accelerate cocaine metabolism, producing biologically inactive metabolites by administration of an efficient cocaine-specific exogenous enzyme. Our recent studies have led to the discovery of the desirable, highly efficient cocaine hydrolases (CocHs) that can efficiently detoxify and inactivate cocaine without affecting normal functions of the CNS. Preclinical and clinical data have demonstrated that these CocHs are safe for use in humans and are effective for accelerating cocaine metabolism. However, the actual therapeutic use of a CocH in cocaine addiction treatment is limited by its short biological half-life (e.g., 8 h or shorter in rats). Here we demonstrate a novel CocH form, a catalytic antibody analog, which is a fragment crystallizable (Fc)-fused CocH dimer (CocH-Fc) constructed by using CocH to replace the Fab region of human IgG1. The CocH-Fc not only has a high catalytic efficiency against cocaine but also, like an antibody, has a considerably longer biological half-life (e.g., ∼107 h in rats). A single dose of CocH-Fc was able to accelerate cocaine metabolism in rats even after 20 d and thus block cocaine-induced hyperactivity and toxicity for a long period. Given the general observation that the biological half-life of a protein drug is significantly longer in humans than in rodents, the CocH-Fc reported in this study could allow dosing once every 2-4 wk, or longer, for treatment of cocaine addiction in humans.


Assuntos
Hidrolases de Éster Carboxílico/administração & dosagem , Hidrolases de Éster Carboxílico/uso terapêutico , Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Animais , Biocatálise , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/toxicidade , Cocaína/metabolismo , Humanos , Hidrólise , Camundongos , Modelos Moleculares , Ratos Sprague-Dawley , Receptores Fc/metabolismo , Fatores de Tempo
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