The Nitrogen Mustards.

The nitrogen mustards are powerful cytotoxic and lymphoablative agents and have been used for more than 60 years. They are employed in the treatment of cancers, sarcomas, and hematologic malignancies. Cyclophosphamide, the most versatile of the nitrogen mustards, also has a place in stem cell transplantation and the therapy of autoimmune diseases. Adverse effects caused by the nitrogen mustards on the central nervous system, kidney, heart, bladder, and gonads remain important issues. Advances in analytical techniques have facilitated the investigation of the pharmacokinetics of the nitrogen mustards, especially the oxazaphosphorines, which are prodrugs requiring metabolic activation. Enzymes involved in the metabolism of cyclophosphamide and ifosfamide are very polymorphic, but a greater understanding of the pharmacogenomic influences on their activity has not yet translated into a personalized medicine approach. In addition to damaging DNA, the nitrogen mustards can act through other mechanisms, such as antiangiogenesis and immunomodulation. The immunomodulatory properties of cyclophosphamide are an area of current exploration. In particular, cyclophosphamide decreases the number and activity of regulatory T cells, and the interaction between cyclophosphamide and the intestinal microbiome is now recognized as an important factor. New derivatives of the nitrogen mustards continue to be assessed. Oxazaphosphorine analogs have been synthesized in attempts to both improve efficacy and reduce toxicity, with varying degrees of success. Combinations of the nitrogen mustards with monoclonal antibodies and small-molecule targeted agents are being evaluated. SIGNIFICANCE STATEMENT: The nitrogen mustards are important, well-established therapeutic agents that are used to treat a variety of diseases. Their role is continuing to evolve.

Three-dimensional cell culture models for anticancer drug screening: Worth the effort?

High attrition of new oncology drug candidates in clinical trials is partially caused by the poor predictive capacity of artificial monolayer cell culture assays early in drug discovery. Monolayer assays do not take the natural three-dimensional (3D) microenvironment of cells into account. As a result, false positive compounds often enter clinical trials, leading to high dropout rates and a waste of time and money. Over the past 2 decades, tissue engineers and cell biologists have developed a broad range of 3D in vitro culturing tools that better represent in vivo cell biology. These tools preserve the 3D architecture of cells and can be used to predict toxicity of and resistance against antitumor agents. Recent progress in tissue engineering further improves 3D models by taking into account the tumor microenvironment, which is important for metastatic progression and vascularization. However, the widespread implementation of 3D cell cultures into cell-based research programs has been limited by various factors, including their cost and reproducibility. In addition, different 3D cell culture techniques often produce spheroids of different size and shape, which can strongly influence drug efficacy and toxicity. Hence, it is imperative to morphometrically characterize multicellular spheroids to avoid generalizations among different spheroid types. Standardized 3D culturing procedures could further reduce data variability and enhance biological relevance. Here, we critically evaluate the benefits and challenges inherent to growing cells in 3D, along with an overview of the techniques used to form spheroids. This is done with a specific focus on antitumor drug screening.

Fast and Reliable Quantitative Peptidomics with labelpepmatch.

The use of stable isotope tags in quantitative peptidomics offers many advantages, but the laborious identification of matching sets of labeled peptide peaks is still a major bottleneck. Here we present labelpepmatch, an R-package for fast and straightforward analysis of LC-MS spectra of labeled peptides. This open-source tool offers fast and accurate identification of peak pairs alongside an appropriate framework for statistical inference on quantitative peptidomics data, based on techniques from other -omics disciplines. A relevant case study on the desert locust Schistocerca gregaria proves our pipeline to be a reliable tool for quick but thorough explorative analyses.

The benefits and limitations of reaction cell and sector field inductively coupled plasma mass spectrometry in the detection and quantification of phosphopeptides.

RATIONALE: The phosphorylation of proteins is one of the most important post-translational modifications in nature. Knowledge of the quantity or degree of protein phosphorylation in biological samples is extremely important. A combination of liquid chromatography (LC) and inductively coupled plasma mass spectrometry (ICP-MS) allows the absolute and relative quantification of the phosphorus signal. METHODS: A comparison between dynamic reaction cell quadrupole ICP-MS (DRC-Q-ICP-MS) and high-resolution sector field ICP-MS (SF-ICP-MS) in detecting signals of phosphorus-containing species using identical capillary LC (reversed-phase technology) and nebulizer settings was performed. RESULTS: A method to diminish the reversed-phase gradient-related signal instability in phosphorus detection with LC/ICP-MS applications was developed. Bis(4-nitrophenyl)phosphate (BNPP) was used as a standard to compare signal-to-noise ratios and limits of detection (LODs) between the two instrumental setups. The LOD reaches a value of 0.8 µg L(-1) when applying the DRC technology in Q-ICP-MS and an LOD of 0.09 µg L(-1) was found with the SF-ICP-MS setup. This BNPP standard was further used to compare the absolute quantification possibilities of phosphopeptides in these two setups. CONCLUSIONS: This one-to-one comparison of two interference-reducing ICP-MS instruments demonstrates that absolute quantification of individual LC-separated phosphopeptides is possible. However, based on the LOD values, SF-ICP-MS has a higher sensitivity in detecting phosphorus signals and thus is preferred in phosphopeptide analysis.

Evaluation of the antibacterial and antibiofilm activities of novel CRAMP-vancomycin conjugates with diverse linkers.

We report the design, synthesis and antibacterial activity analysis of conjugates of vancomycin and cathelicidin-related antimicrobial peptides (CRAMP). Vancomycin inhibits the nascent peptidoglycan synthesis and is highly active against Gram-positive bacteria, whereas Gram-negative bacteria are generally insensitive due to a protective outer membrane. CRAMP is known to translocate across the Gram-negative outer membrane by a self-promoted uptake mechanism. Vancomycin-CRAMP conjugates were synthesized using click chemistry with diverse hydrophilic and hydrophobic linkers, with CRAMP functioning as a carrier peptide for the transfer of vancomycin through the outer membrane. Small hydrophobic linkers with an aromatic group result in the most active conjugates against planktonic Gram-negative bacteria, while maintaining the high activity of vancomycin against Gram-positive bacteria. These conjugates thus show a broad-spectrum activity, which is absent in CRAMP or vancomycin alone, and which is strongly improved compared to an equimolar mixture of CRAMP and vancomycin. In addition, these conjugates also show a strong inhibitory activity against S. Typhimurium biofilm formation.

Systematic identification of hypothetical bacteriophage proteins targeting key protein complexes of Pseudomonas aeruginosa.

Addressing the functionality of predicted genes remains an enormous challenge in the postgenomic era. A prime example of genes lacking functional assignments are the poorly conserved, early expressed genes of lytic bacteriophages, whose products are involved in the subversion of the host metabolism. In this study, we focused on the composition of important macromolecular complexes of Pseudomonas aeruginosa involved in transcription, DNA replication, fatty acid biosynthesis, RNA regulation, energy metabolism, and cell division during infection with members of seven distinct clades of lytic phages. Using affinity purifications of these host protein complexes coupled to mass spectrometric analyses, 37 host complex-associated phage proteins could be identified. Importantly, eight of these show an inhibitory effect on bacterial growth upon episomal expression, suggesting that these phage proteins are potentially involved in hijacking the host complexes. Using complementary protein-protein interaction assays, we further mapped the inhibitory interaction of gp12 of phage 14-1 to the α subunit of the RNA polymerase. Together, our data demonstrate the powerful use of interactomics to unravel the biological role of hypothetical phage proteins, which constitute an enormous untapped source of novel antibacterial proteins. (Data are available via ProteomeXchange with identifier PXD001199.).

Derivatives of the mouse cathelicidin-related antimicrobial peptide (CRAMP) inhibit fungal and bacterial biofilm formation.

We identified a 26-amino-acid truncated form of the 34-amino-acid cathelicidin-related antimicrobial peptide (CRAMP) in the islets of Langerhans of the murine pancreas. This peptide, P318, shares 67% identity with the LL-37 human antimicrobial peptide. As LL-37 displays antimicrobial and antibiofilm activity, we tested antifungal and antibiofilm activity of P318 against the fungal pathogen Candida albicans. P318 shows biofilm-specific activity as it inhibits C. albicans biofilm formation at 0.15 µM without affecting planktonic survival at that concentration. Next, we tested the C. albicans biofilm-inhibitory activity of a series of truncated and alanine-substituted derivatives of P318. Based on the biofilm-inhibitory activity of these derivatives and the length of the peptides, we decided to synthesize the shortened alanine-substituted peptide at position 10 (AS10; KLKKIAQKIKNFFQKLVP). AS10 inhibited C. albicans biofilm formation at 0.22 µM and acted synergistically with amphotericin B and caspofungin against mature biofilms. AS10 also inhibited biofilm formation of different bacteria as well as of fungi and bacteria in a mixed biofilm. In addition, AS10 does not affect the viability or functionality of different cell types involved in osseointegration of an implant, pointing to the potential of AS10 for further development as a lead peptide to coat implants.

Colorectal cancer biomarker discovery and validation using LC-MS/MS-based proteomics in blood: truth or dare?

Globally, colorectal cancer (CRC) is the third most common malignant neoplasm. However, highly sensitive, specific, noninvasive tests that allow CRC diagnosis at an early stage are still needed. As circulatory blood reflects the physiological status of an individual and/or the disease status for several disorders, efforts have been undertaken to identify candidate diagnostic CRC markers in plasma and serum. In this review, the challenges, bottlenecks and promising properties of mass spectrometry (MS)-based proteomics in blood are discussed. More specifically, important aspects in clinical design, sample retrieval, sample preparation, and MS analysis are presented. The recent developments in targeted MS approaches in plasma or serum are highlighted as well.

Differential peptidomics highlights adipokinetic hormone as key player in regulating digestion in anautogenous flesh fly, Sarcophaga crassipalpis.

Females of anautogenous flesh flies, Sarcophaga crassipalpis, need a protein meal in order to produce their first batch of eggs. This protein meal elicits an increase in midgut proteolytic activity that is under neuropeptidergic regulation. Time series of decapitation and rescue experiments of liver fed flies evidenced the need of a peptide factor released by corpora cardiaca (CC) within 4h post protein feeding in order to assure complete protein digestion. Q-Exactive quantitative differential peptidomics analysis on CC of sugar fed flies and flies 5h post protein feeding respectively, showed a unique consistent decrease in the stored amount of adipokinetic hormone (AKH) ranging between 16% up to 63%. Injection of AKH into liver fed decapitated flies as well as sugar fed intact flies resulted in dose dependent enhanced midgut proteolytic activity up to the level of intact protein fed flies. This suggests a key role of AKH in food depended reproduction.

Analysis of the formalin-fixed paraffin-embedded tissue proteome: pitfalls, challenges, and future prospectives.

Formalin-fixed paraffin-embedded (FFPE) tissues are a real treasure for retrospective analysis considering the amount of samples present in hospital archives, combined with pathological, clinical, and outcome information available for every sample. Although unlocking the proteome of these tissues is still a challenge, new approaches are being developed. In this review, we summarize the different mass spectrometry platforms that are used in human clinical studies to unravel the FFPE proteome. The different ways of extracting crosslinked proteins and the analytical strategies are pointed out. Also, the pitfalls and challenges concerning the quality of FFPE proteomic approaches are depicted. We also evaluated the potential of these analytical methods for future clinical FFPE proteomics applications.

A comprehensive summary of LL-37, the factotum human cathelicidin peptide.

Cathelicidins are a group of antimicrobial peptides. Since their discovery, it has become clear that they are an exceptional class of peptides, with some members having pleiotropic effects. Not only do they possess an antibacterial, antifungal and antiviral function, they also show a chemotactic and immunostimulatory/-modulatory effect. Moreover, they are capable of inducing wound healing, angiogenesis and modulating apoptosis. Recent insights even indicate for a role of these peptides in cancer. This review provides a comprehensive summary of the most recent and relevant insights concerning the human cathelicidin LL-37.

A hybrid, de novo based, genome-wide database search approach applied to the sea urchin neuropeptidome.

Peptidomics is the identification and study of the in vivo biologically active peptide profile. A combination of high performance liquid chromatography, mass spectrometry, and bioinformatics tools such as database search engines are commonly used to perform the analysis. We report a methodology based on a database system holding the completed translated genome, whereby de novo sequencing and genome-wide database searching are combined. The methodology was applied to the sea urchin neuropeptidome resulting in a 30% increase in identification rate.

The Phage-Encoded N-Acetyltransferase Rac Mediates Inactivation of Pseudomonas aeruginosa Transcription by Cleavage of the RNA Polymerase Alpha Subunit.

In this study, we describe the biological function of the phage-encoded protein RNA polymerase alpha subunit cleavage protein (Rac), a predicted Gcn5-related acetyltransferase encoded by phiKMV-like viruses. These phages encode a single-subunit RNA polymerase for transcription of their late (structure- and lysis-associated) genes, whereas the bacterial RNA polymerase is used at the earlier stages of infection. Rac mediates the inactivation of bacterial transcription by introducing a specific cleavage in the α subunit of the bacterial RNA polymerase. This cleavage occurs within the flexible linker sequence and disconnects the C-terminal domain, required for transcription initiation from most highly active cellular promoters. To achieve this, Rac likely taps into a novel post-translational modification (PTM) mechanism within the host Pseudomonas aeruginosa. From an evolutionary perspective, this novel phage-encoded regulation mechanism confirms the importance of PTMs in the prokaryotic metabolism and represents a new way by which phages can hijack the bacterial host metabolism.

Spectral clustering in peptidomics studies helps to unravel modification profile of biologically active peptides and enhances peptide identification rate.

When studying the set of biologically active peptides (the so-called peptidome) of a cell type, organ, or entire organism, the identification of peptides is mostly attempted by MS. However, identification rates are often dismally unsatisfactory. A great deal of failed or missed identifications may be attributable to the wealth of modifications on peptides, some of which may originate from in vivo post-translational processes to activate the molecule, whereas others could be introduced during the tissue preparation procedures. Preliminary knowledge of the modification profile of specific peptidome samples would greatly improve identification rates. To this end we developed an approach that performs clustering of mass spectra in a way that allows us to group spectra having similar peak patterns over significant segments. Comparing members of one spectral group enables us to assess the modifications (expressed as mass shifts in Dalton) present in a peptidome sample. The clustering algorithm in this study is called Bonanza, and it was applied to MALDI-TOF/TOF MS spectra from the mouse. Peptide identification rates went up from 17 to 36% for 278 spectra obtained from the pancreatic islets and from 21 to 43% for 163 pituitary spectra. Spectral clustering with subsequent advanced database search may result in the discovery of new biologically active peptides and modifications thereof, as shown by this report indeed.

Comparative peptidomics of Caenorhabditis elegans versus C. briggsae by LC-MALDI-TOF MS.

Neuropeptides are important signaling molecules that function in cell-cell communication as neurotransmitters or hormones to orchestrate a wide variety of physiological conditions and behaviors. These endogenous peptides can be monitored by high throughput peptidomics technologies from virtually any tissue or organism. The neuropeptide complement of the soil nematode Caenorhabditis elegans has been characterized by on-line two-dimensional liquid chromatography and quadrupole time-of-flight tandem mass spectrometry (2D-nanoLC Q-TOF MS/MS). Here, we use an alternative peptidomics approach combining liquid chromatography (LC) with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to map the peptide content of C. elegans and another Caenorhabditis species, Caenorhabditis briggsae. This study allows a better annotation of neuropeptide-encoding genes from the C. briggsae genome and provides a promising basis for further evolutionary comparisons.

Screening of a drug repurposing library with a nematode motility assay identifies promising anthelmintic hits against Cooperia oncophora and other ruminant parasites.

Parasitic nematodes continue to cause significant economic losses in livestock globally. Given the limited number of anthelmintic drugs on the market and the currently increasing drug resistance, there is an urgent need for novel anthelmintics. Most motility assays of anthelmintic activity for parasitic nematodes are laborious and low throughput, and therefore not suitable for screening large compound libraries. Cooperia oncophora accounts for a large proportion of reports on the drug-resistance development of parasites globally. Therefore, using a WMicroTracker instrument, we established a practical, automated and low-cost whole-organism motility assay against exsheathed L3 stages (xL3s) of the ruminant parasite Cooperia oncophora, and screened a repurposing library comprising 2745 molecules. Fourteen known anthelmintics contained in this library were picked up in this blind screen, as well as four novel hits: thonzonium bromide, NH125, physostigmine sulfate, and EVP4593. The four hits were also active against xL3s of Ostertagia ostertagi, Haemonchus contortus and Teladorsagia circumcincta using the same assay. Cytotoxicity testing showed that thonzonium bromide and NH125 (1-Benzyl-3-cetyl-2-methylimidazolium iodide) have significant cytotoxicity. EVP4593 (N(4)-(2-(4-phenoxyphenyl)ethyl)-4,6-quinazolinediamine) demonstrated a potent and broad anthelmintic activity, and a high selectivity index. Moreover, given its novel and unexplored chemical scaffold for anthelmintic activity, EVP4593 is an interesting anthelmintic hit for further optimization.

Advantages and shortcomings of cell-based electrical impedance measurements as a GPCR drug discovery tool.

G Protein-Coupled Receptors (GPCRs) transduce extracellular signals and activate intracellular pathways, usually through activating associated G proteins. Due to their involvement in many human diseases, they are recognized worldwide as valuable drug targets. Many experimental approaches help identify small molecules that target GPCRs, including in vitro cell-based reporter assays and binding studies. Most cell-based assays use one signaling pathway or reporter as an assay readout. Moreover, they often require cell labeling or the integration of reporter systems. Over the last decades, cell-based electrical impedance biosensors have been explored for drug discovery. This label-free method holds many advantages over other cellular assays in GPCR research. The technology requires no cell manipulation and offers real-time kinetic measurements of receptor-mediated cellular changes. Instead of measuring the activity of a single reporter, the impedance readout includes information on multiple signaling events. This is beneficial when screening for ligands targeting orphan GPCRs since the signaling cascade(s) of the majority of these receptors are unknown. Due to its sensitivity, the method also applies to cellular models more relevant to disease, including patient-derived cell cultures. Despite its advantages, remaining issues regarding data comparability and interpretability has limited implementation of cell-based electrical impedance (CEI) in drug discovery. Future optimization must include both full exploitation of CEI response data using various ways of analysis as well as further exploration of its potential to detect biased activities early on in drug discovery. Here, we review the contribution of CEI technology to GPCR research, discuss its comparative benefits, and provide recommendations.

OdK2, a Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae.

The first Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae (OdK2) was purified, sequenced and characterized physiologically. OdK2 consists of 38 amino acids, including six conserved cysteine and a C-terminal lysine residue, as revealed by the unique use of a quadrupole ion cyclotron resonance Fourier-transform mass spectrometer. Based on multiple sequence alignments, OdK2 was classified as alpha-KTX3.11. The pharmacological effects of OdK2 were studied on a panel of eight different cloned K(+) channels (vertebrate Kv1.1-Kv1.6, Shaker IR and hERG) expressed in Xenopus laevis oocytes. Interestingly, OdK2 selectively inhibits the currents through Kv1.3 channels with an IC50 value of 7.2+/-2.7nM.

Abundant plasma protein depletion using ammonium sulfate precipitation and Protein A affinity chromatography.

Plasma is a highly valuable resource for biomarker research since it is easy obtainable and contains a high amount of information on patient health status. Although advancements in the field of proteomics enabled analysis of the plasma proteome, identification of low abundant proteins remains challenging due to high complexity and large dynamic range. In order to reduce the dynamic range of protein concentrations, a tandem depletion technique consisting of ammonium sulfate precipitation and Protein A affinity chromatography was developed. Using this method, 50% of albumin, together with other high abundant proteins such as alpha-1-antitrypsin, was depleted from the plasma sample at 20% to 40% ammonium sulfate saturation levels. In combination with immunoglobulin removal using a Protein A column, this technique delivered up to 40 new low- to medium abundance protein identifications when performing a shotgun mass spectrometry analysis. Compared to non-depleted plasma, 270 additional protein spots were observed during 2D-PAGE analysis. These results illustrate that this tandem depletion method is equivalent to commercial kits which are based on immune-affinity chromatography. Moreover, this method using Protein A immunoglobulin depletion was shown to be highly reproducible and a minimal amount of non-target proteins was depleted. The combination of ammonium sulfate precipitation and Protein A affinity chromatography offers a low cost, efficient, straightforward and reproducible alternative to commercial kits, with proteins remaining in native conformation, allowing protein activity and protein interaction studies.

Iterative Chemical Engineering of Vancomycin Leads to Novel Vancomycin Analogs With a High in Vitro Therapeutic Index.

Vancomycin is a glycopeptide antibiotic that inhibits transpeptidation during cell wall synthesis by binding to the D-Ala-D-Ala termini of lipid II. For long, it has been used as a last resort antibiotic. However, since the emergence of the first vancomycin-resistant enterococci in 1987, vancomycin resistance has become widespread, especially in hospitals. We have synthesized and evaluated 110 vancomycin analogs modified at the C-terminal carboxyl group of the heptapeptide moiety with R2NHR1NH2 substituents. Through iterative optimizations of the substituents, we identified vancomycin analogs that fully restore (or even exceed) the original inhibitory activity against vancomycin-resistant enterococci (VRE), vancomycin-intermediate (VISA) and vancomycin-resistant Staphylococcus aureus (VRSA) strains. The best analogs have improved growth inhibitory activity and in vitro therapeutic indices against a broad set of VRE and methicillin-resistant S. aureus (MRSA) isolates. They also exceed the activity of vancomycin against Clostridium difficile ribotypes. Vanc-39 and Vanc-42 have a low probability to provoke antibiotic resistance, and overcome different vancomycin resistance mechanisms (VanA, VanB, and VanC1).