RESUMO
The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.
Assuntos
Insulina , Penicilina Amidase , Peptídeos , Engenharia de Proteínas , Sequência de Aminoácidos , Humanos , Insulina/análogos & derivados , Insulina/biossíntese , Lisina/química , Penicilina Amidase/química , Penicilina Amidase/genética , Peptídeos/química , Peptídeos/genética , Engenharia de Proteínas/métodosRESUMO
The field of protein conjugation most commonly refers to the chemical, enzymatic, or chemoenzymatic formation of new covalent bonds between two polypeptides, or between a single polypeptide and a new molecule (polymer, small molecule, nucleic acid, carbohydrate, etc.). Due to the modest selectivity of chemical methods for protein conjugation, there are increased efforts to develop biocatalysts that confer regioselectivity for site-specific modification, thereby complementing the existing toolbox of chemical conjugation strategies. This review summarizes key advances in the use of enzymes to functionalize proteins with commercial relevance. The examples put forth have demonstrated value at the industrial level or show promising industrial potential in the laboratory.
Assuntos
Enzimas/metabolismo , Proteínas/metabolismo , Aminoaciltransferases/química , Aminoaciltransferases/metabolismo , Aminoaciltransferases/uso terapêutico , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/uso terapêutico , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/uso terapêutico , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Humanos , Imunoconjugados/química , Imunoconjugados/metabolismo , Oxirredutases/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas/química , Transferases/metabolismoRESUMO
An enantioselective synthesis of the potent anti-HIV nucleoside EFdA is presented. Key features of stereocontrol include construction of the fully substituted 4'-carbon via a biocatalytic desymmetrization of 2-hydroxy-2-((triisopropylsilyl)ethynyl)propane-1,3-diyl diacetate and a Noyori-type asymmetric transfer hydrogenation to control the stereochemistry of the 3'-hydroxyl bearing carbon. The discovery of a selective crystallization of an N-silyl nucleoside intermediate enabled isolation of the desired ß-anomer from the glycosylation step.
Assuntos
Fármacos Anti-HIV/síntese química , Desoxiadenosinas/síntese química , Inibidores da Transcriptase Reversa/síntese química , Catálise , Glicosídeos/química , Glicosilação , Hidrogenação , Estrutura Molecular , Oxirredução , EstereoisomerismoRESUMO
Flavins and related molecules catalyze organic Baeyer-Villiger reactions. Combined experimental and DFT studies indicate that these molecules also catalyze the insertion of oxygen into metal-carbon bonds through a Baeyer-Villiger-like transition state.
Assuntos
Flavinas/metabolismo , Compostos Organometálicos/química , Oxigênio/química , Rênio/química , Alcanos/química , Catálise , Peróxido de Hidrogênio/química , OxirreduçãoRESUMO
Playing a pivotal role in the metabolism of neurotransmitters in the central nervous system, the mitochondrial enzymes monoamine oxidases A and B (MAO A and B) have been for long studied as drug targets for neurodegenerative and neurological diseases. MAO inhibitors (MAOIs) are clinically used to treat Parkinson's disease and depression by blocking the degradation of neuroactive catecholamines and providing a symptomatic relief in the patients. More recent is the idea that the neuroprotective effect of MAOIs may result from the prevention of oxidative stress produced by the MAO reaction rather than being simply related to the inhibition of neurotransmitters degradation. Tranylcypromine and phenelzine are among the first developed MAOI drugs and have been used for years to treat depression. Their usage is now limited to cases of refractory depression because of their negative side effects, which are due to both the lack of MAO A/MAO B selectivity and the inhibition of other enzymes such as the drug-metabolizing cytochromes P450. Although the multi-target action of these MAOIs determines negative implications, the most newly developed compounds have improved properties not only for their specificity relatively to MAO A/MAO B selectivity but also because they function through multiple mechanisms that produce beneficial effects. In particular, safinamide, a MAO B selective inhibitor in clinical trials for Parkinson's disease, is neuroprotective by blocking the voltage-dependent Na+ and Ca2+ channels and the Ca2+-mediated glutamate release processes. Rasagiline is a drug used in combination with L-dopa in the treatment of parkinsonian patients and the metabolic products of its degradation exert neuroprotective effects. Moreover, rasagiline scaffold is used to design analogs by addition of pharmacophores that act on other neurological targets. This multi-target approach may prove successful in order to find new and more effective therapies for the complexity of neurodegenerative diseases.
Assuntos
Inibidores da Monoaminoxidase/farmacologia , Monoaminoxidase/metabolismo , Doenças do Sistema Nervoso/tratamento farmacológico , Animais , Humanos , Inibidores da Monoaminoxidase/química , Doenças do Sistema Nervoso/enzimologia , Relação Estrutura-AtividadeRESUMO
The major structural difference between human monoamine oxidases A (MAO A) and B (MAO B) is that MAO A has a monopartite substrate cavity of ~550 Å(3) volume and MAO B contains a dipartite cavity structure with volumes of ~290 Å(3) (entrance cavity) and ~400 Å(3) (substrate cavity). Ile199 and Tyr326 side chains separate these two cavities in MAO B. To probe the function of these gating residues, Ile199Ala and Ile199Ala-Tyr326Ala mutant forms of MAO B were investigated. Structural data on the Ile199Ala MAO B mutant show no alterations in active site geometries compared with wild-type enzyme while the Ile199Ala-Tyr326Ala MAO B mutant exhibits alterations in residues 100-103 which are part of the loop gating the entrance to the active site. Both mutant enzymes exhibit catalytic properties with increased amine K(M) but unaltered k(cat) values. The altered K(M) values on mutation are attributed to the influence of the cavity structure in the binding and subsequent deprotonation of the amine substrate. Both mutant enzymes exhibit weaker binding affinities relative to wild-type enzyme for small reversible inhibitors. Ile199Ala MAO B exhibits an increase in binding affinity for reversible MAO B specific inhibitors which bridge both cavities. The Ile199Ala-Tyr326Ala double mutant exhibits inhibitor binding properties more similar to those of MAO A than to MAO B. These results demonstrate that the bipartite cavity structure in MAO B plays an important role in substrate and inhibitor recognition to distinguish its specificities from those of MAO A and provide insights into specific reversible inhibitor design for these membrane-bound enzymes.
Assuntos
Inibidores da Monoaminoxidase/química , Monoaminoxidase/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Benzilaminas/metabolismo , Domínio Catalítico , Humanos , Isoleucina/metabolismo , Cinética , Monoaminoxidase/química , Monoaminoxidase/genética , Especificidade por Substrato , Tirosina/metabolismoRESUMO
Crystallographic and biochemical studies have been employed to identify the binding site and mechanism for potentiation of imidazoline binding in human monoamine oxidase B (MAO B). 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) inhibits recombinant human MAO B with a K(i) of 8.3 ± 0.6 µM, whereas tranylcypromine-inhibited MAO B binds 2-BFI with a K(d) of 9 ± 2 nM, representing an increase in binding energy Δ(ΔG) of -3.9 kcal/mol. Crystal structures show the imidazoline ligand bound in a site that is distinct from the substrate-binding cavity. Contributions to account for the increase in binding affinity upon tranylcypromine inhibition include a conformational change in the side chain of Gln(206) and a "closed conformation" of the side chain of Ile(199), forming a hydrophobic "sandwich" with the side chain of Ile(316) on each face of the benzofuran ring of 2-BFI. Data with the I199A mutant of human MAO B and failure to observe a similar binding potentiation with rat MAO B, where Ile(316) is replaced with a Val residue, support an allosteric mechanism where the increased binding affinity of 2-BFI results from a cooperative increase in H-bond strength through formation of a more hydrophobic milieu. These insights should prove valuable in the design of high affinity and specific reversible MAO B inhibitors.
Assuntos
Benzofuranos/metabolismo , Imidazóis/metabolismo , Monoaminoxidase/química , Monoaminoxidase/metabolismo , Proteínas Recombinantes/metabolismo , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Humanos , Monoaminoxidase/genética , Inibidores da Monoaminoxidase/farmacologia , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Tranilcipromina/farmacologiaRESUMO
Previous studies have shown that (E)-8-(3-chlorostyryl)caffeine (CSC) is a specific reversible inhibitor of human monoamine oxidase B (MAO-B) and does not bind to human MAO-A. Since the small molecule isatin is a natural reversible inhibitor of both MAO-B and MAO-A, (E)-5-styrylisatin and (E)-6-styrylisatin analogues were synthesized in an attempt to identify inhibitors with enhanced potencies and specificities for MAO-B. The (E)-styrylisatin analogues were found to exhibit higher binding affinities than isatin with the MAO preparations tested. The (E)-5-styrylisatin analogues bound more tightly than the (E)-6 analogue although the latter exhibits the highest MAO-B selectivity. Molecular docking studies with MAO-B indicate that the increased binding affinity exhibited by the (E)-styrylisatin analogues, in comparison to isatin, is best explained by the ability of the styrylisatins to bridge both the entrance cavity and the substrate cavity of the enzyme. Experimental support for this model is shown by the weaker binding of the analogues to the Ile199Ala mutant of human MAO-B. The lower selectivity of the (E)-styrylisatin analogues between MAO-A and MAO-B, in contrast to CSC, is best explained by the differing relative geometries of the aromatic rings for these two classes of inhibitors.
Assuntos
Isatina/análogos & derivados , Isatina/química , Inibidores da Monoaminoxidase/síntese química , Monoaminoxidase/química , Ligação Competitiva , Encéfalo/metabolismo , Química Farmacêutica/métodos , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Isatina/farmacologia , Cinética , Modelos Químicos , Estrutura Molecular , Inibidores da Monoaminoxidase/farmacologia , Mutação , Relação Estrutura-AtividadeRESUMO
Mechanistic and structural studies have been carried out to investigate the molecular basis for the irreversible inhibition of human MAO-B by mofegiline. Competitive inhibition with substrate shows an apparent K(i) of 28 nM. Irreversible inhibition of MAO-B occurs with a 1:1 molar stoichiometry with no observable catalytic turnover. The absorption spectral properties of mofegiline inhibited MAO-B show features (lambda(max) approximately 450 nm) unlike those of traditional flavin N(5) or C(4a) adducts. Visible and near-UV circular dichroism spectra of the mofegiline-MAO-B adduct shows a negative peak at 340 nm with an intensity similar to that of N(5) flavocyanine adducts. The X-ray crystal structure of the mofegiline-MAO-B adduct shows a covalent bond between the flavin cofactor N(5) with the distal allylamine carbon atom as well as the absence of the fluorine atom. A mechanism to explain these structural and spectral data is proposed.