Tetrahydrophthalazinone Inhibitor of Phosphodiesterase with In Vitro Activity against Intracellular Trypanosomatids.

The phosphodiesterase inhibitor tetrahydrophthalazinone NPD-008 was explored by phenotypic in vitro screening, target validation, and ultrastructural approaches against Trypanosoma cruzi NPD-008 displayed activity against different forms and strains of T. cruzi (50% effective concentration [EC50], 6.6 to 39.5 µM). NPD-008 increased cAMP levels of T. cruzi and its combination with benznidazole gave synergistic interaction. It was also moderately active against intracellular amastigotes of Leishmania amazonensis and Leishmania infantum, confirming a potential activity profile as an antitrypanosomatid drug candidate.

Efficacy of Novel Pyrazolone Phosphodiesterase Inhibitors in Experimental Mouse Models of Trypanosoma cruzi.

Pyrazolones are heterocyclic compounds with interesting biological properties. Some derivatives inhibit phosphodiesterases (PDEs) and thereby increase the cellular concentration of cyclic AMP (cAMP), which plays a vital role in the control of metabolism in eukaryotic cells, including the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease (CD), a major neglected tropical disease. In vitro phenotypic screening identified a 4-bromophenyl-dihydropyrazole dimer as an anti-T. cruzi hit and 17 novel pyrazolone analogues with variations on the phenyl ring were investigated in a panel of phenotypic laboratory models. Potent activity against the intracellular forms (Tulahuen and Y strains) was obtained with 50% effective concentration (EC50) values within the 0.17 to 3.3 µM range. Although most were not active against bloodstream trypomastigotes, an altered morphology and loss of infectivity were observed. Pretreatment of the mammalian host cells with pyrazolones did not interfere with infection and proliferation, showing that the drug activity was not the result of changes to host cell metabolism. The pyrazolone NPD-227 increased the intracellular cAMP levels and was able to sterilize T. cruzi-infected cell cultures. Thus, due to its high potency and selectivity in vitro, and its additive interaction with benznidazole (Bz), NPD-227 was next assessed in the acute mouse model. Oral dosing for 5 days of NPD-227 at 10 mg/kg + Bz at 10 mg/kg not only reduced parasitemia (>87%) but also protected against mortality (>83% survival), hence demonstrating superiority to the monotherapy schemes. These data support these pyrazolone molecules as potential novel therapeutic alternatives for Chagas disease.

Evaluation of phthalazinone phosphodiesterase inhibitors with improved activity and selectivity against Trypanosoma cruzi.

BACKGROUND: Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, needs urgent alternative therapeutic options as the treatments currently available display severe limitations, mainly related to efficacy and toxicity. OBJECTIVES: As phosphodiesterases (PDEs) have been claimed as novel targets against T. cruzi, our aim was to evaluate the biological aspects of 12 new phthalazinone PDE inhibitors against different T. cruzi strains and parasite forms relevant for human infection. METHODS: In vitro trypanocidal activity of the inhibitors was assessed alone and in combination with benznidazole. Their effects on parasite ultrastructural and cAMP levels were determined. PDE mRNA levels from the different T. cruzi forms were measured by quantitative reverse transcription PCR. RESULTS: Five TcrPDEs were found to be expressed in all parasite stages. Four compounds displayed strong effects against intracellular amastigotes. Against bloodstream trypomastigotes (BTs), three were at least as potent as benznidazole. In vitro combination therapy with one of the most active inhibitors on both parasite forms (NPD-040) plus benznidazole demonstrated a quite synergistic profile (xΣ FICI = 0.58) against intracellular amastigotes but no interaction (xΣ FICI = 1.27) when BTs were assayed. BTs treated with NPD-040 presented disrupted Golgi apparatus, a swollen flagellar pocket and signs of autophagy. cAMP measurements of untreated parasites showed that amastigotes have higher ability to efflux this second messenger than BTs. NPD-001 and NPD-040 increase the intracellular cAMP content in both BTs and amastigotes, which is also released into the extracellular milieu. CONCLUSIONS: The findings demonstrate the potential of PDE inhibitors as anti-T. cruzi drug candidates.

Imidazole Derivatives as Promising Agents for the Treatment of Chagas Disease.

More than 100 years after being first described, Chagas disease remains endemic in 21 Latin American countries and has spread to other continents. Indeed, this disease, which is caused by the protozoan parasite Trypanosoma cruzi, is no longer just a problem for the American continents but has become a global health threat. Current therapies, i.e., nifurtimox and benznidazole (Bz), are far from being adequate, due to their undesirable effects and their lack of efficacy in the chronic phases of the disease. In this work, we present an in-depth phenotypic evaluation in T. cruzi of a new class of imidazole compounds, which were discovered in a previous phenotypic screen against different trypanosomatids and were designed as potential inhibitors of cAMP phosphodiesterases (PDEs). The confirmation of several activities similar or superior to that of Bz prompted a synthesis program of hit optimization and extended structure-activity relationship aimed at improving drug-like properties such as aqueous solubility, which resulted in additional hits with 50% inhibitory concentration (IC50) values similar to that of Bz. The cellular effects of one representative hit, compound 9, on bloodstream trypomastigotes were further investigated. Transmission electron microscopy revealed cellular changes, after just 2 h of incubation with the IC50 concentration, that were consistent with induced autophagy and osmotic stress, mechanisms previously linked to cAMP signaling. Compound 9 induced highly significant increases in both cellular and medium cAMP levels, confirming that inhibition of T. cruzi PDE(s) is part of its mechanism of action. The potent and selective activity of this imidazole-based PDE inhibitor class against T. cruzi constitutes a successful repurposing of research into inhibitors of mammalian PDEs.

Activities of psilostachyin A and cynaropicrin against Trypanosoma cruzi in vitro and in vivo.

In vitro and in vivo activities against Trypanosoma cruzi were evaluated for two sesquiterpene lactones: psilostachyin A and cynaropicrin. Cynaropicrin had previously been shown to potently inhibit African trypanosomes in vivo, and psilostachyin A had been reported to show in vivo effects against T. cruzi, albeit in another test design. In vitro data showed that cynaropicrin was more effective than psilostachyin A. Ultrastructural alterations induced by cynaropicrin included shedding events, detachment of large portions of the plasma membrane, and vesicular bodies and large vacuoles containing membranous structures, suggestive of parasite autophagy. Acute toxicity studies showed that one of two mice died at a cynaropicrin dose of 400 mg/kg of body weight given intraperitoneally (i.p.). Although no major plasma biochemical alterations could be detected, histopathology demonstrated that the liver was the most affected organ in cynaropicrin-treated animals. Although cynaropicrin was as effective as benznidazole against trypomastigotes in vitro, the treatment (once or twice a day) of T. cruzi-infected mice (up to 50 mg/kg/day cynaropicrin) did not suppress parasitemia or protect against mortality induced by the Y and Colombiana strains. Psilostachyin A (0.5 to 50 mg/kg/day given once a day) was not effective in the acute model of T. cruzi infection (Y strain), reaching 100% animal mortality. Our data demonstrate that although it is very promising against African trypanosomes, cynaropicrin does not show efficacy compared to benznidazole in acute mouse models of T. cruzi infection.

In vitro and in vivo studies of the antiparasitic activity of sterol 14α-demethylase (CYP51) inhibitor VNI against drug-resistant strains of Trypanosoma cruzi.

Chagas disease affects more than 10 million people worldwide, and yet, as it has historically been known as a disease of the poor, it remains highly neglected. Two currently available drugs exhibit severe toxicity and low effectiveness, especially in the chronic phase, while new drug discovery has been halted for years as a result of a lack of interest from pharmaceutical companies. Although attempts to repurpose the antifungal drugs posaconazole and ravuconazole (inhibitors of fungal sterol 14α-demethylase [CYP51]) are finally in progress, development of cheaper and more efficient, preferably Trypanosoma cruzi-specific, chemotherapies would be highly advantageous. We have recently reported that the experimental T. cruzi CYP51 inhibitor VNI cures with 100% survival and 100% parasitological clearance both acute and chronic murine infections with the Tulahuen strain of T. cruzi. In this work, we further explored the potential of VNI by assaying nitro-derivative-resistant T. cruzi strains, Y and Colombiana, in highly stringent protocols of acute infection. The data show high antiparasitic efficacy of VNI and its derivative (VNI/VNF) against both forms of T. cruzi that are relevant for mammalian host infection (bloodstream and amastigotes), with the in vivo potency, at 25 mg/kg twice a day (b.i.d.), similar to that of benznidazole (100 mg/kg/day). Transmission electron microscopy and reverse mutation tests were performed to explore cellular ultrastructural and mutagenic aspects of VNI, respectively. No mutagenic potential could be seen by the Ames test at up to 3.5 µM, and the main ultrastructural damage induced by VNI in T. cruzi was related to Golgi apparatus and endoplasmic reticulum organization, with membrane blebs presenting an autophagic phenotype. Thus, these preliminary studies confirm VNI as a very promising trypanocidal drug candidate for Chagas disease therapy.

Antiplasmodial, Trypanocidal, and Genotoxicity In Vitro Assessment of New Hybrid α,α-Difluorophenylacetamide-statin Derivatives.

BACKGROUND: Statins present a plethora of pleiotropic effects including anti-inflammatory and antimicrobial responses. A,α-difluorophenylacetamides, analogs of diclofenac, are potent pre-clinical anti-inflammatory non-steroidal drugs. Molecular hybridization based on the combination of pharmacophoric moieties has emerged as a strategy for the development of new candidates aiming to obtain multitarget ligands. METHODS: Considering the anti-inflammatory activity of phenylacetamides and the potential microbicidal action of statins against obligate intracellular parasites, the objective of this work was to synthesize eight new hybrid compounds of α,α-difluorophenylacetamides with the moiety of statins and assess their phenotypic activity against in vitro models of Plasmodium falciparum and Trypanosoma cruzi infection besides exploring their genotoxicity safety profile. RESULTS: None of the sodium salt compounds presented antiparasitic activity and two acetated compounds displayed mild anti-P. falciparum effect. Against T. cruzi, the acetate halogenated hybrids showed moderate effect against both parasite forms relevant for human infection. Despite the considerable trypanosomicidal activity, the brominated compound revealed a genotoxic profile impairing future in vivo testing. CONCLUSIONS: However, the chlorinated derivative was the most promising compound with chemical and biological profitable characteristics, without presenting genotoxicity in vitro, being eligible for further in vivo experiments.

Phenotypic evaluation and in silico ADMET properties of novel arylimidamides in acute mouse models of Trypanosoma cruzi infection.

Arylimidamides (AIAs), previously termed as reversed amidines, present a broad spectrum of activity against intracellular microorganisms. In the present study, three novel AIAs were evaluated in a mouse model of Trypanosoma cruzi infection, which is the causative agent of Chagas disease. The bis-AIAs DB1957, DB1959 and DB1890 were chosen based on a previous screening of their scaffolds that revealed a very promising trypanocidal effect at nanomolar range against both the bloodstream trypomastigotes (BTs) and the intracellular forms of the parasite. This study focused on both mesylate salts DB1957 and DB1959 besides the hydrochloride salt DB1890. Our current data validate the high activity of these bis-AIA scaffolds that exhibited EC50 (drug concentration that reduces 50% of the number of the treated parasites) values ranging from 14 to 78 nM and 190 to 1,090 nM against bloodstream and intracellular forms, respectively, also presenting reasonable selectivity indexes and no mutagenicity profile predicted by in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET). Acute toxicity studies using murine models revealed that these AIAs presented only mild toxic effects such as reversible abdominal contractions and ruffled fur. Efficacy assays performed with Swiss mice infected with the Y strain revealed that the administration of DB1957 for 5 consecutive days, with the first dose given at parasitemia onset, reduced the number of BTs at the peak, ranging between 21 and 31% of decrease. DB1957 was able to provide 100% of animal survival, while untreated animals showed 70% of mortality rates. DB1959 and DB1890B did not reduce circulating parasitism but yielded >80% of survival rates.

In vitro and in vivo investigation of the efficacy of arylimidamide DB1831 and its mesylated salt form--DB1965--against Trypanosoma cruzi infection.

Chagas disease is caused by infection with the intracellular protozoan parasite Trypanosoma cruzi. At present, nifurtimox and benznidazole, both compounds developed empirically over four decades ago, represent the chemotherapeutic arsenal for treating this highly neglected disease. However, both drugs present variable efficacy depending on the geographical area and the occurrence of natural resistance, and are poorly effective against the later chronic stage. As a part of a search for new therapeutic opportunities to treat chagasic patients, pre-clinical studies were performed to characterize the activity of a novel arylimidamide (AIA--DB1831 (hydrochloride salt) and DB1965 (mesylate salt)) against T. cruzi. These AIAs displayed a high trypanocidal effect in vitro against both relevant forms in mammalian hosts, exhibiting a high selectivity index and a very high efficacy (IC(50) value/48 h of 5-40 nM) against intracellular parasites. DB1965 shows high activity in vivo in acute experimental models (mouse) of T. cruzi, showing a similar effect to benznidazole (Bz) when compared under a scheme of 10 daily consecutive doses with 12.5 mg/kg. Although no parasitological cure was observed after treating with 20 daily consecutive doses, a combined dosage of DB1965 (5 mg/kg) with Bz (50 mg/kg) resulted in parasitaemia clearance and 100% animal survival. In summary, our present data confirmed that aryimidamides represent promising new chemical entities against T. cruzi in therapeutic schemes using the AIA alone or in combination with other drugs, like benznidazole.

Uso racional de inibidores de fosfodiesterases como abordagem terapêutica para Doença de Chagas

Descoberta em 1909 pelo médico sanitarista e pesquisador brasileiro Carlos Chagas, a doença de Chagas (DC) faz parte do grupo de vinte agravos conhecidos como Doenças Tropicais Negligenciadas, e é causada pelo protozoário hemoflagelado Trypanosoma cruzi. Os fármacos de referência, benzonidazol (BZ) e nifurtimox, apresentam importantes limitações, como efeitos colaterais consideráveis, eficácia limitada na fase crônica e cepas naturalmente resistentes a ambos. Neste contexto, grupos de pesquisa, incluindo os do consórcio PDE4NPD, vêm trabalhando na identificação, otimização e triagem de inibidores de fosfodiesterases (PDEs), a partir de bibliotecas comerciais focadas em PDEs humanas e baseando-se nas diferenças estruturais e funcionais das isoformas parasitárias. Mesmo com alto grau de conservação, estas enzimas, que se apresentam em cinco isoformas em tripanossomatídeos e têm como função exclusiva a hidrólise dos nucleotídeos cíclicos AMPc e GMPc, importantes mensageiros intracelulares que interferem na osmorregulação, na diferenciação, na infectividade e na sobrevida dos parasitos, o que os torna interessantes alvos para novas abordagens terapêuticas. Desta forma, o objetivo desta tese foi realizar a triagem fenotípica de inibidores de PDEs sobre T. cruzi através de estudos in vitro e in vivo. Este trabalho resultou em sete artigos, nos quais abordamos diferentes aspectos de 66 compostos distribuídos em três classes químicas: pirazolonas, ftalazinonas e imidazois Nos estudos in vitro, consideramos sobre distintas cepas e DTUs (do inglês Discrete Typing Units), a ação anti-T. cruzi, toxicidade e seletividade em relação a células de mamíferos, alterações morfológicas induzidas pelos compostos, potencial efeito sobre a fisiologia de células hospedeiras, impacto da terapia combinada dos compostos junto ao BZ, assim como a validação do alvo mensurada pelos níveis de AMPc em parasitos tratados (ou não) com os inibidores e sequenciamento das isoformas do parasito (cepas Y e Colombiana). Observamos que, com exceção dos imidazois, a maioria dos inibidores apresenta maior atividade sobre as formas intracelulares em relação aos tripomastigotas sanguíneos. Descartamos a possível ação sobre o potencial microbicida da célula hospedeira através da avaliação de efeito pré-infecção, que não resultou em redução da carga parasitária, e da análise sobre amastigotas livres, revelando manutenção da mesma faixa de atividade quando comparadas às intracelulares, o que sugere várias hipóteses: diferenças metabólicas entre as duas formas, cujo biometabolização é mais intensa nas formas proliferativas em relação aos tripomastigotas, e como mostrado presentemente de modo inédito, amastigotas exibem superior efluxo de AMPc, em comparação a formas sanguíneas, além de algumas diferenças acerca da expressão das isoformas de PDEs. Estes fatores podem resultar nos diferentes perfis de susceptibilidade à maioria dos compostos, sendo mais potentes sobre amastigotas Contudo, apesar de não levar à lise das formas sanguíneas, os compostos induziram profundas alterações morfológicas e de viabilidade nos parasitos, gerando impacto em seu potencial infectivo. O candidato mais promissor, a pirazolona NPD-227, foi direcionada para os ensaios in vivo de toxicidade aguda e de eficácia sobre modelos murinos. Confirmamos a baixa toxicidade deste inibidor com NOAEL (do inglês non-observed adverse effects level) de 400 mpk. Sua ação sobre a infecção experimental aguda por T. cruzi, em esquemas de monoterapia e sob co-administração com BZ, revelou um efeito significativamente superior do combo quando comparada à monoterapia, tanto nos níveis de parasitemia como na sobrevida dos animais. As isoformas humanas de PDE desfrutam de uma longa história como alvos farmacológicos para um amplo espectro de condições clínicas e essa realocação de know-how e tecnologia, oferece novas oportunidades para identificação de candidatos terapêuticos para a DC.

Ensaios biológicos in vitro e in vivo de novos compostos sintéticos sobre infecção por Trypanosoma cruzi

Dentre os protozoários causadores de doenças em humanos, destaca-se o Trypanosoma cruzi, agente etiológico da doença de Chagas (DC), descrita em 1909 por Carlos Chagas. Os fármacos atualmente disponíveis para o tratamento desta infecção, benzonidazol (BZ) e nifurtimox, mostram limitada eficácia na fase crônica tardia e estão associados a efeitos adversos graves, ressaltando a urgência no desenvolvimento de novas opções terapêuticas. Neste contexto, nosso grupo vem trabalhando em diferentes frentes na investigação de agentes com potencial antiparasitário. No presente trabalho, foi avaliado um total de 35 compostos, sendo 6 bis-arilimidamidas (AIAs) (DB1966, DB1967, DB1968, DB1979, DB1989 e DB1995), 9 mono-arilimidamidas (DB1996, DB1997, DB1980, DB2001, DB2002, DB2003, DB2004, DB2006 e DB2007) e 20 inibidores de fosfodiesterases (PDE) (VUF11848, VUF13522, VUF13526, VUF13527, VUF13536, VUF14256, VUF14656, VUF14703, VUF14767, VUF14768, VUF14852, VUF14907, VUF14975, VUFH1601, GBR11827, BS9538, IOTA0372, IGS1.1, IGS1.18, IGS5.51), comparando eficácia e segurança com BZ. Os resultados mostram que as AIAs dicatiônicas são significativamente mais ativas tanto sobre tripomastigotas sanguíneos como sobre amastigotas intracelulares do parasita quando comparadas aos compostos monocatiônicos, com valores de EC50 na faixa nanomolar (0.03 a 1 \03BCM). Todas as bis-AIAs exibiram atividade superior à do BZ, com destaque para DB1967 e DB1989 que, in vivo, foram capazes de reduzir significativamente os níveis de parasitemia, apesar de não alcançarem uma proteção contra a mortalidade comparável à do BZ. Já em relação aos inibidores de PDE, foi observada uma atividade diferencial a depender da forma evolutiva estudada, sendo os tripomastigotas menos sensíveis do que as formas intracelulares. GBR11827 se destaca por ser a mais ativa sobre a forma sanguínea e a de ação mais rápida (6 h) e com caráter irreversível, enquanto VUF13527 mantém perfil semelhante de atividade sobre as distintas cepas estudadas (Y, Tulahuen e Colombiana) exibindo EC50 entre 0,1 e 0,5 \03BCM. A atividade sobre amastigotas axênicas e a análise de tratamento das células hospedeiras antes da infecção pelo parasito descartaram ação moduladora dos inibidores sobre a célula hospedeira. O conjunto dos dados contribui para o desenvolvimento racional e otimização de novas moléculas destas classes, com o objetivo de identificar novas alternativas para o tratamento da DC.