Terminal Cyclization Research Articles

Improving In Vivo Brain Delivery of Monoclonal Antibody Using Novel Cyclic Peptides.

Many proteins can be used to treat brain diseases; however, the presence of the blood–brain barrier (BBB) creates an obstacle to delivering them into the brain. Previously, various molecules were delivered through the paracellular pathway of the BBB via its modulation, using ADTC5 and HAV6 peptides. This study goal was to design new cyclic peptides with N-to-C terminal cyclization for better plasma stability and modulation of the BBB. Cyclic HAVN1 and HAVN2 peptides were derived from a linear HAV6 peptide. Linear and N-to-C terminal cyclic ADTHAV peptides were designed by combining the sequences of ADTC5 and HAV6. These novel cyclic peptides were used to deliver an IRdye800CW-labeled IgG monoclonal antibody into the brain. Cyclic HAVN1 and HAVN2 peptides deliver IgG into the brain, while the parent linear HAV6 peptide does not. Cyclic and linear ADTHAV and ADTC5 peptides enhanced brain delivery of IgG mAb, in which cyclic ADTHAV peptide was better than linear ADTHAV (p = 0.07). Cyclic ADTHAV and ADTC5 influenced the distribution of IgG mAb in other organs while HAV6, HAVN1 and HAVN2 did not. In summary, the novel cyclic peptides are generally better BBB modulators than their linear counterparts for delivering IgG mAb into the brain.

Crystal structure of TchmY from Actinoplanes teichomyceticus.

Moenomycin-type antibiotics are phosphoglycolipids that are notable for their unique modes of action and have proven to be useful in animal nutrition. The gene clusters tchm from Actinoplanes teichomyceticus and moe from Streptomyces are among a limited number of known moenomycin-biosynthetic pathways. Most genes in tchm have counterparts in the moe cluster, except for tchmy and tchmz, the functions of which remain unknown. Sequence analysis indicates that TchmY belongs to the isoprenoid enzyme C2-like superfamily and may serve as a prenylcyclase. The enzyme was proposed to be involved in terminal cyclization of the moenocinyl chain in teichomycin, leading to the diumycinol chain of moenomycin isomers. Here, recombinant TchmY protein was expressed in Escherichia coli and its crystal structure was solved by SIRAS. Structural analysis and comparison with other prenylcyclases were performed. The overall fold of TchmY consists of an (α/α)6-barrel, and a potential substrate-binding pocket is found in the central chamber. These results should provide important information regarding the biosynthetic basis of moenomycin antibiotics.

Forced Degradation Study of Monoclonal Antibody Using Two- Dimensional Liquid Chromatography

Monoclonal antibody (mAb) products are extraordinarily heterogeneous due to the presence of a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, glycation, terminal cyclization, aggregation, and fragmentation. Forced degradation study is a common practice to assess the potential modifications and degradation pathways of mAbs upon extreme conditions, including light exposure and extreme local pH. The degraded samples are subject to characterization using a wide array of analyses, including ion exchange chromatography (IEC) for charge variants and size exclusion chromatography (SEC) for size variants. In this proof-of-concept study, a two-dimensional liquid chromatography (2D-LC) approach was successfully applied to mAb forced degradation samples for evaluation of size distribution in the IEC charge profile. The charge variant peaks in first-dimension (1D) IEC were fractioned by a heart-cutting schedule, and the integrated peak parking feature accommodated later-eluting 1D cuts when the previous ones were still under second-dimension (2D) analysis. Thus, for a singly IEC injection, all IEC fractions were acquired by the 2D for SEC analysis. This study demonstrated that 2D SEC was compatible with 1D IEC for 2D-LC analysis. The method sensitivity was sufficient to determine the aggregates and fragments in each individual IEC cut. The calculated total aggregates and fragments in the stressed mAb-1 samples were comparable to those detected by one dimensional SEC. Most importantly, the 2D IEC-SEC approach was capable of bridging the mAb charge and size variants in a real-time and efficient manner. It was observed that aggregates were enriched in the most basic region in the IEC charge profile, especially in the highly degraded samples. This is the first IEC-SEC 2D-LC application for intact antibody analysis as per the authors’ knowledge.

A new tool for monoclonal antibody analysis

Monoclonal antibody (mAb) products are extraordinarily heterogeneous due to the presence of a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, glycation, and terminal cyclization. The modifications in different domains of the antibody molecule can result in different biological consequences. Therefore, characterization and routine monitoring of domain-specific modifications are essential to ensure the quality of the therapeutic antibody products. For this purpose, a rapid and informative methodology was developed to examine the heterogeneity of individual domains in mAb products. A recently discovered endopeptidase, IdeS, cleaves heavy chains below the hinge region, producing F(ab')2 and Fc fragments. Following reduction of disulfide bonds, three antibody domains (LC, Fd, and Fc/2) can be released for further characterization. Subsequent analyses by liquid chromatography/mass spectrometry, capillary isoelectric focusing, and glycan mapping enable domain-specific profiling of oxidation, charge heterogeneity, and glycoform distribution. When coupled with reversed phase chromatography, the unique chromatographic profile of each molecule offers a simple strategy for an identity test, which is an important formal test for biopharmaceutical quality control purposes. This methodology is demonstrated for a number of IgGs of different subclasses (IgG1, IgG2, IgG4), as well as an Fc fusion protein. The presented technique provides a convenient platform approach for scientific and formal therapeutic mAb product characterization. It can also be applied in regulated drug substance batch release and stability testing of antibody and Fc fusion protein products, in particular for identity and routine monitoring of domain-specific modifications.

The role of oxyanion holes in the structure and function of type III polyketide synthases (768.18)

The objective of this study was to combine the tools of protein crystallography and enzyme kinetics to explore the structural mechanisms of type III polyketide synthases (PKSs). Type III PKSs are proteins involved in diverse metabolic processes including cell signaling, pathogen defense and the development of reproductive cells. Mechanistically, these enzymes generate metabolites through the repetitive addition of acetate units onto a starter molecule; a process that produces reactive intermediates that are ultimately cyclized into the final product. How these enzymes control their reactive intermediates and terminal cyclizations is not fully understood and is a major hindrance to protein engineering efforts. We present here our results describing the role of oxyanion holes in the mechanism of type III PKSs. Crystallographic studies of reaction analogs trapped in the active site revealed that remnants of an ancient oxyanion hole are involved in stabilizing reactive intermediates. Unexpectedly, we observed that a small molecule, metazachlor, has different conformations in the active site of functionally distinct PKSs. Enzyme kinetic studies supported our structural findings by establishing that metazachlor differentially inhibits type III PKSs based on their terminal cyclization steps. These structure‐function studies are being used as a guide for altering the terminal cyclization preferences of type III PKSs to build novel compounds.Grant Funding Source: Supported by Howard Hughes Medical Institute and National Science Foundation

Prediction of Monomer Isomery in Florine: A Workflow Dedicated to Nonribosomal Peptide Discovery

Nonribosomal peptides represent a large variety of natural active compounds produced by microorganisms. Due to their specific biosynthesis pathway through large assembly lines called NonRibosomal Peptide Synthetases (NRPSs), they often display complex structures with cycles and branches. Moreover they often contain non proteogenic or modified monomers, such as the D-monomers produced by epimerization. We investigate here some sequence specificities of the condensation (C) and epimerization (E) domains of NRPS that can be used to predict the possible isomeric state (D or L) of each monomer in a putative peptide. We show that C- and E- domains can be divided into 2 sub-regions called Up-Seq and Down-Seq. The Up-Seq region corresponds to an InterPro domain (IPR001242) and is shared by C- and E-domains. The Down-Seq region is specific to the enzymatic activity of the domain. Amino-acid signatures (represented as sequence logos) previously described for complete C-and E-domains have been restricted to the Down-Seq region and amplified thanks to additional sequences. Moreover a new Down-Seq signature has been found for Ct-domains found in fungi and responsible for terminal cyclization of the peptides. The identification of these signatures has been included in a workflow named Florine, aimed to predict nonribosomal peptides from NRPS sequence analyses. In some cases, the prediction of isomery is guided by genus-specific rules. Florine was used on a Pseudomonas genome to allow the determination of the type of pyoverdin produced, the update of syringafactin structure and the identification of novel putative products.

( S)-13-hydroxygeranylgeraniol-derived furanoditerpenes from Bifurcaria bifurcata

Two new diterpenes were isolated from a sample of the brown alga Bifurcaria bifurcata collected from Brittany on the Atlantic coast and their structures established by spectroscopic methods. The new diterpenes are derived from ( S)-13-hydroxygeranylgeraniol by terminal cyclization and oxidation leading to a furan-3-yl ring or a β, γ-epoxy-γ-lactone. One of them showed a cytotoxic effect to fertilized sea urchin eggs. The chemical shifts of the methyl groups and quaternary carbons in ( S)-13-hydroxygeranylgeraniol have been revised to take account of the results obtained in a 2D NMR long-range C H correlation experiment and the absolute configuration at C-13 determined for the first time. The geographical variation in the diterpenoid composition of B. bifurcata was also studied.