The Synthetic Peptide LyeTx I mn∆K, Derived from Lycosa erythrognatha Spider Toxin, Is Active against Methicillin-Resistant Staphylococcus aureus (MRSA) In Vitro and In Vivo.

The urgent global health challenge posed by methicillin-resistant Staphylococcus aureus (MRSA) infections demands effective solutions. Antimicrobial peptides (AMPs) represent promising tools of research of new antibacterial agents and LyeTx I mn∆K, a short synthetic peptide based on the Lycosa erythrognatha spider venom, is a good representative. This study focused on analyzing the antimicrobial activities of LyeTx I mn∆K, including minimum inhibitory and bactericidal concentrations, synergy and resensitization assays, lysis activity, the effect on biofilm, and the bacterial death curve in MRSA. Additionally, its characterization was conducted through isothermal titration calorimetry, dynamic light scattering, calcein release, and finally, efficacy in a mice wound model. The peptide demonstrates remarkable efficacy against planktonic cells (MIC 8-16 µM) and biofilms (>30% of inhibition) of MRSA, and outperforms vancomycin in terms of rapid bactericidal action and anti-biofilm effects. The mechanism involves significant membrane damage. Interactions with bacterial model membranes, including those with lysylphosphatidylglycerol (LysylPOPG) modifications, highlight the versatility and selectivity of this compound. Also, the peptide has the ability to sensitize resistant bacteria to conventional antibiotics, showing potential for combinatory therapy. Furthermore, using an in vivo model, this study showed that a formulated gel containing the peptide proved superior to vancomycin in treating MRSA-induced wounds in mice. Together, the results highlight LyeTx I mnΔK as a promising prototype for the development of effective therapeutic strategies against superficial MRSA infections.

PnPP-15, a Synthetic Peptide Derived from a Toxin from Phoneutria nigriventer Spider Venom, Alleviates Diabetic Neuropathic Pain and Acts Synergistically with Pregabalin in Mice.

Diabetic neuropathic pain is one of the complications that affect a wide variety of the diabetic population and is often difficult to treat. Only a small number of patients experience pain relief, which usually comes with onerous side effects and low levels of satisfaction. The search for new analgesic drugs is necessary, given the limitations that current drugs present. Combining drugs to treat neuropathic pain has been attracting interest to improve their efficacy compared to single-drug monotherapies while also reducing dose sizes to minimize side effects. The aim of our study was to verify the antinociceptive effect of a synthetic peptide, PnPP-15, alone and combined with pregabalin, in male Swiss diabetic mice using the von Frey method. PnPP-15 is a synthetic peptide derived from PnPP19, a peptide representing a discontinuous epitope of the primary structure of the toxin PnTx2-6 from the venom of the spider Phoneutria nigriventer. The antinociceptive activity of both compounds was dose-dependent and showed synergism, which was verified by isobolographic analysis. Treatment with PnPP-15 did not cause spontaneous or forced motor changes and did not cause any damage or signs of toxicity in the analyzed organs (pancreas, lung, heart, kidney, brain, or liver). In conclusion, PnPP-15 is a great candidate for an analgesic drug against neuropathic pain caused by diabetes and exerts a synergistic effect when combined with pregabalin, allowing for even more efficient treatment.

Therapeutic Prospection of Animal Venoms-Derived Antimicrobial Peptides against Infections by Multidrug-Resistant Acinetobacter baumannii: A Systematic Review of Pre-Clinical Studies.

Infections caused by multidrug-resistant Acinetobacter baumannii (MDR-Ab) have become a public health emergency. Due to the small therapeutic arsenal available to treat these infections, health agencies have highlighted the importance of developing new antimicrobials against MDR-Ab. In this context, antimicrobial peptides (AMPs) stand out, and animal venoms are a rich source of these compounds. Here, we aimed to summarize the current knowledge on the use of animal venom-derived AMPs in the treatment of MDR-Ab infections in vivo. A systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The eight studies included in this review identified the antibacterial activity of eleven different AMPs against MDR-Ab. Most of the studied AMPs originated from arthropod venoms. In addition, all AMPs are positively charged and rich in lysine residues. In vivo assays showed that the use of these compounds reduces MDR-Ab-induced lethality and bacterial load in invasive (bacteremia and pneumonia) and superficial (wounds) infection models. Moreover, animal venom-derived AMPs have pleiotropic effects, such as pro-healing, anti-inflammatory, and antioxidant activities, that help treat infections. Animal venom-derived AMPs are a potential source of prototype molecules for the development of new therapeutic agents against MDR-Ab.

Antifungal activity of a shortened analogue of the natural peptide LyeTx I isolated from the venom of the spider Lycosa erythrognatha.

The increase in the incidence of fungal infections associated with the limited therapeutic arsenal available and the increasing rate of resistance of pathogenic fungi reinforce the need for research of new antifungal agents. Thus, this study aims to evaluate the antifungal activity of the peptide LyeTx I mnΔK, a shortened analogue of the natural peptide LyeTx I derived from spider venom, against Candida species. LyeTx I mnΔK showed potent activity against Candida spp. with minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) between 4 and 32 µM. The peptide also completely inhibited the yeast-to-hypha transition (at 2 µM) and broke mature biofilms (67% reduction at 32 µM) of C. albicans. In addition, LyeTx I mnΔK did not induce resistance in C. albicans during 21 days of exposure. Therefore, the LyeTx I mnΔK is a promising prototype for the development of new antifungal agents.

Animal venoms as a source of antiviral peptides active against arboviruses: a systematic review.

Arthropod-borne viruses (arboviruses), such as Zika virus (ZIKV), chikungunya virus (CHIKV), dengue virus (DENV), yellow fever virus (YFV), and West Nile virus (WNV), are pathogens of global importance. Therefore, there has been an increasing need for new drugs for the treatment of these viral infections. In this context, antimicrobial peptides (AMPs) obtained from animal venoms stand out as promising compounds because they exhibit strong antiviral activity against emerging arboviral pathogens. Thus, we systematically searched and critically analyzed in vitro and in vivo studies that evaluated the anti-arbovirus effect of peptide derivatives from toxins produced by vertebrates and invertebrates. Thirteen studies that evaluated the antiviral action of 10 peptides against arboviruses were included in this review. The peptides were derived from the venom of scorpions, spiders, wasps, snakes, sea snails, and frogs and were tested against DENV, ZIKV, YFV, WNV, and CHIKV. Despite the high structural variety of the peptides included in this study, their antiviral activity appears to be associated with the presence of positive charges, an excess of basic amino acids (mainly lysine), and a high isoelectric point (above 8). These peptides use different antiviral mechanisms, the most common of which is the inhibition of viral replication, release, entry, or fusion. Moreover, peptides with virucidal and cytoprotective (pre-treatment) effects were also identified. In conclusion, animal-venom-derived peptides stand out as a promising alternative in the search and development of prototype antivirals against arboviruses.

LyeTxI-b, a Synthetic Peptide Derived From a Spider Venom, Is Highly Active in Triple-Negative Breast Cancer Cells and Acts Synergistically With Cisplatin.

Breast cancer is the most common cancer that affects women globally and is among the leading cause of women's death. Triple-negative breast cancer is more difficult to treat because hormone therapy is not available for this subset of cancer. The well-established therapy against triple-negative breast cancer is mainly based on surgery, chemotherapy, and immunotherapy. Among the drugs used in the therapy are cisplatin and carboplatin. However, they cause severe toxicity to the kidneys and brain and cause nausea. Therefore, it is urgent to propose new chemotherapy techniques that provide new treatment options to patients affected by this disease. Nowadays, peptide drugs are emerging as a class of promising new anticancer agents due to their lytic nature and, apparently, a minor drug resistance compared to other conventional drugs (reviewed in Jafari et al., 2022). We have recently reported the cytotoxic effect of the antimicrobial peptide LyeTx I-b against glioblastoma cells (Abdel-Salam et al., 2019). In this research, we demonstrated the cytotoxic effect of the peptide LyeTx I-b, alone and combined with cisplatin, against triple-negative cell lines (MDA-MD-231). LyeTx-I-b showed a selectivity index 70-fold higher than cisplatin. The peptide:cisplatin combination (P:C) 1:1 presented a synergistic effect on the cell death and a selective index value 16 times greater than the cisplatin alone treatment. Therefore, an equi-effective reduction of cisplatin can be reached in the presence of LyeTx I-b. Cells treated with P:C combinations were arrested in the G2/M cell cycle phase and showed positive staining for acridine orange, which was inhibited by bafilomycin A1, indicating autophagic cell death (ACD) as a probable cell death mechanism. Furthermore, Western blot experiments indicated a decrease in P21 expression and AKT phosphorylation. The decrease in AKT phosphorylation is indicative of ACD. However, other studies are still necessary to better elucidate the pathways involved in the cell death mechanism induced by the peptide and the drug combinations. These findings confirmed that the peptide LyeTx I-b seems to be a good candidate for combined chemotherapy to treat breast cancer. In addition, in vivo studies are essential to validate the use of LyeTx I-b as a therapeutic drug candidate, alone and/or combined with cisplatin.

From the PnTx2-6 Toxin to the PnPP-19 Engineered Peptide: Therapeutic Potential in Erectile Dysfunction, Nociception, and Glaucoma.

The venom of the "armed" spider Phoneutria nigriventer comprises several potent toxins. One of the most toxic components from this venom is the neurotoxin PnTx2-6 (LD50 = ∼ 0.7 µg/mouse, 48 residues, five disulfide bridges, MW = 5,289.31 Da), which slows down the inactivation of various Na+ channels. In mice and rats, this toxin causes priapism, an involuntary and painful erection, similar to what is observed in humans bitten by P. nigriventer. While not completely elucidated, it is clear that PnTx2-6 potentiates erectile function via NO/cGMP signaling, but it has many off-target effects. Seeking to obtain a simpler and less toxic molecule able to retain the pharmacological properties of this toxin, we designed and synthesized the peptide PnPP-19 (19 residues, MW = 2,485.6 Da), representing a discontinuous epitope of PnTx2-6. This synthetic peptide also potentiates erectile function via NO/cGMP, but it does not target Na+ channels, and therefore, it displays nontoxic properties in animals even at high doses. PnPP-19 effectively potentiates erectile function not only after subcutaneous or intravenous administration but also following topical application. Surprisingly, PnPP-19 showed central and peripheral antinociceptive activity involving the opioid and cannabinoid systems, suggesting applicability in nociception. Furthermore, considering that PnPP-19 increases NO availability in the corpus cavernosum, this peptide was also tested in a model of induced intraocular hypertension, characterized by low NO levels, and it showed promising results by decreasing the intraocular pressure which prevents retinal damage. Herein, we discuss how was engineered this smaller active non-toxic peptide with promising results in the treatment of erectile dysfunction, nociception, and glaucoma from the noxious PnTx2-6, as well as the pitfalls of this ongoing journey.

LyeTx I-b Peptide Attenuates Tumor Burden and Metastasis in a Mouse 4T1 Breast Cancer Model.

Cationic anticancer peptides have exhibited potent anti-proliferative and anti-inflammatory effects in neoplastic illness conditions. LyeTx I-b is a synthetic peptide derived from Lycosa erythrognatha spider venom that previously showed antibiotic activity in vitro and in vivo. This study focused on the effects of LyeTxI-b on a 4T1 mouse mammary carcinoma model. Mice with a palpable tumor in the left flank were subcutaneously or intratumorally injected with LyeTx I-b (5 mg/kg), which significantly decreased the tumor volume and metastatic nodules. Histological analyses showed a large necrotic area in treated primary tumors compared to the control. LyeTxI-b reduced tumor growth and lung metastasis in the 4T1 mouse mammary carcinoma model with no signs of toxicity in healthy or cancerous mice. The mechanism of action of LyeTx I-b on the 4T1 mouse mammary carcinoma model was evaluated in vitro and is associated with induction of apoptosis and cell proliferation inhibition. Furthermore, LyeTx I-b seems to be an efficient regulator of the 4T1 tumor microenvironment by modulating several cytokines, such as TGF-ß, TNF-α, IL-1ß, IL-6, and IL-10, in primary tumor and lung, spleen, and brain. LyeTx I-b also plays a role in leukocytes rolling and adhesion into spinal cord microcirculation and in the number of circulating leukocytes. These data suggest a potent antineoplastic efficacy ofLyeTx I-b.

Tityus serrulatus (Scorpion): From the Crude Venom to the Construction of Synthetic Peptides and Their Possible Therapeutic Application Against Toxoplasma gondii Infection.

Toxoplasmosis, caused by Toxoplasma gondii, is a major public concern owing to its neurotropic nature and high morbidity and mortality rates in immunocompromised patients and newborns. Current treatment for this disease is inefficient and produces side effects. Inflammatory mediators produced during T. gondii infection (e.g., cytokines and nitric oxide) are crucial in controlling parasite replication. In this context, Tityus serrulatus venom (TsV) induces the production of inflammatory mediators by immune cells. Thus, this study aimed to isolate and identify the components of TsV with potential anti-T. gondii activity. TsV was extracted from scorpions and lyophilized or loaded onto a column to obtain its fractions. TsV subfractions were obtained using chromatography, and its amino acid sequence was identified and applied to peptide design using bioinformatics tools. The C57BL/6 mice and their harvested macrophages were used to test the anti-Toxoplasma activity of TsV components and peptides. TsV and its fraction F6 attenuated the replication of tachyzoites in macrophages and induced nitric oxide and cytokine (IL-12, TNF, and IL-6) production by infected cells, without host cell toxicity. Moreover, Su6-B toxin, a subfraction of F6, demonstrated anti-T. gondii activity. The partially elucidated and characterized amino acid sequence of Sub6-B demonstrated 93% similarity with T. serrulatus 2 toxin (Ts2). Ts2 mimetic peptides ("Pep1," "Pep2a," and "Pep2b") were designed and synthesized. Pep1 and Pep2a, but not Pep2b, reduced the replication of tachyzoites in macrophages. In vivo, treatment of T. gondii-infected mice with Pep1, Pep2a, or Pep2b decreased the number of cerebral cysts and did not induce hepatotoxicity in the animals. Taken together, our data show promising immunomodulatory and antiparasitic activity of TsV that could be explored and applied in future therapies for treating infectious parasitic diseases such as toxoplasmosis.

Synthetic Peptides Derived From Lycosa Erythrognatha Venom: Interaction With Phospholipid Membranes and Activity Against Resistant Bacteria.

Superbugs are a public health problem, increasing the need of new drugs and strategies to combat them. Our group has previously identified LyeTxI, an antimicrobial peptide isolated from Lycosa erythrognatha spider venom. From LyeTxI, we synthesized and characterized a derived peptide named LyeTxI-b, which has shown significant in vitro and in vivo activity. In this work, we elucidate the interaction of LyeTxI-b with artificial membranes as well as its effects on resistant strains of bacteria in planktonic conditions or biofilms. Isothermal titration calorimetry revealed that LyeTxI-b interacts more rapidly and with higher intensity with artificial vesicles, showing higher affinity to anionic vesicles, when compared to synthetic LyeTxI. In calcein experiments, LyeTxI-b caused greater levels of vesicle cleavage. Both peptides showed antibacterial activity at concentrations of µmol L-1 against 12 different clinically isolated strains, in planktonic conditions, in a concentration-dependent manner. Furthermore, both peptides elicited a dose-dependent production of reactive oxygen species in methicillin-resistant Staphylococcus aureus. In S. aureus biofilm assay, LyeTxI-b was more potent than LyeTxI. However, none of these peptides reduced Escherichia coli biofilms. Our results show LyeTxI-b as a promising drug against clinically resistant strains, being a template for developing new antibiotics.

A short synthetic peptide, based on LyeTx I from Lycosa erythrognatha venom, shows potential to treat pneumonia caused by carbapenem-resistant Acinetobacter baumannii without detectable resistance.

The emergence of antibiotic-resistant bacteria, especially carbapenem-resistant Acinetobacter baumannii (CRAB), together with relative stagnation in the development of effective antibiotics, has led to enormous health and economic problems. In this study, we aimed to describe the antibacterial spectrum of LyeTx I mnΔK, a short synthetic peptide based on LyeTx I from Lycosa erythrognatha venom, against CRAB. LyeTx I mnΔK showed considerable antibacterial activity against extensively resistant A. baumannii, with minimum inhibitory and bactericidal concentrations ranging from 1 to 16 µM and 2 to 32 µM, respectively. This peptide significantly increased the release of 260 nm-absorbing intracellular material from CRAB, suggesting bacteriolysis. LyeTx I mnΔK was shown to act synergistically with meropenem and colistin against CRAB. The cytotoxic concentration of LyeTx I mnΔK against Vero cells (CC50 = 55.31 ± 5.00 µM) and its hemolytic activity (HC50 = 77.07 ± 4.00 µM) were considerably low; however, its antibacterial activity was significantly reduced in the presence of human and animal serum and trypsin. Nevertheless, the inhalation of this peptide was effective in reducing pulmonary bacterial load in a mouse model of CRAB infection. Altogether, these results demonstrate that the peptide LyeTx I mnΔK is a potential prototype for the development of new effective and safe antibacterial agents against CRAB.

The synthetic peptide PnPP-19 potentiates erectile function via nNOS and iNOS.

PnPP-19 peptide has a primary sequence design based on molecular modeling studies of PnTx2-6 toxin. It comprises the amino acid residues that are potentially significant for the pharmacological action of PnTx2-6. Ex vivo and in vivo experiments in normotensive, hypertensive, or diabetic murine models have shown a significant improvement in penile erection after administration of PnPP-19. Given the potential use of PnPP-19 in pharmaceutical formulations to treat erectile dysfunction and the lack of information concerning its mode of action, the present work investigates its activities on the nitrergic system. PnPP-19 induced a significant increase in nitric oxide (NO) and cGMP levels in corpus cavernosum (cc). These effects were inhibited by l-NAME, a non-selective inhibitor of nitric oxide synthase (NOS); were partially inhibited by 7- Nitroindazole, a selective inhibitor of neuronal NOS (nNOS); and were abolished by L-NIL, a selective inhibitor of inducible NOS (iNOS). This potentiating effect was not affected by atropine. PnPP-19 also led to changes in mRNA levels, protein expression and phosphorylation at specific sites of NOS, in cc. Assays using cavernous tissue from knockout mice to endothelial NOS (eNOS), nNOS or iNOS showed that PnPP-19 potentiates relaxation only in eNOS-knockout mice, which suggests an essential role for nNOS. Surprisingly, iNOS enhanced the potentiation of erectile function evoked by PnPP-19. Our results demonstrate that this new synthetic peptide potentiates erectile function via nitric oxide activation and reinforce its role as a new pharmacological tool for the treatment of erectile dysfunction.

Tityus serrulatus scorpion venom as a potential drug source for Chagas' disease: Trypanocidal and immunomodulatory activity.

Current chemical therapies for Chagas Disease (CD) lack ability to clear Trypanosoma cruzi (Tc) parasites and cause severe side effects, making search for new strategies extremely necessary. We evaluated the action of Tityus serrulatus venom (TsV) components during Tc infection. TsV treatment increased nitric oxide and pro-inflammatory cytokine production by Tc-infected macrophages (MØ), decreased intracellular parasite replication and trypomastigotes release, also triggering ERK1/2, JNK1/2 and p38 activation. Ts7 demonstrated the highest anti-Tc activity, inducing high levels of TNF and IL-6 in infected MØ. TsV/Ts7 presented synergistic effect on p38 activation when incubated with Tc antigen. KPP-treatment of MØ also decreased trypomastigotes releasing, partially due to p38 activation. TsV/Ts7-pre-incubation of Tc demonstrated a direct effect on parasite decreasing MØ-trypomastigotes releasing. In vivo KPP-treatment of Tc-infected mice resulted in decreased parasitemia. Summarizing, this study opens perspectives for new bioactive molecules as CD-therapeutic treatment, demonstrating the TsV/Ts7/KPP-trypanocidal and immunomodulatory activity during Tc infection.

Neurotoxic and convulsant effects induced by jack bean ureases on the mammalian nervous system.

Ureases are microbial virulence factors either because of the enzymatic release of ammonia or due to many other non-enzymatic effects. Here we studied two neurotoxic urease isoforms, Canatoxin (CNTX) and Jack Bean Urease (JBU), produced by the plant Canavalia ensiformis, whose mechanisms of action remain elusive. The neurotoxins provoke convulsions in rodents (LD50 ∼2 mg/kg) and stimulate exocytosis in cell models, affecting intracellular calcium levels. Here, electrophysiological and brain imaging techniques were applied to elucidate their mode of action. While systemic administration of the toxins causes tonic-clonic seizures in rodents, JBU injected into rat hippocampus induced spike-wave discharges similar to absence-like seizures. JBU reduced the amplitude of compound action potential from mouse sciatic nerve in a tetrodotoxin-insensitive manner. Hippocampal slices from CNTX-injected animals or slices treated in vitro with JBU failed to induce long term potentiation upon tetanic stimulation. Rat cortical synaptosomes treated with JBU released L-glutamate. JBU increased the intracellular calcium levels and spontaneous firing rate in rat hippocampus neurons. MicroPET scans of CNTX-injected rats revealed increased [18]Fluoro-deoxyglucose uptake in epileptogenesis-related areas like hippocampus and thalamus. Curiously, CNTX did not affect voltage-gated sodium, calcium or potassium channels currents, neither did it interfere on cholinergic receptors, suggesting an indirect mode of action that could be related to the ureases' membrane-disturbing properties. Understanding the neurotoxic mode of action of C. ensiformis ureases could help to unveil the so far underappreciated relevance of these toxins in diseases caused by urease-producing microorganisms, in which the human central nervous system is affected.

Participation of Central Muscarinic Receptors on the Nervous Form of Chagas Disease in Mice Infected via Intracerebroventricular with Colombian Trypanosoma cruzi Strain.

Acute chagasic encephalitis is a clinically severe central nervous system (CNS) manifestation. However, the knowledge of the nervous form of Chagas disease is incomplete. The role of the muscarinic acetylcholine receptor (mAChR) on mice behavior and brain lesions induced by Trypanosoma cruzi (Colombian strain) was herein investigated in mice treated with the mAChR agonist and antagonist (carbachol and atropine), respectively. Immunosuppressed or non-immunosuppressed mice were intracerebroventricularly (icv) or intraperitoneally (ip) infected. All groups were evaluated 15 d.p.i. (days post infection). Intraperitoneally infected animals had subpatent parasitemia. Patent parasitemia occurred only in icv infected mice. The blockade of mAChR increased the parasitemia, parasitism and lesions compared to its activation. Infected not treated (INT ip) mice did not present meningitis and encephalitis, regardless of immunosuppression. INT icv brains presented higher cellularity, discrete signs of cellular degeneration, frequent presence of parasites and focal meningitis. The immunosuppressed atropine + icv mice presented increased intracellular parasitism associated with degenerative parenchymal changes, while carbachol + icv mice presented discrete meningitis, preservation of the cortex and absence of relevant parasitism. Cholinergic receptor blockage increased impairment of coordination vs. receptor activation. Muscarinic cholinergic pathway seems to be involved in immune mediated cell invasion events while its blockade favored infection evolution, brain lesions, and behavioral alterations.

Small cyclic sodium channel inhibitors.

Voltage-gated sodium (NaV) channels play crucial roles in a range of (patho)physiological processes. Much interest has arisen within the pharmaceutical industry to pursue these channels as analgesic targets following overwhelming evidence that NaV channel subtypes NaV1.7-NaV1.9 are involved in nociception. More recently, NaV1.1, NaV1.3 and NaV1.6 have also been identified to be involved in pain pathways. Venom-derived disulfide-rich peptide toxins, isolated from spiders and cone snails, have been used extensively as probes to investigate these channels and have attracted much interest as drug leads. However, few peptide-based leads have made it as drugs due to unfavourable physiochemical attributes including poor in vivo pharmacokinetics and limited oral bioavailability. The present work aims to bridge the gap in the development pipeline between drug leads and drug candidates by downsizing these larger venom-derived NaV inhibitors into smaller, more "drug-like" molecules. Here, we use molecular engineering of small cyclic peptides to aid in the determination of what drives subtype selectivity and molecular interactions of these downsized inhibitors across NaV subtypes. We designed a series of small, stable and novel NaV probes displaying NaV subtype selectivity and potency in vitro coupled with potent in vivo analgesic activity, involving yet to be elucidated analgesic pathways in addition to NaV subtype modulation.

Shortened derivatives from native antimicrobial peptide LyeTx I: In vitro and in vivo biological activity assessment.

In the continuing search for novel antibiotics, antimicrobial peptides are promising molecules, due to different mechanisms of action compared to classic antibiotics and to their selectivity for interaction with microorganism cells rather than with mammalian cells. Previously, our research group has isolated the antimicrobial peptide LyeTx I from the venom of the spider Lycosa erythrognatha. Here, we proposed to synthesize three novel shortened derivatives from LyeTx I (LyeTx I mn; LyeTx I mnΔK; LyeTx I mnΔKAc) and to evaluate their toxicity and biological activity as potential antimicrobial agents. Peptides were synthetized by Fmoc strategy and circular dichroism analysis was performed, showing that the three novel shortened derivatives may present membranolytic activity, like the original LyeTx I, once they folded as an alpha helix in 2.2.2-trifluorethanol and sodium dodecyl sulfate. In vitro assays revealed that the shortened derivative LyeTx I mnΔK presents the best score between antimicrobial (↓ MIC) and hemolytic (↑ EC50) activities among the synthetized shortened derivatives, and LUHMES cell-based NeuriTox test showed that it is less neurotoxic than the original LyeTx I (EC50 [LyeTx I mnΔK] ⋙ EC50 [LyeTx I]). In vivo data, obtained in a mouse model of septic arthritis induced by Staphylococcus aureus, showed that LyeTx I mnΔK is able to reduce infection, as demonstrated by bacterial recovery assay (∼10-fold reduction) and scintigraphic imaging (less technetium-99m labeled-Ceftizoxime uptake by infectious site). Infection reduction led to inflammatory process and pain decreases, as shown by immune cells recruitment reduction and threshold nociception increment, when compared to positive control group. Therefore, among the three shortened peptide derivatives, LyeTx I mnΔK is the best candidate as antimicrobial agent, due to its smaller amino acid sequence and toxicity, and its greater biological activity.

Funcionalização do polietilenoglicol metil éter via reação de bromoacetilação: ativação, caracterização por MALDI-TOF e mecanismo de reação / Poly(ethylene glycol) methyl ether functionalization by bromoacetylation reaction: Activation, characterization by MALDI-TOF and reaction mechanism / Funcionalización de polietilenglicol metil éter mediante reacción de bromoacetilación: activación, caracterización por MALDI-TOF y mecanismo de reacción

Resumo A PEGuilação, reação química de conjugação com a molécula de polietilenoglicol (PEG) ou polietilenoglicol metil éter (mPEG), tem sido amplamente aplicada pelas indústrias farmacêuticas como estratégia de melhoria das propriedades farmaco-cinéticas de compostos bioativos. O PEG é um polímero que possui um esqueleto de poliéter quimicamente inerte e que apresenta grupos hidroxilas (-OH) em suas extremidades. Assim, o PEG para tornar-se apto como reagente de conjugação deve ser ativado com um grupo funcional que seja reativo. Nesse sentido, a bromoaceti-lação apresenta-se como uma alternativa para a funcionalização do PEG. Portanto, nesse trabalho objetivamos descrever em detalhes os procedimentos e o mecanismo de reação envolvida na funcionalização do mPEG, através da reação de bromoacetilação. Além do mais, estudamos a aplicação do MALDI-ToF para a caracterização do produto ativado. Após a bromoacetilação, por um procedimento adaptado, obteve-se o bromoacetil-mPEG-éster, com rendimento bruto de 56,78%. Análises posteriores, por espectrometria de massas por MALDI-ToF, possibilitaram identificar e caracterizar o produto bromoacetilado. Entre as condições de reação, o controle de temperatura (-10 °C a 0 °C) mostrou-se eficaz favorecendo a adição nucleofílica essencial à bromoacetilação. Assim, concluímos que o controle da baixa temperatura reacional é um fator chave para o favorecimento da adição nucleofílica à carbonila e, portanto, essencial na obtenção do mPEG funcionalizado via bromoacetilação. Estudos posteriores serão necessários, no entanto, para confirmar se o mPEG esterificado, nessas condições, poderá ser utilizado na conjugação com moléculas de natureza proteica ou peptídica, por meio de substituição nucleofílica bimolecular.

SUMMARY PEGylation, a chemical reaction of conjugation with the polyethylene glycol molecule (PEG), has been widely applied by the pharmaceutical industries as a strategy to improve the pharmacokinetic properties of bioactive compounds. PEG is a polymer that has a chemically inert polyether backbone and hydroxyl groups (-OH) at its ends. Thus, PEG to become fit as a reagent for conjugation must be activated with a functional group that is reactive. In this sense, bromoacetylation presents itself as an alternative for the functionalization of PEG. Therefore, in this study we aim to describe in detail the procedures and reaction mechanism involved in the functionalization of mPEG through the bromoacetylation reaction. In addition, we used the spectrometric technique, by MALDI-ToF, for the characterization of the activated product. After applying an adapted bromoacetylation procedure, bromoacetyl-mPEG-ester was obtained with a yield of 56.78%. Subsequent analyzes of MALDI-ToF mass spectrometry were able to correctly identify and characterize the bromoacety-lated product. Among the reaction conditions, temperature control (from -10 °C to 0 °C) was effective in favoring the essential nucleophilic addition to bromoacetylation. Thus, we conclude that the control of the low reaction temperature is a key factor in favoring the nucleophilic addition to carbonyl and, therefore, obtaining a favorable conversion to functionalized PEG via bromoacetylation. Further studies, however, will be necessary to confirm whether PEG esterified with these conditions can be used in conjunction with molecules of a protein or peptide nature by means of bimolecular nucleophilic substitution.

RESUMEN La PEGilación, una reacción química de conjugación con la molécula de polietilenglicol (PEG), ha sido ampliamente aplicada por las industrias farmacéuticas como una estrategia para mejorar las propiedades farmacocinéticas de los compuestos bioactivos. El PEG es un polímero formado por un esqueleto de poliéter químicamente inerte con grupos hidroxilo (-OH) en sus extremos. Por lo tanto, para usar el PEG como reactivo de conjugación debe activarse con un grupo funcional que sea reactivo. En este sentido, la bromoacetilación es una alternativa para la funcionalización de PEG. De esta manera, en este trabajo nuestro objetivo es describir en detalle los procedimientos y el mecanismo de reacción involucrados en la funcionalización de PEG a través de la reacción de bromoacetilación. Además, estudiamos la aplicación de MALDI-ToF para la caracterización del producto activado. Después de aplicar un procedimiento de bromoacetilación adaptado, se obtuvo bromoacetil-mPEG-éster con un rendimiento bruto de 56,78%. Los análisis posteriores de espectrometría de masas por MALDI-ToF pudieron identificar y caracterizar correctamente el producto bromoacetilado. Entre las condiciones de reacción, el control de la temperatura (desde -10 °C hasta 0 °C) fue eficaz para favorecer la adición nucleofílica esencial a la bromoacetilación. Así, concluimos que el control de la baja temperatura de reacción es un factor clave para favorecer la adición nucleofílica al carbonilo y, por lo tanto, esencial para obtener el mPEG funcionalizado mediante la bromoacetilación. Sin embargo, serán necesarios más estudios para confirmar si el mPEG esterificado en estas condiciones puede usarse junto con moléculas de naturaleza proteica o peptídica por medio de la sustitución nucleófila bimolecular.