RESUMO
The function of endogenous cell-cell signaling peptides relies on their interactions with cognate receptors, which in turn are influenced by the peptides' structures, necessitating a comprehensive understanding of the suite of post-translational modifications of the peptide. Herein, we report the initial characterization of putative peptide isomerase enzymes extracted from R. norvegicus, A. californica, and B. taurus tissues. These enzymes are both tissue and substrate-specific across all three organisms. Notably, the lungs of the mammalian species, and the central nervous system of the mollusk displayed the highest isomerase activity among the examined tissues. In vitro enzymatic conversion was observed for several endogenous peptides, such as the tetrapeptide GFFD in A. californica, and mammalian neuropeptide FF in R. norvegicus and B. taurus. To understand their mode of action, we explored the effects of several inhibitors on these enzymes, which suggest common active site residues. While further characterization of these enzymes is required, the investigations emphasize a widespread and overlooked enzyme activity related to the creation of bioactive peptides.
Assuntos
Oligopeptídeos , Animais , Especificidade por Substrato , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Isomerases/metabolismo , Isomerases/química , Processamento de Proteína Pós-Traducional , Sequência de AminoácidosRESUMO
D-amino acid-containing peptides (DAACPs) in animals are a class of bioactive molecules formed via the posttranslational modification of peptides consisting of all-L-amino acid residues. Amino acid residue isomerization greatly impacts the function of the resulting DAACP. However, because isomerization does not change the peptide's mass, this modification is difficult to detect by most mass spectrometry-based peptidomic approaches. Here we describe a method for the identification of DAACPs that can be used to systematically survey peptides extracted from a tissue sample in a nontargeted manner.
Assuntos
Aminoácidos , Espectrometria de Massa com Cromatografia Líquida , Animais , Aminoácidos/química , Cromatografia Líquida , Espectrometria de Massas em Tandem/métodos , PeptídeosRESUMO
Determination of stereoisomers is an integral part of pharmaceutical analysis. Chiral liquid chromatography (LC) method development is typically initiated through screening of chiral stationary phases (CSPs) and mobile phases (MPs) since chiral separation is difficult to predict. We have previously reported a screening strategy using chiral reversed-phase (RP) LC as two primary tiers due to its versatility for enantiorecognition and compatibility with diverse sample matrices. Here we focus on developing a normal-phase (NP) LC screening strategy as a secondary tier for chiral method screening. A database was constructed from 60 NPLC screens performed on up to 18 CSPs and 3 MPs using gradient elution. This was used to investigate the effectiveness of NPLC compared to RPLC screening, as well as the impact of MP composition and the selectivity of different CSPs in NPLC screening. A success hit rate of 90% was observed in NPLC compared to 84% in RPLC screening for Bristol Myers Squibb compounds. Importantly, NPLC screening generated successful hit(s) in 81% of the cases that failed in RPLC, demonstrating the value of NPLC as a complementary screening tier. After optimizing the CSP/MP selection, we proposed a NPLC screening workflow with several user-options according to method requirements and instrument capacity. Among these, the most comprehensive NPLC screening consisted of ten CSPs (AD, AS, AY, AZ, OD, OJ, IC, IE, IG, O1) with three MPs. When combined with RPLC, an overall success rate of 97% was achieved for the diverse set of pharmaceutical compounds.
Assuntos
Cromatografia de Fase Reversa , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Cromatografia de Fase Reversa/métodos , Indicadores e Reagentes , Preparações Farmacêuticas , EstereoisomerismoRESUMO
A highly stereoselective synthesis of a cyclic dinucleotide (CDN) STING agonist containing two chiral thiophosphoramidate linkages is described. These rare yet key functional groups were, for the first time, installed efficiently and with high diastereoselectivity using a specially designed P(V) reagent. By utilizing this strategy, the CDN was prepared in greater than 16-fold higher yield than the prior P(III) approach, with fewer hazardous reagents and chromatographic purifications.
Assuntos
Proteínas de Membrana , Indicadores e Reagentes , Proteínas de Membrana/químicaRESUMO
Chirality control plays a critical role in developing stereoisomeric drugs. Due to the complexity and lack of predictability in chiral separations, column screening remains the gold standard to initiate chiral method development for active pharmaceutical ingredients (APIs) and synthetic intermediates. Chiral reversed-phase (RP) liquid chromatography (LC) has gained favor over other modes due to its versatility and compatibility in analyzing a wide range of chiral compounds in various matrices. Herein, we established a tier-based chiral RPLC screen strategy by constructing and analyzing a database of 101 chiral screens with a total of 3,401 entries (unique LC runs) for proprietary APIs or intermediates at Bristol Myers Squibb. Up to 17 polysaccharide-based chiral stationary phases (CSPs) and four mobile phases (MPs) have been screened with gradient elution. A selection of ten CSPs with two MPs was found sufficient to achieve successful separation for 82% of the total screens. Two RPLC screen tiers (Tier 1: AZ, OD, ID, and IG) and (Tier 2: AY, OJ, OZ, IA, IC, and IH) were proposed along with two MPs (acidic and neutral) to target ~70% hit rate for Tier 1, and ~80% for the combined set. We also implemented a user-friendly workflow to enable walk-up chiral RPLC screening with automated reports and system suitability tests.
Assuntos
Cromatografia de Fase Reversa/métodos , Preparações Farmacêuticas/análise , Polissacarídeos/química , Preparações Farmacêuticas/química , Preparações Farmacêuticas/isolamento & purificação , EstereoisomerismoRESUMO
D-Amino acid-containing peptides (DAACPs) in animals are a class of bioactive molecules formed via the posttranslational modification of peptides consisting of all-L-amino acid residues. Amino acid residue isomerization greatly impacts the function of the resulting DAACP. However, because isomerization does not change the peptide's mass, this modification is difficult to detect by most mass spectrometry-based peptidomic approaches. Here we describe a method for the identification of DAACPs that can be used to systematically survey peptides extracted from a tissue sample in a non-targeted manner.
Assuntos
Aminoácidos/análise , Antígenos CD13/metabolismo , Cromatografia Líquida/métodos , Ensaios de Triagem em Larga Escala/métodos , Fragmentos de Peptídeos/análise , Espectrometria de Massas em Tandem/métodos , Aminoácidos/química , AnimaisRESUMO
Ribosomally synthesized and post-translationally modified peptide (RiPP) natural products are attractive for genome-driven discovery and re-engineering, but limitations in bioinformatic methods and exponentially increasing genomic data make large-scale mining of RiPP data difficult. We report RODEO (Rapid ORF Description and Evaluation Online), which combines hidden-Markov-model-based analysis, heuristic scoring, and machine learning to identify biosynthetic gene clusters and predict RiPP precursor peptides. We initially focused on lasso peptides, which display intriguing physicochemical properties and bioactivities, but their hypervariability renders them challenging prospects for automated mining. Our approach yielded the most comprehensive mapping to date of lasso peptide space, revealing >1,300 compounds. We characterized the structures and bioactivities of six lasso peptides, prioritized based on predicted structural novelty, including one with an unprecedented handcuff-like topology and another with a citrulline modification exceptionally rare among bacteria. These combined insights significantly expand the knowledge of lasso peptides and, more broadly, provide a framework for future genome-mining efforts.
Assuntos
Produtos Biológicos/metabolismo , Mineração de Dados , Genoma/genética , Genômica , Peptídeos/metabolismo , Produtos Biológicos/química , Vias Biossintéticas/genética , Aprendizado de Máquina , Cadeias de Markov , Família Multigênica/genética , Peptídeos/química , Peptídeos/genéticaRESUMO
Natural products (NPs) serve important roles as drug candidates and as tools for chemical biology. However, traditional NP discovery, largely based on bioassay-guided approaches, is biased toward abundant compounds and rediscovery rates are high. Orthogonal methods to facilitate discovery of new NPs are thus needed, and herein we describe an isotope tag-based expansion of reactivity-based NP screening to address these shortcomings. Reactivity-based screening is a directed discovery approach in which a specific reactive handle on the NP is targeted by a chemoselective probe to enable its detection by mass spectrometry. In this study, we have developed an aminooxy-containing probe to guide the discovery of aldehyde- and ketone-containing NPs. To facilitate the detection of labeling events, the probe was dibrominated, imparting a unique isotopic signature to distinguish labeled metabolites from spectral noise. As a proof of concept, the probe was then utilized to screen a collection of bacterial extracts, leading to the identification of a new analogue of antipain, deimino-antipain. The bacterial producer of deimino-antipain was sequenced and the responsible biosynthetic gene cluster was identified by bioinformatic analysis and heterologous expression. These data reveal the previously undetermined genetic basis for a well-known family of aldehyde-containing, peptidic protease inhibitors, including antipain, chymostatin, leupeptin, elastatinal, and microbial alkaline protease inhibitor, which have been widely used for over 40 years.
Assuntos
Aldeídos/química , Produtos Biológicos/análise , Produtos Biológicos/metabolismo , Cetonas/química , Streptomyces/química , Aldeídos/metabolismo , Biologia Computacional , Cetonas/metabolismo , Estrutura Molecular , Streptomyces/isolamento & purificação , Streptomyces/metabolismoRESUMO
A receptor binding class of d-amino acid-containing peptides (DAACPs) is formed in animals from an enzymatically mediated post-translational modification of ribosomally translated all-l-amino acid peptides. Although this modification can be required for biological actions, detecting it is challenging because DAACPs have the same mass as their all-l-amino acid counterparts. We developed a suite of mass spectrometry (MS) protocols for the nontargeted discovery of DAACPs and validated their effectiveness using neurons from Aplysia californica. The approach involves the following three steps, with each confirming and refining the hits found in the prior step. The first step is screening for peptides resistant to digestion by aminopeptidase M. The second verifies the presence of a chiral amino acid via acid hydrolysis in deuterium chloride, labeling with Marfey's reagent, and liquid chromatography-mass spectrometry to determine the chirality of each amino acid. The third involves synthesizing the putative DAACPs and comparing them to the endogenous standards. Advantages of the method, the d-amino acid-containing neuropeptide discovery funnel, are that it is capable of detecting the d-form of any common chiral amino acid, and the first two steps do not require peptide standards. Using these protocols, we report that two peptides from the Aplysia achatin-like neuropeptide precursor exist as GdYFD and SdYADSKDEESNAALSDFA. Interestingly, GdYFD was bioactive in the Aplysia feeding and locomotor circuits but SdYADSKDEESNAALSDFA was not. The discovery funnel provides an effective means to characterize DAACPs in the nervous systems of animals in a nontargeted manner.
Assuntos
Aminoácidos/análise , Aplysia/química , Neuropeptídeos/análise , Aminoácidos/metabolismo , Animais , Aplysia/citologia , Aplysia/metabolismo , Antígenos CD13/metabolismo , Espectrometria de Massas , Neurônios/química , Neurônios/citologia , Neurônios/metabolismo , Neuropeptídeos/metabolismoRESUMO
TAR DNA-binding protein-43 (TDP-43) is a nuclear RNA-binding protein involved in many cellular pathways. TDP-43-positive inclusions are a hallmark of amyotrophic lateral sclerosis (ALS). The major clinical presentation of ALS is muscle weakness due to the degeneration of motor neurons. Mislocalization of TDP-43 from the nucleus to the cytoplasm is an early event of ALS. In this study, we demonstrate that cytoplasmic mislocalization of TDP-43 was accompanied by increased activation of AMP-activated protein kinase (AMPK) in motor neurons of ALS patients. The activation of AMPK in a motor neuron cell line (NSC34) or mouse spinal cords induced the mislocalization of TDP-43, recapitulating this characteristic of ALS. Down-regulation of AMPK-α1 or exogenous expression of a dominant-negative AMPK-α1 mutant reduced TDP-43 mislocalization. Suppression of AMPK activity using cAMP-simulating agents rescued the mislocalization of TDP-43 in NSC34 cells and delayed disease progression in TDP-43 transgenic mice. Our findings demonstrate that activation of AMPK-α1 plays a critical role in TDP-43 mislocalization and the development of ALS; thus, AMPK-α1 may be a potential drug target for this devastating disease.