Current Protein and Peptide Science - Volume 20, Issue 11, 2019

Small molecule spectrophotometric probes for DNA/RNA and proteins are of the utmost importance for diagnostics in biochemical and biomedical research. Both, naturally occurring and synthetic probes, often include peptide sequence responsible for the selectivity toward the particular target; however, commercially available dyes are restricted to single point attachment to the peptide (having one reactive group). Here presented are our recent advances in the development of novel amino acidfluorophore probes, with the unique characteristic of free N- and C-terminus available for incorporation at any peptide backbone position. Intriguingly, already monomeric amino acid-fluorophores showed recognition among various DNA/RNA, whereby steric impact and contribution of halogens is systematically studied. Moreover, some dyes revealed intracellular mitochondria specificity. Further, several hetero-dimeric chromophore systems were prepared, demonstrating that synergistic effect can lead to simultaneous DNA, RNA and protein fluorimetric recognition, combined with enzyme inhibition. Also, homodimeric cyanines equipped with chlorine revealed intriguing DNA/RNA selectivity in respect to well-known parent TOTO and YOYO dyes.

Lactoferrin was isolated and purified for the first time over 50-years ago. Since then, extensive studies on the structure and function of this protein have been performed and the research is still being continued. In this mini-review we focus on presenting recent scientific efforts towards the elucidation of the role and therapeutic potential of lactoferrin saturated with iron(III) or manganese(III) ions. The difference in biological activity of metal-saturated lactoferrin vs. the unmetalated one is emphasized. The strategies for oral delivery of lactoferrin, are also reviewed, with particular attention to the metalated protein.

Ruthenium(II) polypyridyl complexes have been extensively studied for the past few decades as promising anticancer agents. Despite the expected intravenous route of administration, the interaction between Ru(II) polypyridyl compounds and serum proteins is not well characterized and vast majority of the available literature data concerns determination of the binding constant. Ru-protein adducts can modify the biological effects of the Ru complexes influencing their cytotoxic and antimicrobial activity as well as introduce significant changes in their photophysical properties. More extensive research on the interaction between serum proteins and Ru(II) polypyridyl complexes is important for further development of Ru(II) polypyridyl compounds towards their application in anticancer therapy and diagnostics and can open new opportunities for already developed complexes.

Single stranded microbial DNA fragments with unmethylated deoxycytidylyldeoxyguanosine dinucleotide (CpG) motifs are interpreted as danger signals by the innate immune system via recognition by the Toll-like Receptor 9 (TLR9). Their synthetic analogues, Oligodeoxynucleotides (ODN) comprise a promising class of immune modulators with potential applications in the treatment of multiple diseases, such as cancer, autoimmune diseases or allergy. ODN molecules contain a core hexamer sequence, which is species specific consisting of GACGTT and AACGT for mouse and GTCGTT in humans. Assessment of structural features of different type of ODNs is highly challenging. NMR spectroscopic insights were gained for a short, single CpG motif containing ODN 1668. The structural basis of ODN recognition by TLR9 recently started to unravel as crystal structures of TLR9 orthologues in complex with ODN 1668 were solved. Systematic investigations of ODN sequences revealed that ODNs with a single CpG motif are capable of activating mouse TLR9, but two closely positioned CpG motifs are necessary for activation of human TLR9. Furthermore, longer ODNs with TCC and TCG sequences at the 5’ end were shown to activate TLR9 with higher efficiency. It was revealed that 5’-xCx motif containing short ODNs (sODN) are able to augment the immune response of short, single CpG containing ODNs, which are incapable of activating of TLR9 alone. All these observations pointed to the existence of a second binding site on TLR9, which was characterized in crystal structures that delivered further insights of the nucleic acid recognition of the innate immune system by TLR9.

The concept of ligand potency is briefly discussed here as well as why this is still a challenge for its complete comprehension. In this context, we explain also the meaning of ligand efficiency (LE), which has been greeted with both enthusiasm and criticism among the drug design audience. A full understanding of LE requires the complex interpretation of the potency concept presenting the uncertainty similar to this of the Zeno paradox. In reality, the efficiency of LE is caused by the high degree of preference for slim pharma drug candidates.

The recent high-resolution structures of amyloid fibrils show that the organization of peptide segments into amyloid aggregate architecture is a general process, though the morphology is more complex and intricate than suspected previously. The amyloid fibrils are often cytotoxic, accumulating as intracellular inclusions or extracellular plaques and have the ability to interfere with cellular physiology causing various cellular malfunctions. At the same time, the highly ordered amyloid structures also present an opportunity for nature to store and protect peptide chains under extreme conditions – something that might be used for designing storage, formulation, and delivery of protein medications or for contriving bio-similar materials of great resistance or structure-ordering capacity. Here we summarize amyloid characteristics; discussing the basic morphologies, sequential requirements and 3D-structure that are required for the understanding of this newly (re)discovered protein structure – a prerequisite for developing either inhibitors or promoters of amyloid-forming processes

Enzymes of the prolyl oligopeptidase family (S9 family) recognize their substrates not only by the specificity motif to be cleaved but also by size - they hydrolyze oligopeptides smaller than 30 amino acids. They belong to the serine-protease family, but differ from classical serine-proteases in size (80 kDa), structure (two domains) and regulation system (size selection of substrates). This group of enzymes is an important target for drug design as they are linked to amnesia, schizophrenia, type 2 diabetes, trypanosomiasis, periodontitis and cell growth. By comparing the structure of various members of the family we show that the most important features contributing to selectivity and efficiency are: (i) whether the interactions weaving the two domains together play a role in stabilizing the catalytic triad and thus their absence may provide for its deactivation: these oligopeptidases can screen their substrates by opening up, and (ii) whether the interaction-prone β-edge of the hydrolase domain is accessible and thus can guide a multimerization process that creates shielded entrance or intricate inner channels for the size-based selection of substrates. These cornerstones can be used to estimate the multimeric state and selection strategy of yet undetermined structures.

Calmodulin (CaM) is a highly conserved eukaryotic Ca2+ sensor protein that is able to bind a large variety of target sequences without a defined consensus sequence. The recognition of this diverse target set allows CaM to take part in the regulation of several vital cell functions. To fully understand the structural basis of the regulation functions of CaM, the investigation of complexes of CaM and its targets is essential. In this minireview we give an outline of the different types of CaM - peptide complexes with 3D structure determined, also providing an overview of recently determined structures. We discuss factors defining the orientations of peptides within the complexes, as well as roles of anchoring residues. The emphasis is on complexes where multiple binding modes were found.

Several endocrine glands produce steroid hormones. Thanks to the work of chemists and biochemists, the main synthetic as well as metabolic pathways of steroid hormones were included in the textbooks more than 50 years ago and the classical endocrine gland functions were identified. Later on, evidence of steroid hormone effects beyond the classical endocrine gland function has been accumulating. Testosterone was shown to participate in the stress response and may influence coping with stressors. We have shown a decrease in testosterone concentrations in saliva in children undergoing a school exam compared to values on a non-exam school day. Testosterone has been associated with different cognitive functions in both adults and children. Circulating testosterone has been linked to negative symptoms of schizophrenia. Aldosterone is acting via mineralocorticoid receptors, which are thought to be fully occupied by glucocorticoids in the brain. Until now, an action of aldosterone in the brain has not been considered at all, because the enzyme 11-beta-hydroxysteroid dehydrogenase type 2, which would enable aldosterone to bind to receptors is absent in most of the brain areas. We have brought evidence that aldosterone can act in the brain and produce anxiogenic and depressogenic effects. To facilitate the translation of animal findings into clinical research, we have developed methodology for measurement of salivary aldosterone and obtained first data on a relationship between salivary aldosterone and trait anxiety. We have shown that salivary aldosterone concentrations reflect treatment outcome in patients with major depressive disorder.

Background: As the bacterial resistance to antibacterial chemotherapeutics is one of the greatest problems in modern medicine, efforts are made to develop new antimicrobial drugs. Compounds with a piperazine ring have proved to be promising agents against various pathogens. Objective: The aim of the study was to prepare a series of new N-phenylpiperazines and determine their activity against various pathogens. Method: Target compounds were prepared by multi-step synthesis starting from an appropriate substituted acid to an oxirane intermediate reacting with 1-(4-nitrophenyl)piperazine. Lipophilicity and pKa values were experimentally determined. Other molecular parameters were calculated. The inhibitory activity of the target compounds against Staphylococcus aureus, four mycobacteria strains, Bipolaris sorokiniana, and Fusarium avenaceum was tested. In vitro antiproliferative activity was determined on a THP-1 cell line, and toxicity against plant was determined using Nicotiana tabacum. Results: In general, most compounds demonstrated only moderate effects. 1-(2-Hydroxy-3-{[4-(propan- 2-yloxy)benzoyl]oxy}propyl)-4-(4-nitrophenyl)piperazinediium dichloride and 1-{3-[(4-butoxybenzoyl)- oxy]-2-hydroxypropyl}-4-(4-nitrophenyl)piperazinediium dichloride showed the highest inhibition activity against M. kansasii (MIC = 15.4 and 15.0 μM, respectively) and the latter also against M. marinum (MIC = 15.0 μM). 1-(2-Hydroxy-3-{[4-(2-propoxyethoxy)benzoyl]oxy}propyl)-4-(4-nitrophenyl)piperazinediium dichloride had the highest activity against F. avenaceum (MIC = 14.2 μM). All the compounds showed only insignificant toxic effects on human and plant cells. Conclusion: Ten new 1-(4-nitrophenyl)piperazine derivatives were prepared and analyzed, and their antistaphylococcal, antimycobacterial, and antifungal activities were determined. The activity against M. kansasii was positively influenced by higher lipophilicity, the electron-donor properties of substituent R and a lower dissociation constant. The exact mechanism of action will be investigated in follow-up studies.