Trastuzumab does not bind rat or mouse ErbB2/neu: implications for selection of non-clinical safety models for trastuzumab-based therapeutics

Purpose Assessment of non-clinical safety signals relies on understanding species selectivity of antibodies. This is particularly important with antibody–drug conjugates, where it is key to determine target-dependent versus target-independent toxicity. Although it appears to be widely accepted that trastuzumab does not bind mouse or rat HER2/ErbB2/neu, numerous investigators continue to use mouse models to investigate safety signals of trastuzumab and trastuzumab emtansine (T-DM1). We, therefore, conducted a broad array of both binding and biologic studies to demonstrate selectivity of trastuzumab for human HER2 versus mouse/rat neu. Methods Binding of anti-neu and anti-HER2 antibodies was assessed by ELISA, FACS, IHC, Scatchard, and immunoblot methods in human, rat, and mouse cell lines. In human hepatocytes, T-DM1 uptake and catabolism were measured by LC-MS/MS; cell viability changes were determined using CellTiter-Glo. Results Our data demonstrate, using different binding methods, lack of trastuzumab binding to rat or mouse neu. Structural studies show important amino acid differences in the trastuzumab-HER2 binding interface between mouse/rat and human HER2 ECD. Substitution of these rodent amino acid residues into human HER2 abolish binding of trastuzumab. Cell viability changes, uptake, and catabolism of T-DM1 versus a DM1 non-targeted control ADC were comparable, indicating target-independent effects of the DM1-containing ADCs. Moreover, trastuzumab binding to human or mouse hepatocytes was not detected. Conclusions These data, in total, demonstrate that trastuzumab, and by extension T-DM1, do not bind rat or mouse neu, underscoring the importance of species selection for safety studies investigating trastuzumab or trastuzumab-based therapeutics.

A quantitative comparison of human embryonic and induced stem cell proteomes

Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. However, a clear overview of their differences at the protein level is still incomplete. In this study we characterise the proteomes of hiPSC and hESC lines, where we find that they express a similar set of proteins but show consistent quantitative differences that can be masked by the normalisation methods. hiPSCs have a higher protein content, with over 1,500 proteins showing over two-fold increased expression. They also display proteomic differences in their mitochondria, with increased expression of mitochondrial transporters and metabolic proteins as well as mitochondrial translation machinery. The hiPSCs also show higher expression of important amino acid transporters, secreted proteins, and growth factors with potential to affect neighbouring cells, coupled with a systematic reduction in the expression levels of H1 histone variants. We conclude that despite hiPSCs and hESCs being highly similar cell types, they show important differences in protein expression that may be relevant for their use in clinical research.

Adipocytic Glutamine Synthetase Upregulation via Altered Histone Methylation Promotes 5FU Chemoresistance in Peritoneal Carcinomatosis of Colorectal Cancer

The development of resistance to 5-fluorouracil (5FU) chemotherapy is a major handicap for sustained effective treatment in peritoneal carcinomatosis (PC) of colorectal cancer (CRC). Metabolic reprogramming of adipocytes, a component of the tumor microenvironment and the main composition of peritoneum, plays a significant role in drug resistance of PC, with the mechanisms being not fully understood. By performing metabolomics analysis, we identified glutamine (Gln), an important amino acid, inducing resistance to 5FU-triggered tumor suppression of CRC-PC through activating mTOR pathway. Noteworthily, genetic overexpression of glutamine synthetase (GS) in adipocytes increased chemoresistance to 5FU in vitro and in vivo while this effect was reversed by pharmacological blockage of GS. Next, we showed that methionine metabolism were enhanced in amino acid omitted from CRC-PC of GS transgenic (TgGS) mice, increasing intracellular levels of S-carboxymethy-L-cys. Moreover, loss of dimethylation at lysine 4 of histone H3 (H3k4me2) was found in adipocytes in vitro, which may lead to increased expression of GS. Furthermore, biochemical inhibition of lysine specific demethylase 1 (LSD1) restored H3k4me2, thereby reducing GS-induced chemoresistance to 5FU. Our findings indicate that GS upregulation-induced excessive of Gln in adipocytes via altered histone methylation is potential mediator of resistance to 5FU chemotherapy in patients with CRC-PC.

Epithelial argininosuccinate synthetase is dispensable for intestinal regeneration and tumorigenesis

The epithelial signaling pathways involved in damage and regeneration, and neoplastic transformation are known to be similar. We noted upregulation of argininosuccinate synthetase (ASS1) in hyperproliferative intestinal epithelium. Since ASS1 leads to de novo synthesis of arginine, an important amino acid for the growth of intestinal epithelial cells, its upregulation can contribute to epithelial proliferation necessary to be sustained during oncogenic transformation and regeneration. Here we investigated the function of ASS1 in the gut epithelium during tissue regeneration and tumorigenesis, using intestinal epithelial conditional Ass1 knockout mice and organoids, and tissue specimens from colorectal cancer patients. We demonstrate that ASS1 is strongly expressed in the regenerating and Apc-mutated intestinal epithelium. Furthermore, we observe an arrest in amino acid flux of the urea cycle, which leads to an accumulation of intracellular arginine. However, loss of epithelial Ass1 does not lead to a reduction in proliferation or increase in apoptosis in vivo, also in mice fed an arginine-free diet. Epithelial loss of Ass1 seems to be compensated by altered arginine metabolism in other cell types and the liver.

Citrulline, Biomarker of Enterocyte Functional Mass and Dietary Supplement. Metabolism, Transport, and Current Evidence for Clinical Use

L-Citrulline is a non-essential but still important amino acid that is released from enterocytes. Because plasma levels are reduced in case of impaired intestinal function, it has become a biomarker to monitor intestinal integrity. Moreover, oxidative stress induces protein citrullination, and antibodies against anti-citrullinated proteins are useful to monitor rheumatoid diseases. Citrullinated histones, however, may even predict a worse outcome in cancer patients. Supplementation of citrulline is better tolerated compared to arginine and might be useful to slightly improve muscle strength or protein balance. The following article shall provide an overview of L-citrulline properties and functions, as well as the current evidence for its use as a biomarker or as a therapeutic supplement.

Molecular Dynamics Analysis of Glucose Oxidase Stability against Temperature

Glucose oxidase (GOD) from local isolated Aspergillus niger IPBCC.08.610 shows a widespread application, specifically as a bioanode in glucose-based biofuel cells. Enzymes with adequate thermal stability are necessary for enhancing product efficiency. Also, evaluating the structural dynamics to improve temperature helps to determine the residue. The molecular dynamics simulation of GOD_IPBCC_1CF3 at temperatures of 300, 400, and 500 K was carried out to analyze important amino acid residues for thermal stability. The results showed that the amino acid residues responsible for thermal stability were dispersed into several essential regions, including D576 at the C terminal, E266-R250, and E38-R237 in the FAD-binding domain E485-R470 in the substrate-binding antiparallel beta system. However, the FAD molecular flexibility against temperature depends on conserve E48 by stabilizing the ribose sugar moiety.

Pharmacophore-Based Screening & Modification of Amiloride Analogs for targeting the NhaP-type Cation-Proton Antiporter in Vibrio cholerae

Structural and mutational analysis of Vc-NhaP2 identified a putative cation binding pocket formed by antiparallel extended regions of two transmembrane segments (TMSs V/XII) along with TMS VI. Molecular Dynamics (MD) simulations suggested that the flexibility of TMS-V/XII is crucial for the intra-molecular conformational events in Vc-NhaP2. In this study, we developed some putative Vc-NhaP2 inhibitors from Amiloride analogs (AAs). Molecular docking of the modified AAs revealed promising binding. The four selected drugs potentially interacted with functionally important amino acid residues located on the cytoplasmic side of TMS VI, the extended chain region of TMS V and TMS XII and the loop region between TMSs VIIII and IX. Molecular dynamics simulations revealed that binding of the selected drugs can potentially destabilize the Vc-NhaP2 and alters the flexibility of the functionally important TMS VI. The work presents the utility of in silico approaches for the rational identification of potential targets and drugs that could target NhaP2 cation proton antiporter to control Vibrio cholerae. The goal is to identify potential drugs that can be validated in future experiments.

Highly Diastereoselective Construction of Carbon– Heteroatom Quaternary Stereogenic Centers in the Synthesis of Analogs of Bioactive Compounds: From Monofluorinated Epoxyalkylphosphonates to α-Fluoro-, β-, or γ-Amino Alcohol Derivatives of Alkylphosphonates

The synthesis of the stable surrogates of an important amino acid (R)-4-amino-3-hydroxybutyric acid (GABOB) such as substituted hydroxy aminophosphonic acids bearing a quaternary stereogenic center is presented. Highly diastereoselective formations of fluorinated spiroepoxy alkylphosphonate or related tertiary carbon-containing oxiranes from β-keto phosphonates possessing methyl, phenyl, or cyclohexenyl substituents, are reported. Stereoselective acid-promoted epoxide opening by bromide or azide followed by reduction/protection afforded tertiary bromides or N-Boc derivatives of β-amino-γ-hydroxy alkylphosphonates in most cases, while the reactions of oxiranes with different amines yielded their β-hydroxy-γ-amino regioisomers. Surprisingly, during the synthesis of amino phosphonic acids, we observe that the acid-induced rearrangement proceeded in a high diastereospecific manner, leading finally to substituted β-hydroxy-γ-aminoalkylphosphonic acids.

Directed evolution improves the enzymatic synthesis of L-5-hydroxytryptophan by an engineered tryptophan synthase

L-5-Hydroxytryptophan is an important amino acid that is widely used in food and medicine. In this study, L-5-hydroxytryptophan was synthesized by a modified tryptophan synthase. A direct evolution strategy was applied to engineer tryptophan synthase from Escherichia coli to improve the efficiency of L-5-hydroxytryptophan synthesis. Tryptophan synthase was modified by error-prone PCR. A high activity mutant enzyme (V231A/K382G) was obtained by a high-throughput screening method. The activity of mutant enzyme (V231A/K382G) is 3.79 times higher than that of its parent, and kcat/Km of the mutant enzyme (V231A/K382G) was 4.36 mM− 1∙s− 1. The mutant enzyme (V231A/K382G) reaction conditions for the production of L-5-hydroxytryptophan were 100 mmol/L L-serine at pH 8.5 and 35°C for 15 h, reaching a yield of L-5-hydroxytryptophan of 86.7%. Directed evolution is an effective strategy to increase the activity of tryptophan synthase.

Impact of D2O on peptidization of L-histidine

This is our sixth consecutive study carried out in an order to collect an experimental evidence on the impact of heavy water (D2O) on spontaneous peptidization of the proteinogenic α-amino acids and this time it is L-histidine (L-His). Scientists have not yet achieved a full consensus regarding the source of this very important amino acid in human and mammalian tissues, and on this particular question rather contradictory answers in form of experimental results are produced, equally supporting its exogenous and endogenous origin. Although this issue still remains unsolved, for practical demands of life sciences the two UN agencies, FAO and WHO, have both tentatively accepted that L-His is an exogenous α-amino acid. As analytical techniques, in our studies we employed high-performance liquid chromatography with the diode array detection (HPLC–DAD), mass spectrometry (MS), and scanning electron microscopy (SEM). Spontaneous peptidization of L-His dissolved in methanol + H2O, 7:3 (v/v) was carried out at 22 ± 0.5 °C in the darkness for a relatively long period of 314 h, and its progress was chromatographically checked by targeting concentration of the L-His monomer in the 12-min intervals. This investigation revealed alternating yet non-periodic concentration changes, indicating changeable formation and hydrolytic decay of the L-His-derived oligopeptides in the function of time, and a fast net concentration fall of the L-His monomer (witnessing to quite vigorous peptidization). Moreover, the MS results confirmed formation of the relatively high oligopeptides, falling within the range of two or more dozen L-His monomer units. Impact of D2O on peptidization of L-His was traced with use of MS and SEM for the L-His samples dissolved in aqueous methanol solvents containing 5, 10, 20, and 30% D2O, and also in pure D2O. Similar to the results earlier presented for five other proteinogenic α-amino acids, heavy water exerts a powerful inhibitory effect on spontaneous peptidization of L-His, equally perceptible when assessed with aid of mass spectrometry (with the mass spectra in the first instance playing the role of quasi-quantitative fingerprints), and based on purely qualitative micrographs derived with use of SEM.