Desenvolvimento de novas entidades químicas e formulações para o tratamento de dermatomicoses

As dermatomicoses são infecções fúngicas da pele, ocasionadas principalmente por dermatófitos e Candida spp. As dermatofitoses, denominação específica para dermatomicoses de origem dermatofítica, são as doenças cutâneas mais prevalentes da atualidade, possuindo como agentes etiológicos fungos filamentosos dermatofíticos de três gêneros anamórficos: Microsporum, Trichophyton e Epidermophyton. A resistência dos dermatófitos à terapêutica convencional vem sendo cada vez mais relatada, contribuindo para as comuns recidivas da micose. Infecções (muco) cutâneas e também sistêmicas podem ser causadas por leveduras oportunistas do gênero Candida - C. albicans e espécies de C. não-albicans (CNA; tais como C. tropicalis, C. glabrata, C. parapsilosis, C. dubliniensis e C. krusei). As espécies emergentes de CNA apresentam resistência mais pronunciada aos fármacos de escolha para o tratamento de candidíases, o que implica em falhas na terapia clínica e aumento considerável das taxas de morbimortalidade. Neste contexto, faz-se necessária a pesquisa por novos agentes antifúngicos com amplo espectro de ação e toxicidade seletiva para o patógeno, que superem as limitações da terapêutca atual. Assim, este trabalho teve como objetivo principal investigar, em uma ampla triagem, a atividade antifúngica de cento e oitenta e nove compostos sintéticos (de variadas classes químicas). Além disso, determinar as relações de estrutura-atividade, caracterizar o mecanismo de ação e perfil toxicológico das moléculas, a fim de prospectarem-se novas alternativas eficazes e seguras para o tratamento de infecções, primordialmente associadas a dermatófitos e Candida spp.. Em relação aos tiocianatos alílicos (TAs), 1ª classe investigada, o composto cloro-derivado apresentou um amplo espectro de atividade antifúngica e também atividade antiquimiotáxica, com 100% de redução da migração de leucócitos. A faixa de concentração inibitória mínima (CIM) dos compostos foi de 25 a 50 μg/mL, com mecanismo de ação relacionado à complexação com ergosterol fúngico. A nanoemulsão desenvolvida com o composto mais eficaz melhorou a atividade antifúngica in vitro, diminuindo os valores de CIM em até 64 vezes para dermatófitos e 4 vezes para Candida spp.. O composto cloro-derivado mais promissor não foi mutagênico, nem ocasionou inviabilidade celular em leucócitos humanos, embora tenha apresentado dano ao DNA dose-dependente. Além disso, não foi irritante (ensaio da membrana cório-alantoide - HET-CAM) e inibiu totalmente o crescimento fúngico em um modelo alternativo de dermatofitose. Para as δlactonas (δ-Ls), 2ª classe, nenhum dos compostos foi mutagênico, genotóxico ou irritante, quando avaliados em concentrações mais elevadas que a CIM. Dois dos compostos da série apresentaram os menores valores de CIM (25-50 μg/mL) e um espectro mais amplo de atividade antifúngica contra fungos filamentosos e leveduras. O mecanismo de ação foi relacionado ao dano na parede e membrana celular, com ação alvo específica dependente do tipo de halogênio presente na estrutura. O dano às células fúngicas foi corroborado por imagens de microscopia eletrônica de varredura, que destacaram células lisadas e completamente alteradas em sua morfologia, após tratamento in vitro com δ-Ls. Para os 1,4-benzenodióis 2-substituídos (1,4-BZs), 3ª classe – com síntese inédita, seis moléculas (2, 5, 6, 8, 11 e 12) apresentaram ação antimicrobiana de amplo espectro, incluindo efeito contra espécies resistentes e multirresistentes de dermatófitos (T. mentagrophytes), CNA e bactérias. As faixas de CIMs destes compostos para fungos e bactérias foram 25 a 50 μg/mL e 8 a 128 μg/mL, respectivamente, sendo o mecanismo de ação antifúngico relacionado à parede e membrana celular. O composto 8, mais promissor em relação ao efeito antifúngico, não ocasionou genotoxicidade e mutagenicidade em células leucocitárias humanas e nem hemólise. Complementarmente, os compostos não foram irritantes (HET-CAM). Por fim, para os derivados substituídos de 1,3-bisariloxipropano (1,3-BXPs), representando o último grupo de moléculas investigadas, encontrou-se o melhor perfil de atividade antifúngica/toxicidade, dentre todas as classes químicas analisadas neste estudo, com ênfase para o composto 2j (1,3-bis(3,4-diclorofenoxi)propan-2-amino-cloreto). 2j foi o fungicida mais ativo contra dermatófitos e Candida spp., em concentrações fungicidas mínimas (CFMs) muito baixas (0,39 - 3,12 μg/mL), incluindo ação contra isolados clínicos resistentes e multirresistentes. O perfil de toxicidade de 2j foi promissor, apresentando índice de seletividade > 10, em relação a linfócitos humanos. O composto foi classificado como não irritante pelo teste HET-CAM e não causou alterações histopatológicas na pele da orelha de porco, apresentando uma excelente perspectiva para aplicação tópica. 2j tem como alvo a parede celular fúngica, o que foi confirmado por imagens de microscopia eletrônica de varredura. O composto foi incorporado em um hidrogel com potencial bioadesivo. Os resultados da permeação cutânea humana mostraram que 2j permaneceu significativamente na epiderme, o que é o ideal para o tratamento de dermatomicoses. Adicionalmente, em relação à candidíase sistêmica em Drosophila melanogaster Toll-deficientes, 2j apresentou eficácia estatisticamente comparável ao fluconazol no controle do processo infeccioso. Portanto, o composto 2j demonstrou potencial para o desenvolvimento de fármacos antifúngicos, com um mecanismo de ação elucidado e já aplicado em uma formulação semissólida, visando uma nova opção terapêutica para infecções fúngicas da pele não responsivas ao tratamento convencional.Dermatomycoses are fungal infections of the skin, mainly caused by dermatophytes and Candida spp. Dermatophytoses, a specific name for dermatomycoses of dermatophytic origin, are the most prevalent skin diseases today, with dermatophytic filamentous fungi of three anamorphic genera as the etiological agents: Microsporum, Trichophyton and Epidermophyton. The resistance of dermatophytes to conventional therapy has been increasingly reported, contributing to the common recurrence of mycosis. (Mucus) cutaneous and also systemic infections may be caused by opportunistic yeasts of the genus Candida - C. albicans and species of non-albicans Candida (NAC; such as C. tropicalis, C. glabrata, C. parapsilosis, C. dubliniensis and C. krusei). Emerging NAC species show more pronounced resistance to the drugs of choice for the treatment of candidiasis, which implies failures in clinical therapy and a considerable increase in morbimortality rates. In this context, it is necessary to search for new antifungal agents with a broad spectrum of action and selective toxicity for the pathogen, which overcome the limitations of current therapy. Thus, the main objective of this work was to investigate, in a wide screening, the antifungal activity of one hundred and eighty-nine synthetic compounds (of various chemical classes). In addition, to determine the structure-activity relationships, characterize the mechanism of action and toxicological profile of the molecules, in order to prospect new effective and safe alternatives for the treatment of infections primarily associated with dermatophytes and Candida spp. In relation to allylic thiocyanates (ATs), 1st class investigated, the chloro-derivative presented a broad spectrum of antifungal activity and also antichemotactic activity, with a 100% reduction in leukocyte migration. The minimum inhibitory concentration (MIC) range of the compounds was 25 to 50 μg/mL, with a mechanism of action related to complexation with fungal ergosterol. The nanoemulsion developed with the most effective compound improved the in vitro antifungal activity, reducing MIC values by up to 64-fold for dermatophytes and 4-fold for Candida spp. The most promising chloro derivative was not mutagenic, nor did it cause cellular inviability of human leukocytes, although it did present dose-dependent damage to the DNA. In addition, it was not irritant (chorioallantoic membrane assay - HET-CAM) and totally inhibited fungal growth in an alternative model of dermatophytosis. For δ-lactones (δ-Ls), 2nd class, none of the compounds were mutagenic, genotoxic or irritant when evaluated at higher concentrations than MIC. Two of the compounds in the series had the lowest MIC values (25-50 μg/mL) and a broader spectrum of antifungal activity against filamentous fungi and yeasts. The mechanism of action was related to the damage in the cell wall and membrane, with specific target action dependent on the type of halogen present in the structure. Damage to fungal cells was corroborated by scanning electron microscopy images, which highlighted lysed cells and completely altered their morphology after in vitro treatment with δ-Ls. For the 2-substituted 1,4-benzenediols (1,4-BZs), six molecules (2, 5, 6, 8, 11 and 12) presented broad spectrum of antimicrobial action, including effect against resistant and multidrug-resistant species of dermatophytes (T. mentagrophytes), NAC and bacteria. The MIC ranges of these compounds for fungi and bacteria were 25 to 50 μg/mL and 8 to 128 μg/mL, respectively, and the mechanism of antifungal action was related to cell wall and membrane. Compound 8, which is more promising regarding the antifungal effect, did not cause genotoxicity and mutagenicity in human leukocyte cells nor hemolysis. In addition, the compounds were non-irritating (HET-CAM). Finally, for the substituted 1,3-bisaryloxypropane derivatives (1,3-BXPs), representing the last group of molecules investigated, the best profile of antifungal activity/toxicity was found among all the chemical classes analyzed in this study, with emphasis for the compound 2j (1,3-bis(3,4-dichlorophenoxy)propan-2-aminium chloride). 2j was the most active fungicide against dermatophytes and Candida spp., in very low minimal fungicidal concentrations (MFCs) (0.39 - 3.12 μg/mL), including action against resistant and multidrug-resistant clinical isolates. The toxicity profile of 2j was promising, with a selectivity index > 10, in relation to human lymphocytes. The compound was classified as non-irritant by the HET-CAM test and did not cause histopathological changes in pig ear skin, presenting an excellent perspective for topical application. 2j targets the fungal cell wall, which was confirmed by scanning electron microscopy images. The compound was incorporated into a hydrogel with bioadhesive potential. The results of human skin permeation showed that 2j remained significantly in the epidermis, ideally for the treatment of dermatomycosis. In addition, in relation to systemic candidiasis in Toll-deficient Drosophila melanogaster, 2j presented a statistically comparable efficacy to fluconazole in the control of the infectious process. Therefore, compound 2j has demonstrated potential for the development of antifungal drugs with a mechanism of action elucidated and already applied in a semi-solid formulation aimed at a new therapeutic option for fungal skin infections not responsive to conventional treatment

Fungal Pathogenesis in Humans: The Growing Threat

Cancer survival rates and successful organ transplantation in patients continues to increase due to improvements in early diagnosis and treatments. Since immuno-suppressive therapies are frequently used, the mortality rate due to secondary infections has become an ever-increasing problem. Opportunistic fungal infections are probably the deadliest threat to these patients due to their difficult early diagnosis, the limited effect of antifungal drugs and the appearance of resistances. In recent years, a considerable effort has been devoted to investigating the role of many virulence traits in the pathogenic outcome of fungal infections. New virulence factors (hypoxia adaptation, CO2 sensing, pH regulation, micronutrient acquisition, secondary metabolites, immunity regulators, etc.) have been reported and their molecular mechanisms of action are being thoroughly investigated. The recent application of gene-editing technologies such as CRISPr-Cas9, has opened a whole new window to the discovery of new fungal virulence factors. Accurate fungal genotyping, Next Generation Sequencing and RNAseq approaches will undoubtedly provide new clues to interpret the plethora of molecular interactions controlling these complex systems. Unraveling their intimate regulatory details will provide insights for a more target-focused search or a rational design of more specific antifungal agents. This Special Issue is show significant discoveries, proofs of concept of new theories or relevant observations in fungal pathogenesis and its regulation

Identification and characterization of novel putative virulence factors in Candida albicans

The C. albicans community is currently laying the foundation of understanding how this human pathogen causes infection. C. albicans infections represent a major medical and economic burden for today’s society with an estimated 400,000 blood stream infections worldwide and direct costs exceeding 1$ billion dollar a year in the U.S. alone. Although finding the biological causes of this disease seemed to be beyond our reach in the past, various aspects of the infection have been recently unveiled including its pathology, immunology, histology, and epidemiology. Here we explored the genetic components of this disease by studying the complex host-pathogen dynamics through a series of in vivo, ex vivo and in vitro experiments. By using a pathogen unbiased reverse genetic approach and a host gene candidate strategy we uncovered some of the genes and pathways that are important for pathogenicity and immunity. In particular we explored the complex host-pathogen dynamics using a C. albicans - C. elegans model system and identified four novel putative virulence factors. We focused on Zcf15, a C. albicans transcription factor that has been poorly characterized in the literature and that plays an important role in the pathogen’s ability to resist host generated reactive oxygen species (ROS). By leveraging the power of RNASeq and ChIP-Seq we identified Zcf15 transcriptional targets and DNA binding sites. These studies suggest that Zcf15 plays a critical role in carbon metabolism and that it exerts its ability to protect the pathogen from ROS by controlling the expression of thiol- peroxidases and other detoxifying enzymes. We also showed here that in C. elegans, the host’s ability to counteract the infection relies on the MAPK pathway, evidence that mirrors what has been found by others in mammals and that emphasizes the usefulness of studying C. albicans infections in smaller genetically traceable organisms like C. elegans. The nematode model is also shown here to be a powerful tool not only to study the genetic bases that drive infection and immunity but also to identify new compounds that can be used for therapeutic intervention. This model was instrumental in identifying filastatin, a small molecule that was subsequently found by our collaborators to be capable of reducing virulence in mammals. The antifungal properties of filastatin are currently undertaking further preclinical testing. Overall this thesis shed light on the complex mechanisms of C. albicans pathogenicity and host immunity and identified novel virulence determinants that can be used by the larger community for further biological studies or even drug development

Fungal Reproduction and Growth

One of the largest and most diverse kingdoms in eukaryotes is fungi, which consists of approximately 2.2–3.8 million species. This book provides readers with an in-depth understanding of fungi diversity and the role of fungi in the ecosystem. Chapters address such topics as fungi reproduction and pathology, fungal mycotoxicity, fungi mating mechanisms, and much more

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