Mechanistic insights on the antibacterial action of the kyotorphin peptide derivatives revealed by in vitro studies and Galleria mellonella proteomic analysis.

OBJECTIVES: The selected kyotorphin derivatives were tested to improve their antimicrobial and antibiofilm activity. The antimicrobial screening of the KTP derivatives were ascertained in the representative strains of bacteria, including Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa. METHODS: Kyotorphin derivatives, KTP-NH2, KTP-NH2-DL, IbKTP, IbKTP-NH2, MetKTP-DL, MetKTP-LD, were designed and synthesized to improve lipophilicity and resistance to enzymatic degradation. Peptides were synthesized by standard solution or solid-phase peptide synthesis and purified using RP-HPLC, which resulted in >95 % purity, and were fully characterized by mass spectrometry and 1H NMR. The minimum inhibitory concentrations (MIC) determined for bacterial strains were between 20 and 419 µM. The direct effect of IbKTP-NH2 on bacterial cells was imaged using scanning electron microscopy. The absence of toxicity, high survival after infection and an increase in the hemocytes count was evaluated by injections of derivatives in Galleria mellonella larvae. Proteomics analyses of G. mellonella hemolymph were performed to investigate the underlying mechanism of antibacterial activity of IbKTP-NH2 at MIC. RESULTS: IbKTP-NH2 induces morphological changes in bacterial cell, many differentially expressed proteins involved in DNA replication, synthesis of cell wall, and virulence were up-regulated after the treatment of G. mellonella with IbKTP-NH2. CONCLUSION: We suggest that this derivative, in addition to its physical activity on the bacterial membranes, can elicit a cellular and humoral immune response, therefore, it could be considered for biomedical applications.

Antifungal and anti-biofilm activity of designed derivatives from kyotorphin.

Kyotorphin (KTP, l-tyrosyl-l-arginine) is an endogenous analgesic neuropeptide first isolated from bovine brain in 1979. Previous studies have shown that kyotorphins possess anti-inflammatory and antimicrobial activity. Six kyotorphins-KTP-NH2, KTP-NH2-DL, ibuprofen-conjugated KTP (IbKTP), IbKTP-NH2, N-methyl-D-Tyr-L-Arg, and N-methyl-L-Tyr-D-Arg-were designed and synthesized to improve lipophilicity and resistance to enzymatic degradation. This study assessed the antimicrobial and antibiofilm activity of these peptides. The antifungal activity of kyotorphins was determined in representative strains of Candida species, including Candida albicans ATCC 10231, Candida krusei ATCC 6258, and six clinical isolates-Candida dubliniensis 19-S, Candida glabrata 217-S, Candida lusitaniae 14-S, Candida novergensis 51-S, Candida parapsilosis 63, and Candida tropicalis 140-S-obtained from the oral cavity of HIV-positive patients. The peptides were synthesized by standard solution or solid-phase synthesis, purified by RP-HPLC (purity >95 %), and characterized by nuclear magnetic resonance. The results of the broth microdilution assay and scanning electron microscopy showed that IbKTP-NH2 presented significant antifungal activity against Candida strains and antibiofilm activity against the clinical isolates. The absence of toxic activity and survival after infection was assessed after injecting the peptide in larvae of Galleria mellonella as experimental infection model. Furthermore, IbKTP-NH2 had strong antimicrobial activity against multidrug-resistant bacteria and fungi and was not toxic to G. mellonella larvae up to a concentration of 500 mM. These results suggest that IbKTP-NH2, in addition to its known effect on cell membranes, can elicit a cellular immune response and, therefore, is promising for biomedical application.

Amidated and Ibuprofen-Conjugated Kyotorphins Promote Neuronal Rescue and Memory Recovery in Cerebral Hypoperfusion Dementia Model.

Chronic brain ischemia is a prominent risk factor for neurological dysfunction and progression for dementias, including Alzheimer's disease (AD). In rats, permanent bilateral common carotid artery occlusion (2VO) causes a progressive neurodegeneration in the hippocampus, learning deficits and memory loss as it occurs in AD. Kyotorphin (KTP) is an endogenous antinociceptive dipeptide whose role as neuromodulator/neuroprotector has been suggested. Recently, we designed two analgesic KTP-derivatives, KTP-amide (KTP-NH2) and KTP-NH2 linked to ibuprofen (IbKTP-NH2) to improve KTP brain targeting. This study investigated the effects of KTP-derivatives on cognitive/behavioral functions (motor/spatial memory/nociception) and hippocampal pathology of female rats in chronic cerebral hypoperfusion (2VO-rat model). 2VO-animals were treated with KTP-NH2 or IbKTP-NH2 for 7 days at weeks 2 and 5 post-surgery. After behavioral testing (week 6), coronal sections of hippocampus were H&E-stained or immunolabeled for the cellular markers GFAP (astrocytes) and NFL (neurons). Our findings show that KTP-derivatives, mainly IbKTP-NH2, enhanced cognitive impairment of 2VO-animals and prevented neuronal damage in hippocampal CA1 subfield, suggesting their potential usefulness for the treatment of dementia.

The anti-inflammatory action of the analgesic kyotorphin neuropeptide derivatives: insights of a lipid-mediated mechanism.

Recently, a designed class of efficient analgesic drugs derived from an endogenous neuropeptide, kyotorphin (KTP, Tyr-Arg) combining C-terminal amidation (KTP-NH2) and N-terminal conjugation to ibuprofen (Ib), IbKTP-NH2, was developed. The Ib moiety is an enhancer of KTP-NH2 analgesic action. In the present study, we have tested the hypothesis that KTP-NH2 is an enhancer of the Ib anti-inflammatory action. Moreover, the impact of the IbKTP-NH2 conjugation on microcirculation was also evaluated by a unified approach based on intravital microscopy in the murine cremasteric muscle. Our data show that KTP-NH2 and conjugates do not cause damage on microcirculatory environment and efficiently decrease the number of leukocyte rolling induced by lipopolysaccharide (LPS). Isothermal titration calorimetry showed that the drugs bind to LPS directly thus contributing to LPS aggregation and subsequent elimination. In a parallel study, molecular dynamics simulations and NMR data showed that the IbKTP-NH2 tandem adopts a preferential "stretched" conformation in lipid bilayers and micelles, with the simulations indicating that the Ib moiety is anchored in the hydrophobic core, which explains the improved partition of IbKTP-NH2 to membranes and the permeability of lipid bilayers to this conjugate relative to KTP-NH2. The ability to bind glycolipids concomitant to the anchoring in the lipid membranes through the Ib residue explains the analgesic potency of IbKTP-NH2 given the enriched glycocalyx of the blood-brain barrier cells. Accumulation of IbKTP-NH2 in the membrane favors both direct permeation and local interaction with putative receptors as the location of the KTP-NH2 residue of IbKTP-NH2 and free KTP-NH2 in lipid membranes is the same.

Correlation between membrane translocation and analgesic efficacy in kyotorphin derivatives.

Amidated kyotorphin (L-Tyr-L-Arg-NH2; KTP-NH2) causes analgesia when systemically administered. The lipophilic ibuprofen-conjugated derivative of KTP-NH2 has improved analgesic efficacy. However, fast degradation by peptidases impacts negatively in the pharmacodynamics of these drugs. In this work, selected derivatives of KTP and KTP-NH2 were synthesized to combine lipophilicity and resistance to enzymatic degradation. Eight novel structural modifications were tested for the potential to transverse lipid membranes and to evaluate their efficacy in vivo. The rationale behind the design of the pool of the eight selected molecules consisted in the addition of individual group at the N-terminus, namely the tert-butyloxycarbonyl (Boc), γ-aminobutyric acid (GABA), acetyl, butanoyl, and propanoyl or in the substitution of the tyrosine residue by an indole moiety and in the replacement of the peptidic bond by a urea-like bond in some cases. All the drugs used in the study are intrinsically fluorescent, which enables the use of spectrofluorimetry to sample the drugs in the permeation assays. The results show that the BOC and indolyl derivatives of KTP-NH2 have maximal ability to permeate membranes with concomitant maximal analgesic power. Overall, the results demonstrate that membrane permeation is correlated with analgesic efficacy. However, this is not the only factor accounting for analgesia. KTP-NH2 for instance has low passive permeation but is known to have central action. In this case, hypothetical transcytosis over the blood-brain barrier seems to depend on dipeptide transporters.

Antimicrobial properties of analgesic kyotorphin peptides unraveled through atomic force microscopy.

Antimicrobial peptides (AMPs) are promising candidates as alternatives to conventional antibiotics for the treatment of resistant pathogens. In the last decades, new AMPs have been found from the cleavage of intact proteins with no antibacterial activity themselves. Bovine hemoglobin hydrolysis, for instance, results in AMPs and the minimal antimicrobial peptide sequence was defined as Tyr-Arg plus a positively charged amino acid residue. The Tyr-Arg dipeptide alone, known as kyotorphin (KTP), is an endogenous analgesic neuropeptide but has no antimicrobial activity itself. In previous studies new KTP derivatives combining C-terminal amidation and Ibuprofen (Ib) - KTP-NH(2), IbKTP, IbKTP-NH(2) - were designed in order to improve KTP brain targeting. Those modifications succeeded in enhancing peptide-cell membrane affinity towards fluid anionic lipids and higher analgesic activity after systemic injection resulted therefrom. Here, we investigated if this affinity for anionic lipid membranes also translates into antimicrobial activity because bacteria have anionic membranes. Atomic force microscopy revealed that KTP derivatives perturbed Staphylococcus aureus membrane structure by inducing membrane blebbing, disruption and lysis. In addition, these peptides bind to red blood cells but are non-hemolytic. From the KTP derivatives tested, amidated KTP proves to be the most active antibacterial agent. The combination of analgesia and antibacterial activities with absence of toxicity is highly appealing from the clinical point of view and broadens the therapeutic potential and application of kyotorphin peptides.