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1.
MAbs ; 13(1): 1987180, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34693867

RESUMO

The global health crisis and economic tolls of COVID-19 necessitate a panoply of strategies to treat SARS-CoV-2 infection. To date, few treatment options exist, although neutralizing antibodies against the spike glycoprotein have proven to be effective. Because infection is initiated at the mucosa and propagates mainly at this site throughout the course of the disease, blocking the virus at the mucosal milieu should be effective. However, administration of biologics to the mucosa presents a substantial challenge. Here, we describe bifunctional molecules combining single-domain variable regions that bind to the polymeric Ig receptor (pIgR) and to the SARS-CoV-2 spike protein via addition of the ACE2 extracellular domain (ECD). The hypothesis behind this design is that pIgR will transport the molecule from the circulation to the mucosal surface where the ACE ECD would act as a decoy receptor for the nCoV2. The bifunctional molecules bind SARS-Cov-2 spike glycoprotein in vitro and efficiently transcytose across the lung epithelium in human tissue-based analyses. Designs featuring ACE2 tethered to the C-terminus of the Fc do not induce antibody-dependent cytotoxicity against pIgR-expressing cells. These molecules thus represent a potential therapeutic modality for systemic administration of neutralizing anti-SARS-CoV-2 molecules to the mucosa.


Assuntos
Anticorpos Antivirais , Tratamento Farmacológico da COVID-19 , Receptores de Imunoglobulina Polimérica , SARS-CoV-2/imunologia , Anticorpos de Cadeia Única , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , Células CHO , COVID-19/genética , COVID-19/imunologia , Cricetulus , Cães , Feminino , Humanos , Células Madin Darby de Rim Canino , Camundongos , Mucosa Bucal/imunologia , Domínios Proteicos , Receptores de Imunoglobulina Polimérica/genética , Receptores de Imunoglobulina Polimérica/imunologia , Receptores de Imunoglobulina Polimérica/uso terapêutico , SARS-CoV-2/genética , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacocinética , Anticorpos de Cadeia Única/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/genética , Suínos
2.
Org Biomol Chem ; 8(6): 1280-3, 2010 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-20204194

RESUMO

A micro-scale technique has been developed for the process development of transaminase catalysed reactions. This pH indicator based, colorimetric assay can be used to investigate and optimise reaction conditions at 100 microL scale. Enzyme activity and stability as a function of various reaction parameters, including temperature, pH and co-solvent concentration, have been determined. Additionally, reactions have been scaled up from 100 microL to 25 mL under the optimal reaction conditions identified by the micro-scale process development activities. Excellent conversion (>99%) and enantioselectivity (>99% ee) were obtained.


Assuntos
Biocatálise , Ensaios Enzimáticos/métodos , Transaminases/metabolismo , Colorimetria , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Solventes/química , Temperatura , Transaminases/química
3.
Chem Commun (Camb) ; (16): 2127-9, 2009 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-19360168

RESUMO

A range of enantiomerically pure (R)- and (S)-configured chiral amines has been prepared in excellent e.e. (99%) by combining a transaminase enzyme with an amino acid oxidase and catalytic quantities of pyruvate.


Assuntos
Aminas/química , Aminoácido Oxirredutases/química , Transaminases/química , Catálise , Cinética , Estereoisomerismo
4.
Org Biomol Chem ; 7(2): 395-8, 2009 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-19109687

RESUMO

A rapid, high-throughput screening methodology has been developed for the determination of transaminase activity. This pH based, colorimetric assay can also be used to scale reactions directly from 100 microL screening scale to 25 mL development scale. Additionally, three techniques have been developed to drive transamination reactions toward complete conversion. The first method uses lactate dehydrogenase to remove the inhibitory pyruvate keto acid by-product from the reaction and drive reaction equilibrium toward the desired amine. The second method is a single enzyme system, and uses a large excess of isopropylamine to drive the transamination. Method three requires only a catalytic amount of amine donor, as an amino acid dehydrogenase is employed to regenerate the amine donor in situ using ammonia. All three systems have been demonstrated for the production of optically pure methylbenzylamine from acetophenone. An enantiomeric excess of >99% was achieved for both the R- and S-methylbenzylamine products.


Assuntos
Transaminases/química , Transaminases/metabolismo , Acetofenonas/química , Acetofenonas/metabolismo , Catálise , Cinética , Estereoisomerismo , Transaminases/antagonistas & inibidores
5.
Science ; 363(6424)2019 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-30655413

RESUMO

Innovations in synthetic chemistry have enabled the discovery of many breakthrough therapies that have improved human health over the past century. In the face of increasing challenges in the pharmaceutical sector, continued innovation in chemistry is required to drive the discovery of the next wave of medicines. Novel synthetic methods not only unlock access to previously unattainable chemical matter, but also inspire new concepts as to how we design and build chemical matter. We identify some of the most important recent advances in synthetic chemistry as well as opportunities at the interface with partner disciplines that are poised to transform the practice of drug discovery and development.


Assuntos
Química Farmacêutica/tendências , Descoberta de Drogas , Preparações Farmacêuticas/síntese química , Biocatálise , Indústria Farmacêutica , Enzimas/química , Ensaios de Triagem em Larga Escala , Invenções , Aprendizado de Máquina , Fotoquímica
6.
Science ; 366(6470): 1255-1259, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31806816

RESUMO

Enzyme-catalyzed reactions have begun to transform pharmaceutical manufacturing, offering levels of selectivity and tunability that can dramatically improve chemical synthesis. Combining enzymatic reactions into multistep biocatalytic cascades brings additional benefits. Cascades avoid the waste generated by purification of intermediates. They also allow reactions to be linked together to overcome an unfavorable equilibrium or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the number of steps of the previously reported routes.


Assuntos
Biocatálise , Desoxiadenosinas/química , Inibidores da Transcriptase Reversa/química , Biotecnologia/métodos , Preparações Farmacêuticas/síntese química , Estereoisomerismo
7.
Org Lett ; 10(5): 741-4, 2008 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-18247494

RESUMO

Herein we report the asymmetric synthesis of 1,2-dipyridyl-1,2-diarylethanes via an unusual Cu(I)-catalyzed dimerization reaction. Subjection of a variety of enantioenriched substituted 2-pyridyl alcohols to a one-pot protocol generates the desired products in good yields and diastereoselectivities and with ee's up to >99%.


Assuntos
Cobre/química , Etano/análogos & derivados , Etano/síntese química , Hidrocarbonetos Bromados/síntese química , Catálise , Etano/química , Hidrocarbonetos Bromados/química , Estrutura Molecular , Estereoisomerismo
8.
ACS Synth Biol ; 7(7): 1730-1741, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-29782150

RESUMO

Directed evolution experiments designed to improve the activity of a biocatalyst have increased in sophistication from the early days of completely random mutagenesis. Sequence-based and structure-based methods have been developed to identify "hotspot" positions that when randomized provide a higher frequency of beneficial mutations that improve activity. These focused mutagenesis methods reduce library sizes and therefore reduce screening burden, accelerating the rate of finding improved enzymes. Looking for further acceleration in finding improved enzymes, we investigated whether two existing methods, one sequence-based (Protein GPS) and one structure-based (using Bioluminate and MOE), were sufficiently predictive to provide not just the hotspot position, but also the amino acid substitution that improved activity at that position. By limiting the libraries to variants that contained only specific amino acid substitutions, library sizes were kept to less than 100 variants. For an initial round of ATA-117 R-selective transaminase evolution, we found that the methods used produced libraries where 9% and 18% of the amino acid substitutions chosen were amino acids that improved reaction performance in lysates. The ability to create combinations of mutations as part of the initial design was confounded by the relatively large number of predicted mutations that were inactivating (30% and 45% for the sequence-based and structure-based methods, respectively). Despite this, combining several mutations identified within a given method produced variant lysates 7- and 9-fold more active than the wild-type lysate, highlighting the capability of mutations chosen this way to generate large advances in activity in addition to the reductions in screening.


Assuntos
Evolução Molecular Direcionada , Mutagênese/genética , Mutação/genética
9.
Org Lett ; 9(2): 335-8, 2007 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-17217298

RESUMO

The synthesis of diarylmethanols via the reduction of a range of substituted benzophenone and benzoylpyridine derivatives with ketoreductase enzymes (KREDs) has afforded chiral products with high yield (>90%) and ee (up to >99%). Ortho, meta, and para substitutions with a variety of electron-donating, electron-withdrawing, and halogen groups were examined. Substitution at the ortho position and/or highly electronically dissymmetric molecules were not required for good selectivity, as is the case with conventional chemical catalyst reductions. [reaction: see text].


Assuntos
Cetonas/química , Metanol/análogos & derivados , Metanol/síntese química , Oxirredutases/química , Catálise , Metanol/química , Estrutura Molecular , Oxirredução , Estereoisomerismo
10.
ACS Med Chem Lett ; 8(5): 476-480, 2017 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-28523096

RESUMO

The use of biocatalysis in the pharmaceutical industry continues to expand as a result of increased access to enzymes and the ability to engineer those enzymes to meet the demands of industrial processes. However, we are still just scratching the surface of potential biocatalytic applications. The time pressures present in pharmaceutical process development are incompatible with the long lead times required for engineering a suitable biocatalyst. Dramatic increases in the speed of protein engineering are needed to deliver on the ever increasing opportunities for industrial biocatalytic processes.

11.
Org Lett ; 16(10): 2716-9, 2014 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-24786635

RESUMO

A novel synthesis of vernakalant is described. Using inexpensive and readily available reagents, the key transformations involve (1) an efficient zinc-amine-promoted etherification, (2) a highly stereoselective enzyme-catalyzed dynamic asymmetric transamination to set up the two contiguous chiral centers in the cyclohexane ring, and (3) a pyrrolidine ring formation via alkyl-B(OH)2-catalyzed amidation and subsequent imide reduction.


Assuntos
Anisóis/síntese química , Pirrolidinas/síntese química , Aminação , Aminas/química , Anisóis/química , Catálise , Cloretos/química , Cicloexanos , Estrutura Molecular , Pirrolidinas/química , Estereoisomerismo , Compostos de Zinco/química
13.
Chem Commun (Camb) ; 47(2): 773-5, 2011 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-21072390

RESUMO

A fast and sensitive method for screening transaminase activity and enantioselectivity, using D- and L-amino acid oxidases, allows new amine substrates to be rapidly identified.


Assuntos
Transaminases/metabolismo , Aminas/química , Aminas/metabolismo , Calorimetria , L-Aminoácido Oxidase/metabolismo , Estereoisomerismo , Especificidade por Substrato
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