Crosstalk between mismatch repair and base excision repair in human gastric cancer
By Simonelli Valeria; Leuzzi Giuseppe; Basile Giorgia; D’Errico Mariarosaria; Fortini Paola; Franchitto Annapaola; Parlanti Eleonora; Giuliani Alessandro; Dogliotti Eugenia; Viti Valentina; et al
From Oncotarget (2017), 8(49), 84827-84840, Language: English, Database: MEDLINE
DNA repair gene expression in a set of gastric cancers suggested an inverse association between the expression of the mismatch repair (MMR) gene MLH1 and that of the base excision repair (BER) gene DNA polymerase β (Polβ). To gain insight into possible crosstalk of these two repair pathways in cancer, we analysed human gastric adenocarcinoma AGS cells over-expressing Polβ or Polβ active site mutants, alone or in combination with MLH1 silencing. Next, we investigated the cellular response to the alkylating agent methyl methanesulfonate (MMS) and the purine analogue 6-thioguanine (6-TG), agents that induce lesions that are substrates for BER and/or MMR. AGS cells over-expressing Polβ were resistant to 6-TG to a similar extent as when MLH1 was inactivated while inhibition of O(6)-methylguanine-DNA methyltransferase (MGMT) was required to detect resistance to MMS. Upon either treatment, the association with MLH1 down-regulation further amplified the resistant phenotype. Moreover, AGS cells mutated in Polβ were hypersensitive to both 6-TG and MMS killing and their sensitivity was partially rescued by MLH1 silencing. We provide evidence that the critical lethal lesions in this new pathway are double strand breaks that are exacerbated when Polβ is defective and relieved when MLH1 is silenced. In conclusion, we provide evidence of crosstalk between MLH1 and Polβ that modulates the response to alkylation damage. These studies suggest that the Polβ/MLH1 status should be taken into consideration when designing chemotherapeutic approaches for gastric cancer.
Structure-Based Identification of HIV-1 Nucleocapsid Protein Inhibitors Active against Wild-Type and Drug-Resistant HIV-1 Strains.
Mori M, Kovalenko L, Malancona S, Saladini F, De Forni D, Pires M, Humbert N, Real E, Botzanowski T, Cianférani S, Giannini A, Dasso Lang MC, Cugia G, Poddesu B, Lori F, Zazzi M, Harper S, Summa V, Mely Y, Botta M. (2017)
ACS Chem Biol. 2017 Dec 28.
Phosphorylation of huntingtin at residue T3 is decreased in Huntington’s disease and modulates mutant huntingtin protein conformation.
By Cariulo, Cristina; Azzollini, Lucia; Verani, Margherita; Martufi, Paola; Boggio, Roberto; Chiki, Anass; Deguire, Sean M.; Cherubini, Marta; Gines, Silvia; Marsh, J. Lawrence; et al
From Proceedings of the National Academy of Sciences of the United States of America (2017), 114(50), E10809-E10818. Language: English, Database: CAPLUS, DOI:10.1073/pnas.1705372114
Posttranslational modifications can have profound effects on the biol. and biophys. properties of proteins assocd. with misfolding and aggregation. However, their detection and quantification in clin. samples and an understanding of the mechanisms underlying the pathol. properties of misfolding- and aggregation-prone proteins remain a challenge for diagnostics and therapeutics development. We have applied an ultrasensitive immunoassay platform to develop and validate a quant. assay for detecting a posttranslational modification (phosphorylation at residue T3) of a protein assocd. with polyglutamine repeat expansion, namely Huntingtin, and characterized its presence in a variety of preclin. and clin. samples. We find that T3 phosphorylation is greatly reduced in samples from Huntington’s disease models and in Huntington’s disease patients, and we provide evidence that bona-fide T3 phosphorylation alters Huntingtin exon 1 protein conformation and aggregation properties. These findings have significant implications for both mechanisms of disease pathogenesis and the development of therapeutics and diagnostics for Huntington’s disease.
Validation of Ultrasensitive Mutant Huntingtin Detection in Human Cerebrospinal Fluid by Single Molecule Counting Immunoassay.
By Fodale, Valentina; Boggio, Roberto; Daldin, Manuel; Cariulo, Cristina; Spiezia, Maria Carolina; Byrne, Lauren M.; Leavitt, Blair R.; Wild, Edward J.; Macdonald, Douglas; Weiss, Andreas; et al
From Journal of Huntington’s Disease (2017), 6(4), 349-361. Language: English, Database: CAPLUS, DOI:10.3233/jhd-170269
The measurement of disease-relevant biomarkers has become a major component of clin. trial design, but in the absence of rigorous clin. and anal. validation of detection methodol., interpretation of results may be misleading. In Huntington’s disease (HD), measurement of the concn. of mutant huntingtin protein (mHTT) in cerebrospinal fluid (CSF) of patients may serve as both a disease progression biomarker and a pharmacodynamic readout for HTT-lowering therapeutic approaches. We recently published the quantification of mHTT levels in HD patient CSF by a novel ultrasensitive immunoassay-based technol. and here anal. validate it for use. Objective: : This work aims to anal. and clin. validate our ultrasensitive assay for mHTT measurement in human HD CSF, for application as a pharmacodynamic biomarker of CNS mHTT lowering in clin. trials. Methods: : The single mol. counting (SMC) assay is an ultrasensitive bead-based immunoassay where upon specific recognition, dye-labeled antibodies are excited by a confocal laser and emit fluorescent light as a readout. The detection of mHTT by this technol. was clin. validated following established Food and Drug Administration and European Medicine Agency guidelines. Results: : The SMC assay was demonstrated to be accurate, precise, specific, and reproducible. While no matrix influence was detected, a list of interfering substances was compiled as a guideline for proper collection and storage of patient CSF samples. In addn., a set of recommendations on result interpretation is provided. Conclusions: : This SMC assay is a robust and ultrasensitive method for the relative quantification of mHTT in human CSF.
Largazole Analogues as Histone Deacetylase Inhibitors and Anticancer Agents: An Overview of Structure-Activity Relationships.
By Poli Giulio; Botta Maurizio; Di Fabio Romano; Ferrante Luca; Di Fabio Romano; Summa Vincenzo
From ChemMedChem (2017), 12(23), 1917-1926, Language: English, Database: MEDLINE
Since the time of its identification, the natural compound largazole rapidly caught the attention of the medicinal chemistry community for its impressive potency as an inhibitor of histone deacetylases (HDACs) and its strong antiproliferative activity against a broad panel of cancer cell lines. The design of largazole analogues is an expanding field of study, due to their remarkable potential as novel anticancer therapeutics. At present, a large ensemble of largazole analogues has been reported, allowing the identification of important structure-activity relationships (SAR) that can guide the design of novel compounds with improved HDAC inhibitory profiles, anticancer activity, and pharmacokinetic properties. The aim of this review is to concisely summarize the information obtained by biological evaluations of the various largazole analogues reported to date, with particular attention given to the latest analogues, as well as to analyze the various SAR obtained from this data, with the purpose of providing useful guidelines for the development of novel potent and selective HDAC inhibitors to be used as anticancer agents.
Insights on the conformational dynamics of human frataxin through modifications of loop-1.
By Noguera Martin E; Vazquez Diego S; Herrera Maria Georgina; Roman Ernesto A; Aran Martin; Smal Clara; Alaimo Nadine; Gallo Mariana; Santos Javier
From Archives of biochemistry and biophysics (2017), 636123-137, Language: English, Database: MEDLINE
Human frataxin (FXN) is a highly conserved mitochondrial protein involved in iron homeostasis and activation of the iron-sulfur cluster assembly. FXN deficiency causes the neurodegenerative disease Friedreich’s Ataxia. Here, we investigated the effect of alterations in loop-1, a stretch presumably essential for FXN function, on the conformational stability and dynamics of the native state. We generated four loop-1 variants, carrying substitutions, insertions and deletions. All of them were stable and well-folded proteins. Fast local motions (ps-ns) and slower long-range conformational dynamics (μs-ms) were altered in some mutants as judged by NMR. Particularly, loop-1 modifications impact on the dynamics of a distant region that includes residues from the β-sheet, helix α1 and the C-terminal. Remarkably, all the mutants retain the ability to activate cysteine desulfurase, even when two of them exhibit a strong decrease in iron binding, revealing a differential sensitivity of these functional features to loop-1 perturbation. Consequently, we found that even for a small and relatively rigid protein, engineering a loop segment enables to alter conformational dynamics through a long-range effect, preserving the native-state structure and important aspects of function.
Vitamin B12 ameliorates the phenotype of a mouse model of DiGeorge syndrome.
By Lania Gabriella; Colonna Vincenza; Baldini Antonio; Bresciani Alberto; Bisbocci Monica; Francone Alessandra; Altamura Sergio; Baldini Antonio
From Human molecular genetics (2017), 26(22), 4540, Language: English, Database: MEDLINE
Pathological conditions caused by reduced dosage of a gene, such as gene haploinsufficiency, can potentially be reverted by enhancing the expression of the functional allele. In practice, low specificity of therapeutic agents, or their toxicity reduces their clinical applicability. Here, we have used a high throughput screening (HTS) approach to identify molecules capable of increasing the expression of the gene Tbx1, which is involved in one of the most common gene haploinsufficiency syndromes, the 22q11.2 deletion syndrome. Surprisingly, we found that one of the two compounds identified by the HTS is the vitamin B12. Validation in a mouse model demonstrated that vitamin B12 treatment enhances Tbx1 gene expression and partially rescues the haploinsufficiency phenotype. These results lay the basis for preclinical and clinical studies to establish the effectiveness of this drug in the human syndrome.
Discovery by organism based high-throughput screening of new multi-stage compounds affecting Schistosoma mansoni viability, egg formation and production.
By Guidi Alessandra; Lalli Cristiana; Gimmelli Roberto; Saccoccia Fulvio; Ruberti Giovina; Nizi Emanuela; Andreini Matteo; Harper Steven; Gennari Nadia; Bresciani Alberto
From PLoS neglected tropical diseases (2017), 11(10), e0005994, Language: English, Database: MEDLINE
Schistosomiasis, one of the most prevalent neglected parasitic diseases affecting humans and animals, is caused by the Platyhelminthes of the genus Schistosoma. Schistosomes are the only trematodes to have evolved sexual dimorphism and the constant pairing with a male is essential for the sexual maturation of the female. Pairing is required for the full development of the two major female organs, ovary and vitellarium that are involved in the production of different cell types such as oocytes and vitellocytes, which represent the core elements of the whole egg machinery. Sexually mature females can produce a large number of eggs each day. Due to the importance of egg production for both life cycle and pathogenesis, there is significant interest in the search for new strategies and compounds not only affecting parasite viability but also egg production. Here we use a recently developed high-throughput organism-based approach, based on ATP quantitation in the schistosomula larval stage of Schistosoma mansoni for the screening of a large compound library, and describe a pharmacophore-based drug selection approach and phenotypic analyses to identify novel multi-stage schistosomicidal compounds. Interestingly, worm pairs treated with seven of the eight compounds identified show a phenotype characterized by defects in eggshell assemblage within the ootype and egg formation with degenerated oocytes and vitelline cells engulfment in the uterus and/or oviduct. We describe promising new molecules that not only impair the schistosomula larval stage but also impact juvenile and adult worm viability and egg formation and production in vitro.
Nuclear factor (erythroid-derived 2)-like 2 (NRF2) drug discovery: Biochemical toolbox to develop NRF2 activators by reversible binding of Kelch-like ECH-associated protein 1 (KEAP1).
By Bresciani, Alberto; Missineo, Antonino; Gallo, Mariana; Cerretani, Mauro; Fezzardi, Paola; Tomei, Licia; Cicero, Daniel Oscar; Altamura, Sergio; Santoprete, Alessia; Ingenito, Raffaele; et al
From Archives of Biochemistry and Biophysics (2017), 631, 31-41. Language: English, Database: CAPLUS, DOI:10.1016/j.abb.2017.08.003
Mechanisms that activate innate antioxidant responses, as a way to mitigate oxidative stress at the site of action, hold much therapeutic potential in diseases, such as Parkinson’s disease, Alzheimer’s disease and Huntington’s disease, where the use of antioxidants as monotherapy has not yielded pos. results. The nuclear factor NRF2 is a transcription factor whose activity upregulates the expression of cell detoxifying enzymes in response to oxidative stress. NRF2 levels are modulated by KEAP1, a sensor of oxidative stress. KEAP1 binds NRF2 and facilitates its ubiquitination and subsequent degrdn. Recently, compds. that reversibly disrupt the NRF2-KEAP1 interaction have been described, opening the field to a new era of safer NRF2 activators. This paper describes a set of new, robust and informative biochem. assays that enable the selection and optimization of non-covalent KEAP1 binders. These include a time-resolved fluorescence resonance energy transfer (TR-FRET) primary assay with high modularity and robustness, a surface plasmon resonance (SPR) based KEAP1 direct binding assay that enables the quantification and anal. of full kinetic binding parameters and finally a 1H-15N heteronuclear single quantum coherence (HSQC) NMR assay suited to study the interaction surface of KEAP1 with residue-specific information to validate the interaction of ligands in the KEAP1 binding site.
Polyglutamine expansion affects huntingtin conformation in multiple Huntington’s disease models.
By Daldin, Manuel; Fodale, Valentina; Cariulo, Cristina; Azzollini, Lucia; Verani, Margherita; Martufi, Paola; Spiezia, Maria Carolina; Deguire, Sean M.; Cherubini, Marta; Macdonald, Douglas; et al
From Scientific Reports (2017), 7(1), 1-15. Language: English, Database: CAPLUS, DOI:10.1038/s41598-017-05336-7
Conformational changes in disease-assocd. or mutant proteins represent a key pathol. aspect of Huntington’s disease (HD) and other protein misfolding diseases. Using immunoassays and biophys. approaches, we and others have recently reported that polyglutamine expansion in purified or recombinantly expressed huntingtin (HTT) proteins affects their conformational properties in a manner dependent on both polyglutamine repeat length and temp. but independent of HTT protein fragment length. These findings are consistent with the HD mutation affecting structural aspects of the amino-terminal region of the protein, and support the concept that modulating mutant HTT conformation might provide novel therapeutic and diagnostic opportunities. We now report that the same conformational TR-FRET based immunoassay detects polyglutamine- and temp.-dependent changes on the endogenously expressed HTT protein in peripheral tissues and post-mortem HD brain tissue, as well as in tissues from HD animal models. We also find that these temp.- and polyglutamine-dependent conformational changes are sensitive to bona-fide phosphorylation on S13 and S16 within the N17 domain of HTT. These findings provide key clin. and preclin. relevance to the conformational immunoassay, and provide supportive evidence for its application in the development of therapeutics aimed at correcting the conformation of polyglutamine-expanded proteins as well as the pharmacodynamics readouts to monitor their efficacy in preclin. models and in HD patients.
High-Affinity “Click” RGD Peptidomimetics as Radiolabeled Probes for Imaging av ß3 Integrin.
By Piras Monica; Testa Andrea; Fleming Ian N; Dall’Angelo Sergio; Andriu Alexandra; Zanda Matteo; Menta Sergio; Menta Sergio; Mori Mattia; Brown Gavin D; et al
From ChemMedChem (2017), 12(14), 1142-1151, Language: English, Database: MEDLINE
Nonpeptidic Arg-Gly-Asp (RGD)-mimic ligands were designed and synthesized by click chemistry between an arginine-azide mimic and an aspartic acid-alkyne mimic. Some of these molecules combine excellent in vitro properties (high αv β3 affinity, selectivity, drug-like logD, high metabolic stability) with a variety of radiolabeling options (e.g., tritium and fluorine-18, plus compatibility with radio-iodination), not requiring the use of chelators or prosthetic groups. The binding mode of the resulting triazole RGD mimics to αv β3 or αIIb β3 receptors was investigated by molecular modeling simulations. Lead compound 12 was successfully radiofluorinated and used for in vivo positron emission tomography/computed tomography (PET/CT) studies in U87 tumor models, which showed only modest tumor uptake and retention, owing to rapid excretion. These results demonstrate that the novel click RGD mimics are excellent radiolabeled probes for in vitro and cell-based studies on αv β3 integrin, whereas further optimization of their pharmacokinetic and dynamic profiles is necessary for successful use in in vivo imaging.
PINK1 and BECN1 relocalize at mitochondria-associated membranes during mitophagy and promote ER-mitochondria tethering and autophagosome formation.
By Gelmetti Vania; Valente Enza Maria; De Rosa Priscilla; Torosantucci Liliana; Marini Elettra Sara; Romagnoli Alessandra; Di Rienzo Martina; Fimia Gian Maria; Di Rienzo Martina; Arena Giuseppe; et al
From Autophagy (2017), 13(4), 654-669, Language: English, Database: MEDLINE
Mitophagy is a highly specialized process to remove dysfunctional or superfluous mitochondria through the macroautophagy/autophagy pathway, aimed at protecting cells from the damage of disordered mitochondrial metabolism and apoptosis induction. PINK1, a neuroprotective protein mutated in autosomal recessive Parkinson disease, has been implicated in the activation of mitophagy by selectively accumulating on depolarized mitochondria, and promoting PARK2/Parkin translocation to them. While these steps have been characterized in depth, less is known about the process and site of autophagosome formation upon mitophagic stimuli. A previous study reported that, in starvation-induced autophagy, the proautophagic protein BECN1/Beclin1 (which we previously showed to interact with PINK1) relocalizes at specific regions of contact between the endoplasmic reticulum (ER) and mitochondria called mitochondria-associated membranes (MAM), from which the autophagosome originates. Here we show that, following mitophagic stimuli, autophagosomes also form at MAM; moreover, endogenous PINK1 and BECN1 were both found to relocalize at MAM, where they promoted the enhancement of ER-mitochondria contact sites and the formation of omegasomes, that represent autophagosome precursors. PARK2 was also enhanced at MAM following mitophagy induction. However, PINK1 silencing impaired BECN1 enrichment at MAM independently of PARK2, suggesting a novel role for PINK1 in regulating mitophagy. MAM have been recently implicated in many key cellular events. In this light, the observed prevalent localization of PINK1 at MAM may well explain other neuroprotective activities of this protein, such as modulation of mitochondrial calcium levels, mitochondrial dynamics, and apoptosis.
Identification by High-Throughput Screening of Pseudomonas Acyl-Coenzyme A Synthetase Inhibitors.
By Turcano, Lorenzo; Visaggio, Daniela; Frangipani, Emanuela; Missineo, Antonino; Andreini, Matteo; Altamura, Sergio; Visca, Paolo; Bresciani, Alberto
From SLAS Discovery (2017), 22(7), 897-905. Language: English, Database: CAPLUS, DOI:10.1177/2472555216689283
Pseudomonas infections are common among hospitalized, immunocompromised, and chronic lung disease patients. These infections are recalcitrant to common antibacterial therapies due to inherent antibiotic resistance. To meet the need of new anti-Pseudomonas drugs, a sensitive, homogenous, and robust assay was developed with the aim of identifying inhibitors of acyl-CoA synthetases (ACSs) from Pseudomonas. Given the importance of fatty acids for in vivo nutrition of Pseudomonas, such inhibitors might have the potential to reduce the bacterial fitness during infection. The assay, based on a coupled reaction between the Pseudomonas spp. ACS and the firefly luciferase, allowed the identification of three classes of inhibitors by screening of a diverse compd. collection. These compds. were confirmed to reversibly bind ACS with potencies in the micromolar range. Two classes were found to compete with acyl-CoA, while the third one was competitive with fatty acid binding. Although these compds. inhibit the bacterial ACS in cell-free assays, they show modest or no effect on Pseudomonas growth in vitro.
Understanding the Molecular Determinant of Reversible Human Monoamine Oxidase B Inhibitors Containing 2H-Chromen-2-One Core: Structure-Based and Ligand-Based Derived Three-Dimensional Quantitative Structure-Activity Relationships Predictive Models.
By Mladenovic Milan; Patsilinakos Alexandros; Pirolli Adele; Sabatino Manuela; Ragno Rino; Patsilinakos Alexandros; Ragno Rino; Pirolli Adele
From Journal of chemical information and modeling (2017), 57(4), 787-814, Language: English, Database: MEDLINE
Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of aryalkylamines neurotransmitters with concomitant reduction of oxygen to hydrogen peroxide. Consequently, the enzyme’s malfunction can induce oxidative damage to mitochondrial DNA and mediates development of Parkinson’s disease. Thus, MAO B emerges as a promising target for developing pharmaceuticals potentially useful to treat this vicious neurodegenerative condition. Aiming to contribute to the development of drugs with the reversible mechanism of MAO B inhibition only, herein, an extended in silico-in vitro procedure for the selection of novel MAO B inhibitors is demonstrated, including the following: (1) definition of optimized and validated structure-based three-dimensional (3-D) quantitative structure-activity relationships (QSAR) models derived from available cocrystallized inhibitor-MAO B complexes; (2) elaboration of SAR features for either irreversible or reversible MAO B inhibitors to characterize and improve coumarin-based inhibitor activity (Protein Data Bank ID: 2V61 ) as the most potent reversible lead compound; (3) definition of structure-based (SB) and ligand-based (LB) alignment rule assessments by which virtually any untested potential MAO B inhibitor might be evaluated; (4) predictive ability validation of the best 3-D QSAR model through SB/LB modeling of four coumarin-based external test sets (267 compounds); (5) design and SB/LB alignment of novel coumarin-based scaffolds experimentally validated through synthesis and biological evaluation in vitro. Due to the wide range of molecular diversity within the 3-D QSAR training set and derived features, the selected N probe-derived 3-D QSAR model proves to be a valuable tool for virtual screening (VS) of novel MAO B inhibitors and a platform for design, synthesis and evaluation of novel active structures. Accordingly, six highly active and selective MAO B inhibitors (picomolar to low nanomolar range of activity) were disclosed as a result of rational SB/LB 3D QSAR design; therefore, D123 (IC50 = 0.83 nM, Ki = 0.25 nM) and D124 (IC50 = 0.97 nM, Ki = 0.29 nM) are potential lead candidates as anti-Parkinson’s drugs.
Mitotic cell death induction by targeting the mitotic spindle with tubulin-inhibitory indole derivative molecules.
By Di Cesare Erica; Verrico Annalisa; Miele Andrea; Giubettini Maria; Rovella Paola; Cundari Enrico; Lavia Patrizia; Miele Andrea; Giubettini Maria; Coluccia Antonio; et al
From Oncotarget (2017), 8(12), 19738-19759, Language: English, Database: MEDLINE
Tubulin-targeting molecules are widely used cancer therapeutic agents. They inhibit microtubule-based structures, including the mitotic spindle, ultimately preventing cell division. The final fates of microtubule-inhibited cells are however often heterogeneous and difficult to predict. While recent work has provided insight into the cell response to inhibitors of microtubule dynamics (taxanes), the cell response to tubulin polymerization inhibitors remains less well characterized. Arylthioindoles (ATIs) are recently developed tubulin inhibitors. We previously identified ATI members that effectively inhibit tubulin polymerization in vitro and cancer cell growth in bulk cell viability assays. Here we characterise in depth the response of cancer cell lines to five selected ATIs. We find that all ATIs arrest mitotic progression, yet subsequently yield distinct cell fate profiles in time-lapse recording assays, indicating that molecules endowed with similar tubulin polymerization inhibitory activity in vitro can in fact display differential efficacy in living cells. Individual ATIs induce cytological phenotypes of increasing severity in terms of damage to the mitotic apparatus. That differentially triggers MCL-1 down-regulation and caspase-3 activation, and underlies the terminal fate of treated cells. Collectively, these results contribute to define the cell response to tubulin inhibitors and pinpoint potentially valuable molecules that can increase the molecular diversity of tubulin-targeting agents.
An efficient liquid chromatography-high resolution mass spectrometry approach for the optimization of the metabolic stability of therapeutic peptides.
By Esposito, Simone; Mele, Riccardo; Ingenito, Raffaele; Bianchi, Elisabetta; Bonelli, Fabio; Monteagudo, Edith; Orsatti, Laura
From Analytical and Bioanalytical Chemistry (2017), 409(10), 2685-2696. Language: English, Database: CAPLUS, DOI:10.1007/s00216-017-0213-1
In drug discovery, there is increasing interest in peptides as therapeutic agents due to several appealing characteristics that are typical of this class of compds., including high target affinity, excellent selectivity, and low toxicity. However, peptides usually present also some challenging ADME (absorption, distribution, metab., and excretion) issues such as limited metabolic stability, poor oral bioavailability, and short half-lives. In this context, early preclin. in vitro studies such as plasma metabolic stability assays are crucial to improve developability of a peptidic drug. In order to speed up the optimization of peptide metabolic stability, a strategy was developed for the integrated semi-quant. detn. of metabolic stability of peptides and qual. identification/structural elucidation of their metabolites in preclin. plasma metabolic stability studies using liq. chromatog.-high-resoln. Orbitrap mass spectrometry (LC-HRMS). Sample prepn. was based on protein pptn.: exptl. conditions were optimized after evaluating and comparing different org. solvents in order to obtain an adequate extn. of the parent peptides and their metabolites and to minimize matrix effect. Peptides and their metabolites were analyzed by reverse-phase liq. chromatog.: a template gradient (total run time, 6 min) was created to allow retention and good peak shape for peptides of different polarity and isoelec. points. Three LC columns were selected to be systematically evaluated for each series of peptides. Targeted and untargeted HRMS data were simultaneously acquired in pos. full scan + data-dependent MS/MS acquisition mode, and then processed to calc. plasma half-life and to identify the major cleavage sites, this latter by using the software Biopharma Finder. Finally, as an example of the application of this workflow, a study that shows the plasma stability improvement of a series of antimicrobial peptides is described. This approach was developed for the evaluation of in vitro plasma metabolic stability studies of peptides, but it could also be applied to other in vitro metabolic stability models (e.g., whole blood, hepatocytes).