M2R: a Python add-on to cobrapy for modifying human genome-scale metabolic reconstruction using the gut microbiota models.

MOTIVATION: The gut microbiota is the human body's largest population of microorganisms that interact with human intestinal cells. They use ingested nutrients for fundamental biological processes and have important impacts on human physiology, immunity and metabolome in the gastrointestinal tract. RESULTS: Here, we present M2R, a Python add-on to cobrapy that allows incorporating information about the gut microbiota metabolism models to human genome-scale metabolic models (GEMs) like RECON3D. The idea behind the software is to modify the lower bounds of the exchange reactions in the model using aggregated in- and out-fluxes from selected microbes. M2R enables users to quickly and easily modify the pool of the metabolites that enter and leave the GEM, which is particularly important for those looking into an analysis of the metabolic interaction between the gut microbiota and human cells and its dysregulation. AVAILABILITY AND IMPLEMENTATION: M2R is freely available under an MIT License at https://github.com/e-weglarz-tomczak/m2r. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

GEM-Based Metabolic Profiling for Human Bone Osteosarcoma under Different Glucose and Glutamine Availability.

Cancer cell metabolism is dependent on cell-intrinsic factors, such as genetics, and cell-extrinsic factors, such nutrient availability. In this context, understanding how these two aspects interact and how diet influences cellular metabolism is important for developing personalized treatment. In order to achieve this goal, genome-scale metabolic models (GEMs) are used; however, genetics and nutrient availability are rarely considered together. Here, we propose integrated metabolic profiling, a framework that allows enriching GEMs with metabolic gene expression data and information about nutrients. First, the RNA-seq is converted into Reaction Activity Score (RAS) to further scale reaction bounds. Second, nutrient availability is converted to Maximal Uptake Rate (MUR) to modify exchange reactions in a GEM. We applied our framework to the human osteosarcoma cell line (U2OS). Osteosarcoma is a common and primary malignant form of bone cancer with poor prognosis, and, as indicated in our study, a glutamine-dependent type of cancer.

Helix-loop-helix peptide foldamers and their use in the construction of hydrolase mimetics.

De novo designed helix-loop-helix peptide foldamers containing cis-2-aminocyclopentanecarboxylic acid residues were evaluated for their conformational stability and possible use in enzyme mimetic development. The correlation between hydrogen bond network size and conformational stability was demonstrated through CD and NMR spectroscopies. Molecules incorporating a Cys/His/Glu triad exhibited enzyme-like hydrolytic activity.

Controlling the Helix Handedness of ααß-Peptide Foldamers through Sequence Shifting.

Peptide foldamers containing both cis-ß-aminocyclopentanecarboxylic acid and α-amino acid residues combined in various sequence patterns (ααß, αααß, αßααß, and ααßαααß) were screened using CD and NMR spectroscopy for the tendency to form helices. ααß-Peptides were found to fold into an unprecedented and well-defined 16/17/15/18/14/17-helix. By extending the length of the sequence or shifting a fragment of the sequence from one terminus to another in ααß-peptides, the balance between left-handed and right-handed helix populations present in the solution can be controlled. Engineering of the peptide sequence could lead to compounds with either a strong propensity for the selected helix sense or a mixture of helical conformations of opposite senses.

Discovery of potent and selective inhibitors of human aminopeptidases ERAP1 and ERAP2 by screening libraries of phosphorus-containing amino acid and dipeptide analogues.

A collection of fifty phosphonic and phosphinic acids was screened for inhibition of ERAP1 and ERAP2, the human endoplasmic reticulum aminopeptidases. The cooperative action of these enzymes is manifested by trimming a variety of antigenic precursors to be presented on the cell surface by major histocompatibility class I. The SAR studies revealed several potent compounds, particularly among the phosphinic dipeptide analogues, that were strong inhibitors of ERAP2 (Ki=100-350nM). A wide structural diversity of the applied organophosphorus compounds, predominantly non-proteinogenic analogues, allowed identification of representatives selective toward only one form of ERAP. For example, N'-substituted α,ß-diaminophosphonates and phosphinates exhibited potency only toward ERAP2, which is in agreement with the P1 basic substrate-oriented specificity. Such discriminating ligands are invaluable tools for elucidating the precise role of a particular aminopeptidase in the concerted function of antigen processing and in human diseases.

Identification of methionine aminopeptidase 2 as a molecular target of the organoselenium drug ebselen and its derivatives/analogues: Synthesis, inhibitory activity and molecular modeling study.

A collection of twenty-six organoselenium compounds, ebselen and its structural analogues, provided a novel approach for inhibiting the activity of human methionine aminopeptidase 2 (MetAP2). This metalloprotease, being responsible for the removal of the amino-terminal methionine from newly synthesized proteins, plays a key role in angiogenesis, which is essential for the progression of diseases, including solid tumor cancers. In this work, we discovered that ebselen, a synthetic organoselenium drug molecule with anti-inflammatory, anti-oxidant and cytoprotective activity, inhibits one of the main enzymes in the tumor progression pathway. Using three-step synthesis, we obtained twenty-five ebselen derivatives/analogues, ten of which are new, and tested their inhibitory activity toward three neutral aminopeptidases (MetAP2, alanine and leucine aminopeptidases). All of the tested compounds proved to be selective, slow-binding inhibitors of MetAP2. Similarly to ebselen, most of its analogues exhibited a moderate potency (IC50=1-12µM). Moreover, we identified three strong inhibitors that bind favorably to the enzyme with the half maximal inhibitory concentration in the submicromolar range.

Fusion-fission-mitophagy cycling and metabolic reprogramming coordinate nerve growth factor (NGF)-dependent neuronal differentiation.

Neuronal differentiation is regulated by nerve growth factor (NGF) and other neurotrophins. We explored the impact of NGF on mitochondrial dynamics and metabolism through time-lapse imaging, metabolomics profiling, and computer modeling studies. We show that NGF may direct differentiation by stimulating fission, thereby causing selective mitochondrial network fragmentation and mitophagy, ultimately leading to increased mitochondrial quality and respiration. Then, we reconstructed the dynamic fusion-fission-mitophagy cycling of mitochondria in a computer model, integrating these processes into a single network mechanism. Both the computational model and the simulations are able to reproduce the proposed mechanism in terms of mitochondrial dynamics, levels of reactive oxygen species (ROS), mitophagy, and mitochondrial quality, thus providing a computational tool for the interpretation of the experimental data and for future studies aiming to detail further the action of NGF on mitochondrial processes. We also show that changes in these mitochondrial processes are intertwined with a metabolic function of NGF in differentiation: NGF directs a profound metabolic rearrangement involving glycolysis, TCA cycle, and the pentose phosphate pathway, altering the redox balance. This metabolic rewiring may ensure: (a) supply of both energy and building blocks for the anabolic processes needed for morphological reorganization, as well as (b) redox homeostasis.

Simultaneous Integration of Gene Expression and Nutrient Availability for Studying the Metabolism of Hepatocellular Carcinoma Cell Lines.

How cancer cells utilize nutrients to support their growth and proliferation in complex nutritional systems is still an open question. However, it is certainly determined by both genetics and an environmental-specific context. The interactions between them lead to profound metabolic specialization, such as consuming glucose and glutamine and producing lactate at prodigious rates. To investigate whether and how glucose and glutamine availability impact metabolic specialization, we integrated computational modeling on the genome-scale metabolic reconstruction with an experimental study on cell lines. We used the most comprehensive human metabolic network model to date, Recon3D, to build cell line-specific models. RNA-Seq data was used to specify the activity of genes in each cell line and the uptake rates were quantitatively constrained according to nutrient availability. To integrated both constraints we applied a novel method, named Gene Expression and Nutrients Simultaneous Integration (GENSI), that translates the relative importance of gene expression and nutrient availability data into the metabolic fluxes based on an observed experimental feature(s). We applied GENSI to study hepatocellular carcinoma addiction to glucose/glutamine. We were able to identify that proliferation, and lactate production is associated with the presence of glucose but does not necessarily increase with its concentration when the latter exceeds the physiological concentration. There was no such association with glutamine. We show that the integration of gene expression and nutrient availability data into genome-wide models improves the prediction of metabolic phenotypes.

Identification of ebselen and its analogues as potent covalent inhibitors of papain-like protease from SARS-CoV-2.

An efficient treatment against a COVID-19 disease, caused by the novel coronavirus SARS-CoV-2 (CoV2), remains a challenge. The papain-like protease (PLpro) from the human coronavirus is a protease that plays a critical role in virus replication. Moreover, CoV2 uses this enzyme to modulate the host's immune system to its own benefit. Therefore, it represents a highly promising target for the development of antiviral drugs. We used Approximate Bayesian Computation tools, molecular modelling and enzyme activity studies to identify highly active inhibitors of the PLpro. We discovered organoselenium compounds, ebselen and its structural analogues, as a novel approach for inhibiting the activity of PLproCoV2. Furthermore, we identified, for the first time, inhibitors of PLproCoV2 showing potency in the nanomolar range. Moreover, we found a difference between PLpro from SARS and CoV2 that can be correlated with the diverse dynamics of their replication, and, putatively to disease progression.

Estimating kinetic constants in the Michaelis-Menten model from one enzymatic assay using Approximate Bayesian Computation.

The Michaelis-Menten equation is one of the most extensively used models in biochemistry for studying enzyme kinetics. However, this model requires at least a couple (e.g., eight or more) of measurements at different substrate concentrations to determine kinetic parameters. Here, we report the discovery of a novel tool for calculating kinetic constants in the Michaelis-Menten equation from only a single enzymatic assay. As a consequence, our method leads to reduced costs and time, primarily by lowering the amount of enzymes, since their isolation, storage and usage can be challenging when conducting research.

Neutral metalloaminopeptidases APN and MetAP2 as newly discovered anticancer molecular targets of actinomycin D and its simple analogs.

The potent transcription inhibitor Actinomycin D is used with several cancers. Here, we report the discovery that this naturally occurring antibiotic inhibits two human neutral aminopeptidases, the cell-surface alanine aminopeptidase and intracellular methionine aminopeptidase type 2. These metallo-containing exopeptidases participate in tumor cell expansion and motility and are targets for anticancer therapies. We show that the peptide portions of Actinomycin D and Actinomycin X2 are not required for effective inhibition, but the loss of these regions changes the mechanism of interaction. Two structurally less complex Actinomycin D analogs containing the phenoxazone chromophores, Questiomycin A and Actinocin, appear to be competitive inhibitors of both aminopeptidases, with potencies similar to the non-competitive macrocyclic parent compound (Ki in the micromolar range). The mode of action for all four compounds and both enzymes was demonstrated by molecular modeling and docking in the corresponding active sites. This knowledge gives new perspectives to Actinomycin D's action on tumors and suggests new avenues and molecules for medical applications.

A structural insight into the P1S1 binding mode of diaminoethylphosphonic and phosphinic acids, selective inhibitors of alanine aminopeptidases.

N'-substituted 1,2-diaminoethylphosphonic acids and 1,2-diaminoethylphosphinic dipeptides were explored to unveil the structural context of the unexpected selectivity of these inhibitors of M1 alanine aminopeptidases (APNs) versus M17 leucine aminopeptidase (LAP). The diaminophosphonic acids were obtained via aziridines in an improved synthetic procedure that was further expanded for the phosphinic pseudodipeptide system. The inhibitory activity, measured for three M1 and one M17 metalloaminopeptidases of different sources (bacterial, human and porcine), revealed several potent compounds (e.g., Ki = 65 nM of 1u for HsAPN). Two structures of an M1 representative (APN from Neisseria meningitidis) in complex with N-benzyl-1,2-diaminoethylphosphonic acid and N-cyclohexyl-1,2-diaminoethylphosphonic acid were determined by the X-ray crystallography. The analysis of these structures and the models of the phosphonic acid complexes of the human ortholog provided an insight into the role of the additional amino group and the hydrophobic substituents of the ligands within the S1 active site region.

Structure-guided, single-point modifications in the phosphinic dipeptide structure yield highly potent and selective inhibitors of neutral aminopeptidases.

Seven crystal structures of alanyl aminopeptidase from Neisseria meningitides (the etiological agent of meningitis, NmAPN) complexed with organophosphorus compounds were resolved to determine the optimal inhibitor-enzyme interactions. The enantiomeric phosphonic acid analogs of Leu and hPhe, which correspond to the P1 amino acid residues of well-processed substrates, were used to assess the impact of the absolute configuration and the stereospecific hydrogen bond network formed between the aminophosphonate polar head and the active site residues on the binding affinity. For the hPhe analog, an imperfect stereochemical complementarity could be overcome by incorporating an appropriate P1 side chain. The constitution of P1'-extended structures was rationally designed and the lead, phosphinic dipeptide hPhePψ[CH2]Phe, was modified in a single position. Introducing a heteroatom/heteroatom-based fragment to either the P1 or P1' residue required new synthetic pathways. The compounds in the refined structure were low nanomolar and subnanomolar inhibitors of N. meningitides, porcine and human APNs, and the reference leucine aminopeptidase (LAP). The unnatural phosphinic dipeptide analogs exhibited a high affinity for monozinc APNs associated with a reasonable selectivity versus dizinc LAP. Another set of crystal structures containing the NmAPN dipeptide ligand were used to verify and to confirm the predicted binding modes; furthermore, novel contacts, which were promising for inhibitor development, were identified, including a π-π stacking interaction between a pyridine ring and Tyr372.

An integrated approach to the ligand binding specificity of Neisseria meningitidis M1 alanine aminopeptidase by fluorogenic substrate profiling, inhibitory studies and molecular modeling.

Neisseria meningitides is a gram-negative diplococcus bacterium and is the main causative agent of meningitis and other meningococcal diseases. Alanine aminopeptidase from N. meningitides (NmAPN) belongs to the family of metallo-exopeptidase enzymes, which catalyze the removal of amino acids from the N-terminus of peptides and proteins, and are found among all the kingdoms of life. NmAPN is suggested to be mostly responsible for proteolysis and nutrition delivery, similar to the orthologs from other bacteria. To explore the possibility of NmAPN being a potential drug target for inhibition and development of novel therapeutic agents, the specificity of the S1 and S1' binding sites was explored using an integrated approach. Initially, an extensive library consisting of almost 100 fluorogenic substrates derived from both natural and unnatural amino acids, were used to obtain a detailed substrate fingerprint of the S1 pocket of NmAPN. A broad substrate tolerance of NmAPN was revealed, with bulky basic and hydrophobic ligands being the most favored substrates. Additionally, the potency of a set of organophosphorus inhibitors of neutral aminopeptidases, amino acid and dipeptide analogs was determined. Inhibition constants in the nanomolar range, determined for phosphinic dipeptides, proves the positive increase in inhibition impact of the P1' ligand elongation. The results were further verified via molecular modeling and docking of canonical aminopeptidase phosphinic dipeptide inhibitors in the NmAPN active site. These studies present comprehensive characterization of interactions responsible for specific ligand binding. This knowledge provides invaluable insight into understanding of the enzyme and development of novel NmAPN inhibitors.