Tryptophan deficiency induced by indoleamine 2,3-dioxygenase 1 results in glucose transporter 1-dependent promotion of aerobic glycolysis in pancreatic cancer.

Indoleamine 2,3-dioxygenase 1 (IDO1), the key enzyme in the catabolism of the essential amino acid tryptophan (Trp) through kynurenine pathway, induces immune tolerance and is considered as a critical immune checkpoint, but its impacts as a metabolism enzyme on glucose and lipid metabolism are overlooked. We aim to clarify the potential role of IDO1 in aerobic glycolysis in pancreatic cancer (PC). Analysis of database revealed the positive correlation in PC between the expressions of IDO1 and genes encoding important glycolytic enzyme hexokinase 2 (HK2), pyruvate kinase (PK), lactate dehydrogenase A (LDHA) and glucose transporter 1 (GLUT1). It was found that IDO1 could modulate glycolysis and glucose uptake in PC cells, Trp deficiency caused by IDO1 overexpression enhanced glucose uptake by stimulating GLUT1 translocation to the plasma membrane of PC cells. Besides, Trp deficiency caused by IDO1 overexpression suppressed the apoptosis of PC cells via promoting glycolysis, which reveals the presence of IDO1-glycolysis-apoptosis axis in PC. IDO1 inhibitors could inhibit glycolysis, promote apoptosis, and exhibit robust therapeutic efficacy when combined with GLUT1 inhibitor in PC mice. Our study reveals the function of IDO1 in the glucose metabolism of PC and provides new insights into the therapeutic strategy for PC.

Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry.

Aggregates are recognized as one of the most critical product-related impurities in monoclonal antibody (mAb)-based therapeutics due to their negative impact on the stability and safety of the drugs. So far, investigational efforts have primarily focused on understanding the causes and effects of mAb self-aggregation, including both internal and external factors. In this study, we focused on understanding mAb stability in the presence of its monovalent fragment, formed through hinge cleavage and loss of one Fab unit (referred to as "Fab/c"), a commonly observed impurity during manufacturing and stability. The Fab/c fragments were generated using a limited IgdE digestion that specifically cleaves above the IgG1 mAb hinge region, followed by hydrophobic interaction chromatographic (HIC) enrichment. Two IgG1 mAbs containing different levels of Fab/c fragments were incubated under thermally accelerated conditions. A method based on size exclusion chromatography coupled with native mass spectrometry (SEC-UV-native MS) was developed and used to characterize the stability samples and identified the formation of heterogeneous dimers, including intact dimer, mAb-Fab/c dimer, Fab/c-Fab/c dimer, and mAb-Fab dimer. Quantitative analyses on the aggregation kinetics suggested that the impact of Fab/c fragment on the aggregation rate of individual dimer differs between a glycosylated mAb (mAb1) and a non-glycosylated mAb (mAb2). An additional study of deglycosylated mAb1 under 25°C accelerated stability conditions suggests no significant impact of the N-glycan on mAb1 total aggregation rate. This study also highlighted the power of SEC-UV-native MS method in the characterization of mAb samples with regard to separating, identifying, and quantifying mAb aggregates and fragments.

Evaluating protein cross-linking as a therapeutic strategy to stabilize SOD1 variants in a mouse model of familial ALS.

Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment. We hypothesized that cyclic thiosulfinate cross-linkers, which selectively target a rare, 2 cysteine-containing motif, can stabilize fALS-causing SOD1 variants in vivo. We created a library of chemically diverse cyclic thiosulfinates and determined structure-cross-linking-activity relationships. A pre-lead compound, "S-XL6," was selected based upon its cross-linking rate and drug-like properties. Co-crystallographic structure clearly establishes the binding of S-XL6 at Cys 111 bridging the monomers and stabilizing the SOD1 dimer. Biophysical studies reveal that the degree of stabilization afforded by S-XL6 (up to 24°C) is unprecedented for fALS, and to our knowledge, for any protein target of any kinetic stabilizer. Gene silencing and protein degrading therapeutic approaches require careful dose titration to balance the benefit of diminished fALS SOD1 expression with the toxic loss-of-enzymatic function. We show that S-XL6 does not share this liability because it rescues the activity of fALS SOD1 variants. No pharmacological agent has been proven to bind to SOD1 in vivo. Here, using a fALS mouse model, we demonstrate oral bioavailability; rapid engagement of SOD1G93A by S-XL6 that increases SOD1G93A's in vivo half-life; and that S-XL6 crosses the blood-brain barrier. S-XL6 demonstrated a degree of selectivity by avoiding off-target binding to plasma proteins. Taken together, our results indicate that cyclic thiosulfinate-mediated SOD1 stabilization should receive further attention as a potential therapeutic approach for fALS.

Preparation of astaxanthin-loaded composite micelles with coaxial electrospray technology for enhanced oral bioavailability and improved antioxidation capability.

BACKGROUND: Astaxanthin (AST) is approved by the US Food and Drug Administration (FDA) as a safe dietary supplement for humans. As a potent lipid-soluble keto-carotenoid, it is widely used in food, cosmetics, and the pharmaceutical industry. However, its low solubility limits its powerful biological activity and its application in these fields. This study aims to develop a delivery system to address the low solubility and bioavailability of AST and to enhance its antioxidant capacity. RESULTS: Astaxanthin-loaded composite micelles were successfully prepared via coaxial electrospray technology. Astaxanthin existed in the amorphous state in the electro-sprayed formulation with an approximate particle size of 186.28 nm and with a polydispersity index of 0.243. In this delivery system, Soluplus and copovidone (PVPVA 64) were the main polymeric matrix for AST, which then released the drug upon contact with aqueous media, resulting in an overall increase in drug solubility and a release rate of 94.08%. Meanwhile, lecithin, and Polyethylene glycol-grafted Chitosan (PEG-g-CS) could support the absorption of AST in the gastrointestinal tract, assisting transmembrane transport. The relative bioavailability reached about 308.33% and the reactive oxygen species (ROS) scavenging efficiency of the formulation was 44.10%, which was 1.57 times higher than that of free astaxanthin (28.10%) when both were at the same concentration level based on astaxanthin. CONCLUSION: Coaxial electrospray could be applied to prepare a composite micelles system for the delivery of poorly water-soluble active ingredients in functional food, cosmetics, and medicine. © 2023 Society of Chemical Industry.

Quantification of diaphragmatic dynamic dysfunction in septic patients by bedside ultrasound.

Although diaphragmatic dysfunction is an important indicator of severity of illness and poor prognosis in ICU patients, there is no convenient and practical method to monitor diaphragmatic function. This study was designed to analyze diaphragmatic dynamic dysfunction by bedside ultrasound in septic patients and provide quantitative evidence to assess diaphragm function systematically. This prospective observational study was conducted from October 2019 to January 2021 in the Department of Respiratory and Critical Care Medicine. 74 patients suffered from sepsis were recruited and divided into two groups, sepsis group 1 (2 ≤ SOFA ≤ 5, n = 41) and sepsis group 2 (SOFA > 5, n = 33). 107 healthy volunteers were randomly recruited as the control group. In all participants, the diaphragmatic thickness and excursion were measured directly and the dynamic parameters including thickening fraction (TF), EQB/EDB, Contractile velocity, and area under diaphragmatic movement curve (AUDMC) were calculated by bedside ultrasound during quiet breathing (QB) and deep breathing (DB). Each parameter among three groups was analyzed separately by covariance analysis, which was adjusted by age, sex, body mass index, MAP, hypertension, and diabetes. First, contractile dysfunction occurred before diaphragmatic atrophy both in sepsis group 1 and sepsis group 2. Second, compared with the control group, the dynamic parameters showed significant decrease in sepsis group 1 and more obvious change in sepsis group 2, including TF, EQB/EDB. Third, the maximum contractile velocity decreased in sepsis group 1, reflecting the damage of intrinsic contraction efficiency accurately. Finally, per breathing AUDMC in two septic groups were lower than those in control group. However, per minute AUDMC was compensated by increasing respiratory rate in sepsis group 1, whereas it failed to be compensated which indicated gradual failure of diaphragm in sepsis group 2. Diaphragmatic ultrasound can be used to quantitatively evaluate the severity of sepsis patients whose contractile dysfunction occurred before diaphragmatic atrophy. As dynamic parameters, TF and EQB/EDB are early indicator associated with diaphragmatic injury. Furthermore, maximum contractile velocity can reflect intrinsic contraction efficiency accurately. AUDMC can evaluate diaphragmatic breathing effort and endurance to overcome resistance loads effectively.

Development and Application of a Liquid Chromatography-Mass Spectrometry Method for Residual Iodixanol Quantification in AAV-Based Gene Therapy Product Development.

Adeno-associated viruses (AAVs) are nonenveloped viruses that have become popular gene transfer vectors to deliver DNA to target cells in clinical gene therapy. Iodixanol-based density gradient is one of the widely used purification methods for serotype-independent AAVs. However, residual iodixanol in AAV could be a safety concern, and further purification to remove this process-related impurity is typically needed. An analytical assay with high sensitivity is essential for the detection of residual iodixanol to ensure the safety of AAV products. We developed a liquid chromatography-mass spectrometry method with the limit of quantification of 0.01 µg/mL for residual iodixanol measurement in AAVs. The method also demonstrated linearity over four orders of magnitude that allows quantifying a high iodixanol concentration in in-process samples with excellent recovery and accuracy. In addition, we further explored a highly efficient purification method for removal of the residual iodixanol, to minimize the safety concern from iodixanol as a process impurity.

Predictive value of D-dimer and analysis of risk factors in pregnant women with suspected pulmonary embolism after cesarean section.

BACKGROUND: Acute pulmonary embolism (PE) is one of the leading causes of maternal mortality, and cesarean section is an established independent risk factor for PE. The diagnostic utility of D-dimer for PE in non-pregnant women has been well-established, but its role in women with suspected PE after cesarean section is unclear. Furthermore, the optimal threshold level in this patient population is unknown. Traditional D-dimer levels have low diagnostic specificity, resulting in many pregnant women being exposed to potentially harmful radiation despite negative diagnostic imaging results. This research aimed to optimize the clinical threshold for D-dimer to improve specificity while ensuring high sensitivity and to identify risk factors for PE after cesarean section. METHODS: This retrospective study of 289 women who underwent diagnostic imaging (ventilation/perfusion [V/Q] or computed tomographic pulmonary angiography [CTPA]) for suspected acute PE after cesarean delivery from 2010 to 2021 was conducted. Clinical data and laboratory indicators within 24 h postpartum including D-dimer levels were collected for analyses. RESULTS: The final analysis included 125 patients, among whom 33 were diagnosed with acute PE (incidence of 11.42%, 95% confidence interval 7.7-15.1). The receiver operating characteristic curve analysis suggested that a D-dimer cut-off value of 800 ng/mL had specificity of 25.26% and sensitivity of 100% for detecting PE. The cut-off value was adjusted to 1000 ng/mL with a specificity of 34.74% and a sensitivity of 96.67%. Using a D-dimer cut-off value of 800 ng/mL (instead of the conventional value of 500 ng/mL) increased the number of patients excluded from suspected PE from 9.6 to 18.4% without additional false-negative results. Of note, a history of known thrombophilia was significantly more common in patients with PE than in those without (P < 0.05). No other independent risk factors were noted in our study. CONCLUSIONS: The D-dimer cut-off value of 800 ng/mL ensures high sensitivity and increases specificity compared to the conventional threshold of 500 ng/mL. Utilizing this higher threshold can reduce the number of unnecessary CT and subsequently unnecessary radiation exposure, in women after cesarean delivery. Prospective studies should also be conducted to verify these results.

A Highly Sensitive LC-MS/MS Method for Targeted Quantitation of Lipase Host Cell Proteins in Biotherapeutics.

Identification and accurate quantitation of host cell proteins (HCPs) in biotherapeutic drugs has become increasingly important due to the negative impact of certain HCPs on the safety, stability, and other product quality of biotherapeutics. Recently, several lipase HCPs have been identified to potentially cause the enzymatic degradation of polysorbate, a widely used excipient in the formulation of biotherapeutics, which can severely impact the stability and product quality of drug products. In this study, we identified three lipase HCPs that were frequently detected in Chinese hamster ovary (CHO) cell cultures using shotgun proteomics, including phospholipase B-like 2 (PLBL2), lipoprotein lipase (LPL), and lysosomal acid lipase (LIPA). A targeted quantitation method for these three lipase HCPs was developed utilizing liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) with high-resolution multiple-reaction-monitoring (MRMhr) quantitation. The method demonstrated good sensitivity with low limit of quantitation (LLOQ) around 1 ng/mL, and linear dynamic range of three orders of magnitude for the three lipase HCPs. It has been applied for the characterization of process intermediates from various in-house monoclonal antibody (mAb) production. In addition, the method has also been used to evaluate the robustness of clearance for one of the lipase HCPs, PLBL2, under different column purification process conditions.

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

Astragaloside IV attenuates chronic intermittent hypoxia-induced myocardial injury by modulating Ca2+ homeostasis.

Obstructive sleep apnea syndrome (OSAS) is an important consequence of chronic intermittent hypoxia (CIH). Astragaloside IV (AS-IV) exerts multiple protective effects in diverse diseases. However, whether AS-IV can attenuate CIH-induced myocardial injury is unclear. In this study, rats exposed to CIH were established and treated with AS-IV for 4 weeks. In vitro, H9C2 cardiomyocytes subjected to CIH exposure were treated with AS-IV for 48 hours. Then the cardiac function, morphology, fibrosis, apoptosis and Ca2+ homeostasis were determined to assess cardiac damage. Results showed that AS-IV attenuated cardiac dysfunction and histological lesions in CIH rats. The increased TUNEL-positive cells and activated apoptotic proteins in CIH rats were reduced by AS-IV. We also noticed that AS-IV reversed the accumulation of Ca2+ and altered expressions of Ca2+ handling proteins (decreases of SERCA2a and RYR2, and increases of p-CaMKII and NCX1) under CIH exposure. Furthermore, CIH-induced reduction of SERCA2a activity was increased by AS-IV in rats. Similar results were also observed in H9C2 cells. Altogether, these findings indicate that AS-IV modulates Ca2+ homeostasis to inhibit apoptosis, protecting against CIH-induced myocardial injury eventually, suggesting it may be a potential agent for cardiac damage of OSAS patients. SIGNIFICANCE OF THE STUDY: Chronic intermittent hypoxia (CIH) is a great contributor of OSAS, which is closely associated with cardiovascular diseases. It is necessary for developing a promising drug to attenuate CIH-induced myocardial injury. This work suggests that AS-IV can attenuate myocardial apoptosis and calcium disruption, thus protecting against CIH-induced myocardial injury. It may represent a novel therapeutic for cardiac damage of OSAS.

Molecular cloning and characterization of a cathepsin L-like cysteine protease of Angiostrongylus cantonensis.

Angiostrongylus cantonensis is a parasitic nematode dwelling in the heart and pulmonary arteries of rats, which can cause angiostrongyliasis in human by accidental infections, manifested as eosinophilic meningitis or meningoencephalitis. Cysteine proteases are the major class of endopeptidases that are expressed at a high level in A. cantonensis, which suggests it may play key roles in pathogenesis of the disease. In this study, the biological properties of the cathepsin L-like peptidase (Ac-cathL) of A. cantonensis were investigated. The Ac-cathL gene was identified from the fourth stage cDNA library of A. cantonensis, and then cloned and characterized by bioinformatics analysis and heterologous expression. The open reading frame (ORF) of Ac-cathL (1068 bp) encodes a protein of 355 amino acids with an estimated molecular weight of 58.0 kDa. Sequence analysis and multiple sequence alignment demonstrated that Ac-cathL resembles members of cathepsin L family of other parasites and mammals. Stage-dependent mRNA expression analysis showed that Ac-cathL transcripts were expressed in all stages of A. cantonensis, with the highest expression in female stage. The recombinant Ac-cathL (rAc-cathL) expressed in Escherichia coli exhibited protease activity in acidic pH as demonstrated by gelatin zymography, as well as hydrolytic activity against natural substrates, including BSA, human IgG and human fibrinogen. Immunolocalization revealed that Ac-cathL is localized in tegument of the 18 days post infection stage and uterus of the female adult stage. Therefore, these results implied that the Ac-cathL plays important roles in host tissue migration, nutrition uptake and immune evasion.

Application of a label-free and domain-specific free thiol method in monoclonal antibody characterization.

Characterization of free thiol variants in antibody therapeutics is important for biopharmaceutical development, as the presence of free thiols may have an impact on aggregate formation, structural and thermal stability, as well as antigen-binding potency of monoclonal antibodies. Most current methods for free thiol quantification involve labeling of free thiol groups by different tagging molecules followed by UV, fluorescence or mass spectrometry (MS) detection. Here, we optimized a label-free liquid chromatography (LC)-UV/MS method for free thiol quantification at a subunit level and compared this method with two orthogonal and conventional approaches, Ellman's assay and peptide mapping with differential alkylation. This subunit unit approach was demonstrated to be able to provide domain-specific free thiol quantification and comparable results with labeling approaches, using a relatively simple and efficient workflow.

Discovery and Characterization of Histidine Oxidation Initiated Cross-links in an IgG1 Monoclonal Antibody.

Novel cross-links between an oxidized histidine and intact histidine, lysine, or cysteine residues were discovered and characterized from high-molecular weight (HMW) fractions of an IgG1 monoclonal antibody (mAb). The mAb HMW fractions were collected using preparative size-exclusion chromatography (SEC) and extensively characterized to understand the mechanism of formation of the nonreducible and covalently linked portion of the HMWs. The HMW fractions were IdeS digested, reduced, and analyzed by size-exclusion chromatography coupled with mass spectrometry (SEC-MS). The nonreducible cross-links were found to be enriched in the fragment crystallizable (Fc) region of the heavy chain, with a net mass increase of 14 Da. Detailed peptide mapping revealed as many as seven covalent cross-links in the HMW fractions, where oxidized histidines react with intact histidine, lysine, and free cysteine to form cross-links. It is the first time that histidine-cysteine (His-Cys) and histidine-lysine (His-Lys) in addition to histidine-histidine (His-His) cross-links were discovered in monoclonal antibody HMW species. The histidine oxidation hot spots were identified, which include conserved histidine residues His292 and His440 in the Fc region and His231 in the hinge region of the IgG1 mAb heavy chain. Their cross-linking partners include His231, His292, His440, and Cys233 in the hinge region and Lys297 in the Fc region. A cross-linking mechanism has been proposed that involves nucleophilic addition by histidine, cysteine, or lysine residues to the carbonyl-containing histidine oxidation intermediates to form the cross-links.

Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LC-MS/MS-Based Proteomics.

Liquid chromatography-mass spectrometry (LC-MS)-based proteomics is a powerful technique for the profiling of protein expression in cells in a high-throughput fashion. Herein we report a protocol using LC-MS/MS-based proteomics for the screening of enzymes involved in natural product biosynthesis, such as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) from bacterial strains. Taking advantage of the large size of modular NRPSs and PKSs (often >200 kDa), size-based separation (SDS-PAGE) is employed prior to LC-MS/MS analysis. Based upon the protein identifications obtained through software search, we can accurately pinpoint the expressed NRPS and/or PKS gene clusters from a given strain and growth condition. The proteomics screening result can be used to guide the discovery of potentially new nonribosomal peptide and polyketide natural products.

Mechanism of Inactivation of Neuronal Nitric Oxide Synthase by (S)-2-Amino-5-(2-(methylthio)acetimidamido)pentanoic Acid.

Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine to l-citrulline and the second messenger nitric oxide. Three mechanistic pathways are proposed for the inactivation of neuronal NOS (nNOS) by (S)-2-amino-5-(2-(methylthio)acetimidamido)pentanoic acid (1): sulfide oxidation, oxidative dethiolation, and oxidative demethylation. Four possible intermediates were synthesized. All compounds were assayed with nNOS, their IC50, KI, and kinact values were obtained, and their crystal structures were determined. The identification and characterization of the products formed during inactivation provide evidence for the details of the inactivation mechanism. On the basis of these studies, the most probable mechanism for the inactivation of nNOS involves oxidative demethylation with the resulting thiol coordinating to the cofactor heme iron. Although nNOS is a heme-containing enzyme, this is the first example of a NOS that catalyzes an S-demethylation reaction; the novel mechanism of inactivation described here could be applied to the design of inactivators of other heme-dependent enzymes.

Proteomics guided discovery of flavopeptins: anti-proliferative aldehydes synthesized by a reductase domain-containing non-ribosomal peptide synthetase.

Due to the importance of proteases in regulating cellular processes, the development of protease inhibitors has garnered great attention. Peptide-based aldehydes are a class of compounds that exhibit inhibitory activities against various proteases and proteasomes in the context of anti-proliferative treatments for cancer and other diseases. More than a dozen peptide-based natural products containing aldehydes have been discovered such as chymostatin, leupeptin, and fellutamide; however, the biosynthetic origin of the aldehyde functionality has yet to be elucidated. Herein we describe the discovery of a new group of lipopeptide aldehydes, the flavopeptins, and the corresponding biosynthetic pathway arising from an orphan gene cluster in Streptomyces sp. NRRL-F6652, a close relative of Streptomyces flavogriseus ATCC 33331. This research was initiated using a proteomics approach that screens for expressed enzymes involved in secondary metabolism in microorganisms. Flavopeptins are synthesized through a non-ribosomal peptide synthetase containing a terminal NAD(P)H-dependent reductase domain likely for the reductive release of the peptide with a C-terminal aldehyde. Solid-phase peptide synthesis of several flavopeptin species and derivatives enabled structural verification and subsequent screening of biological activity. Flavopeptins exhibit sub-micromolar inhibition activities against cysteine proteases such as papain and calpain as well as the human 20S proteasome. They also show anti-proliferative activities against multiple myeloma and lymphoma cell lines.

Gobichelin A and B: Mixed-Ligand Siderophores Discovered Using Proteomics.

"Omic" strategies have been increasingly applied to natural product discovery processes, with (meta-)genome sequencing and mining implemented in many laboratories to date. Using the proteomics-based discovery platform called PrISM (Proteomic Investigation of Secondary Metabolism), we discovered two new siderophores gobichelin A and B from Streptomyces sp. NRRL F-4415, a strain without a sequenced genome. Using the proteomics information as a guide, the 37 kb gene cluster responsible for production of gobichelins was sequenced and its 20 open reading frames interpreted into a biosynthetic scheme. This led to the targeted detection and structure elucidation of the new compounds produced by nonribosomal peptide (NRP) synthesis.

A proteomic survey of nonribosomal peptide and polyketide biosynthesis in actinobacteria.

Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small molecule products of these gene clusters still unknown. Here, we employed a "protein-first" method called PrISM (Proteomic Investigation of Secondary Metabolism) to screen 26 unsequenced actinomycetes using mass spectrometry-based proteomics for the targeted detection of expressed nonribosomal peptide synthetases or polyketide synthases. Improvements to the original PrISM screening approach (Nat. Biotechnol. 2009, 27, 951-956), for example, improved de novo peptide sequencing, have enabled the discovery of 10 NRPS/PKS gene clusters from 6 strains. Taking advantage of the concurrence of biosynthetic enzymes and the secondary metabolites they generate, two natural products were associated with their previously "orphan" gene clusters. This work has demonstrated the feasibility of a proteomics-based strategy for use in screening for NRP/PK production in actinomycetes (often >8 Mbp, high GC genomes) versus the bacilli (2-4 Mbp genomes) used previously.

Directed evolution of the nonribosomal peptide synthetase AdmK generates new andrimid derivatives in vivo.

Many lead compounds in the search for new drugs derive from peptides and polyketides whose similar biosynthetic enzymes have been difficult to engineer for production of new derivatives. Problems with generating multiple analogs in a single experiment along with lack of high-throughput methods for structure-based screening have slowed progress in this area. Here, we use directed evolution and a multiplexed assay to screen a library of >14,000 members to generate three derivatives of the antibacterial compound, andrimid. Another limiting factor in reengineering these mega-enzymes of secondary metabolism has been that commonly used hosts such as Escherichia coli often give lower product titers, so our reengineering was performed in the native producer, Pantoea agglomerans. This integrated in vivo approach can be extended to larger enzymes to create analogs of natural products for bioactivity testing.

Proteomics-based discovery of koranimine, a cyclic imine natural product.

Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are large enzymes responsible for the biosynthesis of medically and ecologically important secondary metabolites. In a previous report, we described a proteomics approach to screen for expressed NRPSs or PKSs from bacteria with or without sequenced genomes. Here we used this proteome mining approach to discover a novel natural product arising from rare adenylation (A) and reductase (Red) domains in its biosynthetic machinery. We also cloned the entire gene cluster and elucidated the biosynthesis of the new compound, which is produced by an unsequenced Bacillus sp. isolated from soil collected in Koran, Louisiana.