Rafaela Scalco Ferreira

Caffeic acid phenethyl ester (CAPE) protects PC12 cells from MPP+ toxicity by inducing the expression of neuron-typical proteins.

Neurite loss is an early event in neurodegenerative diseases; therefore, the regeneration of the network of neurites constitutes an interesting strategy of treatment for such disorders. Neurotrophic factors play a critical role in neuronal regeneration, but their clinical use is limited by their inability to cross the blood brain barrier. Oxidative and inflammatory events are implicated in neurodegeneration and antioxidant compounds have been suggested as potential neuroprotectors. The protective potential of CAPE (caffeic acid phenethyl ester) has been shown in different models of neurotoxicity (in vitro and in vivo) and it has been associated with immune-modulatory, antioxidant and anti-inflammatory properties; however, other mechanisms might be involved. The present study demonstrates that CAPE protects PC12 cells from the cellular death induced by the dopaminergic neurotoxin MPP(+) by increasing the network of neurites. Results showed that CAPE induced the formation, elongation and ramification of neurites in PC12 cells non-stimulated with NGF (nerve growth factor) and inhibited the shortage of neurites induced by the dopaminergic neurotoxin. These effects were associated with increased expression of neuron-typical proteins responsible for axonal growth (GAP-43) and synaptogenesis (synaptophysin and synapsin I). It is noteworthy that, unlike neurotrophins, CAPE would be able to cross the blood brain barrier and exert its neurotrophic effects in the brain. This study corroborates the therapeutic potential of CAPE in neurodegenerative diseases while proposes the involvement of neuroplasticity in the mechanism of neuroprotection.

The neuroprotection of cannabidiol against MPP⁺-induced toxicity in PC12 cells involves trkA receptors, upregulation of axonal and synaptic proteins, neuritogenesis, and might be relevant to Parkinson's disease.

Cannabidiol (CBD) is a non-psychoactive constituent of Cannabis sativa with potential to treat neurodegenerative diseases. Its neuroprotection has been mainly associated with anti-inflammatory and antioxidant events; however, other mechanisms might be involved. We investigated the involvement of neuritogenesis, NGF receptors (trkA), NGF, and neuronal proteins in the mechanism of neuroprotection of CBD against MPP(+) toxicity in PC12 cells. CBD increased cell viability, differentiation, and the expression of axonal (GAP-43) and synaptic (synaptophysin and synapsin I) proteins. Its neuritogenic effect was not dependent or additive to NGF, but it was inhibited by K252a (trkA inhibitor). CBD did not increase the expression of NGF, but protected against its decrease induced by MPP(+), probably by an indirect mechanism. We also evaluated the neuritogenesis in SH-SY5Y cells, which do not express trkA receptors. CBD did not induce neuritogenesis in this cellular model, which supports the involvement of trkA receptors. This is the first study to report the involvement of neuronal proteins and trkA in the neuroprotection of CBD. Our findings suggest that CBD has a neurorestorative potential independent of NGF that might contribute to its neuroprotection against MPP(+), a neurotoxin relevant to Parkinsons disease.

Integração das técnicas de triagem virtual e triagem biológica automatizada em alta escala: oportunidades e desafios em P&D de fármacos

High-throughput screening (HTS) and virtual screening (VS) are useful methods employed in drug discovery, allowing the identification of promising hits for lead optimization. The efficiency of these approaches depends on a number of factors, such as the organization of high quality databases of compounds and the parameterization of essential components of the screen process. This brief review presents the basic principles of the HTS and VS methods, as well as a perspective of the utility and integration of these drug design approaches, highlighting current opportunities and future challenges in medicinal chemistry.

In silico screening strategies for novel inhibitors of parasitic diseases

Introduction: Parasitic diseases are a major global problem causing long-term disability and death, with severe medical and psychological consequences around the world. Despite the prevalence of parasitic disease, the treatment options for many of these illnesses are still inadequate and there is a dire need for new antiparasitic drugs. In silico screening techniques, which are powerful strategies for hit generation, are widely being applied in the design of new ligands for parasitic diseases. Areas covered: This article analyses the application of ligand- and structure-based virtual screening strategies against a variety of parasitic diseases and discusses the benefits of the integration between computational and experimental approaches toward the discovery of new antiparasitic agents. The analysis is illustrated by recent examples, with emphasis on the strategies reported within the past 2 years. Expert opinion: Virtual screening techniques are powerful tools commonly used in drug discovery against parasitic diseases, which have provided new opportunities for the identification of several novel compound classes with antiparasitic activity.

High concentration of trichlorfon (1 mM) disrupts axonal cytoskeleton and decreases the expression of plasticity-related proteins in SH-SY5Y cells

Some organophosphorus compounds (OPs) induce a neurodegenerative disorder known as organophosphate-induced delayed neuropathy (OPIDN), which is related to irreversible inhibition of neuropathy target esterase (NTE) and impairment of neurite outgrowth. The present study addresses the effects of trichlorfon, mipafox (neuropathic model) and paraoxon (non-neuropathic model) on neurite outgrowth and neuroplasticity-related proteins in retinoic-acid-stimulated SH-SY5Y cells, a cellular model widely used to study the neurotoxicity of OPs. Mipafox (20μM) decreased cellular differentiation and the expression of neurofilament 200 (NF-200), growth associated- (GAP-43) and synaptic proteins (synapsin I and synaptophysin); whereas paraoxon (300μM) induced no effect on cellular differentiation, but significant decrease of NF-200, GAP-43, synapsin I and synaptophysin as compared to controls. However, the effects of paraoxon on these proteins were significantly lower than the effects of mipafox. In conclusion, axonal cytoskeletal proteins, as well as axonal plasticity-related proteins are more effectively affected by neuropathic (mipafox) than by non-neuropathic (paraoxon) OPs, suggesting that they might play a role in the mechanism of OPIDN. At high concentration (1mM), trichlorfon induced effects similar to those of the neuropathic OP, mipafox (20μM), but also caused high inhibition of AChE. Therefore, these effects are unlikely to occur in humans at non-lethal doses of trichlorfon.

Non-cytotoxic Concentration of Cisplatin Decreases Neuroplasticity-Related Proteins and Neurite Outgrowth Without Affecting the Expression of NGF in PC12 Cells

Cisplatin is the most effective and neurotoxic platinum chemotherapeutic agent. It induces a peripheral neuropathy characterized by distal axonal degeneration that might progress to degeneration of cell bodies and apoptosis. Most symptoms occur nearby distal axonal branches and axonal degeneration might induce peripheral neuropathy regardless neuronal apoptosis. The toxic mechanism of cisplatin has been mainly associated with DNA damage, but cisplatin might also affect neurite outgrowth. Nevertheless, the neurotoxic mechanism of cisplatin remains unclear. We investigated the early effects of cisplatin on axonal plasticity by using non-cytotoxic concentrations of cisplatin and PC12 cells as a model of neurite outgrowth and differentiation. PC12 cells express NGF-receptors (trkA) and respond to NGF by forming neurites, branches and synaptic vesicles. For comparison, we used a neuronal model (SH-SY5Y cells) that does not express trkA nor responds to NGF. Cisplatin did not change NGF expression in PC12 cells and decreased neurite outgrowth in both models, suggesting a NGF/trkA independent mechanism. It also reduced axonal growth (GAP-43) and synaptic (synapsin I and synaptophysin) proteins in PC12 cells, without inducing mitochondrial damage or apoptosis. Therefore, cisplatin might affect axonal plasticity before DNA damage, NGF/trkA down-regulation, mitochondrial damage or neuronal apoptosis. This is the first study to show that neuroplasticity-related proteins might be early targets of the neurotoxic action of cisplatin and their role on cisplatin-induced peripheral neuropathy should be investigated in vivo.

A synthetic snake-venom-based tripeptide (Glu-Val-Trp) protects PC12 cells from MPP+ toxicity by activating the NGF-signaling pathway

HighlightsA synthetic analogue (p‐BTX‐I) of a snake venom peptide mimics NGF in PC12 cells.p‐BTX‐I increases neuritogenesis and viability of PC12 cells exposed to MPP+.Like NGF, p‐BTX‐I activates trkA, PI3K/AKT and MAPK‐ERK pathways.Like NGF, p‐BTX‐I increases the expression of GAP‐43 and synapsin I.The neurotrophic action of p‐BTX‐I in PC12 cells is not dependent on NGF. ABSTRACT Venom small peptides that target neurotrophin receptors might be beneficial in neurodegeneration, including Parkinsońs disease (PD). Their small size, ease of synthesis, structural stability and target selectivity make them important tools to overcome the limitations of endogenous neurotrophins as therapeutic agents. Additionally, they might be optimized to improve resistance to enzymatic degradation, bioavailability, potency and, mainly, lipophilicity, important to cross the blood brain barrier (BBB). Here, we evaluated the neuroprotective effects and mechanisms of the synthetic snake‐venom‐based peptide p‐BTX‐I (Glu‐Val‐Trp) in PC12 cells treated with MPP+ (1‐methyl‐4‐phenylpyridinium), a dopaminergic neurotoxin that induces Parkinsonism in vivo. The peptide p‐BTX‐I induced neuritogenesis, which was reduced by (i) k252a, antagonist of the NGF‐selective receptor, trkA (tropomyosin receptor kinase A); (ii) LY294002, inhibitor of the PI3 K/AKT pathway and (iii) U0126, inhibitor of the MAPK‐ERK pathway. Besides that, p‐BTX‐I also increased the expression of GAP‐43 and synapsin, which are molecular markers of axonal growth and synaptic communication. In addition, the peptide increased the viability and differentiation of cells exposed to MPP+, known to inhibit neuritogenesis. Altogether, our findings suggest that the synthetic peptide p‐BTX‐I protects PC12 cells from MPP+ toxicity by a mechanism that mimics the neurotrophic action of NGF. Therefore, the molecular structure of p‐BTX‐I might be relevant in the development of drugs aimed at restoring the axonal connectivity in neurodegenerative processes.

Structure-Based Virtual Screening and Biochemical Evaluation for the Identification of Novel Trypanosoma Brucei Aldolase Inhibitors

INTRODUCTION The glycolytic enzyme fructose-1,6-bisphosphate aldolase is a validated molecular target in human African trypanosomiasis (HAT) drug discovery, a neglected tropical disease (NTD) caused by the protozoan Trypanosoma brucei. Herein, a structure-based virtual screening (SBVS) approach to the identification of novel T. brucei aldolase inhibitors is described. Distinct molecular docking algorithms were used to screen more than 500,000 compounds against the X-ray structure of the enzyme. This SBVS strategy led to the selection of a series of molecules which were evaluated for their activity on recombinant T. brucei aldolase. The effort led to the discovery of structurally new ligands able to inhibit the catalytic activity of the enzyme. RESULTS The predicted binding conformations were additionally investigated in molecular dynamics simulations, which provided useful insights into the enzyme-inhibitor intermolecular interactions. CONCLUSION The molecular modeling results along with the enzyme inhibition data generated practical knowledge to be explored in further structure-based drug design efforts in HAT drug discovery.

Structure-Based Drug Design to Overcome Drug Resistance: Challenges and Opportunities

Drug resistance is a common concern for the development of novel antiviral, antimicrobial and anticancer therapies. To overcome this problem, several strategies have been developed, many of which involving the theme of this review, the use of structure-based drug design (SBDD) approaches. These include the successful design of new compounds that target resistant mutant proteins, as well as the development of drugs that target multiple proteins involved in specific biochemical pathways. Finally, drug resistance can also be considered in the early stages of drug discovery, through the use of strategies to delay the development of resistance. The purpose of this brief review is to underline the usefulness of SBDD approaches based on case studies, highlighting present challenges and opportunities in drug design.