Antinociceptive and Cytotoxic Activity of Opioid Peptides with Hydrazone and Hydrazide Moieties at the C-Terminus.

In the present contribution, we analyze the influence that C-terminal extension of short opioid peptide sequences by organic fragments has on receptor affinity, in vivo analgesic activity, and antimelanoma properties. The considered fragments were based on either N-acylhydrazone (NAH) or N'-acylhydrazide motifs combined with the 3,5-bis(trifluoromethyl)phenyl moiety. Eleven novel compounds were synthesized and subject to biological evaluation. The analyzed compounds exhibit a diversified range of affinities for the µ opioid receptor (MOR), rather low δ opioid receptor (DOR) affinities, and no appreciable neurokinin-1 receptor binding. In three out of four pairs, N-acylhydrazone-based derivatives bind MOR better than their N'-acylhydrazide counterparts. The best of the novel derivatives have similar low nanomolar MOR binding affinity as the reference opioids, such as morphine and biphalin. The obtained order of MOR affinities was compared to the results of molecular docking. In vivo, four tested compounds turned out to be relatively strong analgesics. Finally, the NAH-based analogues reduce the number of melanoma cells in cell culture, while their N'-acylhydrazide counterparts do not. The antimelanoma properties are roughly correlated to the lipophilicity of the compounds.

Can Keratin Scaffolds be used for Creating Three-dimensional Cell Cultures?

Three-dimensional (3D) cell cultures were created with the use of fur keratin associated proteins (F-KAPs) as scaffolds. The procedure of preparation F-KAP involves combinations of chemical activation and enzymatic digestion. The best result in porosity and heterogeneity of F-KAP surface was received during pepsin digestion. The F-KAP had a stable structure, no changes were observed after heat treatment, shaking and washing. The 0.15-0.5 mm fraction had positive effect for formation of 3D scaffolds and cell culturing. Living rat mesenchymal cells on the F-KAP with no abnormal morphology were observed by SEM during 32 days of cell culturing.

Molecular Antioxidant Properties and In Vitro Cell Toxicity of the p-Aminobenzoic Acid (PABA) Functionalized Peptide Dendrimers.

BACKGROUND: Exposure to ozone level and ultraviolet (UV) radiation is one of the major concerns in the context of public health. Numerous studies confirmed that abundant free radicals initiate undesired processes, e.g. carcinogenesis, cells degeneration, etc. Therefore, the design of redox-active molecules with novel structures, containing radical quenchers molecules with novel structures, and understanding their chemistry and biology, might be one of the prospective solutions. Methods: We designed a group of peptide dendrimers carrying multiple copies of p-aminobenzoic acid (PABA) and evaluated their molecular antioxidant properties in 1,1'-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) tests. Cytotoxicity against human melanoma and fibroblast cells as well as against primary cerebral granule cells (CGC) alone and challenged by neurotoxic sodium glutamate and production of reactive oxygen species (ROS) in presence of dendrimers were measured. Results: PABA-terminated dendrimers express enhanced radical and radical cation scavenging properties in relation to PABA alone. In cellular tests, the dendrimers at 100 M fully suppress and between 20⁻100 M reduce proliferation of the human melanoma cell line. In concentration 20 M dendrimers generate small amount of the reactive oxygen species (<25%) but even in their presence human fibroblast and mouse cerebellar granule cells remain intact Moreover, dendrimers at 0.2⁻20 µM concentration (except one) increased the percentage of viable fibroblasts and CGC cells treated with 100 M glutamate. Conclusions: Designed PABA-functionalized peptide dendrimers might be a potential source of new antioxidants with cationic and neutral radicals scavenging potency and/or new compounds with marked selectivity against human melanoma cell or glutamate-stressed CGC neurons. The scavenging level of dendrimers depends strongly on the chemical structure of dendrimer and the presence of other groups that may be prompted into radical form. The present studies found different biological properties for dendrimers constructed from the same chemical fragments but the differing structure of the dendrimer tree provides once again evidence that the structure of dendrimer can have a significant impact on drug⁻target interactions.

Bioinspired Amphiphilic Peptide Dendrimers as Specific and Effective Compounds against Drug Resistant Clinical Isolates of E. coli.

Evolution-derived natural compounds have been inspirational for design of numerous pharmaceuticals, e.g., penicillins and tetracyclines. Herein, we present a bioinspired strategy to design peptide dendrimers for the effective therapy of E. coli infections where the selection of appropriate amino acids and the mode of their assembly are based on the information gained from research on membranolytic natural antimicrobial peptides (AMP's). On the molecular level two opposite effects were explored: the effect of multiple positive charges necessary for membrane disintegration was equilibrated by the anchoring role of tryptophanes. Indeed, a series of Trp-terminated dendrimers exhibited high potency against clinical isolates of antibiotic resistant ESBL E. coli strains, stability in human plasma along with very low hemo- and genotoxicity. Investigation of the underlying antimicrobial mechanism indicated that the dendrimers studied at minimal inhibitory concentration showed weak permeability toward membranes. Solid-state 2D NMR studies revealed their presence on and inside the model membranes. Therefore, their biological properties might be explained by targeting of extra- or intracellular receptors. Our results point to a new approach to design novel branched antimicrobials with high therapeutic index.

Hydrazone Linker as a Useful Tool for Preparing Chimeric Peptide/Nonpeptide Bifunctional Compounds.

The area of multitarget compounds, joining two pharmacophores within one molecule, is a vivid field of research in medicinal chemistry. Not only pharmacophoric elements are essential for the design and activity of such compounds, but the type and length of linkers used to connect them are also crucial. In the present contribution, we describe compound 1 in which a typical opioid peptide sequence is combined with a fragment characteristic for neurokinin-1 receptor (NK1R) antagonists through a hydrazone bridge. The compound has a high affinity for µ- and δ-opioid receptors (IC50= 12.7 and 74.0 nM, respectively) and a weak affinity for the NK1R. Molecular modeling and structural considerations explain the observed activities. In in vivo test, intrathecal and intravenous administrations of 1 exhibited a strong analgesic effect, which indicates potential BBB penetration. This letter brings an exemplary application of the hydrazone linker for fast, facile, and successful preparation of chimeric compounds.

Biphalin preferentially recruits peripheral opioid receptors to facilitate analgesia in a mouse model of cancer pain - A comparison with morphine.

The search for new drugs for cancer pain management has been a long-standing goal in basic and clinical research. Classical opioid drugs exert their primary antinociceptive effect upon activating opioid receptors located in the central nervous system. A substantial body of evidence points to the relevance of peripheral opioid receptors as potential targets for cancer pain treatment. Peptides showing limited blood-brain-barrier permeability promote peripheral analgesia in many pain models. In the present study we examined the peripheral and central analgesic effect of intravenously administered biphalin - a dimeric opioid peptide in a mouse skin cancer pain model, developed by an intraplantar inoculation of B16F0 melanoma cells. The effect of biphalin was compared with morphine - a golden standard in cancer pain management. Biphalin produced profound, dose-dependent and naloxone sensitive spinal analgesia. Additionally, the effect in the tumor-bearing paw was largely mediated by peripheral opioid receptors, as it was readily attenuated by the blood-brain-barrier-restricted opioid receptor antagonist - naloxone methiodide. On the contrary, morphine facilitated its analgesic effect primarily by activating spinal opioid receptors. Both drugs induced tolerance in B16F0 - implanted paws after chronic treatment, however biphalin as opposed to morphine, showed little decrease in its activity at the spinal level. Our results indicate that biphalin may be considered a future alternative drug in cancer pain treatment due to an enhanced local analgesic activity as well as lower tolerance liability compared with morphine.

Chronic mild stress facilitates melanoma tumor growth in mouse lines selected for high and low stress-induced analgesia.

Both chronic stress conditions and hyperergic reaction to environmental stress are known to enhance cancer susceptibility. We described two mouse lines that displayed high (HA) and low (LA) swim stress-induced analgesia (SSIA) to investigate the relationship between inherited differences in sensitivity to stress and proneness to an increased growth rate of subcutaneously inoculated melanoma. These lines display several genetic and physiological differences, among which distinct sensitivity to mutagens and susceptibility to cancer are especially noticeable. High analgesic mice display high proneness both to stress and a rapid local spread of B16F0 melanoma. However, stress-resistant LA mice do not develop melanoma tumors after inoculation, or if so, tumors regress spontaneously. We found that the chronic mild stress (CMS) procedure leads to enhanced interlinear differences in melanoma susceptibility. Tumors developed faster in stress conditions in both lines. However, LA mice still displayed a tendency for spontaneous regression, and 50% of LA mice did not develop a tumor, even under stressed conditions. Moreover, we showed that chronic stress, but not tumor progression, induces depressive behavior, which may be an important clue in cancer therapy. Our results clearly indicate how the interaction between genetic susceptibility to stress and environmental stress determine the risk and progression of melanoma. To our knowledge, HA/LA mouse lines are the first animal models of distinct melanoma progression mediated by inherited differences in stress reactivity.