Cathepsin L inhibitors with activity against the liver fluke identified from a focus library of quinoxaline 1,4-di-N-Oxide derivatives

This article belongs to the Special Issue Recent Trends on Enzymes Inhibitors and Activators in Drug Research IIInfections caused by Fasciola species are widely distributed in cattle and sheep causing significant economic losses, and are emerging as human zoonosis with increasing reports of human cases, especially in children in endemic areas. The current treatment is chemotherapeutic, triclabendazole being the drug of preference since it is active against all parasite stages. Due to the emergence of resistance in several countries, the discovery of new chemical entities with fasciolicidal activity is urgently needed. In our continuous search for new fasciolicide compounds, we identified and characterized six quinoxaline 1,4-di-N-oxide derivatives from our in-house library. We selected them from a screening of novel inhibitors against FhCL1 and FhCL3 proteases, two essential enzymes secreted by juvenile and adult flukes. We report compounds C7, C17, C18, C19, C23, and C24 with an IC50 of less than 10 µM in at least one cathepsin. We studied their binding kinetics in vitro and their enzyme-ligand interactions in silico by molecular docking and molecular dynamic (MD) simulations. These compounds readily kill newly excysted juveniles in vitro and have low cytotoxicity in a Hep-G2 cell line and bovine spermatozoa. Our findings are valuable for the development of new chemotherapeutic approaches against fascioliasis, and other pathologies involving cysteine proteases

Cathepsin L3 from fasciola hepatica induces NLRP3 inflammasome alternative activation in murine dendritic cells

The production of IL-1-family cytokines such as IL-1β and IL-18 is finely regulated by inflammasome activation after the recognition of pathogens associated molecular pattern (PAMPs) and danger associated molecular patterns (DAMPs). However, little is known about the helminth-derived molecules capable of activating the inflammasome. In the case of the helminth trematode Fasciola hepatica, the secretion of different cathepsin L cysteine peptidases (FhCL) is crucial for the parasite survival. Among these enzymes, cathepsin L3 (FhCL3) is expressed mainly in the juvenile or invasive stage. The ability of FhCL3 to digest collagen has demonstrated to be critical for intestinal tissue invasion during juvenile larvae migration. However, there is no information about the interaction of FhCL3 with the immune system. It has been shown here that FhCL3 induces a non-canonical inflammasome activation in dendritic cells (DCs), leading to IL-1β and IL-18 production without a previous microbial priming. Interestingly, this activation was depending on the cysteine protease activity of FhCL3 and the NLRP3 receptor, but independent of caspase activation. We also show that FhCL3 is internalized by DCs, promoting pro-IL-1β cleavage to its mature and biologically active form IL-1β, which is released to the extracellular environment. The FhCL3-induced NLRP3 inflammasome activation conditions DCs to promote a singular adaptive immune response, characterized by increased production of IFN-γ and IL-13. These data reveal an unexpected ability of FhCL3, a helminth-derived molecule, to activate the NLRP3 inflammasome, which is independent of the classical mechanism involving caspase activation.Fil: Celias, Daiana Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Corvo, Ileana. Universidad de la República; UruguayFil: Silvane, Leonardo Micael. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Tort, José Francisco. Universidad de la República; UruguayFil: Chiapello, Laura Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Fresno, Manuel. Centro de Biología Molecular Severo Ochoa; EspañaFil: Arranz, Alicia. Centro de Biología Molecular Severo Ochoa; EspañaFil: Motran, Claudia Cristina. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Cervi, Laura Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

Novel and selective inactivators of Triosephosphate isomerase with anti-trematode activity

International audienceTrematode infections such as schistosomiasis and fascioliasis cause signifcant morbidity in an estimated 250 million people worldwide and the associated agricultural losses are estimated at more than US$ 6 billion per year. Current chemotherapy is limited. Triosephosphate isomerase (TIM), an enzyme of the glycolytic pathway, has emerged as a useful drug target in many parasites, including Fasciola hepatica TIM (FhTIM). We identifed 21 novel compounds that selectively inhibit this enzyme. Using microscale thermophoresis we explored the interaction between target and compounds and identifed a potent interaction between the sulfonyl-1,2,4-thiadiazole (compound 187) and FhTIM,which showed an IC50 of 5µM and a Kd of 66nM. In only 4hours, this compound killed the juvenile form of F. hepatica with an IC50 of 3µM, better than the reference drug triclabendazole (TCZ). Interestingly, we discovered in vitro inhibition of FhTIM by TCZ, with an IC50 of 7µM suggesting a previously uncharacterized role of FhTIM in the mechanism of action of this drug. Compound 187 was also active against various developmental stages of Schistosoma mansoni. The low toxicity in vitro in diferent cell types and lack of acute toxicity in mice was demonstrated for this compound, as was demonstrated the efcacy of 187 in vivo in F. hepatica infected mice. Finally, we obtained the frst crystal structure ofFhTIM at 1.9Å resolution which allows us using docking to suggest a mechanism of interaction between compound 187 and TIM. In conclusion, we describe a promising drug candidate to control neglected trematode infections in human and animal health

A nature-inspired design yields a new class of steroids against trypanosomatids

This article belongs to the Special Issue Drug Discovery for Neglected DiseasesChagas disease and Leishmaniasis are neglected endemic protozoan diseases recognized as public health problems by the World Health Organization. These diseases affect millions of people around the world however, efficient and low-cost treatments are not available. Different steroid molecules with antimicrobial and antiparasitic activity were isolated from diverse organisms (ticks, plants, fungi). These molecules have complex structures that make de novo synthesis extremely difficult. In this work, we designed new and simpler compounds with antiparasitic potential inspired in natural steroids and synthesized a series of nineteen steroidal arylideneketones and thiazolidenehydrazines. We explored their biological activity against Leishmania infantum, Leishmania amazonensis, and Trypanosoma cruzi in vitro and in vivo. We also assayed their genotoxicity and acute toxicity in vitro and in mice. The best compound, a steroidal thiosemicarbazone compound 8 (ID_1260) was active in vitro (IC50 200 nM) and in vivo (60% infection reduction at 50 mg/kg) in Leishmania and T. cruzi. It also has low toxicity in vitro and in vivo (LD50 >2000 mg/kg) and no genotoxic effects, being a promising compound for anti-trypanosomatid drug development

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery