Ana Paula Francisco

The multifactorial nature of Alzheimer's disease for developing potential therapeutics

Alzheimers disease (AD) is a multifactorial neurodegenerative disorder with several target proteins contributing to its aetiology. Pathological, genetic, biochemical, and modeling studies all point to a critical role of Aβ aggregation in AD. Though there are still many enigmatic aspects of the Aβ cascade, none of the gaps invalidate the hypothesis. The amyloid hypothesis determines that the production, aggregation and accumulation of Aβ in the brain gives rise to a cascade of neurotoxic events that proceed to neuronal degeneration. Different targets of the disease include APP pathogenic cleavage, cytoskeletal destabilization, neurotransmitter and ion dyshomeostasis, metal ion accumulation, protein misfolding, oxidative stress, neuronal death and gene mutations. Thus, disease-modifying treatments for AD must interfere with the pathogenic steps responsible for the clinical symptoms: the deposition of extracellular Aβ plaques, the intracellular neurofibrillary tangles, inflammation, oxidative stress, iron deregulation, among others. The observations supporting the development of multifunctional compounds in association with the perception that several dual binding site AChEIs were able to reach different targets guided the development of a new drug design strategy, the multi-target-directed-ligand (MTDL) approach. This may be regarded as the buildup of hybrid molecules composed of distinct pharmacophores of different drugs. Thus, each pharmacophore of the new hybrid drug would preserve the capacity of interacting with their specific sites on the targets and, therefore, generate multiple specific pharmacological responses which would enable the treatment of multi-factorial diseases. This review summarizes a few current therapeutic trends on MTDL strategy intended to halt or revert the progression of the disease.

Triazene drug metabolites. Part 17: synthesis and plasma hydrolysis of acyloxymethyl carbamate derivatives of antitumour triazenes

A series of 3-acyloxymethyloxycarbonyl-1-aryl-3-methyltriazenes 5 was synthesised by the sequential reaction of 1-aryl-3-methyltriazenes with (i) chloromethyl chloroformate, (ii) NaI in dry acetone, and (iii) either the silver carboxylate or the carboxylic acids in the presence of silver carbonate. The hydrolysis of these compounds was studied in pH 7.7 isotonic phosphate buffer and in human plasma. Triazene acyloxycarbamates demonstrated their ability to act as substrates for plasma enzymes. For compound 5f, a pH-rate profile was obtained which showed the hydrolysis to involve acid-base catalysis. The reaction is also buffer catalysed. Thus, at pH 7.7, pH-independent, base-catalysed and buffer-catalysed processes all contribute to the hydrolysis reaction. The sensitivity of the hydrolysis reaction to various structural parameters in the substrates indicates that hydrolysis occurs at the ester rather than the carbamate functionality. In plasma, the rates of hydrolysis correlate with partition coefficients, the most lipophilic compounds being the most stable. An aspirin derivative suffers two consecutive enzymatic reactions, the scission of the aspirin acetyl group being followed by the scission of the acyloxy ester group. These results indicate that triazene acyloxymethyl carbamates are prodrugs of the antitumour monomethyltriazenes. They combine chemical stability with a rapid enzymatic hydrolysis, and are consequently good candidates for further prodrug development. Moreover, this type of derivative allowed the synthesis of mutual prodrugs, associating the antitumour monomethyltriazenes with anti-inflammatory NSAIDs as well as with the anticancer agent butyric acid.

Design and discovery of mushroom tyrosinase inhibitors and their therapeutic applications

Introduction: Tyrosinase inhibitors could have a huge importance in medicine, cosmetics and agriculture. Although many tyrosinase inhibitors are available, they have demonstrated only mild efficacy and safety concerns. This has led to the discovery of novel tyrosinase inhibitors that are more safe, potent and efficacious. Areas covered: The authors provide an overview of the recent scientific accounts describing the design of new molecules. These compounds belong to different chemical families. The review emphasizes the rationale behind the discovery, the study of structure–activity relationships, the study of the mechanism and kinetic of inhibition and the cellular effect of the inhibitors. The article is based on the literature published from 2007 onward related with the development of synthetic tyrosinase inhibitors. Expert opinion: Although a great number of tyrosinase inhibitors have been published in the literature, none, as of yet, have reached the potency and safety requirements needed to enter clinical trials. The emergence of new in vitro and in vivo tests will finally allow the arrival of new compounds that are more potent and safe.

Targeting KRAS Oncogene in Colon Cancer Cells with 7-Carboxylate Indolo[3,2-b]quinoline Tri-Alkylamine Derivatives.

Background A guanine-rich strand within the promoter of the KRAS gene can fold into an intra-molecular G-quadruplex structure (G4), which has an important role in the regulation of KRAS transcription. We have previously identified indolo[3,2-b]quinolines with a 7-carboxylate group and three alkylamine side chains (IQ3A) as effective G4 stabilizers and promising selective anticancer leads. Herein we investigated the anticancer mechanism of action of these compounds, which we hypothesized due to stabilization of the G4 sequence in the KRAS promoter and subsequent down-regulation of gene expression. Methodology/Principal Findings IQ3A compounds showed greater stabilization of G4 compared to duplex DNA structures and reduced KRAS promoter activity in a dual luciferase reporter assay. Moreover, IQ3A compounds showed high anti-proliferative activity in HCT116 and SW620 colon cancer cells (IC50 < 2.69 μM), without eliciting cell death in non-malignant HEK293T human embryonic kidney, and human colon fibroblasts CCD18co. IQ3A compounds significantly reduced KRAS mRNA and protein steady-state levels at IC50 concentrations, and increased p53 protein steady-state levels and cell death by apoptosis in HCT116 cells (mut KRAS, wt p53). Furthermore, KRAS silencing in HCT116 p53 wild-type (p53(+/+)) and null (p53(-/-)) isogenic cell lines induced a higher level of cell death, and a higher IQ3A-induced cell death in HCT116 p53(+/+) compared to HCT116 p53(-/-). Conclusions Herein we provide evidence that G4 ligands such as IQ3A compounds can target G4 motifs present in KRAS promoter, down-regulate the expression of the mutant KRAS gene through inhibition of transcription and translation, and induce cell death by apoptosis in colon cancer cell lines. Thus, targeting KRAS at the genomic level with G4 ligands may be a new anticancer therapy strategy for colon cancer.

Dopamine- and tyramine-based derivatives of triazenes: activation by tyrosinase and implications for prodrug design.

A range of triazene derivatives were synthesized and investigated as prodrug candidates for melanocyte-directed enzyme prodrug therapy (MDEPT). The prodrugs contained a tyramine or dopamine promoiety required for tyrosinase activation and this was joined via a urea functional group to the cytotoxic triazene. The stability of each of the prodrugs in phosphate buffer, human plasma and in mushroom tyrosinase is discussed. The identification of the main peak formed after the tyrosinase reaction was attempted by LC-MS and the conversion of prodrug to the quinone was confirmed.

In vitro antimicrobial activity of royleanone derivatives against Gram-positive bacterial pathogens.

Infections caused by multiresistant bacterial pathogens are a significant problem worldwide, turning the search for natural compounds to act as alternatives to antibiotics of major importance. The aim of the present study was to investigate the in vitro antimicrobial activity of 7α‐acetoxy‐6β‐hydroxyroyleanone (1), isolated from Plectranthus grandidentatus (Lamiaceae), and 11 additional royleanone abietane derivatives of 1 (2–12) against important Gram‐positive human bacterial pathogens. Results showed that the aromatic and alkylic esters 2, 3 and 5 are more active than 1 against Enterococcus and Staphylococcus (minimum inhibitory concentration (MIC) values ranging from 0.98 to 62.50 µg/mL). Moreover, 7α‐acetoxy‐6β‐hydroxy‐12‐O‐(4‐chloro)benzoylroyleanone (2) gave rise to a new antibacterial‐prototype (MIC values of 3.91–15.63 µg/mL against Staphylococcus and of 0.98–3.91 µg/mL against Enterococcus). The results showed that the compounds under analysis also present antimicrobial activity against resistant bacteria. The hydrophobic extra‐interactions with bacterial targets seem to play an important role on the activity of royleanones derivatives. Copyright

Targets, structures, and recent approaches in malignant melanoma chemotherapy.

Malignant metastatic melanoma is one of the oncologic diseases with the worst clinical prognosis, due primarily to resistance phenomena against chemotherapeutic agents in current use. However, over the last few years, characterization of the molecular mechanisms involved in the development and progression of the disease has contributed to elucidation of the main pathways by which tissue invasion and metastasis can occur. More importantly, the identification of abnormalities in signaling cascades in melanoma cells has facilitated new therapeutic approaches against malignant melanoma through the design of highly potent and selective drugs with low associated toxicity. Ultimately, recognition of the restricted applicability of new chemotherapies in certain genetic contexts has led to significant improvements in the results of clinical trials, anticipating the existing need for investment in personalized therapies, and taking into account the molecular alterations observed in tumors. Although significant advances have been made in terms of extending the median overall survival rate and improving the quality of life for patients, the mechanisms that compromise in vivo drug efficacy remain poorly understood, particularly those concerning therapeutic resistance phenomena. This review summarizes recently validated targets from the perspective of the medicinal chemistry carried out in the design of the most promising structures.

Development of triazene prodrugs for ADEPT strategy: new insights into drug delivery system based on carboxypeptidase G2 activation.

Six novel urea triazene prodrugs have been synthesized to apply in antibody-directed enzyme prodrug therapy (ADEPT). The chemical and plasmatic stability of l-glutamate triazene prodrugs were evaluated and the chemical reactivity was mainly attributed to an intramolecular catalysis promoted by the neighbouring carboxylate group of the glutamic moiety. These prodrugs showed an elevated binding to plasma proteins. The L-glutamate triazenes were evaluated as prodrugs of the alkylating agents monomethyltriazenes, by activation of the bacterial enzyme carboxypeptidase G2 (CPG2). The synthesized prodrugs have been shown to be good substrates for CPG2, and therefore new candidates for ADEPT strategy.

Synthesis and evaluation of N-acylamino acids derivatives of triazenes. Activation by tyrosinase in human melanoma cell lines

In this research work we report the synthesis of a new series of triazene prodrugs designed for Melanocyte-Directed Enzyme Prodrug Therapy (MDEPT). These compounds are derived from the N-acyltyrosine amino acid - a good enzyme substrate for the tyrosinase enzyme, which is significantly overexpressed in melanoma cells. We analysed their chemical stability and plasma enzymatic hydrolysis, and we also evaluated the release of the antitumoral drug in the presence of the tyrosinase. Subsequently, we performed the evaluation of the prodrug cytotoxicity in melanoma cell lines with different levels of tyrosinase activity. Prodrug 5c showed the highest cytotoxicity against melanoma cell lines, and this effect correlated well with the tyrosinase activity suggesting that prodrug cytotoxicity is tyrosinase-dependent.

The Selective Cytotoxicity of New Triazene Compounds to Human Melanoma Cells

Metastatic melanoma still remains one the most difficult cancers to overcome. The aim of our research was the design of anti-tumour triazene compounds 3 for application to a melanoma-specific therapy. The strategy exploits the unique enzyme pathway of melanin biosynthesis for conversion of non-toxic prodrugs into toxic drugs in the melanoma cell. The compounds 3 were designed by coupling two active moieties, the alkylating triazenes and different tyrosinase substrates. All compounds 3 revealed to be chemically stable in isotonic phosphate buffer (PBS) at physiologic pH (t½≥48h), and most of them showed to be slowly hydrolysed in human plasma (1.5≤t½ (h)≤161). Compounds 3c-n revealed to be excellent tyrosinase substrates (0.74≤t½ (min)≤6) with the best tyrosinase substrate 3l releasing MMT 45s after tyrosinase activation. Structure-activity relationship studies allowed the identification of the better structural features for enzyme affinity. Furthermore, the derivatives 3l and 3m showed cell selectivity with significant cytotoxic effects (IC50 values of 46-65μM) against melanoma cell lines with tyrosinase overexpression MNT-1 and B16F10.