Design and synthesis of bioactive small molecules by traditional and innovative methods

2016 - 2017My PhD research plan concerns the exploration of the structural requirements determining TRPM8 modulation. We prepared a series of N-substituted tryptamines and identified two compounds acting, respectively, as an activator (21) or an inhibitor (12) of calcium influx in HEK293 cells. To develop more potent and selective derivatives we designed and synthetized new series of potential tetrahydroisoquinoline- and tetrahydro-β-carboline-based TRPM8 modulators. The synthetic approach used for the preparation of these compounds led to indole-fused aminoacetal derivatives. Optimization of reaction conditions allow us to obtain ring-fused aminals, which could be useful for preparing analogues of biologically active natural and synthetic products. At the same time I worked on the synthesis of potential varicella zoster virus replication inhibitors: tryptamine derivative 17a was found to have a selective activity against this herpesvirus family member. A second part of my PhD was dedicated to the identification of small heterocyclic compounds as GRK2 inhibitors. Lastly, I spent an abroad semester in the University of Graz, where I collaborated to the development of a continuous flow difluoromethylation protocol employing fluoroform as reagent. The protocol is applicable for the direct Cα-difluoromethylation of protected α-amino acids, and enables a highly atom efficient synthesis of the active pharmaceutical ingredient eflornithine. [edited by author]XVI n.s

The Metabolomic Profile in Amyotrophic Lateral Sclerosis Changes According to the Progression of the Disease: An Exploratory Study

Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative pathology of the upper or lower motor neuron. Evaluation of ALS progression is based on clinical outcomes considering the impairment of body sites. ALS has been extensively investigated in the pathogenetic mechanisms and the clinical profile; however, no molecular biomarkers are used as diagnostic criteria to establish the ALS pathological staging. Using the source-reconstructed magnetoencephalography (MEG) approach, we demonstrated that global brain hyperconnectivity is associated with early and advanced clinical ALS stages. Using nuclear magnetic resonance (1H-NMR) and high resolution mass spectrometry (HRMS) spectroscopy, here we studied the metabolomic profile of ALS patients’ sera characterized by different stages of disease progression—namely early and advanced. Multivariate statistical analysis of the data integrated with the network analysis indicates that metabolites related to energy deficit, abnormal concentrations of neurotoxic metabolites and metabolites related to neurotransmitter production are pathognomonic of ALS in the advanced stage. Furthermore, analysis of the lipidomic profile indicates that advanced ALS patients report significant alteration of phosphocholine (PCs), lysophosphatidylcholine (LPCs), and sphingomyelin (SMs) metabolism, consistent with the exigency of lipid remodeling to repair advanced neuronal degeneration and inflammatio

Anticancer Therapies Based on Oxidative Damage: Lycium barbarum Inhibits the Proliferation of MCF-7 Cells by Activating Pyroptosis through Endoplasmic Reticulum Stress

Abstract: Lycium barbarum, commonly recognized as goji berry or wolfberry, is highly appreciated not only for its organoleptic and nutritional properties but also as an important source of bioactive compounds such as polysaccharides, carotenoids, phenolics, and various other non-nutritive compounds. These constituents give it a multitude of health benefits, including antioxidant, antiinflammatory, and anticancer properties. However, the precise biochemical mechanisms responsible for its anticancer effects remain unclear, and the comprehensive composition of goji berry extracts is often insufficiently explored. This study aimed to investigate the biochemical pathways modulated in breast cancer cells by an ethanolic extract of Lycium barbarum fruit (LBE). Following metabolomic profiling using UHPLC-HRMS/MS, we assessed the antitumoral properties of LBE on different breast cancer cell lines. This investigation revealed that LBE exhibited cytotoxic effects, inducing a pro-oxidant effect that triggered pyroptosis activation through endoplasmic reticulum (ER) stress and subsequent activation of the P-IRE1α/XBP1/NLRP3 axis in MCF-7 cells. In addition, LBE did not display cytotoxicity toward healthy human cells but demonstrated antioxidant properties by neutralizing ROS generated by doxorubicin. These findings underscore the potential of LBE as a highly promising natural extract in cancer therap

Peptidomimetics as potent dual SARS-CoV-2 cathepsin-L and main protease inhibitors: In silico design, synthesis and pharmacological characterization

In this paper we present the design, synthesis, and biological evaluation of a new series of peptidomimetics acting as potent anti-SARS-CoV-2 agents. Starting from our previously described Main Protease (MPro) and Papain Like Protease (PLPro) dual inhibitor, CV11, here we disclose its high inhibitory activity against cathepsin L (CTSL) (IC50 = 19.80 ± 4.44 nM), an emerging target in SARS-CoV-2 infection machinery. An in silico design, inspired by the structure of CV11, led to the development of a library of peptidomimetics showing interesting activities against CTSL and Mpro, allowing us to trace the chemical requirements for the binding to both enzymes. The screening in Vero cells infected with 5 different SARS-CoV-2 variants of concerns, highlighted sub-micromolar activities for most of the synthesized compounds (13, 15, 16, 17 and 31) in agreement with the enzymatic inhibition assays results. The compounds showed lack of activity against several different RNA viruses except for the 229E and OC43 human coronavirus strains, also characterized by a cathepsin-L dependent release into the host cells. The most promising derivatives were also evaluated for their chemical and metabolic in-vitro stability, with derivatives 15 and 17 showing a suitable profile for further preclinical characterization

Estudo comparativo histologico e morfometrico de biopsias do intestino delgado em criancas, obtidas por capsula de succao e por pinca endoscopica

A biopsia o intestino delgado e extremamente importante na avaliacao diagnostica e terapeutica de Enteropatias em criancas, principalmente na Doenca Celiaca na qual e essencial para o diagnostico. O metodo padrao empregado rotineiramente para a obtencao de especimes de mucosa intestinal na maioria dos servicos de Gastroenterologia Pediatrica e o de biopsia por capsula de succao. Esse metodo, consolidado desde a decada de 60, fornece fragmentos adequados para a analise histologica, entretanto, alem de demorado, tem alto indice de falha tecnica e requer controle radiologico para localizacao da capsula. A biopsia do intestino delgado por pinca endoscopica, embora muito usada em adultos, permanece, ainda, controversa e de uso limitado em criancas. Este trabalho realizou um estudo comparativo histologico e morfometrico de biopsias do intestino delgado de criancas com Enteropatia, com a finalidade de avaliar a qualidade e a adequacao histologica dos especimes obtidos. Foram submetidas a analise histologica 157 biopsias do intestino delgado obtidas por capsula de succao e 84 biopsias obtidas por pinca endoscopica. Avaliou-se a orientacao do fragmento, a profundidade, a extensao, a presenca de glandulas de Brunner e a presenca de artefato de esmagamento, sendo os especimes, posteriormente, graduados qualitativamente em otimo, bom, regular ou ruim. A analise morfometrica foi realizada em 86 biopsias por capsula e em 58 biopsias endoscopicas, medindo a espessura total da mucosa, a altura das vilosidades e a extensao da zona criptica. As biopsias obtidas por capsula de succao e as obtidas por pinca endoscopica foram adequadas para interpretacao histologica, respectivamente, em 82,16 % ( 26,11% consideradas ootimaso ) e em 96,43 % ( ootimaso em 42,86% ), sendo estas diferencas estatisticamente significantes. A analise morfometrica demonstrou que a espessura total da mucosa, assim como a altura das vilosidades diminuem e a extensao da zona criptica aumenta, quanto maior o grau de Enteropatia, sendo que os graus extremos assemelham-se entre si, ou seja, o grau 0 foi semelhante ao grau I e o grau III semelhante ao grau IV. Ambos os metodos fornecem material apropriado para diagnostico anatomo-patologico, porem, os especimes de mucosa obtidos por pinca endoscopica, comparados aos obtidos por capsula de succao, foram melhores e mais adequados para exame histologicoBV UNIFESP: Teses e dissertaçõe

On the modulation of TRPM channels: Current perspectives and anticancer therapeutic implications

The transient melastatin receptor potential (TRPM) ion channel subfamily functions as cellular sensors and transducers of critical biological signal pathways by regulating ion homeostasis. Some members of TRPM have been cloned from cancerous tissues, and their abnormal expressions in various solid malignancies have been correlated with cancer cell growth, survival, or death. Recent evidence also highlights the mechanisms underlying the role of TRPMs in tumor epithelial-mesenchymal transition (EMT), autophagy, and cancer metabolic reprogramming. These implications support TRPM channels as potential molecular targets and their modulation as an innovative therapeutic approach against cancer. Here, we discuss the general characteristics of the different TRPMs, focusing on current knowledge about the connection between TRPM channels and critical features of cancer. We also cover TRPM modulators used as pharmaceutical tools in biological trials and an indication of the only clinical trial with a TRPM modulator about cancer. To conclude, the authors describe the prospects for TRPM channels in oncology

Utilization of fluoroform for difluoromethylation in continuous flow: a concise synthesis of α-difluoromethyl-amino acids

Fluoroform (CHF3) can be considered as an ideal reagent for difluoromethylation reactions. However, due to the low reactivity of fluoroform, only very few applications have been reported so far. Herein we report a continuous flow difluoromethylation protocol on sp(3) carbons employing fluoroform as a reagent. The protocol is applicable for the direct C-alpha-difluoromethylation of protected alpha-amino acids, and enables a highly atom efficient synthesis of the active pharmaceutical ingredient eflornithine

In Silico Methods for the Discovery of Kv7.2/7.3 Channels Modulators: A Comprehensive Review

The growing interest in Kv7.2/7.3 agonists originates from the involvement of these channels in several brain hyperexcitability disorders. In particular, Kv7.2/7.3 mutants have been clearly associated with epileptic encephalopathies (DEEs) as well as with a spectrum of focal epilepsy disorders, often associated with developmental plateauing or regression. Nevertheless, there is a lack of available therapeutic options, considering that retigabine, the only molecule used in clinic as a broad-spectrum Kv7 agonist, has been withdrawn from the market in late 2016. This is why several efforts have been made both by both academia and industry in the search for suitable chemotypes acting as Kv7.2/7.3 agonists. In this context, in silico methods have played a major role, since the precise structures of different Kv7 homotetramers have been only recently disclosed. In the present review, the computational methods used for the design of Kv.7.2/7.3 small molecule agonists and the underlying medicinal chemistry are discussed in the context of their biological and structure-function properties