RESUMO
The biological activity of antimicrobial peptides and proteins is closely related to their structural aspects and is sensitive to certain post-translational modifications such as glycosylation, lipidation and PEGylation. However, PEGylation of protein and peptide drugs has expanded in recent years due to the reduction of their toxicity. Due to their size, the PEGylation process can either preserve or compromise the overall structure of these biopolymers and their biological properties. The antimicrobial peptide LyeTx I-bcys was synthesized by Fmoc strategy and coupled to polyethylene glycol 2.0 kDa. The conjugates were purified by HPLC and characterized by MALDI-ToF-MS analysis. Microbiological assays with LyeTx I-bcys and LyeTx I-bPEG were performed against Staphylococcus aureus (ATCC 33591) and Escherichia coli (ATCC 25922) in liquid medium. MIC values of 2.0 and 1.0 µM for LyeTx I-bcys and 8.0 and 4.0 µM for LyeTx I-bPEG were observed against S. aureus and E. coli, respectively. PEGylation of LyeTx I-bcys (LyeTx I-bPEG) decreased the cytotoxicity determined by MTT method for VERO cells compared to the non-PEGylated peptide. In addition, structural and biophysical studies were performed to evaluate the effects of PEGylation on the nature of peptide-membrane interactions. Surface Plasmon Resonance experiments showed that LyeTx I-b binds to anionic membranes with an association constant twice higher than the PEGylated form. The three-dimensional NMR structures of LyeTx I-bcys and LyeTx I-bPEG were determined and compared with the LyeTx I-b structure, and the hydrodynamic diameter and zeta potential of POPC:POPG vesicles were similar upon the addition of both peptides. The mPEG-MAL conjugation of LyeTx I-bcys gave epimers, and it, together with LyeTx I-bPEG, showed clear α-helical profiles. While LyeTx I-bcys showed no significant change in amphipathicity compared to LyeTx I-b, LyeTx I-bPEG was found to have a slightly less clear separation between hydrophilic and hydrophobic faces. However, the similar conformational freedom of LyeTx I-b and LyeTx I-bPEG suggests that PEGylation does not cause significant structural changes. Overall, our structural and biophysical studies indicate that the PEGylation does not alter the mode of peptide interaction and maintains antimicrobial activity while minimizing tissue toxicity, which confirmed previous results obtained in vivo. Interestingly, significantly improved proteolytic resistance to trypsin and proteinase K was observed after PEGylation.
RESUMO
INTRODUCTION: With the aim of overcoming the high toxicity of PnTx2-6 (or δ-CNTX-Pn2a), a toxin from the venom of the armed spider (Phoneutria nigriventer), the 19-aminoacid peptide, PnPP-19 (P nigriventer potentiator peptide), was synthesized based on molecular modeling studies of PnTx2-6. PnPP-19 improved the erectile function of normotensive rats and mice, without eliciting side effects, and no signs of toxicity were observed. In addition, PnPP-19 was able to potentiate the effect of sildenafil. AIM: To evaluate the efficacy of PnPP-19 in hypertensive and diabetic mouse/rat models in restoring erectile function, after topical administration; verify the biodistribution of PnPP-19 administration (topical and intravenous), permeation, and cyclic guanosine monophosphate (cGMP)/nitric oxide via implication. METHODS: Corpus cavernosum relaxation was evaluated using cavernous strips from male spontaneous hypertensive rats (SHR) and from streptozotocin (STZ)-diabetic mice contracted with phenylephrine and submitted to electrical field stimulation before and after incubation with PnPP-19 (10-8 mol/L, 10 minutes) or vehicle. This procedure was also used to determine cGMP/nitric oxide levels, at 8 Hz and to check the effect of PnPP-19 with sildenafil citrate. Biodistribution assays were performed using iodine 123-radiolabeled PnPP-19. In vivo erectile function was evaluated using intracavernosal pressure/main arterial pressure ratio in STZ-diabetic rats after PnPP-19 topical administration. MAIN OUTCOME MEASURES: PnPP-19 may become a new drug able to fill the gap in the pharmacologic treatment of erectile dysfunction, especially for hypertensive and diabetic individuals RESULTS: PnPP-19 potentiated corpus cavernosum relaxation, in both control and SHR rats. SHR-cavernosal tissue treated with PnPP-19 (1-32 Hz) reached the same relaxation levels as control Wistar rats (16 and 32 Hz). PnPP-19 treatment improved cavernosal tissue relaxation in STZ-diabetic mice and rats. PnPP-19 enhanced cGMP levels in STZ-diabetic mice corpus cavernosum strips. After topical or intravenous administration in rats, 123I-PnPP-19 was mainly recruited to the penis. When topically administered (400 µg/rat), PnPP-19 restores erectile function in STZ-diabetic rats, also improving it in healthy rats by increasing the intracavernosal pressure/main arterial pressure ratio. PnPP-19 exhibited an additive effect when co-administered with sildenafil, showing a novel mode of action regardless of phosphodiesterase type 5 inhibition. CLINICAL IMPLICATIONS: PnPP-19 seems to be an indicated drug to be tested to treat ED in diabetic and hypertensive patients. STRENGTH & LIMITATIONS: PnPP-19, although active by topical application and showing safety to human beings (not shown), has low permeability, about 10% of the applied dose. CONCLUSION: Our results showed that PnPP-19 may emerge as a potent new drug that can be topically administered, becoming a promising alternative for erectile dysfunction treatment. Nunes da Silva C, Pedrosa Nunes K, De Marco Almeida F, et al. PnPP-19 Peptide Restores Erectile Function In Hypertensive And Diabetic Animals Through Intravenous And Topical Administration. J Sex Med 2019;16:365-374.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Disfunção Erétil/tratamento farmacológico , Peptídeos/farmacologia , Venenos de Aranha/farmacologia , Administração Intravenosa , Administração Tópica , Animais , GMP Cíclico/metabolismo , Disfunção Erétil/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Ereção Peniana/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Ratos Wistar , Citrato de Sildenafila/farmacologia , Estreptozocina , Distribuição TecidualRESUMO
In order to better understand the relationship between the primary structure of TsHpt-I - a bradykinin-potentiating peptide (BPP) isolated from the venom of the yellow scorpion Tityus serrulatus, with a non-canonical Lys residue prior to the conservative Pro-Pro doublet - and its cardiovascular effects, a series of ladder peptides were synthesized using the C-terminal portion of TsHpt-I as a template. All synthetic peptides having the Pro-Pro doublet at their C-terminal were able to potentiate the hypotensive effect of bradykinin. Conversely, only those analogues having Lys residue could induce a transient hypotension when intravenously administrated in male rats, indicating that the positive charge located toward the radical of this amino acid residue is crucial for this cardiovascular effect. Differently from all known BPPs, TsHpt-I acts as an agonist of the B(2) receptor and does not inhibit angiotensin-converting enzyme. The capacity of this peptide to activate this subtype of kinin receptor, releasing NO, was also affected by the absence of Lys' side-chain positive charge. Moreover, this study has demonstrated that the minimization of the primary structure of TsHpt-I does not significantly alter the biological effects of this native peptide, which could be of interest for biotechnological purposes.