A bioanalytical screening method for Enterococcus faecalis RNPP-type quorum sensing peptides in murine feces.

Background: Bacteria coordinate their behavior as a group via communication with their peers, known as 'quorum sensing'. Enterococcus faecalis employs quorum sensing via RNPP-peptides which were not yet reported to be present in mammalian biofluids. Results: Solid phase extraction of murine feces was performed, followed by ultra high performance liquid chromatography (UHPLC-MS/MS) in multiple reaction monitoring (MRM) mode (in total <90 min/sample) for the nine known RNPP peptides. Limits of detection ranged between 0.045 and 52 nM. Adequate identification criteria allowed detection of RNPP quorum sensing peptides in 2/20 wild-type murine feces samples (i.e., cAM373 and cOB1). Conclusion: A fit-for-purpose UHPLC-MS/MS method detected these RNPP peptides in wild-type murine feces samples.

Chemical Classification of Cyclic Depsipeptides.

Cyclic depsipeptides (CDPs) are a family of cyclic peptide-related compounds, of which the ring is mainly composed of amino- and hydroxy acid residues joined by amide and ester bonds (at least one), leading to a wide diversity of fascinating chemical structures. They differ in both their ring structure and their side chains, especially by the nature of the unusual and non-amino acid building blocks. To date, however, there is no overall uniform chemical classification system available for CDPs and naming of the diverse family members is done rather arbitrarily. Therefore, a broad evaluation of different CDP structures is done, i.e., 1348 naturally occurring CDPs were included, and a straightforward chemical classification system using apparent chemical characteristics is proposed in order to organize the currently scattered CDP data. The overall validity of the classification approach is verified and the compounds categorized in the same groups are considered to be structurally related. This evaluation also revealed that traditionally formed CDP subfamilies, like the dolastatins, might be misleading from a chemical point of view given the structural differences in this subfamily. This up-to-date CDP overview enables peptide and natural product scientists to study the wide diversity in CDP structures, their chemical interrelationships and identification of existing and newly found CDPs. Together with the available information on the species producing these CDPs and their reported biological activities, this paper provides a useful tool to gain new insights into this diverse group of peptides.

Androgenic and spermatogenic activity of alkylamide-rich ethanol solution extract of Anacyclus pyrethrum DC.

Anacyclus pyrethrum (A. pyrethrum) has been used as Vajikaran Rasayana (aphrodisiac) in traditional Indian ayurvedic medicine to treat male sexual dysfunction, including infertility. Aphrodisiac activity may be due to an increase in the production or effect of androgens, so this study sought to evaluate the androgenic and spermatogenic potential of the alkylamide-rich ethanol solution extract. Male Wistar strain rats weighing between 150 and 180 g were completely randomized divided into five groups. The ethanol solution extract of A. pyrethrum was administered to groups of rats in 50, 100, and 150 mg/kg doses for a period of 28 days, and the action was compared with control and testosterone-treated rats. Thirteen N-alkylamides were detected in the extract by using HPLC/UV/electrospray ionization mass spectrometry method. Extract administration at all the doses produced significant increase in body weight, sperm count, motility, and viability along with serum testosterone, luteinizing hormone, and follicle-stimulating hormone concentrations. Histoarchitecture of testis revealed increased spermatogenic activities. Seminal fructose content was also significantly increased after 28 days of treatment. Our results suggest that the ethanol solution extract of the roots of A. pyrethrum has androgenic potential and may improve male fertility by enhancing spermatogenesis.

A critical quality parameter in quantitative fused-core chromatography: The injection volume.

As part of the method development, the injection volume as a critical quality attribute in fast fused-core chromatography was evaluated. Spilanthol, a pharmaceutically interesting N-alkylamide currently under investigation in our laboratory, was chosen as the model compound. Spilanthol was dissolved in both PBS and MeOH/H2O (70/30, v/v) and subsequently analyzed using a fused-core system hereby selecting five chromatographic characteristics (retention time, area, height, theoretical plates and symmetry factor) as responses. We demonstrated that the injection volume significantly influenced both the qualitative and quantitative performance of fused-core chromatography, a phenomenon which is confounded with the nature of the used sample solvent. From 2 µL up to 100 µL injection volume with PBS as solvent, the symmetry factor decreased favorably by 20%. Moreover, the theoretical plates and the quantitative parameters (area and height) increased up to 30%. On the contrary, in this injection volume range, the theoretical plates for the methanol-based samples decreased by more than 60%, while the symmetry factor increased and the height decreased, both by 30%. The injection volume is thus a critical and often overlooked parameter in fused-core method description and validation.

The influence of peptide impurity profiles on functional tissue-organ bath response: the 11-mer peptide INSL6[151-161] case.

Bioactive peptides have great pharmaceutical potential as nutraceuticals, diagnostics, and therapeutic drugs in several clinical areas. Thus, the search for novel lead peptides with a biological function has attracted renewed interest. Crude peptide material (i.e., ~70% purity) of INSL6[151-161] (NH2-FRSLFWGNHSQ-COOH) was found to trigger a contractile response in guinea pig ileum longitudinal smooth muscle preparations using tissue-organ baths. However, the purified peptide (i.e., ≥ 95% purity) had no effect on this model. Further investigation with crude materials from other suppliers, with purities ranging between 50% and 80%, indicated that the crude products gave a false-positive functional tissue-organ bath conclusion. These observations question the functionality conclusions when using crude-purity peptide materials; during the initial research or discovery phase, peptide quality is generally neglected, possibly leading to misinterpretation of biological results due to by-products from peptide synthesis and, thus, wrong fail/pass decisions. Therefore, we strongly recommend appropriate quality control testing before using any peptides for initial biomedical research or discovery purposes.

Desirability function combining metabolic stability and functionality of peptides.

The evaluation of peptides as potential therapeutic or diagnostic agents requires the consideration of several criteria that are targeted around two axes: functionality and metabolic stability. Most often, a compromise has to be made between these mutually opposing characteristics. In this study, Derringer's desirability function, a multi-criteria decision-making method, was applied to determine the best peptide for opioid studies in a single figure-of-merit. The penetration of the blood-brain barrier (BBB) determines the biological functionality of neuropeptides in the brain target tissue, and consists of an influx and an efflux component. The metabolic stability in the two concerned tissues, i.e. plasma and brain, are taken into consideration as well. The overall selection of the peptide drug candidate having the highest BBB-drugability is difficult due to these conflicting responses as well as the different scalings of the four biological parameters under consideration. The highest desirability, representing the best BBB-drugability, was observed for dermorphin. This peptide is thus the most promising drug candidate from the set of eight opioid peptides that were investigated. The least desirable candidate, with the worst BBB influx and/or metabolic stability, was found to be CTAP. Validation of the desirability function by in vivo medical imaging showed that dermorphin and DAMGO penetrate the BBB, whereas EM-1 and TAPP did not. These results are thus consistent with those obtained with the desirability evaluation. To conclude, the multi-criteria decision method was proven to be useful in biomedical research, where a selection of the best candidate based on opposing characteristics is often required.