Improving metabolite identification for complex peptides using MassMetaSite

Improving metabolite identification for complex peptides using MassMetaSite

70th ASMS Conference on Mass Spectrometry. June 2022

Ismael Zamora; Tatiana Radchenko; Fabien Fontaine; Albert Garriga

Lead Molecular Design, S.L., Sant Cugat del Valles, Spain

Abstract

Introduction

A commonly used strategy in the peptide therapeutics field to introduce chemical modifications such as cyclisation, changing the stereochemistry of an amino acid, substitution of natural amino acids to chemically modified ones and others to improve their efficacy and ADME profile. The study of the metabolic degradation products for synthetically modified therapeutic peptides using LC-MS is a challenging issue. Different chemoinformatics approaches are used for automated metabolite identification. These tools propose metabolite structures based on the combination of metabolite prediction and analysis of MS data. This makes building the virtual set of all potential metabolites time and computational intensive. Fragmentation analysis requires even more computation time. Finally, a third challenge related to the depiction of the parent and the metabolites.

Methods

New algorithms that address the challenges in highly modified therapeutic peptide structure elucidation have been developed. The peak detection algorithm was improved to use the Most Abundant Isotope for parent and potential metabolites. A new algorithm is producing all virtual metabolites applying the library of chemical reactions to each monomer while maintaining the connection to the atoms. A third improvement is the fragmentation algorithm that has two layers of analysis, one at the monomer level and the other one at the bond level. Finally, we will also show the results of the implemented algorithm for the analysis results visualization.

Preliminary data

Using MassMetaSite we analyzed a collection of experimental data for a set of peptides where the data was collected on a Q-Exactive Thermo instrument. The metabolite identification study was performed using a peptide set that included eight compounds: somatostatin and its seven synthetic analogues. All test compounds were incubated in serum. These peptides are all cyclic peptides and seven of them had unnatural amino acids. The structural assignments were performed for 17 degradation products with high mass accuracy (ppm<3). Most of the metabolites resulting from one or two metabolic modifications were produced by amide hydrolysis and were detected by the new algorithm. Each of these compounds showed a suitable fragmentation pattern that could be compared to the parent by the fragmentation algorithm to assign the shifted and non-shifted fragments. All the metabolites were checked manually including a review of the assigned fragments. Metabolites were considered as reliable because the fragmentation was adequate, isotope pattern was as expected, the small differences between the m/z of observed and theoretical, and the mass score was high. As previously reported in the literature, the first two most abundant metabolites with assigned structure correspond to cleavage of the linear part of the somatostatin. Finally, we analyzed experimental data for the insulin and semaglutide data where the data was collected on a Waters QToF. Insulin is a cyclic peptide that contains 3 disulfide bridges and semaglutide is a linear peptide that contains linkages and fatty acid. The visualization algorithm developed allows to show results for these complex structures in a such a manner that it is easy to interpret due to the constraint structure alignment between the substrate and the metabolite (keeping same orientation), a possibility to combine monomer and atom/bond notation. Therefore, the metabolic changes in the structure can be easily seen by the User.

 

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Comparison of CID and EAD fragmentation with automated assignment for small molecule structure elucidation

Comparison of CID and EAD fragmentation with automated assignment for small molecule structure elucidation

70th ASMS Conference on Mass Spectrometry. June 2022

Ismael Zamora1; Christopher Kochansky2; Fabien Fontaine1; Kevin P Bateman2; Jason Causon3; Jose Castro-Perez4; Rolf Kern5

1Lead Molecular Design, S.L., Sant Cugat del Valles, Spain; 2Merck & Co., Inc., Kenilworth, NJ; 3SCIEX, Concord, ON; 4Sciex, Framingham, MA; 5SCIEX, Redwood City, CA

 

Abstract

Introduction

Collisional-induced dissociation (CID) has been the main workhorse for small molecule structure elucidation in drug metabolite identification studies.  Software tools, such as Massmetasite, have been developed to assist in the automatic interpretation of CID MS/MS spectra.  The challenge with CID is that for many drug metabolites, non-informative fragmentation occurs, resulting in a lack of useful structural assignments for these metabolites.  Commercialization of electron activated dissociation (EAD) on a quadrupole time of flight mass spectrometer provides a potentially powerful new tool for small molecule structure elucidation in drug discovery.  Automated interpretation of EAD MS/MS spectra using existing algorithms needs to be tested, modified, and implemented. A comparison of CID and EAD fragmentation using automated interpretation will be presented in this work.

Methods

Small molecule drugs were incubated in rat hepatocytes at 1 µM. Time points:  0, 30 and 120 min were pulled from the incubation and quenched with 1 volume of CH3CN. Samples were vortexed, centrifuged, and the supernatant transferred to an HPLC vial for analysis.

LC separation was performed on a Phenomenex Luna Omega Polar C18, 150 mm column using 0.5µL or 5 µL injection volumes. Gradient separation 0.1% formic acid in water and acetonitrile was performed over 4.75 minutes from 5%B to 95%B with a total of 6.5 minutes.

The samples were analyzed using ZenoTOF 7600 CID-IDA(DDA) and EIEIO IDA(DDA). TOFMS was scanned between m/z 100-1000, CID/EAD MS/MS from 60-1000.  Data was processed in MassMetaSite with CID and EAD fragmentation.

Preliminary data

EAD is a free electron fragmentation mode available recently introduced to accurate mass LC-MS/MS. It utilizes high energy electrons which allows for the dissociation of singly charged precursors, in this work an electron kinetic energy of 10 eV was utilized.  The MS/MS spectra show significant increases in the number of fragment ions observed when going from Zeno CID to Zeno EAD spectra.  The larger number of fragment ions makes it even more important for automated assignment using software tools such as Massmetasite.  Many of the new fragments are the result of radical bond cleavage driven by electron-impact excitation of ions from organics (EIEIO) mechanism.  Typical software algorithms for MS/MS focus on even electron species, typical of CID fragmentation.  With EAD and the production of odd electron fragments, modification of the algorithm is required.  The results to date show much richer fragmentation spectra using EAD versus CID and that the modified algorithm can assign the new odd electron fragments.

 

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Predicting drug metabolism: a site of metabolism prediction tool applied to the cytochrome P450 2C9

Predicting drug metabolism: a site of metabolism prediction tool applied to the cytochrome P450 2C9

June 2003.

Zamora, Ismael; Afzelius, Lovisa; Cruciani, Gabriele

Abstract

The aim of the present study is to develop a method for predicting the site at which molecules will be metabolized by CYP 2C9 (cytochrome P450 2C9) using a previously reported protein homology model of the enzyme. Such a method would be of great help in designing new compounds with a better pharmacokinetic profile, or in designing prodrugs where the compound needs to be metabolized in order to become active.

The methodology is based on a comparison between alignment-independent descriptors derived from GRID Molecular Interaction Fields for the CYP 2C9 active site, and a distance-based representation of the substrate. The predicted site of metabolism is reported as a ranking list of all the hydrogen atoms of each substrate molecule. Eighty-seven CYP 2C9-catalyzed oxidative reactions reported in the literature have been analyzed. In more than 90% of these cases, the hydrogen atom ranked at the first, second, or third position was the experimentally reported site of oxidation.

MetaSite:  Understanding Metabolism in Human Cytochromes from the Perspective of the Chemist

MetaSite:  Understanding Metabolism in Human Cytochromes from the Perspective of the Chemist

September 2005.

Cruciani G, Carosati E, De Boeck B, Ethirajulu K, Mackie C, Howe T, Vianello R

Abstract

Identification of metabolic biotransformations can significantly affect the drug discovery process. Since bioavailability, activity, toxicity, distribution, and final elimination all depend on metabolic biotransformations, it would be extremely advantageous if this information could be produced early in the discovery phase. Once obtained, this information can help chemists to judge whether a potential candidate should be eliminated from the pipeline or modified to improve chemical stability or safety of new compounds. The use of in silico methods to predict the site of metabolism in phase I cytochrome-mediated reactions is a starting point in any metabolic pathway prediction.

This paper presents a new method, specifically designed for chemists, that provides the cytochrome involved and the site of metabolism for any human cytochrome P450 (CYP) mediated reaction acting on new substrates. The methodology can be applied automatically to all the cytochromes for which 3D structure is known and can be used by chemists to detect positions that should be protected in order to avoid metabolic degradation or to check the suitability of a new scaffold or prodrug.

The fully automated procedure is also a valuable new tool in early ADME-Tox assays (absorption, distribution, metabolism, and excretion toxicity assays), where drug safety and metabolic profile patterns must be evaluated as soon, and as early, as possible.

Comparison of methods for the prediction of the Metabolic sites for CYP3A4 – Mediated metabolic reactions

Comparison of methods for the prediction of the Metabolic sites for CYP3A4 – Mediated metabolic reactions

June 2006.

Diansong ZhouLovisa AfzeliusScott W. GrimmTommy B. AnderssonRandy J. Zauhar and Ismael Zamora

Abstract

Predictions of the metabolic sites for new chemical entities, synthesized or only virtual, are important in the early phase of drug discovery to guide chemistry efforts in the synthesis of new compounds with reduced metabolic liability. This information can now be obtained from in silico predictions, and therefore, a thorough and unbiased evaluation of the computational techniques available is needed. Several computational methods to predict the metabolic hot spots are emerging.

In this study, metabolite identification using MetaSite and a docking methodology, GLUE, were compared. Moreover, the published CYP3A4 crystal structure and computed CYP3A4 homology models were compared for their usefulness in predicting metabolic sites. A total of 227 known CYP3A4 substrates reported to have one or more metabolites adding up to 325 metabolic pathways were analyzed. Distance-based fingerprints and four-point pharmacophore derived from GRID molecular interaction fields were used to characterize the substrate and protein in MetaSite and the docking methodology, respectively. The CYP3A4 crystal structure and homology model with the reactivity factor enabled achieved a similar prediction success (78%) using the MetaSite method.

The docking method had a relatively lower prediction success (∼57% for the homology model), although it still may provide useful insights for interactions between ligand and protein, especially for uncommon reactions. The MetaSite methodology is automated, rapid, and has relatively accurate predictions compared with the docking methodology used in this study.