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1.
J Nat Prod ; 84(9): 2568-2574, 2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34496568

RESUMO

Twelve guaianolide-type sesquiterpene oligomers with diverse structures were isolated from the whole plants of Ainsliaea fragrans, including a novel trimer (1) and two new dimers (2, 3). The chemical structures of the new compounds were elucidated through spectroscopic data interpretation and computational calculations. Ainsfragolide (1) is an unusual guaianolide sesquiterpene trimer generated with a novel C-C linkage at C2'-C15″, which may be biosynthesized prospectively through a further Michael addition. Cytotoxicity results showed that ainsfragolide (1) was the most potent compound against five cancer cell lines with IC50 values in the range of 0.4-8.3 µM.

2.
Angew Chem Int Ed Engl ; 60(40): 21959-21965, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34351032

RESUMO

Benzoylecgonine (BZE) is the major toxic metabolite of cocaine and is responsible for the long-term cocaine-induced toxicity owing to its long residence time in humans. BZE is also the main contaminant following cocaine consumption. Here, we identified the bacterial cocaine esterase (CocE) as a BZE-metabolizing enzyme (BZEase), which can degrade BZE into biological inactive metabolites (ecgonine and benzoic acid). CocE was redesigned by a reactant-state-based enzyme design theory. An encouraging mutant denoted as BZEase2, presented a >400-fold improved catalytic efficiency against BZE compared with wild-type (WT) CocE. In vivo, a single dose of BZEase2 (1 mg kg-1 , IV) could eliminate nearly all BZE within only two minutes, suggesting the enzyme has the potential for cocaine overdose treatment and BZE elimination in the environment by accelerating BZE clearance. The crystal structure of a designed BZEase was also determined.

3.
Immunity ; 54(8): 1853-1868.e7, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34331873

RESUMO

Antibodies elicited by infection accumulate somatic mutations in germinal centers that can increase affinity for cognate antigens. We analyzed 6 independent groups of clonally related severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) Spike receptor-binding domain (RBD)-specific antibodies from 5 individuals shortly after infection and later in convalescence to determine the impact of maturation over months. In addition to increased affinity and neutralization potency, antibody evolution changed the mutational pathways for the acquisition of viral resistance and restricted neutralization escape options. For some antibodies, maturation imposed a requirement for multiple substitutions to enable escape. For certain antibodies, affinity maturation enabled the neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses.


Assuntos
Afinidade de Anticorpos/imunologia , COVID-19/imunologia , COVID-19/virologia , Interações Hospedeiro-Patógeno/imunologia , Mutação , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Epitopos/química , Epitopos/imunologia , Humanos , Modelos Moleculares , Testes de Neutralização , Ligação Proteica , Conformação Proteica , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Relação Estrutura-Atividade , Virulência/genética
4.
J Biol Chem ; 297(2): 100909, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34171358

RESUMO

The human cytidine deaminase family of APOBEC3s (A3s) plays critical roles in both innate immunity and the development of cancers. A3s comprise seven functionally overlapping but distinct members that can be exploited as nucleotide base editors for treating genetic diseases. Although overall structurally similar, A3s have vastly varying deamination activity and substrate preferences. Recent crystal structures of ssDNA-bound A3s together with experimental studies have provided some insights into distinct substrate specificities among the family members. However, the molecular interactions responsible for their distinct biological functions and how structure regulates substrate specificity are not clear. In this study, we identified the structural basis of substrate specificities in three catalytically active A3 domains whose crystal structures have been previously characterized: A3A, A3B- CTD, and A3G-CTD. Through molecular modeling and dynamic simulations, we found an interdependency between ssDNA substrate binding conformation and nucleotide sequence specificity. In addition to the U-shaped conformation seen in the crystal structure with the CTC0 motif, A3A can accommodate the CCC0 motif when ssDNA is in a more linear (L) conformation. A3B can also bind both U- and L-shaped ssDNA, unlike A3G, which can stably recognize only linear ssDNA. These varied conformations are stabilized by sequence-specific interactions with active site loops 1 and 7, which are highly variable among A3s. Our results explain the molecular basis of previously observed substrate specificities in A3s and have implications for designing A3-specific inhibitors for cancer therapy as well as engineering base-editing systems for gene therapy.

5.
Ann Palliat Med ; 10(6): 6936-6947, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34118862

RESUMO

Cocaine abuse is a serious global public health and social problem, and cocaine detoxification remains a challenge. Benzoylecgonine (BE) is the main toxic metabolite after cocaine consumption, with a longer retention time in the body and environment than cocaine itself. According to many studies, the toxicity of BE to humans is as significant as cocaine itself. Moreover, BE is recognized as an addictive drug contaminant in the environment, especially the freshwater system, leading to worries of its ecotoxicity. Extensive studies on the adverse effects of BE on both humans and ecology have been conducted, showing a marked sub-lethal toxicity of BE to diverse organisms. To eliminate BE in vivo and in vitro, various elimination methods have been developed and their BE removal capacity were evaluated. In this review, we aimed to summarize information in the literature to understand better BE toxicity and elimination that may facilitate the clinical treatment of cocaine abuse. By studying the critical role of BE in cocaine abuse, we propose that the ideal treatment for cocaine abuse should not only detoxify cocaine itself but also remove or degrade BE. Emphasizing the necessity of developing effective BE elimination methods is significant for the development of potential clinical treatments and environmental protections.


Assuntos
Cocaína , Cocaína/análogos & derivados , Humanos
6.
Nat Prod Res ; : 1-6, 2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-33949250

RESUMO

Two new terragine analogs (1‒2) with special succinimide and aminopentane moieties were isolated from the fermentation broth of Bacillus sp. SH-1.2-ROOT-18, an endophyte previously discovered from the root of Dendrobium officinale. The structures were elucidated base on comprehensive 1 D/2D NMR and MS data analysis. Complete NMR assignments for the first reported naturally occurring metabolite 3 was also provided.

7.
Pharmaceuticals (Basel) ; 14(3)2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33671080

RESUMO

Meprin α and ß are zinc-dependent proteinases implicated in multiple diseases including cancers, fibrosis, and Alzheimer's. However, until recently, only a few inhibitors of either meprin were reported and no inhibitors are in preclinical development. Moreover, inhibitors of other metzincins developed in previous years are not effective in inhibiting meprins suggesting the need for de novo discovery effort. To address the paucity of tractable meprin inhibitors we developed ultrahigh-throughput assays and conducted parallel screening of >650,000 compounds against each meprin. As a result of this effort, we identified five selective meprin α hits belonging to three different chemotypes (triazole-hydroxyacetamides, sulfonamide-hydroxypropanamides, and phenoxy-hydroxyacetamides). These hits demonstrated a nanomolar to micromolar inhibitory activity against meprin α with low cytotoxicity and >30-fold selectivity against meprin ß and other related metzincincs. These selective inhibitors of meprin α provide a good starting point for further optimization.

8.
Chem Rev ; 121(6): 3238-3270, 2021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33410674

RESUMO

Drug resistance is prevalent across many diseases, rendering therapies ineffective with severe financial and health consequences. Rather than accepting resistance after the fact, proactive strategies need to be incorporated into the drug design and development process to minimize the impact of drug resistance. These strategies can be derived from our experience with viral disease targets where multiple generations of drugs had to be developed to combat resistance and avoid antiviral failure. Significant efforts including experimental and computational structural biology, medicinal chemistry, and machine learning have focused on understanding the mechanisms and structural basis of resistance against direct-acting antiviral (DAA) drugs. Integrated methods show promise for being predictive of resistance and potency. In this review, we give an overview of this research for human immunodeficiency virus type 1, hepatitis C virus, and influenza virus and the lessons learned from resistance mechanisms of DAAs. These lessons translate into rational strategies to avoid resistance in drug design, which can be generalized and applied beyond viral targets. While resistance may not be completely avoidable, rational drug design can and should incorporate strategies at the outset of drug development to decrease the prevalence of drug resistance.


Assuntos
Antivirais/química , Inibidores Enzimáticos/química , Preparações Farmacêuticas/química , Proteínas Virais/química , Viroses/tratamento farmacológico , Antivirais/metabolismo , Antivirais/farmacologia , Biologia Computacional , Desenho de Fármacos , Farmacorresistência Viral , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , HIV-1/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Humanos , Aprendizado de Máquina , Mutação , Orthomyxoviridae/efeitos dos fármacos , Preparações Farmacêuticas/metabolismo , Ligação Proteica , Transdução de Sinais , Relação Estrutura-Atividade
9.
Curr Opin Struct Biol ; 67: 195-204, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33486429

RESUMO

APOBEC3 enzymes are key enzymes in our innate immune system regulating antiviral response in HIV and unfortunately adding diversity in cancer as they deaminate cytosine. Seven unique single and double domain APOBEC3s provide them with unique activity and specificity profiles for this deamination. Recent crystal and NMR structures of APOBEC3 complexes are unraveling the variety of epitopes involved in binding nucleic acids, including at the catalytic site, elsewhere on the catalytic domain and in the inactive N-terminal domain. The interplay between these diverse interactions is critical to uncovering the mechanisms by which APOBEC3s recognize and process their substrates.


Assuntos
Desaminases APOBEC , DNA , RNA , Desaminases APOBEC/metabolismo
10.
Nat Commun ; 12(1): 559, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495478

RESUMO

Broadly neutralizing antibodies (bnAbs) targeting conserved influenza A virus (IAV) hemagglutinin (HA) epitopes can provide valuable information for accelerating universal vaccine designs. Here, we report structural details for heterosubtypic recognition of HA from circulating and emerging IAVs by the human antibody 3I14. Somatic hypermutations play a critical role in shaping the HCDR3, which alone and uniquely among VH3-30 derived antibodies, forms contacts with five sub-pockets within the HA-stem hydrophobic groove. 3I14 light-chain interactions are also key for binding HA and contribute a large buried surface area spanning two HA protomers. Comparison of 3I14 to bnAbs from several defined classes provide insights to the bias selection of VH3-30 antibodies and reveals that 3I14 represents a novel structural solution within the VH3-30 repertoire. The structures reported here improve our understanding of cross-group heterosubtypic binding activity, providing the basis for advancing immunogen designs aimed at eliciting a broadly protective response to IAV.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Epitopos/química , Epitopos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A/metabolismo , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia
11.
J Mol Biol ; 432(23): 6042-6060, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33098858

RESUMO

APOBEC3G (A3G) is a single-stranded DNA (ssDNA) cytosine deaminase that can restrict HIV-1 infection by mutating the viral genome. A3G consists of a non-catalytic N-terminal domain (NTD) and a catalytic C-terminal domain (CTD) connected by a short linker. While the CTD catalyzes cytosine deamination, the NTD is believed to provide additional affinity for ssDNA. Structures of both A3G domains have been solved individually; however, a full-length A3G structure has been challenging. Recently, crystal structures of full-length rhesus macaque A3G variants were solved which suggested dimerization mechanisms and RNA binding surfaces, whereas the dimerization appeared to compromise catalytic activity. We determined the crystal structure of a soluble variant of human A3G (sA3G) at 2.5 Å and from these data generated a model structure of wild-type A3G. This model demonstrated that the NTD was rotated 90° relative to the CTD along the major axis of the molecule, an orientation that forms a positively charged channel connected to the CTD catalytic site, consisting of NTD loop-1 and CTD loop-3. Structure-based mutations, in vitro deamination and DNA binding assays, and HIV-1 restriction assays identify R24, located in the NTD loop-1, as essential to a critical interaction with ssDNA. Furthermore, sA3G was shown to bind a deoxy-cytidine dinucleotide near the catalytic Zn2+, yet not in the catalytic position, where the interactions between deoxy-cytidines and CTD loop-1 and loop-7 residues were different from those formed with substrate. These new interactions suggest a mechanism explaining why A3G exhibits a 3' to 5' directional preference in processive deamination.


Assuntos
Desaminase APOBEC-3G/ultraestrutura , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/ultraestrutura , Conformação Proteica , Desaminase APOBEC-3G/química , Desaminase APOBEC-3G/genética , Animais , Domínio Catalítico/genética , Cristalografia por Raios X , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Humanos , Macaca mulatta/genética , Mutação/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Zinco/química
12.
Nat Commun ; 11(1): 4198, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826914

RESUMO

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity, or as a therapeutic, has yet been developed to SARS-CoV-2. In this study, we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks ACE2 receptor binding, by overlapping the ACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in 293 cells expressing ACE2. When converted to secretory IgA, MAb326 also neutralizes authentic SARS-CoV-2 virus while the IgG isotype shows no neutralization. Our results suggest that SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Imunoglobulina A/imunologia , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Chlorocebus aethiops , Reações Cruzadas , Epitopos , Células HEK293 , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina A Secretora/imunologia , Imunoglobulina A Secretora/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Vírus da SARS/imunologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
13.
Nat Metab ; 2(9): 934-945, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32719542

RESUMO

Type 1 diabetes (T1D) is caused by the autoimmune destruction of pancreatic beta cells. Pluripotent stem cells can now be differentiated into beta cells, thus raising the prospect of a cell replacement therapy for T1D. However, autoimmunity would rapidly destroy newly transplanted beta cells. Using a genome-scale CRISPR screen in a mouse model for T1D, we show that deleting RNLS, a genome-wide association study candidate gene for T1D, made beta cells resistant to autoimmune killing. Structure-based modelling identified the U.S. Food and Drug Administration-approved drug pargyline as a potential RNLS inhibitor. Oral pargyline treatment protected transplanted beta cells in diabetic mice, thus leading to disease reversal. Furthermore, pargyline prevented or delayed diabetes onset in several mouse models for T1D. Our results identify RNLS as a modifier of beta cell vulnerability and as a potential therapeutic target to avert beta cell loss in T1D.


Assuntos
Sistemas CRISPR-Cas , Diabetes Mellitus Tipo 1/tratamento farmacológico , Estudo de Associação Genômica Ampla , Células Secretoras de Insulina/efeitos dos fármacos , Monoaminoxidase/efeitos dos fármacos , Animais , Autoimunidade/efeitos dos fármacos , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Estresse do Retículo Endoplasmático , Inibidores Enzimáticos/farmacologia , Feminino , Células-Tronco Pluripotentes Induzidas/imunologia , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Transplante das Ilhotas Pancreáticas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Mutação , Pargilina/farmacologia
14.
bioRxiv ; 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32511396

RESUMO

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity or as a therapeutic has yet been developed to SARS-CoV-2. In this study we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks hACE2 receptor binding, by completely overlapping the hACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in human epithelial cells expressing hACE2. SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.

15.
Ann Transl Med ; 8(7): 455, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32395499

RESUMO

Background: Endophyte has now become a potential source for the discovery of novel natural products, as they participate in biochemical pathways of their hosts and produce analogous or novel bioactive compounds. As an epiphytic plant, Dendrobium officinale is one of precious Chinese medicines with various activities. It is well known for containing diverse endophytes, but so far not much is known about their secondary metabolites. Methods: the plant tissues were cut and cultured on agar plates to isolate and purify the endophytic bacteria from Dendrobium officinale. Taxonomical identification of strains was performed by 16s rRNA. At the same time, the crude extracts of the strains were tested for antibacterial and cytotoxic activities to screen out one endophyte, Streptomyces sp. SH-1.2-R-15 for further study. After scale-up fermentation, isolation, purification and structure elucidation by using MS, 1D/2D-NMR spectroscopic method, secondary metabolites were identified and submitted for biological activity test. Results: Fifty-eight endophytic strains representing 9 genera were obtained, with 50% of strains were Streptomyces. One of the most active strain, Streptomyces sp. 1.2-R-15, was selected for bioassay-guided isolation, which led to the discovery of two new peptide-type compounds 1 and 2, as well as a bioactive chartreusin, and four other known natural products. Their structures were determined by comprehensive spectroscopic techniques. Chartreusin showed potent cytotoxicity against Hep3B2.1-7 (IC50 =18.19 µM) and H1299 (IC50 =19.74 µM) cancer cell lines, and antibacterial activity against S. aureus (IC50 =23.25 µM). Conclusions: This study highlights the endophytic bacteria from medical plant D. officinale have potential bioactivity and natural product diversity, thus implicates them as a valuable source for new anticancer and antibiotics agents.

16.
Org Lett ; 22(10): 3739-3743, 2020 05 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.


Assuntos
Lactonas/química , Naftoquinonas/química , Policetídeos/química , Streptomyces/química , Vias Biossintéticas , Cristalografia por Raios X , Estrutura Molecular , Naftoquinonas/isolamento & purificação , Plantas Medicinais , Policetídeos/isolamento & purificação , Análise Espectral
17.
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.


Assuntos
Ácidos Aminoisobutíricos/metabolismo , Antivirais/metabolismo , Ciclopropanos/metabolismo , Hepacivirus/enzimologia , Lactamas Macrocíclicas/metabolismo , Leucina/análogos & derivados , Prolina/análogos & derivados , Quinoxalinas/metabolismo , Serina Proteases/metabolismo , Inibidores de Serino Proteinase/metabolismo , Sulfonamidas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Amidas/química , Amidas/metabolismo , Ácidos Aminoisobutíricos/química , Antivirais/química , Carbamatos/química , Carbamatos/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Ciclopropanos/química , Lactamas Macrocíclicas/química , Leucina/química , Leucina/metabolismo , Mutação , Prolina/química , Prolina/metabolismo , Ligação Proteica , Quinoxalinas/química , Serina Proteases/química , Serina Proteases/genética , Inibidores de Serino Proteinase/química , Sulfonamidas/química , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
18.
Bioconjug Chem ; 30(12): 3021-3027, 2019 12 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.


Assuntos
Hidrolases de Éster Carboxílico/química , Estabilidade Enzimática/efeitos dos fármacos , Polietilenoglicóis/química , Animais , Proteínas de Bactérias , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/farmacocinética , Desenho de Fármacos , Escherichia coli/genética , Meia-Vida , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/imunologia , Proteínas Mutantes/química , Proteínas Mutantes/farmacocinética , Polietilenoglicóis/farmacologia , Proteólise/efeitos dos fármacos , Ratos
19.
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
20.
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.


Assuntos
Acetiltiocolina/química , Proteínas de Bactérias/química , Hidrolases de Éster Carboxílico/química , Colinesterases/química , Cocaína/química , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/uso terapêutico , Biocatálise , Hidrolases de Éster Carboxílico/farmacologia , Hidrolases de Éster Carboxílico/uso terapêutico , Colinesterases/farmacologia , Colinesterases/uso terapêutico , Cocaína/metabolismo , Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Overdose de Drogas/tratamento farmacológico , Humanos , Hidrólise , Inativação Metabólica , Especificidade por Substrato
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